Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods [3 ed.] 0128150416, 9780128150412

Table of contents :
Cover
Toxoplasma Gondii: The Model
Apicomplexan—Perspectives
and Methods
Copyright
Dedication
Contents
List of contributors
Preface to the third edition
1 The history and life cycle of Toxoplasma gondii
1.1 Introduction
1.2 The etiological agent
1.3 Parasite morphology and life cycle
1.3.1 Tachyzoites
1.3.2 Bradyzoite and tissue cysts
1.3.3 Enteroepithelial asexual and sexual stages
1.4 Transmission
1.4.1 Congenital
1.4.2 Carnivorism
1.4.3 Fecal–oral
1.5 Toxoplasmosis in humans
1.5.1 Congenital toxoplasmosis
1.5.2 Acquired toxoplasmosis
1.5.2.1 Children
1.5.2.2 Toxoplasmosis in adults
1.5.2.2.1 Lymphadenopathy
1.5.2.2.2 Ocular disease
1.5.2.2.3 Acquired immunodeficiency syndrome epidemic
1.6 Toxoplasmosis in other animals
1.7 Diagnosis
1.7.1 Sabin–Feldman dye test
1.7.2 Detection of IgM antibodies
1.7.3 Direct agglutination test
1.7.4 Detection of Toxoplasma gondii DNA
1.8 Treatment
1.9 Prevention and control
1.9.1 Serologic screening during pregnancy
1.9.2 Hygiene measures
1.9.3 Animal production practices
1.9.4 Vaccination
References
Further reading
2 The ultrastructure of Toxoplasma gondii
2.1 Invasive stage ultrastructure and genesis
2.1.1 Basic ultrastructural morphology
2.1.2 Comparison of the invasive stages
2.1.3 Host cell invasion
2.1.4 Parasitophorous vacuole, intracellular development
2.1.5 Endodyogeny
2.1.5.1 Mitosis
2.1.5.2 Zoite biogenesis
2.2 Coccidian development in the definitive host
2.2.1 Host–parasite relationship
2.2.2 Asexual development
2.2.3 Sexual development
2.2.3.1 Microgametogony and the microgamete
2.2.3.2 Macrogametogony and the macrogamete
2.2.4 Oocyst wall formation
2.2.5 Fertilization
2.2.6 Oocyst and extracellular sporulation
2.2.7 Excystation
2.3 Development in the intermediate host
2.3.1 Tachyzoite development
2.3.2 Stage conversion: tachyzoite to bradyzoite
2.3.3 Structure of the tissue cyst and bradyzoite
2.3.4 Inflammatory changes in the brains of infected mice
2.3.5 Cyst rupture in immune competent hosts
2.3.6 Development in vitro
2.3.6.1 Tachyzoite development in vitro
2.3.6.2 Bradyzoite development in vitro
References
3 Molecular epidemiology and population structure of Toxoplasma gondii
3.1 Introduction
3.2 Genetic markers
3.2.1 Microsatellites
3.2.2 Polymerase chain reaction restriction fragment length polymorphism
3.2.3 Multilocus DNA sequence typing
3.2.4 Serotyping
3.2.5 Whole-genome sequencing
3.2.6 Correspondence between haplogroups, polymerase chain reaction restriction fragment length polymorphism, and microsate...
3.3 Evolutionary history
3.4 Global diversity and population structure
3.4.1 Geographical distribution
3.4.1.1 Europe
3.4.1.2 Africa
3.4.1.3 Asia
3.4.1.4 Australia
3.4.1.5 North America
3.4.1.6 Central and South America
3.4.2 Factors affecting transmission and genetic exchange
3.4.2.1 Biological factors
3.4.2.2 Dynamics of transmission between different environments or hosts
3.4.2.3 Environmental and human factors
3.5 Outbreak investigations
3.6 Toxoplasma genotype and biological characteristics
3.7 Toxoplasma gondii genotype and human disease
3.7.1 Circumstances of isolation and genetic typing
3.7.2 Congenital toxoplasmosis
3.7.3 Postnatally acquired toxoplasmosis in immunocompetent patients
3.7.3.1 Ocular toxoplasmosis
3.7.3.2 Disseminated toxoplasmosis
3.7.4 Postnatally acquired toxoplasmosis in immunocompromised patients
3.8 Conclusion and perspective on Toxoplasma genotype and human disease
References
4 Human Toxoplasma infection
4.1 Clinical manifestations and course
4.1.1 Introduction and history
4.1.2 Postnatally acquired infection in children and adults
4.1.2.1 Adults and older children with primary, acute acquired Toxoplasma gondii infection
4.1.2.2 The special problem of primary infection during gestation
4.1.2.3 Postnatally acquired infection in older children and adults—the chronic infection
4.1.3 Congenital infection
4.1.3.1 The fetus, infant, and older child
4.1.3.2 Congenital toxoplasmosis in different countries
4.1.3.2.1 France and Belgium
4.1.3.2.2 Austria, Germany, The Netherlands, and Italy
4.1.3.2.3 United States
4.1.3.2.4 Brazil
4.1.4 The special problem of ocular disease
4.1.5 Immune-compromised patients
4.1.5.1 HIV-infected patients
4.1.5.2 Persons with cardiac and renal transplants
4.1.5.3 Bone marrow and hematopoietic stem cell transplantation
4.2 Diagnosis of infection with Toxoplasma gondii
4.2.1 Toxoplasma antigens and diagnostic assays
4.2.2 The development of diagnostic assays
4.2.3 Diagnosis of Toxoplasma gondii infection in pregnant women
4.2.3.1 IgG avidity index
4.2.3.2 Combined, two-test strategies
4.2.4 Improvement of enzyme immunoassay tests for Toxoplasma-specific IgG and IgM antibodies
4.2.5 Recombinant IgG assays—adults
4.2.6 Recombinant IgM and IgG assays—newborns
4.2.7 The Toxoplasma-specific IgG avidity index
4.2.8 Molecular and other diagnostic techniques
4.2.9 Diagnosis of Toxoplasma gondii infection in newborn infants
4.2.10 Prompt diagnosis during gestation to facilitate treatment with unique spillover benefits
4.2.11 Immune-compromised patients
4.3 Treatment
4.3.1 Asymptomatic infection or latent infection
4.3.2 Acute/acquired toxoplasmosis
4.3.3 Acute/acquired toxoplasmosis during pregnancy
4.3.4 Congenital toxoplasmosis
4.3.5 Ocular toxoplasmosis
4.3.6 Toxoplasma infection in immune-compromised persons
4.3.7 Future development of newer improved anti–T. gondii agents
4.4 Prevention
4.5 Other considerations of pathogenesis in human infections
4.5.1 Recent studies of clinically identified associations of human brain or other diseases and presence of Toxoplasma infe...
4.5.2 Structural and functional neuroimaging in uninfected versus infected persons without recognized clinical symptoms
4.5.3 Genetic analyses: candidate human genes in cohort and transmission disequilibrium testing studies
4.5.3.1 National Collaborative Chicago-Based, Congenital Toxoplasmosis Study (sometimes EMSCOT) gestational and congenital ...
4.5.3.2 Case report and literature review concerning mutations and susceptibility to severe disease when infected with Toxo...
4.5.3.3 Brazil
4.5.3.4 Colombia
4.5.3.5 Poland
4.5.4 Signature pathways in neuronal stem cells, peripheral blood monocytic cells, and retinal cells modified by Toxoplasma...
4.6 Conclusion, unifying concepts, and toward the future
References
Further reading
5 Ocular disease due to Toxoplasma gondii
5.1 Introduction
5.2 Historical landmarks in ocular toxoplasmosis
5.3 Epidemiology
5.4 Pathophysiology: lessons from animal models and clinical studies
5.5 Host factors
5.6 Parasite factors
5.7 Animal models
5.8 Clinical characteristics
5.8.1 Recurrence
5.8.2 Congenital ocular toxoplasmosis
5.8.3 Ocular presentation in the elderly
5.8.4 Atypical presentations of ocular toxoplasmosis
5.8.4.1 Immunocompromised patients
5.8.4.2 Acute retinal necrosis
5.8.4.3 Punctate outer retinal toxoplasmosis
5.8.4.4 Other atypical clinical presentations
5.8.5 Classification systems for uveitis and retinochoroiditis
5.8.5.1 Anterior uveitis
5.8.5.2 Vitritis
5.8.5.3 Retinochoroiditis
5.8.6 Optic nerve involvement in ocular toxoplasmosis
5.8.7 Toxoplasma and glaucoma
5.9 Diagnostic tests
5.9.1 Histopathology
5.9.2 Ocular biopsies
5.9.3 Serology
5.9.4 Immunoblotting
5.9.5 Polymerase chain reaction
5.9.6 Clinical tissue culture systems
5.9.7 Ocular imaging
5.9.7.1 Fundus color photographs
5.9.7.2 Fluorescein angiography and indocyanine green angiography
5.9.7.3 Confocal scanning laser ophthalmoscopy
5.9.7.4 Fundus autofluorescence
5.9.7.5 Optical coherent tomography
5.9.7.6 Ultrasonography
5.10 Differential diagnosis
5.11 The treatment and management of ocular toxoplasmosis
5.11.1 Drug treatment of ocular toxoplasmosis
5.11.2 Corticosteroids
5.11.3 Laser treatment
5.11.4 Subconjunctival therapy
5.11.5 Surgical therapy
5.11.6 Intravitreal therapy
5.11.7 Prophylactic therapy
5.12 Conclusion
References
6 Toxoplasmosis in wild and domestic animals
6.1 Introduction
6.2 Toxoplasmosis in wildlife
6.2.1 Felids
6.2.2 Canids
6.2.3 Bears
6.2.4 Raccoons
6.2.5 Squirrels
6.2.6 Rabbits and hares
6.2.7 Skunks and fisher
6.2.8 Beavers
6.2.9 Woodchuck and other large rodents
6.2.10 Insectivores
6.2.11 Bats
6.2.12 White-tailed and mule deer
6.2.13 Other deer
6.2.14 Other wild ruminants
6.2.15 Sea otters and other marine mammals
6.2.16 New world monkeys
6.2.17 Old world monkeys
6.2.18 American marsupials
6.2.19 Australian marsupials
6.2.20 African wildlife
6.2.21 Wild rodents
6.2.22 Wild birds
6.3 Toxoplasmosis in zoos
6.4 Toxoplasma gondii and endangered species
6.5 Toxoplasmosis in pets
6.5.1 Cats
6.5.2 Dogs
6.5.3 Ferrets
6.6 Domestic farm animals
6.6.1 Mink
6.6.2 Horses
6.6.3 Swine
6.6.4 Cattle
6.6.5 Sheep
6.6.6 Goats
6.6.7 Buffalos
6.6.8 Camels
6.6.9 Llamas, alpaca, and vicunas
6.6.10 Chickens
6.6.11 Turkeys
6.6.12 Ducks and geese
6.7 Fish, reptiles, and amphibians
References
Further reading
7 Toxoplasma animal models and therapeutics
7.1 Introduction
7.2 Congenital toxoplasmosis
7.2.1 Mouse
7.2.2 Rat
7.2.3 Calomys callosus
7.2.4 Hamster
7.2.5 Guinea pig
7.2.6 Primate
7.2.7 Rabbit
7.2.8 Other animals
7.3 Ocular toxoplasmosis
7.3.1 Models based on local eye infection
7.3.2 Models based on infection via the carotid artery
7.3.3 Models based on systemic infection
7.4 Cerebral toxoplasmosis
7.4.1 Acute infection models
7.4.2 Localized brain infection models
7.4.3 Progressive Toxoplasma encephalitis models
7.4.4 Chronic relapsing infection models (reactivated toxoplasmosis)
7.4.5 Latent infection models
References
8 Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake
8.1 Introduction
8.2 Fatty acids
8.2.1 Fatty acid biosynthetic pathways—generalities
8.2.2 Fatty acid synthesis in Toxoplasma
8.2.3 Fatty acid salvage by Toxoplasma
8.2.4 Fatty acid fluxes in Toxoplasma
8.3 Glycerophospholipids
8.3.1 Phospholipid biosynthetic pathways—generalities
8.3.2 Phospholipid composition and physiological relevance in Toxoplasma
8.3.3 Phospholipid synthesis in Toxoplasma
8.3.4 Phospholipid salvage by Toxoplasma
8.4 Acylglycerols
8.4.1 Acylglycerol biosynthetic pathways—generalities
8.5 Acylglycerol synthesis and storage in Toxoplasma
8.6 Sterols and steryl esters
8.6.1 Sterol lipid biosynthetic pathways—generalities
8.6.2 Sterol salvage and transport in Toxoplasma
8.6.3 Sterol storage in Toxoplasma
8.7 Sphingolipids
8.7.1 Sphingolipid biosynthetic pathways—generalities
8.7.2 Sphingolipid synthesis in Toxoplasma
8.7.3 Sphingolipid salvage by Toxoplasma
8.8 Isoprenoid derivatives
8.8.1 Isoprenoid biosynthetic pathways—generalities
8.8.2 Isoprenoid synthesis in Toxoplasma
8.8.3 Isoprenoid salvage by Toxoplasma
8.9 Concluding remarks
References
Further reading
9 Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicom...
9.1 Introduction
9.2 Purines
9.2.1 Capture and transport
9.2.1.1 Genome analysis of purine transporters in Apicomplexa
9.2.1.2 Model of purine acquisition in Toxoplasma gondii
9.2.1.3 Properties of purine transporters in Apicomplexa
9.2.2 Purine transport in the parasitized host cell
9.2.3 Purine interconversion and salvage pathways in Apicomplexa
9.2.3.1 Purine salvage pathways in Toxoplasma gondii
9.2.3.2 Purine salvage pathways in Cryptosporidium parvum
9.2.3.3 Purine salvage pathways in Plasmodium falciparum
9.2.3.4 Alternative purine pathways in Apicomplexa
9.2.3.5 Polyamines in Apicomplexa
9.3 Pyrimidines
9.3.1 De novo pyrimidine synthesis in Apicomplexa
9.3.1.1 Organization and regulation of carbamoyl phosphate synthetase II in Apicomplexa
9.3.1.2 Pyrimidine biosynthetic pathways in Apicomplexa
9.3.1.3 Indirect inhibition of pyrimidine biosynthesis
9.3.2 Pyrimidine salvage in Apicomplexa
9.3.2.1 Salvage of pyrimidines in Cryptosporidium parvum
9.3.2.2 Salvage of pyrimidines in Plasmodium falciparum
9.3.2.3 Salvage of pyrimidines in Toxoplasma gondii
9.3.3 Pyrimidine synthesis and salvage pathways related to parasite niche
9.3.3.1 Plasmodium falciparum and Cryptosporidium parvum
9.3.3.2 Toxoplasma gondii
9.3.4 Folate pathways and synthesis of thymine nucleotides
9.3.4.1 Biosynthesis of folates in Apicomplexa
9.3.4.2 Antifolate chemotherapy and antifolate resistance
9.3.5 Toxoplasma gondii pyrimidine genetic selection strategies
9.3.6 Uracil auxotrophy, vaccination, and immunity
References
Further reading
10 Metabolic networks and metabolomics
10.1 Introduction
10.2 Genome-scale metabolic modeling
10.2.1 Systems biology approaches for understanding metabolism
10.2.2 Metabolic modeling and analysis of T. gondii
10.2.3 Harmonization of metabolic models with experimental data
10.2.4 Future perspectives
10.3 Central carbon metabolism
10.3.1 Glycolysis
10.3.2 Gluconeogenesis
10.3.3 Pentose phosphate pathway
10.3.4 Tricarboxylic acid cycle, 2-MCC, and the γ-aminobutyric acid shunt
10.3.5 Oxidative phosphorylation
10.3.6 Fatty-acid biosynthesis
10.3.7 Beta-oxidation
10.4 Carbohydrate metabolism
10.4.1 Sugar nucleotide synthesis
10.4.2 Glycosylation pathways in the secretory pathway
10.4.2.1 N-Glycans
10.4.2.2 Glycosylphosphatidylinositol glycolipids
10.4.2.3 O-Glycosylation
10.4.2.4 Nucleocytoplasmic glycosylation
10.4.3 Amylopectin
10.4.3.1 Synthesis and turnover of amylopectin
10.4.3.2 Regulation of amylopectin turnover
10.4.3.3 Amylopectin function
10.5 Vitamins and cofactor metabolism
10.5.1 Overview of vitamins and cofactors
10.5.2 Vitamins: thiamine B1, flavins B2, niacin B3, pantothenate B5, pyridoxal B6, biotin B7, Myo-inositol B8, and folates B9
10.5.2.1 Thiamine biosynthesis
10.5.2.2 Flavins biosynthesis
10.5.2.3 Niacin metabolism
10.5.2.4 Pantothenate biosynthesis for CoA production
10.5.2.5 Pyridoxal-phosphate metabolism
10.5.2.6 Folate and biopterins biosynthesis
10.5.2.7 Myo-inositol and biotin uptake and utilization
10.5.3 Cofactors: shikimate and chorismate, ubiquinone, heme, lipoic-acid, S-adenosyl-methionine, and glutathione
10.5.3.1 Shikimate and chorismate biosynthesis
10.5.3.2 Ubiquinone biosynthesis
10.5.3.3 Heme biosynthesis
10.5.3.4 Lipoic acid metabolism
10.5.3.5 S-Adenosyl-methionine biosynthesis
10.5.3.6 Glutathione biosynthesis and redox metabolism
10.6 Metabolomics approaches
10.6.1 Intracellular metabolite levels
10.6.2 Metabolic foot-printing
10.6.3 Stable isotope labeling approaches
10.6.4 Metabolomic analysis of host tissues
10.7 Discussion and outlook
10.7.1 Computational modeling
10.7.2 Molecular biology
10.7.3 Metabolomics
References
11 The apicoplast and mitochondrion of Toxoplasma gondii
11.1 Introduction
11.2 The apicoplast
11.2.1 History
11.2.2 Evolution
11.2.3 The apicoplast genome
11.2.4 Expression and translation of the apicoplast genome
11.2.5 Apicoplast genome replication
11.2.6 Apicoplast division
11.2.7 Protein trafficking to the apicoplast
11.2.7.1 Targeting sequences
11.2.7.2 Trafficking mechanisms
11.2.8 Drug sensitivities and the phenomenon of “delayed death”
11.2.9 Apicoplast metabolism
11.3 The mitochondrion
11.3.1 Appearance and ultrastructure
11.3.2 Evolution
11.3.3 Replication and expression of the mitochondrial genome
11.3.4 Protein trafficking to the mitochondrion
11.3.5 Oxidative phosphorylation and energy metabolism
11.3.6 Biosynthetic pathways in the mitochondrion
11.3.7 The mitochondrion as a drug target
11.4 Conclusion
References
12 Calcium storage and homeostasis in Toxoplasma gondii
12.1 Introduction
12.2 Fluorescent methods to study calcium in Toxoplasma
12.2.1 Probes for measuring calcium in Toxoplasma gondii
12.2.2 Ca2+ buffers
12.2.3 Genetic indicators
12.3 Regulation of [Ca2+]i in Toxoplasma gondii
12.3.1 Ca2+ transport across the plasma membrane
12.3.2 Calcium storage
12.3.2.1 Endoplasmic reticulum
12.3.2.2 Mitochondria
12.3.2.3 Acidocalcisomes
12.3.2.4 Plant-like vacuole/vacuolar compartment
12.4 Transducing Ca2+ signals
12.4.1 Calcium-binding proteins
12.4.2 Calcium-dependent protein kinases and their function
12.5 Conclusion
References
13 Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii
13.1 Introduction
13.2 Motility
13.3 Regulated secretion of micronemes
13.4 Release of intracellular calcium as a regulatory cascade
13.5 Calcium-dependent protein kinases
13.6 Nucleotide cyclases and cyclic nucleotide phosphodiesterases
13.6.1 Adenylate cyclases
13.6.2 Phosphodiesterases
13.6.3 Cyclic GMP-dependent protein kinase(PKG)
13.6.4 Cyclic AMP-dependent protein kinase (PKA)
13.7 Conclusion and future directions
References
14 Toxoplasma secretory proteins and their roles in parasite cell cycle and infection
14.1 Introduction
14.2 Motility and invasion
14.2.1 Rapid and active processes unique to apicomplexan parasites
14.2.1.1 Motility
14.2.1.2 Invasion
14.2.1.3 Kinematic analysis of invasion process
14.2.1.4 Alternative routes of invasion
14.2.2 Motility and invasion: central role of micronemes
14.2.3 Moving junction formation: cooperative role between micronemes and rhoptries
14.3 Parasitophorous vacuole formation and maturation
14.3.1 Parasitophorous vacuole formation: role of rhoptries
14.3.2 Maturation of the vacuole: a prominent role of dense granules
14.3.2.1 A complex network of tubules and vesicles
14.3.2.2 Pore inside the parasitophorous vacuole membrane
14.3.2.3 Attraction of host organelles and structures to the parasitophorous vacuole membrane
14.3.2.4 Targeting ROPs and GRAs to the PVM and host cell to neutralize host defense
14.4 Egress
14.5 Micronemes
14.5.1 Trafficking of MICs and the biogenesis of microneme subpopulations
14.5.2 Microneme subpopulations
14.5.3 Microneme proteins
14.5.3.1 MICs sharing homologies with structural domains of eukaryotic proteins involved in protein–protein or protein–carb...
14.5.3.1.1 I- or A-domain
14.5.3.1.2 Thrombospondin type 1 (TSR) repeat domain
14.5.3.1.3 Epidermal growth factor-like domain
14.5.3.1.4 Plasminogen, apple, nematode/apple module
14.5.3.1.5 The chitin-binding-like domain
14.5.3.1.6 Galectin-like domain
14.5.3.1.7 Microneme adhesive repeat domain
14.5.3.2 Other MICs
14.5.3.3 MICs assemble in complexes
14.5.3.4 Cytosolic domain of transmembrane MICs
14.5.4 Microneme secretion
14.5.5 Postsecretory traffic of MICs
14.5.5.1 Parasite surface exposition and posterior capping of MICs
14.5.5.2 Proteolytic cleavages during invasion
14.5.6 Why does Toxoplasma gondii exhibit this patchwork of MICs?
14.5.6.1 MIC2: role in attachment and motility
14.5.6.2 MIC8 and claudin-like apicomplexan microneme protein: potential role in triggering rhoptry secretion
14.5.6.3 AMA1 and AMA1 homologs: role in moving junction formation
14.6 Rhoptries
14.6.1 Biogenesis of rhoptries—clustering and tethering to the apical end
14.6.1.1 Rhoptry: a complex organelle with subcompartments
14.6.1.2 Reshaping of the endosomal pathway for rhoptry biogenesis
14.6.1.3 Rhoptry-targeting signals
14.6.1.4 Rhoptry morphogenesis and clustering to apical end
14.6.2 ROPs and RONs processing
14.6.3 Secretion of rhoptries
14.6.4 Rhoptry proteins and functions
14.6.4.1 Rhoptry proteins associated with the cytosolic face of the rhoptry
14.6.4.1.1 Rab11a
14.6.4.1.2 ARO and its partners AIP and ACβ: apical targeting of rhoptries
14.6.4.1.3 Carbonic anhydrase–related protein
14.6.4.2 Integral membrane proteins
14.6.4.2.1 Acyltransferase DHHC7
14.6.4.2.2 Transporters
Na+/H+ exchanger
Transporter facilitator proteins: TFP2 and TFP3
RON11
14.6.4.3 Luminal rhoptry proteins
14.6.4.3.1 Rhoptry neck complex RON2/RON4/RON5/RON8/RON4L1: role in moving junction formation and invasion
14.6.4.3.2 The rhoptry kinase family (ROPKs): effectors to disarm the host immune response
Parasitophorous vacuole membrane–associated ROPKs
ROPK targeted to the host nucleus
14.6.4.3.3 Toxofilin: control of host cell actin polymerization
14.6.4.3.4 Other RONs/ROPs with less characterized functions
14.6.5 Stage-specific expression of ROPs/RONs
14.6.6 Rhoptry lipids
14.7 Dense granules
14.7.1 The dense granule organelles
14.7.2 The dense granule proteins: GRAs and others
14.7.3 Biogenesis of dense granules: features of both constitutive and regulated secretory pathways
14.7.4 Exocytosis of dense granules
14.7.5 Postsecretory trafficking of GRAs
14.7.6 Dense granule protein function
14.7.6.1 GRAs
14.7.6.2 Other dense granule proteins
14.7.7 Stage-specific expression of dense granule proteins
14.7.7.1 Bradyzoite tissue cyst and GRA proteins
14.7.7.2 Merozoite GRA proteins
14.7.7.3 Sporozoite GRA proteins
14.8 Conclusion
References
15 Endomembrane trafficking pathways in Toxoplasma
15.1 Introduction
15.2 Sorting signals of secretory proteins
15.2.1 Trafficking of rhoptry proteins
15.2.2 Trafficking of micronemal proteins
15.2.3 The role of proteolytic maturation of secretory proteins for their transport
15.2.4 Recycling of maternal organelles during replication
15.3 Coding complement of the Toxoplasma gondii membrane-trafficking system
15.3.1 Overview of trafficking in the apicomplexa
15.3.2 Ras-related protein from brain (Rab) GTPases
15.3.3 Other GTPases
15.3.4 Tethers
15.3.5 Soluble N-ethylmaleimide-sensitive factor attachment protein receptors
15.3.6 Endosomal sorting complexes required for transport complexes
15.3.7 Coats
15.3.8 Adaptor proteins and cargo adapters
15.4 Organization of the Toxoplasma gondii membrane trafficking system
15.4.1 Overview
15.4.2 The endoplasmic reticulum
15.4.3 The Golgi
15.4.4 The dense granules
15.4.5 The endosomal system (micronemes, rhoptries, and the vacuolar compartment/plant-like vacuole)
15.5 An integrated model of exocytic trafficking through the membrane trafficking system
15.6 Dynamics of the endolysosomal system
15.6.1 Overview
15.6.2 Fragmentation and reformation of the vacuolar compartment/plant-like vacuole
15.6.3 Interactions between the vacuolar compartment/plant-like vacuole and endosomal-like compartments
15.7 Endocytosis and endocytic trafficking
15.7.1 Overview
15.7.2 Endocytosis of sulfated glycans
15.7.3 Endocytosis of lipids and surface proteins
15.7.4 Endocytosis of host-derived protein
15.8 Comparison of Toxoplasma gondii endosomal trafficking to model systems
15.8.1 Overview of yeast, mammalian, and plant systems
15.8.2 Similarities and distinctions of Toxoplasma gondii versus model systems
15.9 Autophagy
15.9.1 Coding capacity of the core Toxoplasma gondii autophagy machinery
15.9.2 Autophagy in Toxoplasma gondii
15.9.2.1 Canonical degradative autophagy
15.9.2.2 Evidence for stress-activated canonical degradative autophagy
15.9.2.3 Autophagy as part of an integrated stress response
15.9.2.4 A role for canonical autophagy in parasite virulence
15.9.3 Autophagy and differentiation
15.9.4 Noncanonical function of autophagy-related proteins at the apicoplast
15.10 Final remarks
Glossary
References
16 The Toxoplasma cytoskeleton: structures, proteins, and processes
16.1 Morphology
16.1.1 Life cycle and parasite appearance
16.1.2 Inner membrane complex and pellicle-associated structures
16.1.3 Apical structures
16.1.4 Basal structures
16.1.5 The nucleus
16.1.6 Centrioles, centrosomes, and basal bodies
16.2 Cytoskeletal elements
16.2.1 Tubulin, microtubules, microtubule-associated proteins, motors, and MTOC
16.2.2 Alveolins, glideosome-associated proteins with multiple membrane spans, and other inner membrane complex proteins
16.2.3 Actin, actin-like and actin-related proteins, and actin-binding proteins
16.2.4 Myosin motors, the glideosome, and other associated factors
16.3 Putting it all together: processes
16.3.1 Replication
16.3.1.1 Endodyogeny and endopolygeny
16.3.1.2 Nuclear division
16.3.1.3 Assembly of daughter cytoskeleton buds
16.3.1.4 Emergence of daughter parasites
16.3.1.5 The mature basal complex
16.3.2 Motility, invasion, and egress
16.3.2.1 Glideosome assembly, activation, and regulation
16.3.2.2 Actin polymerization for gliding motility in particular
16.3.2.3 Mechanism of conoid extrusion
16.3.2.4 The role of the host cell in invasion and egress
16.3.3 Other critical roles for Toxoplasma actin
16.4 Summary: a story of adaptation, loss, and novel components
References
17 Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages
17.1 Background
17.2 Rhoptry effectors—a potent class of host manipulators
17.3 Dense granule effectors—a second wave of manipulation
17.4 Conclusion
Acknowledgments
References
18 Bradyzoite and sexual stage development
18.1 Introduction
18.2 Bradyzoite and tissue cyst morphology and biology
18.3 The development of tissue cysts and bradyzoites in vitro
18.4 The cell cycle and bradyzoite development
18.5 The stress response and signaling pathways for bradyzoite formation
18.6 Heat shock proteins
18.7 Transcriptional control of bradyzoite genes
18.8 Cyst wall and matrix antigens
18.9 Surface antigens
18.10 Metabolic differences between bradyzoites and tachyzoites
18.11 Genetic studies on bradyzoite biology
18.12 Sexual stage morphology, biology, and antigens
18.13 Sexual stage development in cell culture
18.14 Sexual stage development in a mouse model
18.15 Summary
References
Further reading
19 Development and application of classical genetics in Toxoplasma gondii
19.1 Summary
19.2 Biology of Toxoplasma
19.2.1 Life cycle
19.2.2 Defining the sexual phase
19.2.3 Population structure and major strain types
19.3 Establishment of transmission genetics
19.3.1 Intra-strain crosses and meiosis
19.3.2 Genetic crosses between different lineages
19.3.3 Implications of selfing versus outcrossing for population structure
19.4 Development of genetic mapping
19.4.1 Advances in molecular genetic tools
19.4.2 Development of linkage maps for forward genetic analysis
19.4.3 Limitation of the current linkage maps
19.5 Mapping phenotypic traits by classical genetics
19.5.1 Mapping drug resistance
19.5.2 Mapping quantitative traits
19.5.3 Genetic approaches for defining virulence genes
19.5.3.1 Mapping differences in the type 1×3 cross
19.5.3.2 Mapping differences in the type 2×3 cross
19.5.3.3 Mapping differences in the type 1×2 cross
19.5.3.4 Mapping differences in crosses to “exotic” lineages
19.5.4 Expression quantitative trait locus mapping
19.5.4.1 Using eQTL mapping to characterize mechanisms of strain-specific gene regulation in Toxoplasma
19.5.4.2 Cross-species eQTL mapping: identifying Toxoplasma loci that affect host gene expression
19.5.5 Summary of differences between lineages
19.5.6 Relevance of the mouse model to other species
19.6 Future challenges
19.6.1 Overcoming current limitations
19.6.2 Expanding phenotypic analyses
References
20 Genetic manipulation of Toxoplasma gondii
20.1 Introduction
20.2 The mechanics of making transgenic parasites
20.2.1 Transient transfection
20.2.2 Stable transformation and positive and negative selectable markers
20.2.3 Homologous recombination and random integration
20.2.4 Enhanced genetic manipulation through CRISPR/Cas9
20.3 Using transgenic parasites to study the function of parasite genes
20.3.1 Tagging subcellular compartments
20.3.2 Tagging of parasite proteins
20.3.3 Genetic analysis of essential genes
20.3.3.1 Tetracycline inducible systems
20.3.3.2 Regulation of protein stability
20.3.3.2.1 Destabilization domain (ddFKBP)
20.3.3.2.2 Auxin-based degron system
20.3.3.3 Site-specific recombination
20.3.3.3.1 Excision of LoxP flanked genes
20.3.3.3.2 U1 small nuclear ribonucleic particles–mediated gene silencing
20.3.4 Insertional mutagenesis and promoter trapping as tools of functional genetic analysis
20.3.5 Forward genetic analysis using chemical mutagenesis and complementation cloning
20.4 Perspectives
20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization
20.5.1 Propagation of Toxoplasma tachyzoites in tissue culture
20.5.1.1 Maintenance of human foreskin fibroblast cells
20.5.1.2 Maintenance of tachyzoites
20.5.1.3 Cryopreservation of host cells and parasites
20.5.1.4 Mycoplasma detection and removal
20.5.1.5 Passaging Toxoplasma tachyzoites/bradyzoite cysts in animal
20.5.2 Transfection and stable transformation protocols
20.5.2.1 Transient transfection
20.5.2.2 Selection of stable transformants
20.5.2.3 Restriction enzyme-mediated integration
20.5.2.4 Cloning of transgenic lines by limiting dilution in 96 well plates
20.5.3 Measuring parasite survival and growth
20.5.3.1 Plaque assay
20.5.3.2 Fluorescence assay
20.5.3.3 β-Galactosidase (LacZ) assay
20.5.3.4 Uracil incorporation assay
20.5.4 Live-cell and indirect immunofluorescence microscopy
20.5.5 Cytometry of parasites and infected cells
20.5.6 Disruption of nonessential genes
20.5.6.1 Disruption of nonessential genes using a CAT/YFP positive/negative selection
20.5.6.2 Disruption of nonessential genes using CRISPR/Cas9
20.5.7 Disruption of essential genes
20.5.7.1 Tetracycline inducible systems
20.5.7.1.1 Two-step strategy
20.5.7.1.2 Single-step approach
20.5.7.2 Regulation of protein stability
20.5.7.2.1 Destabilization domain (ddFKBP)
20.5.7.2.2 Auxin-based degron system
20.5.8 Insertional mutagenesis and tag rescue
20.5.9 Chemical mutagenesis
20.5.10 Complementation cloning using Toxoplasma gondii genomic libraries
20.5.11 Recombinering cosmids of Toxoplasma gondii genomic libraries
20.5.12 Safety concerns working with Toxoplasma gondii
References
21 Regulation of gene expression in Toxoplasma gondii
21.1 Introduction
21.2 Transcription in Toxoplasma
21.2.1 The parasite transcriptome and transcriptional regulation
21.2.2 Gene-specific cis-elements
21.2.3 The evolution of APETALA2-related proteins
21.2.4 ApiAP2 structure determination and DNA binding
21.2.5 The function of ApiAP2 proteins
21.2.6 Other factors that regulate gene expression
21.3 Epigenetics in Toxoplasma
21.3.1 Chromatin and chromatin remodeling
21.3.2 Mapping the Toxoplasma epigenome
21.3.2.1 Chromatin signatures in Toxoplasma biology
21.3.3 Histone-modifying enzymes
21.3.3.1 Histone acetylation
21.3.3.2 Histone methylation
21.3.3.3 Other histone covalent modifications
21.3.3.4 SWI2/SNF2 ATPases
21.3.4 Epigenetic mechanisms as drug targets
21.4 Posttranscriptional mechanisms in Toxoplasma
21.4.1 Translational control
21.4.2 Noncoding and small RNA
21.4.3 Other posttranscriptional mechanisms
21.5 Conclusion and future directions
References
22 Proteomics and posttranslational protein modifications in Toxoplasma gondii
22.1 Introduction to Toxoplasma gondii proteomics
22.2 Toxoplasma gondii global proteomics
22.3 Toxoplasma gondii subproteomes
22.4 Toxoplasma gondii posttranslational modifications
22.4.1 Phosphorylation
22.4.2 Ubiquitination
22.4.3 Palmitoylation
22.4.4 Glycosylation
22.4.5 Methylation
22.4.6 Acetylation
22.4.7 Succinyllysine
22.4.8 SUMOylation
22.5 Studies on the function of posttranslational modifications in Toxoplasma. gondii biology
22.5.1 Posttranslational modifications in motility, invasion, and egress
22.5.2 Posttranslational modifications of the inner membrane complex
22.5.3 Posttranslational modifications in transcriptional and posttranscriptional regulation
22.5.4 Posttranslational modifications as regulators of parasite differentiation
22.5.5 Host–parasite interactions
22.6 Interactions of Toxoplasma gondii posttranslational modifications
22.7 Conclusion
References
23 ToxoDB: the functional genomic resource for Toxoplasma and related organisms*
23.1 Introduction
23.2 Data content
23.3 Genome in ToxoDB
23.4 Functional data in ToxoDB
23.5 The ToxoDB home page
23.6 The search strategy system
23.6.1 Running your first search
23.6.2 Understanding and configuring the results page
23.6.3 Building a multistep search strategy
23.6.4 Defining genes based on their phylogenetic profile
23.7 Genomic colocation
23.8 The genome browser
23.9 Data analysis and integration into ToxoDB
23.9.1 Gene list analysis
23.9.2 Analyze my experiment
23.9.3 Galaxy result integration (my datasets)
23.10 Future directions
References
24 Cerebral toxoplasmosis
24.1 Introduction
24.2 Models for understanding cerebral toxoplasmosis
24.3 Mouse and parasite genotype affect central nervous system outcomes
24.4 Overview of the central nervous system
24.5 Parasite entry into the central nervous system
24.5.1 Toxoplasma gondii dissemination to the central nervous system
24.5.2 Unique features of the blood–brain barrier
24.5.3 Breaching the blood–brain barrier
24.6 Brain regions and host cells infected in the brain
24.6.1 Human toxoplasmosis
24.6.2 Rodent cerebral toxoplasmosis
24.7 Control of cerebral toxoplasmosis
24.7.1 Parenchymal central nervous system cells
24.7.1.1 Neurons
24.7.1.2 Astrocytes
24.7.1.3 Microglia
24.7.2 Systemic immune cells
24.7.2.1 Immune cell infiltration into the central nervous system
24.7.2.2 Innate immune cells
24.7.2.2.1 Monocyte-derived macrophages and dendritic cells
24.7.2.2.2 Neutrophils and other granulocytes
24.7.2.3 Adaptive immune cells
24.7.2.3.1 T cells
24.7.2.3.2 Regulatory T cells
24.7.2.3.3 B cells
24.8 Physiologic effects of Toxoplasma gondii on the central nervous system
24.8.1 Effects on animal behavior
24.8.2 Effects on rodent neurophysiology and structure
24.8.3 Effects on human behavior
24.9 Conclusion
References
25 Innate immunity to Toxoplasma gondii
25.1 Introduction
25.2 The intimate relationship between Toxoplasma gondii and its host cells
25.3 Establishment of infection and mucosal immunity
25.4 The role of IL-12-dependent IFN-γ production for innate resistance
25.5 Antigen processing and presentation
25.6 Molecular basis for innate recognition of Toxoplasma gondii
25.6.1 Toll-like receptor and MyD88
25.6.2 Inflammasome-mediated caspase activation
25.7 IFN-γ-dependent cell autonomous immunity
25.7.1 IFN-γ-induced nitrosative and oxidative defense
25.7.2 IFN-γ-induced restriction of nutrients
25.7.3 IFN-γ-inducible GTPases
25.7.3.1 Immunity-related GTPases (IRGs)
25.7.3.2 Guanylate-binding protein IFN-γ-inducible p65 GTPases
25.7.4 Autophagic processes
25.7.5 Cofactors for IFN-γ-dependent effector mechanisms
25.8 Additional immune pathways altered by Toxoplasma gondii
25.8.1 Parasite utilization of host cell pathways
25.8.2 Modulation of signal transducer and activator of transcription pathways
25.8.3 GRA proteins
25.9 Conclusion and perspectives
References
26 Adaptive immunity
26.1 Introduction
26.1.1 αβ T cells
26.1.2 Other adaptive cell types
26.1.3 Dendritic cells: innate sentinels that initiate and shape adaptive immunity
26.2 How is Toxoplasma gondii “seen” by the adaptive immune system?
26.2.1 Antigen presentation by major histocompatibility complex molecules to T cells
26.2.2 Major histocompatibility complex class I presentation
26.2.2.1 The classical major histocompatibility complex I presentation pathway
26.2.2.2 The major histocompatibility complex I cross-presentation (or exogenous) pathways
26.2.3 Major histocompatibility complex I presentation of Toxoplasma gondii antigens
26.2.3.1 The role of secretion
26.2.3.2 The role of actively infected cells
26.2.3.3 The impact of antigen biochemical properties and trafficking
26.2.4 Modulation of the major histocompatibility complex I presentation pathway by Toxoplasma gondii
26.3 Initiation (priming) of T cell responses by dendritic cells
26.4 Major histocompatibility complex class II presentation
26.4.1 Major histocompatibility complex II presentation of Toxoplasma gondii antigens
26.4.2 Modulation of the major histocompatibility complex II presentation pathway by Toxoplasma gondii
26.5 Adaptive immune responses in the intestinal mucosa and associated lymphoid tissues
26.5.1 Early dissemination in the small intestine
26.5.2 Intestinal humoral responses to Toxoplasma gondii
26.5.3 Toxoplasma gondii acute ileitis: a T cell–mediated immune pathology
26.5.4 Th1/Th17 CD4+ T cells are main effectors of intestinal pathology
26.5.5 Treg and intraepithelial lymphocytes protect the host from gut pathology
26.5.6 Intestinal adaptive immunity in chronic phase
26.6 Lymphoid system
26.6.1 The pivotal role of the IL-12/IFN-γ axis
26.6.2 Immunoregulation during Toxoplasma gondii infection
26.6.3 IL-27
26.6.4 IL-10
26.6.5 Glucocorticoids and anti-inflammatory lipids
26.6.6 CD8+ T cells
26.6.6.1 CD8+ T cells play a prominent role in controlling Toxoplasma gondii
26.6.6.2 CD8+ T cell response in susceptible and resistant mouse strains
26.6.6.3 T cell dynamics during infection in vivo
26.6.7 CD4+ T cells
26.6.7.1 Help for CD8+ T cells
26.6.7.2 Immunosuppression and regulatory CD4+ T cells (Treg)
26.6.7.3 Antibody production and T follicular helper cells
26.7 Adaptive immunity in the brain
26.7.1 T cell entry and behavior in the Toxoplasma gondii–infected brain
26.7.1.1 Three ways to enter the brain
26.7.1.2 T cell entry in the Toxoplasma gondii–infected brain
26.7.1.3 Dynamics of Toxoplasma gondii–specific T cells in brain
26.7.1.4 T cell recirculation in the chronically infected brain
26.7.2 Th1 cytokines and cytotoxicity are essential for parasite control in the central nervous system
26.7.3 Roles of CD4+ and CD8+ T cells in infected brain
26.7.4 Resistance to encephalitis is mediated by CD8+ T cells that efficiently recognize tachyzoite-infected neurons
26.7.5 T cell exhaustion
26.7.6 Tissue-resident memory T cells
26.8 Adaptive immunity in the muscle
26.9 Conclusion
References
Appendix A The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection
References
Epilogue
Index
Back Cover

Citation preview

TOXOPLASMA GONDII

TOXOPLASMA GONDII The Model Apicomplexan—Perspectives and Methods THIRD EDITION Edited by

Louis M. Weiss Department of Medicine, Albert Einstein College of Medicine, New York, NY, United States Department of Pathology, Albert Einstein College of Medicine, New York, NY, United States

Kami Kim Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States Global Health Infectious Diseases Research Program, College of Public Health, University of South Florida, Tampa, FL, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/ permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-815041-2 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Cover photograph: Coccidian stage of Toxoplasma gondii in the cat intestine undergoing endopolygeny (asexual division) showing the formation of multiple daughters within the mother cell cytoplasm, while located within an intestinal epithelial cell. Original magnification 8000X. Image provided by Dr. David Ferguson, Oxford University.

Publisher: Andre Gerhard Wolff Acquisitions Editor: Kattie Washington Editorial Project Manager: Xun Wang Production Project Manager: Sreejith Viswanathan Cover Designer: Miles Hitchen Typeset by MPS Limited, Chennai, India

Dedication We would like to thank our families (Lisa, Hannah, Talia, and Oren; Tom, Clayton, and Vaughan) for their patience, understanding, and tolerance during the completion of this book. In addition, we want to thank the Toxoplasma research community for their enthusiasm and contributions to this project. The Toxoplasma research community is legendary for its generosity toward colleagues and new investigators. It has been a unique pleasure to be involved with such a welcoming and intellectually stimulating group of researchers. There have been many key research groups and individual researchers who have contributed to the development of the critical knowledge base required for progress on this pathogen. This book is a testament to these researchers. We would like to dedicate this book to Elmer Pfefferkorn, PhD, Dartmouth College. Elmer’s work paved the way for the explosion in molecular biology, cell biology, and genomic research associated with this organism. Elmer’s intellectual rigor and deep thinking has had a significant influence on current researchers on Toxoplasma gondii, and we are all indebted to his generosity of spirit and profound insights into this pathogen. Louis M. Weiss and Kami Kim Bronx, NY, 2007 Once again we are deeply grateful for the support of our families, especially Tom and Lisa who again displayed patience, understanding, and tolerance during the completion of this revised edition. The second edition of this book is dedicated to the Toxoplasma scientific community, whose dedication and productivity have made this updated volume a necessity. Louis M. Weiss and Kami Kim Bronx, NY, 2013 Yet again we are profoundly grateful for the patience, understanding, and tolerance of our families which was critical for the completion of this revised edition. The third edition of this book is dedicated to the memory of all of our colleagues who have passed away in the last several years. The work that these various researchers performed laid the foundation for the incredible explosion of knowledge seen in this updated edition. They will be deeply missed by the research community. Louis M. Weiss Bronx, NY, 2019 Kami Kim Tampa, FL, 2019

Contents 3.6 Toxoplasma genotype and biological characteristics 95 3.7 Toxoplasma gondii genotype and human disease 97 3.8 Conclusion and perspective on Toxoplasma genotype and human disease 102 Acknowledgments 103 References 103

List of contributors xiii Preface to the third edition xvii 1. The history and life cycle of Toxoplasma gondii J.P. DUBEY

1.1 Introduction 1 1.2 The etiological agent 1 1.3 Parasite morphology and life cycle 1 1.4 Transmission 5 1.5 Toxoplasmosis in humans 8 1.6 Toxoplasmosis in other animals 10 1.7 Diagnosis 11 1.8 Treatment 12 1.9 Prevention and control 12 Acknowledgments 13 References 13 Further reading 19

4. Human Toxoplasma infection RIMA MCLEOD, WILLIAM COHEN, SAMANTHA DOVGIN, LAUREN FINKELSTEIN AND KENNETH M. BOYER

4.1 4.2 4.3 4.4 4.5

Clinical manifestations and course 117 Diagnosis of infection with Toxoplasma gondii 143 Treatment 159 Prevention 172 Other considerations of pathogenesis in human infections 174 4.6 Conclusion, unifying concepts, and toward the future 201 Acknowledgments 201 References 202 Further reading 225

2. The ultrastructure of Toxoplasma gondii DAVID J.P. FERGUSON AND JEAN-FRANC ¸ OIS DUBREMETZ

2.1 Invasive stage ultrastructure and genesis 21 2.2 Coccidian development in the definitive host 32 2.3 Development in the intermediate host 48 References 58

5. Ocular disease due to Toxoplasma gondii JORGE ENRIQUE GOMEZ-MARIN AND ALEJANDRA DE-LA-TORRE

5.1 5.2 5.3 5.4

3. Molecular epidemiology and population structure of Toxoplasma gondii MARIE-LAURE DARDE´, AURE´LIEN MERCIER, CHUNLEI SU, ASIS KHAN AND MICHAEL E. GRIGG

3.1 3.2 3.3 3.4 3.5

Introduction 63 Genetic markers 64 Evolutionary history 77 Global diversity and population structure Outbreak investigations 90

5.5 5.6 5.7 5.8 5.9 5.10

82

vii

Introduction 229 Historical landmarks in ocular toxoplasmosis 229 Epidemiology 231 Pathophysiology: lessons from animal models and clinical studies 234 Host factors 236 Parasite factors 237 Animal models 240 Clinical characteristics 241 Diagnostic tests 257 Differential diagnosis 267

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5.11 The treatment and management of ocular toxoplasmosis 268 5.12 Conclusion 276 Acknowledgments 276 References 276

6. Toxoplasmosis in wild and domestic animals DAVID S. LINDSAY AND J.P. DUBEY

6.1 6.2 6.3 6.4

Introduction 293 Toxoplasmosis in wildlife 293 Toxoplasmosis in zoos 305 Toxoplasma gondii and endangered species 307 6.5 Toxoplasmosis in pets 308 6.6 Domestic farm animals 310 6.7 Fish, reptiles, and amphibians 312 References 312 Further reading 320

7. Toxoplasma animal models and therapeutics ¨ DER, UTZ REICHARD AND UWE GROß CARSTEN G.K. LU

7.1 Introduction 321 7.2 Congenital toxoplasmosis 322 7.3 Ocular toxoplasmosis 333 7.4 Cerebral toxoplasmosis 342 References 354

8. Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake ISABELLE COPPENS AND CYRILLE BOTTE´

8.1 8.2 8.3 8.4 8.5

Introduction 367 Fatty acids 368 Glycerophospholipids 375 Acylglycerols 381 Acylglycerol synthesis and storage in Toxoplasma 381 8.6 Sterols and steryl esters 383 8.7 Sphingolipids 386 8.8 Isoprenoid derivatives 388 8.9 Concluding remarks 390 Acknowledgments 390 References 390 Further reading 395

9. Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa BARBARA A. FOX AND DAVID J. BZIK

9.1 Introduction 397 9.2 Purines 399 9.3 Pyrimidines 420 References 436 Further reading 449

10. Metabolic networks and metabolomics JOACHIM KLOEHN, AARTI KRISHNAN, CHRISTOPHER J. TONKIN, MALCOLM J. MCCONVILLE AND DOMINIQUE SOLDATI-FAVRE

10.1 Introduction 451 10.2 Genome-scale metabolic modeling 452 10.3 Central carbon metabolism 456 10.4 Carbohydrate metabolism 468 10.5 Vitamins and cofactor metabolism 475 10.6 Metabolomics approaches 487 10.7 Discussion and outlook 491 References 492

11. The apicoplast and mitochondrion of Toxoplasma gondii FRANK SEEBER, JEAN E. FEAGIN, MARILYN PARSONS AND GIEL G. VAN DOOREN

11.1 Introduction 499 11.2 The apicoplast 500 11.3 The mitochondrion 528 11.4 Conclusion 539 Acknowledgments 539 References 539

12. Calcium storage and homeostasis in Toxoplasma gondii DOUGLAS A. PACE, SILVIA N.J. MORENO AND SEBASTIAN LOURIDO

12.1 Introduction 547 12.2 Fluorescent methods to study calcium in Toxoplasma 548 12.3 Regulation of [Ca21]i in Toxoplasma gondii 12.4 Transducing Ca21 signals 561 12.5 Conclusion 568

555

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Acknowledgments References 569

569

13. Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii KEVIN M. BROWN, CHRISTOPHER J. TONKIN, OLIVER BILLKER AND L. DAVID SIBLEY

13.1 13.2 13.3 13.4

Introduction 577 Motility 579 Regulated secretion of micronemes 582 Release of intracellular calcium as a regulatory cascade 584 13.5 Calcium-dependent protein kinases 585 13.6 Nucleotide cyclases and cyclic nucleotide phosphodiesterases 590 13.7 Conclusion and future directions 598 Acknowledgments 598 References 599

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection MARYSE LEBRUN, VERN B. CARRUTHERS AND MARIE-FRANCE CESBRON-DELAUW

14.1 Introduction 607 14.2 Motility and invasion 609 14.3 Parasitophorous vacuole formation and maturation 614 14.4 Egress 618 14.5 Micronemes 618 14.6 Rhoptries 642 14.7 Dense granules 668 14.8 Conclusion 684 Acknowledgments 684 References 684

15. Endomembrane trafficking pathways in Toxoplasma SE´BASTIEN BESTEIRO, CHRISTEN M. KLINGER, MARKUS MEISSNER AND VERN B. CARRUTHERS

15.1 Introduction 705 15.2 Sorting signals of secretory proteins 706 15.3 Coding complement of the Toxoplasma gondii membrane-trafficking system 709 15.4 Organization of the Toxoplasma gondii membrane trafficking system 713

15.5 An integrated model of exocytic trafficking through the membrane trafficking system 716 15.6 Dynamics of the endolysosomal system 721 15.7 Endocytosis and endocytic trafficking 722 15.8 Comparison of Toxoplasma gondii endosomal trafficking to model systems 724 15.9 Autophagy 726 15.10 Final remarks 732 Acknowledgments 733 Glossary 733 References 734

16. The Toxoplasma cytoskeleton: structures, proteins, and processes NAOMI MORRISSETTE AND MARC-JAN GUBBELS

16.1 16.2 16.3 16.4

Morphology 743 Cytoskeletal elements 750 Putting it all together: processes 769 Summary: a story of adaptation, loss, and novel components 779 Acknowledgments 779 References 779

17. Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages JOHN C. BOOTHROYD AND MOHAMED-ALI HAKIMI

17.1 Background 789 17.2 Rhoptry effectors—a potent class of host manipulators 792 17.3 Dense granule effectors—a second wave of manipulation 796 17.4 Conclusion 801 Acknowledgments 802 References 802

18. Bradyzoite and sexual stage development ANTHONY P. SINAI, LAURA J. KNOLL AND LOUIS M. WEISS

18.1 Introduction 807 18.2 Bradyzoite and tissue cyst morphology and biology 809 18.3 The development of tissue cysts and bradyzoites in vitro 814 18.4 The cell cycle and bradyzoite development 818

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18.5 The stress response and signaling pathways for bradyzoite formation 820 18.6 Heat shock proteins 823 18.7 Transcriptional control of bradyzoite genes 826 18.8 Cyst wall and matrix antigens 829 18.9 Surface antigens 834 18.10 Metabolic differences between bradyzoites and tachyzoites 836 18.11 Genetic studies on bradyzoite biology 839 18.12 Sexual stage morphology, biology, and antigens 841 18.13 Sexual stage development in cell culture 842 18.14 Sexual stage development in a mouse model 844 18.15 Summary 844 Acknowledgments 845 References 845 Further reading 857

19. Development and application of classical genetics in Toxoplasma gondii MICHAEL S. BEHNKE, JEROEN P.J. SAEIJ AND JON P. BOYLE

19.1 19.2 19.3 19.4 19.5

Summary 859 Biology of Toxoplasma 860 Establishment of transmission genetics 863 Development of genetic mapping 867 Mapping phenotypic traits by classical genetics 871 19.6 Future challenges 889 Acknowledgments 890 References 890

20. Genetic manipulation of Toxoplasma gondii DAMIEN JACOT, SEBASTIAN LOURIDO, MARKUS MEISSNER, LILACH SHEINER, DOMINIQUE SOLDATI-FAVRE AND BORIS STRIEPEN

20.1 Introduction 897 20.2 The mechanics of making transgenic parasites 898 20.3 Using transgenic parasites to study the function of parasite genes 907 20.4 Perspectives 918 20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization 918 Acknowledgments 934 References 934

21. Regulation of gene expression in Toxoplasma gondii KAMI KIM, VICTORIA JEFFERS AND WILLIAM J. SULLIVAN JR.

21.1 21.2 21.3 21.4

Introduction 941 Transcription in Toxoplasma 942 Epigenetics in Toxoplasma 954 Posttranscriptional mechanisms in Toxoplasma 966 21.5 Conclusion and future directions 970 Acknowledgments 971 References 971

22. Proteomics and posttranslational protein modifications in Toxoplasma gondii LOUIS M. WEISS, JONATHAN WASTLING, VICTORIA JEFFERS, WILLIAM J. SULLIVAN JR AND KAMI KIM

22.1 22.2 22.3 22.4

Introduction to Toxoplasma gondii proteomics 983 Toxoplasma gondii global proteomics 988 Toxoplasma gondii subproteomes 990 Toxoplasma gondii posttranslational modifications 993 22.5 Studies on the function of posttranslational modifications in Toxoplasma. gondii biology 999 22.6 Interactions of Toxoplasma gondii posttranslational modifications 1004 22.7 Conclusion 1009 Acknowledgments 1010 References 1010

23. ToxoDB: the functional genomic resource for Toxoplasma and related organisms OMAR S. HARB, JESSICA C. KISSINGER AND DAVID S. ROOS

23.1 Introduction 1021 23.2 Data content 1022 23.3 Genome in ToxoDB 1023 23.4 Functional data in ToxoDB 1023 23.5 The ToxoDB home page 1026 23.6 The search strategy system 1027 23.7 Genomic colocation 1033 23.8 The genome browser 1034 23.9 Data analysis and integration into ToxoDB 1037 23.10 Future directions 1039 Acknowledgments 1039 References 1040

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CONTENTS

24. Cerebral toxoplasmosis ANITA A. KOSHY, TAJIE H. HARRIS AND MELISSA B. LODOEN

24.1 Introduction 1043 24.2 Models for understanding cerebral toxoplasmosis 1043 24.3 Mouse and parasite genotype affect central nervous system outcomes 1044 24.4 Overview of the central nervous system 1045 24.5 Parasite entry into the central nervous system 1045 24.6 Brain regions and host cells infected in the brain 1048 24.7 Control of cerebral toxoplasmosis 1050 24.8 Physiologic effects of Toxoplasma gondii on the central nervous system 1060 24.9 Conclusion 1062 Acknowledgement 1062 References 1062

25. Innate immunity to Toxoplasma gondii DANA G. MORDUE AND CHRISTOPHER A. HUNTER

25.1 Introduction 1075 25.2 The intimate relationship between Toxoplasma gondii and its host cells 1076 25.3 Establishment of infection and mucosal immunity 1077 25.4 The role of IL-12-dependent IFN-γ production for innate resistance 1078 25.5 Antigen processing and presentation 1080 25.6 Molecular basis for innate recognition of Toxoplasma gondii 1081 25.7 IFN-γ-dependent cell autonomous immunity 1085

25.8 Additional immune pathways altered by Toxoplasma gondii 1091 25.9 Conclusion and perspectives 1094 References 1095

26. Adaptive immunity NICOLAS BLANCHARD, ANNA SALVIONI AND ELLEN A. ROBEY

26.1 Introduction 1107 26.2 How is Toxoplasma gondii “seen” by the adaptive immune system? 1111 26.3 Initiation (priming) of T cell responses by dendritic cells 1118 26.4 Major histocompatibility complex class II presentation 1118 26.5 Adaptive immune responses in the intestinal mucosa and associated lymphoid tissues 1120 26.6 Lymphoid system 1123 26.7 Adaptive immunity in the brain 1131 26.8 Adaptive immunity in the muscle 1138 26.9 Conclusion 1139 References 1139

Appendix A: The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection 1147 CRAIG W. ROBERTS AND STUART WOODS

Epilogue 1183 Index 1185

List of contributors

Michael S. Behnke Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States Se´bastien Besteiro DIMNP, UMR5235 CNRS, University of Montpellier, Montpellier, France Oliver Billker Department of Molecular Biology, Norrland University Hospital, Umea˚, Sweden Nicolas Blanchard Center for Pathophysiology Toulouse-Purpan (CPTP), INSERM, CNRS, University of Toulouse, Toulouse, France John C. Boothroyd Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, CA, United States Cyrille Botte´ Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, Universite´ Grenoble Alpes, Grenoble, France Kenneth M. Boyer Rush University Center, Chicago, IL, United States

Medical

Jon P. Boyle Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, United States Kevin M. Brown Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, United States David J. Bzik Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States Vern B. Carruthers Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, United States

University Bloomberg School of Public Health, Baltimore, MD, United States Marie-Laure Darde´ INSERM, Univ. Limoges, CHU Limoges, UMR 1094, Tropical Neuroepidemiology, Institute of Epidemiology and Tropical Neurology, Limoges, France; Centre National de Re´fe´rence Toxoplasmose/Toxoplasma Biological Resource Center, CHU Limoges, Limoges, France Alejandra de-la-Torre NeURos Group, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogota´, Colombia Samantha Dovgin The University of Chicago, Chicago, IL, United States J.P. Dubey Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD, United States Jean-Franc¸ois Dubremetz UMR CNRS 5235, University of Montpellier, Montpellier, France Jean E. Feagin Department of Global Health, University of Washington, Seattle, WA, United States David J.P. Ferguson Nuffield Department of Clinical Laboratory Science, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom Lauren Finkelstein The University of Chicago, Chicago, IL, United States

Chicago,

Barbara A. Fox Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States

Isabelle Coppens Department of Molecular Microbiology and Immunology, Johns Hopkins

Jorge Enrique Gomez-Marin GEPAMOL Group, Centro de Investigaciones Biome´dicas, Universidad del Quindı´o, Armenia, Colombia

Marie-France Cesbron-Delauw UMR 5525 CNRS, University of Grenoble, Grenoble, France William Cohen The University Chicago, IL, United States

of

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LIST OF CONTRIBUTORS

Michael E. Grigg Laboratory of Parasitic Diseases, Molecular Parasitology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Uwe Groß Institute for Medical Microbiology, University Medical Center, University of Go¨ttingen, Go¨ttingen, Germany Marc-Jan Gubbels Department of Biology, Boston College, Chestnut Hill, MA, United States Mohamed-Ali Hakimi Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions and Immunity to Infection, INSERM U1209, University Grenoble Alpes, Grenoble, France Omar S. Harb Department of Biology, University of Pennsylvania, Philadelphia, PA, United States Tajie H. Harris Department of Neuroscience, Center for Brain Immunology and Glia, University of Virginia, Charlottesville, VA, United States Christopher A. Hunter Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States Damien Jacot Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland Victoria Jeffers Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, United States Asis Khan Laboratory of Parasitic Diseases, Molecular Parasitology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Kami Kim Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States; Global Health Infectious Diseases Research Program, College of Public Health, University of South Florida, Tampa, FL, United States Jessica C. Kissinger Center for Tropical & Emerging Global Diseases, Department of Genetics & Institute of Bioinformatics, University of Georgia, Athens, GA, United States Christen M. Klinger Division of Infectious Diseases, Department of Medicine, University of Alberta, Edmonton, AB, Canada

Joachim Kloehn Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland Laura J. Knoll Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, United States Anita A. Koshy Department of Neurology, University of Arizona, Tucson, AZ, United States; Department of Immunobiology, University of Arizona, Tucson, AZ, United States; BIO5 Institute, University of Arizona, Tucson, AZ, United States Aarti Krishnan Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland Maryse Lebrun UMR 5235 CNRS, University of Montpellier, Montpellier, France David S. Lindsay Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, United States Melissa B. Lodoen Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, United States; Institute for Immunology, University of California, Irvine, CA, United States Sebastian Lourido Whitehead Institute for Biomedical Research, Cambridge, MA, United States; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States Carsten G.K. Lu¨der Institute for Medical Microbiology, University Medical Center, University of Go¨ttingen, Go¨ttingen, Germany Malcolm J. McConville Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia; Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia Rima McLeod The University of Chicago, Chicago, IL, United States Markus Meissner Experimental Parasitology, Department of Veterinary Sciences, Ludwig Maximilians University, Munich, Germany; Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-University, Munich, Germany

LIST OF CONTRIBUTORS

Aure´lien Mercier INSERM, Univ. Limoges, CHU Limoges, UMR 1094, Tropical Neuroepidemiology, Institute of Epidemiology and Tropical Neurology, Limoges, France; Centre National de Re´fe´rence Toxoplasmose/Toxoplasma Biological Resource Center, CHU Limoges, Limoges, France Dana G. Mordue Department of Microbiology and Immunology, New York Medical College, Valhalla, NY, United States Silvia N.J. Moreno Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, United States

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Lilach Sheiner Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary & Life Sciences, Sir Graeme Davies Building, University of Glasgow, Glasgow, United Kingdom L.

David Sibley Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, United States

Anthony P. Sinai Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky, College of Medicine, Lexington, KY, United States

Naomi Morrissette Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, United States

Dominique Soldati-Favre Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland

Douglas A. Pace Department of Biological Sciences, California State University Long Beach, Long Beach, CA, United States

Boris Striepen Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, United States

Marilyn Parsons Department of Global Health, University of Washington, Seattle, WA, United States; Department of Pediatrics, University of Washington, Seattle, WA, United States; Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA, United States

Chunlei Su Department of Microbiology, The University of Tennessee, Knoxville, TN, United States

Utz Reichard Institute for Medical Microbiology, University Medical Center, University of Go¨ttingen, Go¨ttingen, Germany; Amedes MVZ Wagnerstibbe for Medical Microbiology, Infectious Diseases, Hygiene and Tropical Medicine, Go¨ttingen, Germany Craig W. Roberts University Glasgow, United Kingdom

of

Strathclyde,

Ellen A. Robey Department of Molecular and Cell Biology, University of California, Berkeley, CA, United States David S. Roos Department of Biology, University of Pennsylvania, Philadelphia, PA, United States Jeroen P.J. Saeij Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, United States Anna Salvioni Center for Pathophysiology Toulouse-Purpan (CPTP), INSERM, CNRS, University of Toulouse, Toulouse, France Frank Seeber FG16: Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute, Berlin, Germany

William J. Sullivan, Jr. Departments of Pharmacology & Toxicology, Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, IN, United States; Department of Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, IN, United States Christopher J. Tonkin Division of Infectious Disease and Immune Defence, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia Giel G. van Dooren Research School of Biology, Australian National University, Canberra, ACT, Australia Jonathan Wastling Faculty of Natural Sciences, University of Keele, Keele, Staffordshire, United Kingdom Louis M. Weiss Department of Pathology Albert Einstein College of Medicine, Bronx, NY, United States; Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, United States Stuart Woods University of Strathclyde, Glasgow, United Kingdom

Preface to the third edition

Toxoplasma gondii is a ubiquitous, apicomplexan parasite of warm-blooded animals and is one of the most common parasitic infections of humans. Infection can result in encephalitis (especially in immune-compromised hosts), retinochoroiditis, or congenital transmission with fetopathy if a seronegative pregnant woman becomes infected. T. gondii has become a model organism for the study of the Apicomplexa, as it is the most experimentally tractable organism in this important group of intracellular parasites which includes Plasmodium, Eimeria, Cryptosporidium, Neospora, and Theileria. T. gondii is readily amenable to genetic manipulation with refined protocols for both classic and reverse genetics. It has been used for testing the biological or biochemical function of proteins that for one reason or other cannot be readily expressed in other organisms. It is easily propagated and quantified in the laboratory; the mouse animal model is well established; and reagents for study of the host response as well as basic biology of the parasite are widely available. With the recent development of an in vitro method to culture the sexual stage of development, as well as the development of a rodent model that allows oocyte formation, the entire life cycle (tachyzoite, bradyzoite, and oocyst) can be studied in experimental laboratory systems. Immunity to T. gondii is a complex process involving innate and adaptive immune responses. T. gondii has been a useful model system pathogen for understanding the immune response to an intracellular pathogen,

including studies on macrophage function, cell-mediated immunity, dendritic cells, and the gut-associated immune response. The ease with which it can be cultured in vitro, availability of reporter parasite lines, and its pathogenicity in mouse models have facilitated these immunobiology investigations. Because of these experimental advantages, T. gondii has emerged as a major model organism for the study of apicomplexan biology and hostpathogen interactions. Since the publication of the second edition of this book in 2014, the “Omics” revolution has continued to influence all aspects of the Toxoplasma field. Research has been accelerated by the application of CRISPR-Cas9 technology for the genetic manipulation of T. gondii. This has facilitated large-scale screens that have provided, and are providing, critical data on essential genes as well as genes involved in survival within the host and those related to host-pathogen interactions. The application of BioID and other proteomic technologies is further accelerating our understanding of the composition of various structures within this pathogen as well as protein complexes that are essential for its functioning as well as manipulation of its host cell. ToxoDB (https://toxodb. org/toxo/) continues to be a critical resource for the analysis and distribution of these various “omics” datasets, facilitating new insights into the pathobiology of this organism. Unique families of secreted proteins have been identified as modulators of host signaling and gene expression, and the mechanisms by which

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Preface to the third edition

T. gondii alters its host cell and the exquisite dance of host-pathogen interplay is gradually being teased out. Finally, sophisticated imaging techniques are yielding critical insights into the neurobiology and immunology of T. gondii; providing new insights. The first edition of this book was an outgrowth of discussions held at the Seventh International Congress on Toxoplasmosis in Tarrytown, New York in 2003 and the publication of papers and review articles from this congress in March 2004 in the International Journal for Parasitology [volume 34, number 3, pages 249 432]. It was evident at this congress and confirmed at the Eighth International Congress in 2005 (Corsica, France) that the field of study of this pathogen had matured considerably since the First International Congress occurred in 1990 at Dartmouth University (Hanover, NH). This was paralleled by the progressive increase in attendance at this International Congress, which grew from

an initial group of 26 investigators (Fig. P1) to meetings with over 200 participants (see Epilogue Figure E1 from the 15th International Toxoplasmosis Congress), which reflects the increasing number of laboratories working on this organism. The third edition of “Toxoplasma gondii: the model apicomplexan” was assembled to address the numerous advances in the Toxoplasma field in the past 5 years and provide a single volume which would continue to represent the breadth of research efforts and data on this important pathogenic protist. We are fortunate that so many members of the Toxoplasma research community have generously donated their time and expertise to update this book. New authors were added to this edition to revise chapters and provide fresh perspectives on various topics. To this end, the second edition contains other perspectives on several of these topics and provides complementary data to what is found in this

FIGURE P1 Participants in the First International Congress on Toxoplasmosis held 1990 at the Minary Conference Center of Dartmouth College, Squam Lake, Holderness New Hampshire, United States. Bottom: Lloyd Kasper, Rima McLeod, Jean Francois Dubremetz, John Boothroyd, Abott Laboratories (unknown). Middle: Joseph Schwartzman, Elmer Pfefferkorn, Takuro Endo, Louis M. Weiss, Francoise Darcy, Phillippe Thuiliez, Marie France Cesbron, Judy Smith, Alan Johnson, Yasu Suzuki, Takashi Asia, J.P. Dubey. Top: Greg Felice, James L. Fishback, David Sibley, Jack Remington, Alan Sher, Jack Frenkel, David Roos, Keith Joiner, Benjamin Luft, Bill Current.

Preface to the third edition

new edition. Authors of the third edition were encouraged to review older literature comprehensively so that their chapters could serve as free-standing reference articles. In our own laboratory groups, this book has become our standard educational resource. We hope that this new edition will continue to provide important insights into this pathogen, a useful synthesis and summary of the current literature, and suggest new avenues of investigations to current and future investigators in this field.

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We look forward to the ongoing tsunami of research that will enable the field to more thoroughly understand this important parasite and its relationship with its animal and human hosts giving us further insights into how T. gondii has evolved into such a successful intracellular parasite. Louis M. Weiss Bronx, NY, United States, 2019 Kami Kim Tampa, FL, United States, 2019

C H A P T E R

1 The history and life cycle of Toxoplasma gondii J.P. Dubey Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD, United States

based on the morphology (mod. L. toxo 5 arc or bow, plasma 5 life) and the host (Nicolle and Manceaux, 1909). Thus its complete designation is T. gondii (Nicolle and Manceaux, 1908, 1909). In retrospect the correct name for the parasite should have been T. gundii, as Nicolle and Manceaux (1908) had incorrectly identified the host as Ctenodactylus gondi. Splendore (1908, see also English translation Splendore, 2009) discovered the same parasite in a rabbit in Brazil, also erroneously identifying it as Leishmania, but he did not name it. It is a remarkable coincidence that this disease was first recognized in laboratory animals and was first thought to be Leishmania by both groups of investigators.

1.1 Introduction Infections by the protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals on all continents. There are many thousands of references to this parasite in the literature, and it is not possible to give equal treatment to all authors and discoveries (Dubey, 2008). The objective of this chapter is, rather, to provide a history of the milestones in our acquisition of knowledge of the biology of this parasite.

1.2 The etiological agent Nicolle and Manceaux (1908) found a protozoan in tissues of a hamster-like rodent, the gundi, Ctenodactylus gundi, which was being used for leishmaniasis research in the laboratory of Charles Nicolle at the Pasteur Institute in Tunis. They initially believed the parasite to be Leishmania but soon realized that they had discovered a new organism and named it T. gondii

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00001-3

1.3 Parasite morphology and life cycle 1.3.1 Tachyzoites The tachyzoite (Frenkel, 1973) is lunate (Figs. 1.1 and 1.2A) and is the stage that Nicolle and Manceaux (1909) found in the

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© 2020 Elsevier Ltd. All rights reserved.

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FIGURE 1.1

1. The history and life cycle of Toxoplasma gondii

Life cycle of Toxoplasma gondii.

gundi. This stage has also been called trophozoite, the proliferative form, the feeding form, and endozoite. It can infect virtually any cell in the body. It divides by a specialized process called endodyogeny, first described by Goldman et al. (1958). Gustafson et al. (1954) first studied the ultrastructure of the tachyzoite. Sheffield and Melton (1968) provided a complete description of endodyogeny when they fully described its ultrastructure.

1.3.2 Bradyzoite and tissue cysts The term “bradyzoite” (Gr. brady 5 slow) was proposed by Frenkel (1973) to describe the stage encysted in tissues. Bradyzoites are also called cystozoites. Dubey and Beattie (1988) proposed that cysts should be called tissue

cysts (Figs. 1.1, 1.2B, and 1.2C) to avoid confusion with oocysts. It is difficult to determine from the early literature who first identified the encysted stage of the parasite (Lainson, 1958). Levaditi et al. (1928) apparently were the first to report that T. gondii may persist in tissues for many months as “cysts”; however, considerable confusion between the term “pseudocysts” (group of tachyzoites) and tissue cysts existed for many years. Frenkel and Friedlander (1951) and Frenkel (1956) characterized cysts cytologically as containing organisms with a subterminal nucleus and periodic acid Schiff-positive granules (Fig. 1.2C) surrounded by an argyrophilic cyst wall (Fig. 1.2B). Wanko et al. (1962) first described the ultrastructure of the T. gondii cyst and its contents. Jacobs et al. (1960a) first provided a biological characterization of cysts when they

Toxoplasma Gondii

1.3 Parasite morphology and life cycle

3

FIGURE 1.2 Life cycle stages of Toxoplasma gondii. (A) Tachyzoites (arrowhead) in smear. Giemsa stain. Note nucleus dividing into two nuclei (arrow). (B) A small tissue cyst in smear stained with Giemsa and a silver stain. Note the silver-positive tissue cyst wall (arrowhead) enclosing bradyzoites that have a terminal nucleus (arrow). (C) Tissue cyst in section, PAS. Note PAS-positive bradyzoites (arrow) enclosed in a thin PAS-negative cyst wall. (D) Unsporulated oocysts in cat feces. Unstained. PAS, Periodic acid Schiff.

found that the cyst wall was destroyed by pepsin or trypsin, but the cystic organisms were resistant to digestion by gastric juices (pepsinHCl), whereas tachyzoites were destroyed immediately. Thus tissue cysts were shown to be important in the life cycle of T. gondii because carnivorous hosts can become infected by ingesting infected meat. Jacobs et al. (1960b) used the pepsin digestion procedure to isolate viable T. gondii from tissues of chronically infected animals. When T. gondii oocysts were discovered in cat feces in 1970, oocyst excretion was added to the biological description of the cyst (Dubey and Frenkel, 1976). Dubey and Frenkel (1976) performed the first in depth study of the development of tissue cysts and bradyzoites and described their ontogeny and morphology. They found that tissue cysts formed in mice as early as 3 days after their inoculation with tachyzoites.

Cats excrete oocysts (Fig. 1.2D) with a short prepatent period (3 10 days) after ingesting tissue cysts or bradyzoites, whereas after they ingested tachyzoites or oocysts, the prepatent period was longer ($18 days), irrespective of the number of organisms in the inocula (Dubey and Frenkel, 1976; Dubey, 1996, 2001, 2006). Prepatent periods of 11 17 days are thought to result from the ingestion of transitional stages between tachyzoite and bradyzoite (Dubey, 2002, 2005). Wanko et al. (1962) and Ferguson and Hutchison (1987) reported on the ultrastructural of the development of T. gondii tissue cysts. The biology of bradyzoites including morphology, development in cell culture in vivo, conversion of tachyzoites to bradyzoites, and vice versa, tissue cyst rupture, and distribution of tissue cysts in various hosts and tissues was reviewed critically by Dubey et al. (1998).

Toxoplasma Gondii

4

1. The history and life cycle of Toxoplasma gondii

1.3.3 Enteroepithelial asexual and sexual stages Asexual and sexual stages (Figs. 1.3 and 1.4) were reported in the intestine of cats in 1970 (Frenkel, 1970). Dubey and Frenkel (1972) described the asexual and sexual development of T. gondii in enterocytes of the cat and designated the asexual enteroepithelial stages as Types A through E (Figs. 1.3 and 1.4) rather than as generations conventionally known as schizonts in other coccidian parasites. These stages were distinguished morphologically from tachyzoites (Fig. 1.3D) and bradyzoites, which also occur in cat intestine. The challenge was to distinguish different

stages in the cat intestine because there was profuse multiplication of T. gondii 3 days postinfection (Fig. 1.4A). The entire cycle was completed in 66 hours after feeding tissue cysts to cats (Dubey and Frenkel, 1972). There are reports on the ultrastructure of schizonts (Sheffield, 1970; Piekarski et al., 1971; Ferguson et al., 1974), gamonts (Ferguson et al., 1974, 1975; Speer and Dubey, 2005), oocysts, and sporozoites (Christie et al., 1978; Ferguson et al., 1979a,b; Speer et al., 1998; Freppel et al., 2019). In 2005 Speer and Dubey described the ultrastructure of asexual enteroepithelial Types B through E and distinguished their merozoites.

FIGURE 1.3 Asexual and sexual stages of Toxoplasma gondii in sections of small intestine of cats fed tissue cysts. H&E stain. (A) Type C (arrow) schizont with a residual body and a Type B schizont with a hypertrophied host cell nucleus (arrowhead). 52 h p.i. (B) Heavily infected small intestine with schizonts in and gamonts in the epithelium. Five days p.i. (C) Type D and E schizonts (a and d), a mature female gamont (e), young female gamont (b), and two male gamonts (c) in the epithelium. (D) Tachyzoites in the lamina propria (arrows). Types B and D schizonts are below the enterocyte nucleus and often cause hypertrophy of the parasitized cell whereas Types D and E schizonts are always above the enterocyte nucleus and do not cause hypertrophy of the host cell even in hyperparastized cases. Tachyzoites are found in the lamina propria of the cat intestine.

Toxoplasma Gondii

1.4 Transmission

5

animals, particularly sheep, goats, and rodents. Congenital infections can be repeated in some strains of mice (Beverley, 1959), with infected mice producing congenitally infected offspring for at least 10 generations. Beverley discontinued his experiments because of high mortality in some lines of congenitally infected mice and because the progeny from the last generation of infected mice was seronegative and presumed not to be infected with T. gondii. Jacobs (1964) repeated these experiments and found that congenitally infected mice may be infected but not develop antibodies because of immune tolerance. Dubey et al. (1995a) isolated viable T. gondii from seronegative naturally infected mice. These findings are of epidemiological significance.

1.4.2 Carnivorism FIGURE 1.4 Smears of intestinal epithelium of a cat 7 days after feeding tissue cysts (Giemsa stain). (A) Note different sizes of merozoites (a c), schizont with three nuclei (d), schizont with six or more nuclei and merozoites are budding from the surface (e), and a multinucleated schizont (f). (B) Four biflagellated microgametes (arrows) and merozoites (arrowhead) for size comparison.

1.4 Transmission 1.4.1 Congenital The mechanism of transmission of T. gondii remained a mystery until its life cycle was discovered in 1970. Soon after the initial discovery of the organism, it was found that the C. gundi were not infected in the wild and had acquired T. gondii infection in the laboratory. Initially, transmission by arthropods was suspected, but this was never proven (Frenkel, 1970, 1973). Congenital T. gondii infection in a human child was initially described by Wolf et al. (1939a,b) and later found to occur in many species of

Congenital transmission occurs too rarely to explain widespread infection in man and animals worldwide. Weinman and Chandler (1954) suggested that transmission might occur through the ingestion of undercooked meat. Jacobs et al. (1960a) provided evidence to support this idea by demonstrating the resistance to proteolytic enzymes of T. gondii derived from cysts. They found that the cyst wall was immediately dissolved by such enzymes but the released bradyzoites survived long enough to infect the host. This hypothesis of transmission through the ingestion of infected meat was experimentally tested by Desmonts et al. (1965) in an experiment with children in a Paris sanitorium. They compared the acquisition rates of T. gondii infection in children before and after admission to the sanitorium. The 10% yearly acquisition rate of T. gondii antibody rose to 50% after adding two portions of barely cooked beef or horse meat to the daily diet and to a 100% yearly rate after the addition of barely cooked lamb chops. Since the prevalence of T. gondii is much higher in sheep

Toxoplasma Gondii

6

1. The history and life cycle of Toxoplasma gondii

than in horses or cattle, this illustrated the importance of carnivorism in transmission of T. gondii. Epidemiological evidence indicates it is common in humans in some localities where raw meat is routinely eaten. In a survey in Paris, Desmonts et al. (1965) found more than 80% of the adult population sampled had antibodies to T. gondii. Kean et al. (1969) described toxoplasmosis in a group of medical students who had eaten undercooked hamburgers.

1.4.3 Fecal oral While congenital transmission and carnivorism can explain some of the transmission of T. gondii, it does not explain the widespread infection in vegetarians and herbivores. A study in Bombay, India found the prevalence of T. gondii in strict vegetarians to be similar to that in nonvegetarians (Rawal, 1959). Hutchison (1965), a biologist at Strathclyde University in Glasgow, first discovered T. gondii infectivity associated with cat feces. In a preliminary experiment, Hutchison (1965) fed T. gondii cysts to a cat infected with the nematode Toxocara cati and collected feces containing nematode ova. Feces floated in 33% zinc sulfate solution and stored in tap water for 12 months induced toxoplasmosis in mice. This discovery was a breakthrough because, until then, both known forms of T. gondii (i.e., tachyzoites and bradyzoites) were killed by water. Microscopic examination of feces revealed only T. cati eggs and Isospora oocysts. In Hutchison’s report, T. gondii infectivity was not attributed to oocysts or T. cati eggs. He repeated the experiment with two T. cati infected and two T. cati free cats. T. gondii was transmitted only in association with T. cati infection. On this basis, Hutchison (1967) hypothesized that T. gondii was transmitted through nematode ova. He suspected transmission of T. gondii through the eggs of the nematode Toxocara similar to the transmission of the fragile flagellate

Histomonas through Heterakis eggs. Hutchison initially wanted to test the nematode theory using Toxocara canis and T. gondii transmission in the dog but decided on the cat and T. cati model because there was no place to house dogs (J.P. Dubey, 1965, personal communication). Transmission of T. gondii by T. canis eggs made more sense because of the known zoonotic potential of T. canis; T. cati was not, at that time, known to infect humans, but T. canis was. Discovery of the life cycle of T. gondii would have been delayed if Hutchinson had worked with dogs instead of cats. Hutchison’s (1965) report stimulated other investigators to examine fecal transmission of T. gondii through T. cati eggs (Dubey, 1966, 1968; Jacobs, 1967; Hutchison et al., 1968; Frenkel et al., 1969; Sheffield and Melton, 1969). The nematode egg theory of transmission was discarded after Toxoplasma infectivity was dissociated from T. cati eggs (Frenkel et al., 1969), and Toxoplasma infectivity was found in feces of worm-free cats fed T. gondii (Frenkel et al., 1969; Sheffield and Melton, 1969). Finally, in 1970, knowledge of the T. gondii life cycle was completed by discovery of the sexual phase of the parasite in the small intestine of the cat (Frenkel et al., 1970). T. gondii oocysts, the product of schizogony and gametogony, were found in cat feces and characterized morphologically and biologically (Dubey et al., 1970a,b). Several group of workers independently and about the same time found T. gondii oocysts in cat feces (Hutchison et al., 1969, 1970, 1971; Frenkel et al., 1970; Dubey et al., 1970a,b; Sheffield and Melton, 1970; Overdulve, 1970; Weiland and Ku¨hn, 1970; Witte and Piekarski, 1970). The discovery of T. gondii oocyst in cat feces and its implications has been reviewed by Frenkel (1970, 1973) and Garnham (1971). In retrospect the discovery and characterization of the T. gondii oocyst in cat feces was delayed because (1) T. gondii oocysts were

Toxoplasma Gondii

1.4 Transmission

morphologically identical to oocysts of the previously described coccidian parasite of cats and dogs (Dubey et al., 1970a) and (2) until 1970, coccidian oocysts were sporulated in 2.5% potassium dichromate. Chromation of the oocysts wall interfered with excystation of the sporozoites when oocysts were fed to mice and thus the mouse infectivity titer of the oocysts was lower than expected from number of oocysts administered (Dubey et al., 1970a). These findings led to the use of 2% sulfuric acid as the best medium for sporulation and storage of T. gondii oocysts. Unlike dichromate, which was difficult to wash off the oocysts, sulfuric acid could be easily neutralized, and the oocysts could be injected without washing into mice (Dubey et al., 1972). Unlike other coccidians, T. gondii oocysts were found to excyst efficiently when inoculated parenterally into mice and thus alleviated the need for oral inoculation for bioassay of oocysts (Dubey and Frenkel, 1973). Ben Rachid (1970) fed T. gondii oocysts to gundis which died 6 7 days later from toxoplasmosis. This knowledge about the life cycle of T. gondii probably explains how gundis became infected in the laboratory of Nicolle. At least one cat was present in the Pasteur Laboratory in Tunis (Dubey, 1977). Of the many species of animals experimentally infected with T. gondii, only felids excrete T. gondii oocysts (Miller et al., 1972; Jewell et al., 1972; Janitschke and Werner, 1972; Polomoshnov, 1979; Dubey, 2010). Oocysts excreted into the environment have caused several outbreaks of disease in humans (Teutsch et al., 1979; Benenson et al., 1982; Bowie et al., 1997; de Moura et al., 2006). T. gondii oocysts found in the feces of naturally infected cougars (Aramini et al., 1998) were epidemiologically linked to the largest recorded waterborne outbreak of toxoplasmosis in humans (Bowie et al., 1997). Seroepidemiological studies on isolated islands in the Pacific (Wallace, 1969, for full story see

7

Dubey, 2016), Australia (Munday, 1972), and the United States (Dubey et al., 1997) have shown an absence of Toxoplasma on islands without cats, confirming the important role of the cat in the natural transmission of T. gondii. Vaccination of cats with a live mutant strain of T. gondii on eight pig farms in the United States reduced the transmission of T. gondii infection in mice and pigs (Mateus-Pinilla et al., 1999), thus supporting the role of the cat in natural transmission of T. gondii. Historically, before the discovery of the coccidian cycle of T. gondii, coccidian parasites were considered host and site specific and to be transmitted by the fecal oral route. After the discovery of the sexual cycle of T. gondii, several other genera (e.g., Sarcocystis and Besnoitia) were found to be coccidian. Although T. gondii has a wide host range, it has retained the definitivehost specificity restricted to felids. Dr. J.K. Frenkel deserves the credit for initiating testing of many species of animals, including wild felids, for oocyst excretion under difficult (it was not easy handling bob cats and ocelots in cages) housing conditions (Dubey, 2009). Only the felids were found to excrete T. gondii oocysts (Frenkel et al., 1970; Miller et al., 1972). Although T. gondii can be transmitted in several ways, it has adapted to be transmitted most efficiently by carnivorism in the cat and by the fecal oral (oocysts) route in other hosts. Pigs and mice (and presumably humans) can be infected by ingesting even one oocyst (Dubey et al., 1996), whereas 100 oocysts may not infect cats (Dubey, 1996). Cats can excrete millions of oocysts after ingesting only one bradyzoite, while ingestion of 100 bradyzoites may not infect mice orally (Dubey, 2001, 2006). This information has proved very useful in conducting epidemiological studies and for the detection by feeding to cats of low numbers of T. gondii in large samples of meat (Dubey et al., 2005). Understanding the biology of T. gondii oocyst excretion by cats (both wild and domestic is essential for reducing prevalence of

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1. The history and life cycle of Toxoplasma gondii

T. gondii in humans and animals. Although cats are thought to excrete oocysts only once in their life after primary infection, there are no guaranties that they will not do so the second time. Immunity can wane with time (Dubey, 1995); cats have excreted millions of oocysts the second time after challenge with unrelated organisms (Dubey, 1976) or challenge with heterologous T. gondii strains (Zulpo et al., 2018). Therefore pregnant women should avoid contact with cat feces, irrespective of the serological status of the cat. After the discovery of the life cycle of T. gondii in the cat, it became clear why Australasian marsupials and New World monkeys are highly susceptible to clinical toxoplasmosis. The former evolved apparently in the absence of the cat (there were few or no cats in Australia and New Zealand before settlement by Europeans) and the latter lives on tree tops, not exposed to cat feces. In contrast, marsupials in America and Old World monkeys are resistant to clinical toxoplasmosis (Dubey and Beattie, 1988).

1.5 Toxoplasmosis in humans 1.5.1 Congenital toxoplasmosis Three pathologists, Wolf, Cowen, and Paige from New York, United States, first conclusively identified T. gondii in an infant girl who was delivered full term by cesarean section on May 23, 1938, at Babies Hospital, New York (Wolf et al., 1939a,b). The girl developed convulsive seizures at 3 days of age, and lesions were noted in the maculae of both eyes through an ophthalmoscope. She died when a month old and an autopsy was performed. At postmortem, brain, spinal cord, and right eye were removed for examination. Free and intracellular T. gondii were found in lesions of encephalomyelitis and retinitis of the girl. Portions of cerebral cortex and spinal cord

were homogenized in saline and inoculated intracerebrally into rabbits and mice. These animals developed encephalitis, T. gondii was demonstrated in their neural lesions, and T. gondii from these animals was successfully passaged into other mice. Wolf, Cowen, and Paige reviewed in detail their own cases and those reported by others, particularly Janku˚ (1923), and Torres (1927) of T. gondii like encephalomyelitis and chorioretinitis in infants (Wolf and Cowen, 1937, 1938; Wolf et al., 1939a,b, 1940; Cowen et al., 1942; Paige et al., 1942). Janku˚ (1923), an ophthalmologist from Czechoslovakia, was credited earlier with finding a T. gondii like parasite in a human eye. The following description of the case of Janku˚ is taken from the English translation published by Wolf and Cowen (1937): “The patient was born with left microphthalus and became blind at the age of 3 months, and had hydrocephalus. The child died when 11 months old. The eyes and brain were removed at autopsy. Grossly, the child had internal hydrocephalus but the brain was not available for histopathological examination. Chorioretinitis was present in both eyes and cysts-like structures (termed ˚ were seen in the right sporocysts by Janku) eye.” Janku˚ (1923) thought that this parasite was Encephalitozoon (a microsporidia). The material from this case is thought to be destroyed in World War bombing, and so confirmation of these findings is not possible. Torres (1927) found protozoa in lesions of encephalitis in a 2-day-old girl in Rio de Janeiro, Brazil. Numerous organisms were seen, but these were thought to be a new species of Encephalitozoon. This patient also had myocarditis and myositis. In the Netherlands, de Lange (1929) found protozoa in sections of the brain of a 4-month-old child that was born with hydrocephalus. These sections were reexamined by Wolf and Cowen, and a full account was reviewed by Sabin (1942).

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1.5 Toxoplasmosis in humans

Sabin (1942) summarized all that was known of congenital toxoplasmosis in 1942 and proposed typical clinical signs of congenital toxoplasmosis: hydrocephalus or microcephalus, intracerebral calcification, and chorioretinitis. These signs helped in the clinical recognition of congenital toxoplasmosis. Frenkel and Friedlander (1951) published a detailed account of five fatal cases of toxoplasmosis in infants that were born with hydrocephalus; T. gondii was isolated from two of these infants. They described the pathogenesis of internal hydrocephalus as a blockage of the aqueduct of Sylveus due to ventriculitis resulting from a T. gondii antigen antibody reaction. This lesion is unique to human congenital toxoplasmosis and has never been verified in other animals (J.P. Dubey, unpublished). This report was the first in depth description of lesions of congenital toxoplasmosis not only in the central nervous system but also other organs. Hogan (1951) also provided the first detailed clinical description of ocular toxoplasmosis.

1.5.2 Acquired toxoplasmosis 1.5.2.1 Children Sabin (1941) reported toxoplasmosis in a 6-year-old boy from Cincinnati, Ohio. An asymptomatic child with initials of RH was hit with a baseball bat on October 22, 1937. He developed a headache 2 days later and convulsions the day after. He was admitted to the hospital on the 7th day but without obvious clinical signs. Except for lymphadenopathy and enlarged spleen, nothing abnormal was found. He then developed neurological signs and died on the 30th day of illness. The brain and spinal cord were removed for histopathological examination and bioassay. Because of the suspicion of polio virus infection a homogenate of cerebral cortex was inoculated into mice. T. gondii was isolated from the inoculated mice, and this isolate was given the initials of

9

the child and became the famous RH strain. Only small lesions of nonsuppurative encephalitis were found microscopically in the brain of this child. Neither gross lesions and nor any viral or bacterial infections were found. This child most likely had acquired T. gondii infection recently, and the blow to the head was coincidental and unrelated to the onset of symptoms. It is noteworthy that some mice infected with the original RH strain did not die until day 21 postinoculation. By the thirdpassage mice died in 3 5 days after inoculation. The RH strain of T. gondii has since 1938 been passaged in mice in many laboratories. After this prolonged passage, its pathogenicity for mice has been stabilized (Dubey, 1977), and it has lost the capacity to produce oocysts in cats (Frenkel et al., 1976). Additional details of history of toxoplasmosis in humans were given by Dubey (2008) and Weiss and Dubey (2009). 1.5.2.2 Toxoplasmosis in adults Pinkerton and Weinman (1940) identified T. gondii in the heart, spleen, and other tissues of a 22-year-old patient who died in 1937 in Lima, Peru. The patient exhibited fever and concomitant Bartonella sp. infection. Pinkerton and Henderson (1941) isolated T. gondii from blood and tissues of two (50 and 43 years old) individuals who died in St. Louis, Missouri. Recorded symptoms included rash, fever, and malaise. These were the first reports of acute toxoplasmosis in adults without neurological signs. 1.5.2.2.1 Lymphadenopathy

Siim (1956) drew attention to the fact that lymphadenopathy is a frequent sign of acquired toxoplasmosis in adults, and these findings were confirmed by Beverley and Beattie (1958) who reported on the cases of 30 patients. A full appreciation of the clinical symptoms of acquired toxoplasmosis was achieved, when outbreaks of acute toxoplasmosis were reported in adults in the United States (Teutsch et al., 1979) and in Canada (Bowie et al., 1997).

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1. The history and life cycle of Toxoplasma gondii

1.5.2.2.2 Ocular disease

Before 1950 virtually all cases of ocular toxoplasmosis were considered to result from congenital transmission (Perkins, 1961). Wilder (1952) identified T. gondii in eyes that had been enucleated. The significance of this finding lies in the way this discovery was made. These eyes were suspected of being syphilitic, tuberculous, or of having tumors. Wilder was a technician in the registry of Ophthalmic Pathology at Armed Forces Institute of Pathology, Washington, DC, and she routinely microscopically examined the sections that she prepared. She put enormous effort into identify microbes in these “tuberculous” eyes but never identified bacteria or spirochetes by special staining. Then she found T. gondii in the retinas of these eyes. She subsequently collaborated with Jacobs and Cook and found most of these patients with histologically confirmed T. gondii infection had low levels of dye test antibodies (a titer of 1:16), and in one patient antibodies were demonstrable only in undiluted serum (Jacobs et al., 1954a). Jacobs et al. (1954b) made the first isolation of T. gondii from an eye of a 30-year-old male hospitalized at the Walter Reed Army Hospital, Washington, DC. The eye had been enucleated because of pain associated with elevated intraocular pressure. A group of ophthalmologists from southern Brazil initially discovered ocular toxoplasmosis in siblings. Among patients with postnatally acquired toxoplamosis who did not have retinochoidal scars before, 8.3% developed retinal lesions during a 7-year follow-up (Silveira et al., 1988, 2001). Ocular toxoplasmosis was diagnosed in 20 of 95 patients with acute toxoplasmosis associated with the Canadian waterborne outbreak of toxoplasmosis in 1995 (Burnett et al., 1998; also see Holland, 2003). 1.5.2.2.3 Acquired immunodeficiency syndrome epidemic

Before the epidemic of the acquired immunodeficiency syndrome (AIDS) in adults in the

1980s, neurological toxoplasmosis in adults was rarely reported and essentially limited to patients treated for tumors or those given transplants. Luft et al. (1983) reported acute toxoplasmosis-induced encephalitis that was fatal if not treated. In almost all cases, clinical disease occurred as result of reactivation of chronic infection initiated by the depression of intracellular immunity due to HIV infection. Initially, many of these cases of toxoplasmosis in AIDS patients were thought to be lymphoma.

1.6 Toxoplasmosis in other animals Mello (1910) in Turin, Italy, first reported fatal toxoplasmosis in a domestic animal (a 4-month-old dog) that died of acute visceral toxoplasmosis. Over the next 30 years canine toxoplasmosis was reported in Cuba, France, Germany, India, Iraq, Tunisia, USSR, and the United States (Dubey and Beattie, 1988). Campbell et al. (1955) found that most cases of clinical toxoplasmosis were in dogs infected with the canine distemper virus (CDV) infection. Even vaccination with live-attenuated CDV, vaccine can trigger clinical toxoplasmosis in dogs (Dubey et al., 2003a,b). The incidence of clinical toxoplasmosis in dogs has decreased dramatically after vaccination with CDV vaccine became a routine practice. Strangely enough the first case of toxoplasmosis was not reported in a cat until 1942 when Olafson and Monlux found it in a cat from Middletown, New York, United States. In the 1950s and 1960s Galuzo and Zasukhin published in Russian their own studies and those of other researchers on many species of animals from the former USSR. This information was made available to scientists in other countries, when their book was translated into English by Plous Jr. and edited by Fitzgerald (1970). Jı´ra and Kozojed (1970, 1983) published the most comprehensive bibliography of toxoplasmosis, listing more than 12,000 references

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1.7 Diagnosis

and categorizing them by hosts and topics. This work proved useful for literature searches before electronic databases. Toxoplasmosis in sheep deserves special attention because of its economic impact. William Hartley, a well-known veterinary pathologist from New Zealand, and his associates J.L. Jebson and D. McFarlane, discovered T. gondii like organisms in the placentas and fetuses of several unexplained abortions in ewes in New Zealand. They called it New Zealand Type II abortion. Hartley and Marshall (1957) finally isolated T. gondii from aborted fetuses. Hartley (1961) and Jacobs and Hartley (1964) experimentally induced toxoplasmic abortion in ewes. The identification of T. gondii abortion in ewes was a landmark discovery in veterinary medicine. Prior to that protozoa were not recognized as cause of epidemic abortion in livestock. Subsequently, Beverley and Watson (1961) recognized epidemics of ovine abortion in the United Kingdom. Dubey and Towle (1986) and Dubey and Beattie (1988) summarized all that was known about toxoplasmosis in sheep and its impact on agriculture. Millions of lambs are still lost throughout the worldwide due to this infectious disease. Sanger and Cole (1955) were first to isolate T. gondii from a food animal. Dubey and Beattie (1988) and Dubey (2010) reviewed the worldwide literature on toxoplasmosis in humans and other animals. The discovery and naming of two new organisms, Neospora caninum (Dubey et al., 1988) and Sarcocystis neurona (Dubey et al., 1991), that were previously thought to be T. gondii, resulted in new information on the host distribution of T. gondii. We now know that cattle and horses are resistant to clinical T. gondii; there have been no confirmed cases of clinical toxoplasmosis in either cattle or horses (Dubey, 2010). N. caninum was found to be a common cause of abortion in cattle worldwide (Dubey, 2003; Dubey et al., 2017), and S. neurona was found to be a

common cause of fatal encephalomyelitis in horses in the Americas (Dubey et al., 2001). S. neurona was also found to cause systemic disease entities involving eyes, lungs, muscles, and neural tissues in pets and wildlife (Dubey et al., 2015). The finding of T. gondii in marine mammals deserves special mention. Before the discovery of T. gondii oocyst, no one would have suspected that the marine environment would be contaminated with T. gondii and that fisheating marine mammals would be found infected with T. gondii (Dubey et al., 2003a,b; Conrad et al., 2005; Thomas et al., 2007; Miller et al., 2008; Dubey, 2010). Thomas and Cole (1996) and Cole et al. (2000) isolated viable T. gondii from sea otters in the United States. Several reports have now appeared that confirm that T. gondii can occur in many species of marine mammals (Dubey, 2010).

1.7 Diagnosis The current management, diagnosis, and clinical manifestations of T. gondii infection in humans are discussed in Chapter 4, Human Toxoplasma infection.

1.7.1 Sabin Feldman dye test Development of a novel serologic test, the dye test, by Sabin and Feldman (1948), was perhaps the greatest advancement in the field of toxoplasmosis. The dye test is highly sensitive and specific with no evidence for false results in humans. Even titers as low as 1:2 are meaningful for the diagnosis of ocular disease. The ability to identify T. gondii infections based on a simple serological test opened the door for extensive epidemiological studies on the incidence of infection. It became clear that T. gondii infections are widely prevalent in humans in many countries. It also

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1. The history and life cycle of Toxoplasma gondii

demonstrated that the so-called tetrad of clinical signs considered indicative of clinical congenital toxoplasmosis occurred in other diseases and assisted in the differential diagnosis (Sabin and Feldman, 1949; Feldman and Miller, 1956).

1.7.2 Detection of IgM antibodies Remington et al. (1968) first proposed the usefulness of the detection of IgM antibodies in cord blood or infant serum for the diagnosis of congenital toxoplasmosis since IgM antibodies do not cross the placenta, whereas IgG antibodies do cross the placenta. Remington (1969) modified the indirect fluorescent antibody test and the ELISA (Naot and Remington, 1980) to detect IgM in cord blood. Desmonts et al. (1981) developed a modification of IgM-ELISA, combining it with the agglutination test (IgMISAGA) to eliminate the necessity for an enzyme conjugate. Although IgM tests are not perfect, they have proved useful for screening programs (Remington et al., 2001).

1.7.3 Direct agglutination test The development of a simple direct agglutination test has aided tremendously in the serological diagnosis of toxoplasmosis in humans and other animals. In this test, no special equipment or conjugates are needed. This test was initially developed by Fulton (1965) and improved by Desmonts and Remington (1980) and Dubey and Desmonts (1987) who called it the modified agglutination test (MAT). The MAT has been used extensively for the diagnosis of toxoplasmosis in animals (Dubey, 2010). The sensitivity and specificity of MAT has been validated by comparing serologic data and isolation of the parasite from naturally and experimentally infected pigs (Dubey, 1997; Dubey et al., 1995a,b) and naturally infected

chickens (Dubey et al., 2016). Even low MAT titers (,1:10) were found specific.

1.7.4 Detection of Toxoplasma gondii DNA Burg et al. (1989) first reported detection of T. gondii DNA from a single tachyzoite using the B1 gene in a polymerase chain reaction (PCR). Several subsequent PCR tests have been developed using different gene targets. Overall, this technique has proven very useful in the diagnosis of clinical toxoplasmosis (Remington et al., 2011).

1.8 Treatment Sabin and Warren (1942) reported the effectiveness of sulfonamides against murine toxoplasmosis, and Eyles and Coleman (1953) discovered the synergistic effect of combined therapy with sulfonamides and pyrimethamine; the latter is the standard therapy for toxoplasmosis in humans (Remington et al., 2001). Garin and Eyles (1958) found spiramycin to have antitoxoplasmic activity of in mice. Since spiramycin is nontoxic and does not cross placenta, it has been used prophylactically in women during pregnancy to reduce transmission of the parasite from mother to fetus (Desmonts and Couvreur, 1974b).

1.9 Prevention and control 1.9.1 Serologic screening during pregnancy Georges Desmonts initiated studies in Paris, France in the 1960s looking at seroconversion in women during pregnancy and the transmission of T. gondii to the fetus. Blood was obtained at the first visit, at 7 months,

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References

13

and at the time of parturition. Desmonts initiated prophylactic treatment of women who seroconverted during pregnancy. Results of the 15-year study demonstrated that (1) infection acquired during the first two trimesters was most damaging to the fetus, (2) not all women that acquired infection transmitted it to the fetus, (3) women seropositive prior to pregnancy did not transmit infection to the fetus, and (4) treatment with spiramycin reduced congenital transmission, but not clinical disease in infants (Desmonts and Couvreur, 1974a,b). At about the same time, Thalhammer (1973, 1978) initiated a similar screening program for pregnant women in Austria. In addition to scientific knowledge, these screening programs have helped to disseminate information for the prevention of toxoplasmosis. A neonatal serological screening and early treatment for congenital T. gondii infection was initiated in Massachusetts, United States in 1980s (Guerina et al., 1994). The efficacy of treatment of T. gondii infection in the fetus and newborn is not fully delineated, and many issues related to the cost and benefit of screening and treatment in pregnancy and in newborns remain to be examined.

consumption remains the easiest and most economical method of reducing transmission of T. gondii through meat.

1.9.2 Hygiene measures

Acknowledgments

After the discovery of the life cycle of T. gondii in 1970, it became possible to advise pregnant women and other susceptible populations on avoiding contact with oocysts (Frenkel and Dubey, 1972). Studies were conducted to construct thermal curves showing temperatures required to kill T. gondii in infected meat by freezing (Kotula et al., 1991), cooking (Dubey et al., 1990), and by gamma irradiation (Dubey et al., 1986). These data are now used by regulatory agencies to advise consumers about the safety of meat. Freezing of meat overnight in a household freezer before human or animal

I would like to dedicate this paper to the late Profs. J.K.A. Beverley and C.P. Beattie who were my PhD advisors (1964 67) and to the late Prof. J.K. Frenkel who was my postdoctoral studies supervisor (1973 78).

1.9.3 Animal production practices Extensive epidemiological studies on pig farms in the United States in 1990s concluded that keeping cats out of the pig barns and raising pigs indoors can reduce T. gondii infection in pigs (Dubey et al., 1995a,b; Weigel et al., 1995). Because of changes in pig, husbandry prevalence of viable T. gondii in pigs is reduced to ,1% (Dubey et al., 2005). Because ingestion of infected pork is considered the main meat source of T. gondii for humans (at least in the United States), the prevalence of T. gondii in humans in the United States has decreased in the past decade (Jones et al., 2007).

1.9.4 Vaccination Vaccination of sheep with a live cyst-less strain of T. gondii reduces neonatal mortality in lambs, and this vaccine is available commercially (Wilkins and O’Connell, 1983; Buxton and Innes, 1995). To date, there is no vaccine suitable for human use.

References Aramini, J.J., Stephen, C., Dubey, J.P., 1998. Toxoplasma gondii in Vancouver Island cougars (Felis concolor vancouverensis): serology and oocyst shedding. J. Parasitol. 84, 438 440. Benenson, M.W., Takafuji, E.T., Lemon, S.M., Greenup, R.L., Sulzer, A.J., 1982. Oocyst-transmitted toxoplasmosis associated with ingestion of contaminated water. N. Engl. J. Med. 307, 666 669.

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Ben Rachid, M.S., 1970. Contribution a` l’e´tude de la toxoplasmose du gondi. II. Comportement du Ctenodactylus gundi vis-a`-vis de Isospora bigemina. Arch. Inst. Pasteur Tunis 47, 33 35. Beverley, J.K.A., 1959. Congenital transmission of toxoplasmosis through successive generations of mice. Nature 183, 1348 1349. Beverley, J.K.A., Beattie, C.P., 1958. Glandular toxoplasmosis. A survey of 30 cases. Lancet 23, 379 384. Beverley, J.K.A., Watson, W.A., 1961. Ovine abortion and toxoplasmosis in Yorkshire. Vet. Rec. 73, 6 11. Bowie, W.R., King, A.S., Werker, D.H., Isaac-Renton, J.L., Bell, A., Eng, S.B., et al., 1997. Outbreak of toxoplasmosis associated with municipal drinking water. Lancet 350, 173 177. Burg, J.L., Grover, C.M., Pouletty, P., Boothroyd, J.C., 1989. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain-reaction. J. Clin. Microbiol. 27, 1787 1792. Burnett, A.J., Shortt, S.G., Isaac-Renton, J., King, A., Werker, D., Bowie, W.R., 1998. Multiple cases of acquired toxoplasmosis retinitis presenting in an outbreak. Ophthalmology 105, 1032 1037. Buxton, D., Innes, E.A., 1995. A commercial vaccine for ovine toxoplasmosis. Parasitology 110, S11 S16. Campbell, R.S.F., Martin, W.B., Gordon, E.D., 1955. Toxoplamosis as a complication of canine distemper. Vet. Rec. 67, 708 716. Christie, E., Pappas, P.W., Dubey, J.P., 1978. Ultrastructure of excystment of Toxoplasma gondii oocysts. J. Protozool. 25, 438 443. Cole, R.A., Lindsay, D.S., Howe, D.K., Roderick, C.L., Dubey, J.P., Thomas, N.J., et al., 2000. Biological and molecular characterizations of Toxoplasma gondii strains obtained from southern sea otters (Enhydra lutris nereis). J. Parasitol. 86, 526 530. Conrad, P.A., Miller, M.A., Kreuder, C., James, E.R., Mazet, J., Dabritz, H., et al., 2005. Transmission of Toxoplasma: clues from the study of sea otters as sentinels of Toxoplasma gondii flow into the marine environment. Int. J. Parasitol. 35, 1155 1168. Cowen, D., Wolf, A., Paige, B.H., 1942. Toxoplasmic encephalomyelitis. VI. Clinical diagnosis of infantile or congenital toxoplasmosis; survival beyond infancy. Arch. Neurol. Psychiat. 48, 689 739. de Lange, C., 1929. Klinische und pathologischanatomische Mitteilungen u¨ber Hydrocephalus chronicus congenitus und acquisitus. Ztschr. f. d. ges. Neurol. u. Psychiat. 120, 433 500. de Moura, L., Bahia-Oliveira, L.M.G., Wada, M.Y., Jones, J. L., Tuboi, S.H., Carmo, E.H., et al., 2006. Waterborne outbreak of toxoplasmosis in Brazil, from field to gene. Emerg. Infect. Dis. 12, 326 329.

Desmonts, G., Couvreur, J., 1974a. Congenital toxoplasmosis. A prospective study of 378 pregnancies. N. Engl. J. Med. 290, 1110 1116. Desmonts, G., Couvreur, J., 1974b. Toxoplasmosis in pregnancy and its transmission to the fetus. Bull. N. Y. Acad. Med. 50, 146 159. Desmonts, G., Remington, J.S., 1980. Direct agglutination test for diagnosis of Toxoplasma infection: method for increasing sensitivity and specificity. J. Clin. Microbiol. 11, 562 568. Desmonts, G., Couvreur, J., Alison, F., Baudelot, J., Gerbeaux, J., Lelong, M., 1965. E´tude e´pide´miologique sur la toxoplasmose: de l’influence de la cuisson des viandes de boucherie sur la fre´quence de l’infection humaine. Rev. Fr. E´tudes Clin. Biol. 10, 952 958. Desmonts, G., Naot, Y., Remington, J.S., 1981. Immunoglobulin M immunosorbent agglutination assay for diagnosis of infectious diseases. Diagnosis of acute congenital and acquired Toxoplasma infections. J. Clin. Microbiol. 14, 544 549. Dubey, J.P., 1966. Toxoplasmosis and Its Transmission in Cats With Special Reference to Associated Toxocara cati Infestations (Ph.D. thesis). University of Sheffield, England. 1 169. Dubey, J.P., 1968. Isolation of Toxoplasma gondii from the feces of a helminth free cat. J. Protozool. 15, 773 775. Dubey, J.P., 1976. Reshedding of Toxoplasma oocysts by chronically infected cats. Nature 262, 213 214. Dubey, J.P., 1977. Toxoplasma, Hammondia, Besnoitia, Sarcocystis, and other tissue cyst-forming coccidia of man and animals. In: Kreier, J.P. (Ed.), Parasitic Protozoa, vol. 3. Academic Press, New York, pp. 101 237. Dubey, J.P., 1995. Duration of immunity to shedding of Toxoplasma gondii oocysts by cats. J. Parasitol. 81, 410 415. Dubey, J.P., 1996. Infectivity and pathogenicity of Toxoplasma gondii oocysts for cats. J. Parasitol. 82, 957 960. Dubey, J.P., 1997. Validation of the specificity of the modified agglutination test for toxoplasmosis in pigs. Vet. Parasitol. 71, 307 310. Dubey, J.P., 2001. Oocyst shedding by cats fed isolated bradyzoites and comparison of infectivity of bradyzoites of the VEG strain Toxoplasma gondii to cats and mice. J. Parasitol. 87, 215 219. Dubey, J.P., 2002. Tachyzoite-induced life cycle of Toxoplasma gondii in cats. J. Parasitol. 88, 713 717. Dubey, J.P., 2003. Neosporosis in cattle. J. Parasitol. 89 (Suppl.), S42 S46. Dubey, J.P., 2005. Unexpected oocyst shedding by cats fed Toxoplasma gondii tachyzoites: in vivo stage conversion and strain variation. Vet. Parasitol. 133, 289 298.

Toxoplasma Gondii

References

Dubey, J.P., 2006. Comparative infectivity of oocysts and bradyzoites of Toxoplasma gondii for intermediate (mice) and definitive (cats) hosts. Vet. Parasitol. 140, 69 75. Dubey, J.P., 2008. The history of Toxoplasma gondii—the first 100 years. J. Eukaryot. Microbiol. 55, 467 475. Dubey, J.P., 2009. History of the discovery of the life cycle of Toxoplasma gondii. Int. J. Parasitol. 39, 877 882. Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans, second ed. CRC Press, Boca Raton, FL, pp. 1 330, 1 313. Dubey, J.P., 1914 2014. Transmission of Toxoplasma gondii - from land to sea, a personal perspective. A century of paraitology: discoveries, ideas and lessons learned by scientists who published in the Journal of Parasitology, John Wiley & Sons., pp. 148 164. Dubey, J.P., Beattie, C.P., 1988. Toxoplasmosis of Animals and Humans. CRC Press, Boca Raton, FL, pp. 1 220. Dubey, J.P., Carpenter, J.L., Speer, C.A., Topper, M.J., Uggla, A., 1988. Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192, 1269 1285. Dubey, J.P., Desmonts, G., 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Vet. J. 19, 337 339. Dubey, J.P., Frenkel, J.K., 1972. Cyst-induced toxoplasmosis in cats. J. Protozool. 19, 155 177. Dubey, J.P., Frenkel, J.K., 1973. Experimental Toxoplasma infections in mice with strains producing oocysts. J. Parasitol. 59, 505 512. Dubey, J.P., Frenkel, J.K., 1976. Feline toxoplasmosis from acutely infected mice and the development of Toxoplasma cysts. J. Protozool. 23, 537 546. Dubey, J.P., Towle, A., 1986. Toxoplasmosis in Sheep: A Review and Annotated Bibliography. Commonwealth Institute of Parasitology, Herts, UK, pp. 1 152. Dubey, J.P., Miller, N.L., Frenkel, J.K., 1970a. The Toxoplasma gondii oocyst from cat feces. J. Exp. Med. 132, 636 662. Dubey, J.P., Miller, N.L., Frenkel, J.K., 1970b. Characterization of the new fecal form of Toxoplasma gondii. J. Parasitol. 56, 447 456. Dubey, J.P., Swan, G.V., Frenkel, J.K., 1972. A simplified method for isolation of Toxoplasma gondii from feces of cats. J. Parasitol. 58, 1005 1006. Dubey, J.P., Brake, R.J., Murrell, K.D., Fayer, R., 1986. Effects of irradiation on the viability of Toxoplasma gondii cysts in tissues of mice and pigs. Am. J. Vet. Res. 47, 518 522. Dubey, J.P., Kotula, A.W., Sharar, A., Andrews, C.D., Lindsay, D.S., 1990. Effect of high temperature on infectivity of Toxoplasma gondii tissue cysts in pork. J. Parasitol. 76, 201 204. Dubey, J.P., Davis, S.W., Speer, C.A., Bowman, D.D., de Lahunta, A., Granstrom, D.E., et al., 1991. Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic

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agent of equine protozoal myeloencephalitis. J. Parasitol. 77, 212 218. Dubey, J.P., Weigel, R.M., Siegel, A.M., Thulliez, P., Kitron, U.D., Mitchell, M.A., et al., 1995a. Sources and reservoirs of Toxoplasma gondii infection on 47 swine farms in Illinois. J. Parasitol. 81, 723 729. Dubey, J.P., Thulliez, P., Weigel, R.M., Andrews, C.D., Lind, P., Powell, E.C., 1995b. Sensitivity and specificity of various serologic tests for detection of Toxoplasma gondii infection in naturally infected sows. Am. J. Vet. Res. 56, 1030 1036. Dubey, J.P., Lunney, J.K., Shen, S.K., Kwok, O.C.H., Ashford, D.A., Thulliez, P., 1996. Infectivity of low numbers of Toxoplasma gondii oocysts to pigs. J. Parasitol. 82, 438 443. Dubey, J.P., Rollor, E.A., Smith, K., Kwok, O.C.H., Thulliez, P., 1997. Low seroprevalence of Toxoplasma gondii in feral pigs from a remote island lacking cats. J. Parasitol. 83, 839 841. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structure of Toxoplasma gondii tachyzoites, bradyzoites and sporozoites, and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267 299. Dubey, J.P., Lindsay, D.S., Saville, W.J.A., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet. Parasitol. 95, 89 131. Dubey, J.P., Zarnke, R., Thomas, N.J., Wong, S.K., Van Bonn, W., Briggs, M., et al., 2003a. Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and Sarcocystis canis-like infections in marine mammals. Vet. Parasitol. 116, 275 296. Dubey, J.P., Ross, A.D., Fritz, D., 2003b. Clinical Toxoplasma gondii, Hammondia heydorni, and Sarcocystis sp. infections in dogs. Parassitologia 45, 141 146. Dubey, J.P., Hill, D.E., Jones, J.L., Hightower, A.W., Kirkland, E., Roberts, J.M., et al., 2005. Prevalence of viable Toxoplasma gondii in beef, chicken and pork from retail meat stores in the United States: risk assessment to consumers. J. Parasitol. 91, 1082 1093. Dubey, J.P., Howe, D.K., Furr, M., Saville, W.J., Marsh, A. E., Reed, S.M., et al., 2015. An update on Sarcocystis neurona infections in animals and equine protozoal myeloencephalitis (EPM). Vet. Parasitol. 209, 1 42. Dubey, J.P., Laurin, E., Kwok, O.C.H., 2016. Validation of the modified agglutination test for detection of Toxoplasma gondii in free-range chickens by using cat and mouse bioassay. Parasitology 143, 314 319. Dubey, J.P., Hemphill, A., Calero-Bernal, R., Schares, G., 2017. Neosporosis in Animals. CRC Press, Boca Raton, FL, pp. 1 529. Eyles, D.E., Coleman, N., 1953. Synergistic effect of sulfadiazine and daraprim against experimental toxoplasmosis in the mouse. Antibiot. Chemother. 3, 483 490.

Toxoplasma Gondii

16

1. The history and life cycle of Toxoplasma gondii

Feldman, H.A., Miller, L.T., 1956. Serological study of toxoplasmosis prevalence. Am. J. Hyg. 64, 320 335. Ferguson, D.J.P., Hutchison, W.M., 1987. An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitol. Res. 73, 483 491. Ferguson, D.J.P., Hutchison, W.M., Dunachie, J.F., Siim, J. C., 1974. Ultrastructural study of early stages of asexual multiplication and microgametogony of Toxoplasma gondii in the small intestine of the cat. Acta Pathol. Microbiol. Scand. B 82, 167 181. Ferguson, D.J.P., Hutchison, W.M., Siim, J.C., 1975. The ultrastructural development of the macrogamete and formation of the oocyst wall of Toxoplasma gondii. Acta Pathol. Microbiol. Scand. B 83, 491 505. Ferguson, D.J.P., Birch-Andersen, A., Siim, J.C., Hutchison, W.M., 1979a. Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. I. Development of the zygote and formation of sporoblasts. Acta Pathol. Microbiol. Scand. B 87, 171 181. Ferguson, D.J.P., Birch-Andersen, A., Siim, J.C., Hutchison, W.M., 1979b. An ultrastructural study on the excystation of the sporozoites of Toxoplasma gondii. Acta Pathol. Microbiol. Scand. B 87, 277 283. Fitzgerald, P.R., 1970. Toxoplasmosis of Animals. University of Illinois, Urbana, IL, pp. 1 472. Frenkel, J.K., 1956. Pathogenesis of toxoplasmosis and of infections with organisms resembling Toxoplasma. Ann. N.Y. Acad. Sci. 64, 215 251. Frenkel, J.K., 1970. Pursuing Toxoplasma. J. Infect. Dis. 122, 553 559. Frenkel, J.K., 1973. Toxoplasma in and around us. BioScience 23, 343 352. Frenkel, J.K., Dubey, J.P., 1972. Toxoplasmosis and its prevention in cats and man. J. Infect. Dis. 126, 664 673. Frenkel, J.K., Friedlander, S., 1951. Toxoplasmosis: Pathology of Neonatal Disease, Pathogenesis, Diagnosis, and Treatment. Public Health Service Publication No. 141. United States Government Printing Office, Washington, DC, pp. 1 150. Frenkel, J.K., Dubey, J.P., Miller, N.L., 1969. Toxoplasma gondii: fecal forms separated from eggs of the nematode Toxocara cati. Science 164, 432 433. Frenkel, J.K., Dubey, J.P., Miller, N.L., 1970. Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science 167, 893 896. Frenkel, J.K., Dubey, J.P., Hoff, R.L., 1976. Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni. J. Protozool. 23, 421 424. Freppel, W., Ferguson, D.J.P., Shapiro, K., Dubey, J.P., Puech, P.H., Dumtere, A., 2019. Structure, composition, and roles of the Toxoplasma gondii oocyst and sporocyst walls. Cell Surf. 5, 100016.

Fulton, J.D., 1965. Studies on agglutination of Toxoplasma gondii. Trans. R. Soc. Trop. Med. Hyg. 59, 694 704. Garin, J.P., Eyles, D.E., 1958. Le traitement de la toxoplasmose experimentale de la souris par la spiramycine. La Presse Me´dicale 66, 957 958. Garnham, P.C.C., 1971. Progress in Parasitology. University of London, The Athlone Press, London, pp. 1 223. Goldman, M., Carver, R.K., Sulzer, A.J., 1958. Reproduction of Toxoplasma gondii by internal budding. J. Parasitol. 44, 161 171. Guerina, N.G., Hsu, H.W., Meissner, H.C., Maguire, J.H., Lynfield, R., Stechenberg, B., et al., 1994. Neonatal serologic screening and early treatment for congenital Toxoplasma gondii infection. N. Engl. J. Med. 330, 1858 1863. Gustafson, P.V., Agar, H.D., Cramer, D.I., 1954. An electron microscope study of Toxoplasma. Am. J. Trop. Med. Hyg. 3, 1008 1021. Hartley, W.J., 1961. Experimental transmission of toxoplasmosis in sheep. N. Z. Vet. J. 9, 1 6. Hartley, W.J., Marshall, S.C., 1957. Toxoplasmosis as a cause of ovine perinatal mortality. N. Z. Vet. J. 5, 119 124. Hogan, M.J., 1951. Ocular Toxoplasmosis. Columbia University Press, New York, pp. 1 86. Holland, G.N., 2003. Ocular toxoplasmosis: a global reassessment. Part I: Epidemiology and course of disease. Am. J. Ophthalmol. 136, 973 988. Hutchison, W.M., 1965. Experimental transmission of Toxoplasma gondii. Nature 206, 961 962. Hutchison, W.M., 1967. The nematode transmission of Toxoplasma gondii. Trans. R. Soc. Trop. Med. Hyg. 61, 80 89. Hutchison, W.M., Dunachie, J.F., Work, K., 1968. The faecal transmission of Toxoplasma gondii. Acta Pathol. Microbiol. Scand. 74, 462 464. Hutchison, W.M., Dunachie, J.F., Siim, J.C., Work, K., 1969. Life cycle of Toxoplasma gondii. Br. Med. J. 4, 806. Hutchison, W.M., Dunachie, J.F., Siim, J.C., Work, K., 1970. Coccidian-like nature of Toxoplasma gondii. Br. Med. J. 1, 142 144. Hutchison, W.M., Dunachie, J.F., Work, K., Siim, J.C., 1971. The life cycle of the coccidian parasite Toxoplasma gondii, in the domestic cat. Trans. R. Soc. Trop. Med. Hyg. 65, 380 399. Jacobs, L., 1964. The occurrence of Toxoplasma infection in the absence of demonstrable antibodies. Proc. First Int. Congr. Parasitol. 1, 176 177. Jacobs, L., 1967. Toxoplasma and toxoplasmosis. Adv. Parasitol. 5, 1 45. Jacobs, L., Hartley, W.J., 1964. Ovine toxoplasmosis: studies on parasitaemia, tissue infection, and congenital

Toxoplasma Gondii

References

transmission in ewes infected by various routes. Br. Vet. J. 120, 347 364. Jacobs, L., Cook, M.K., Wilder, H.C., 1954a. Serologic data on adults with histologically diagnosed toxoplasmic chorioretinitis. Trans. Am. Acad. Ophthalmol. Otolaryngol. 58, 193 200. Jacobs, L., Fair, J.R., Bickerton, J.H., 1954b. Adult ocular toxoplasmosis. Report of a parasitologically proved case. AMA Arch. Ophthalmol. 52, 63 71. Jacobs, L., Remington, J.S., Melton, M.L., 1960a. The resistance of the encysted form of Toxoplasma gondii. J. Parasitol. 46, 11 21. Jacobs, L., Remington, J.S., Melton, M.L., 1960b. A survey of meat samples from swine, cattle, and sheep for the presence of encysted Toxoplasma. J. Parasitol. 46, 23 28. Janitschke, K., Werner, H., 1972. Untersuchungen uber die Wirtsspezifitat des geschlechtlichen Entwicklungszyklus von Toxoplasma gondii. Z. Parasitenk. 39, 247 254. ˚ J., 1923. Pathogenesa a pathologicka´ anatomie tak Janku, nazvaneho vrozene´ho kolombu zˇ lute´ skvrany voku norma´lne´ velikem a microphthalmicke´m s nalezem parasiˇ ˆ tu v sı´tnici. Casopis le´kaˇreu˚ cˇ eskyck 62, 1021, 1052, 1081, 1111, and 1138. ˚ J., 1959. Die Pathogenese und pathologische Janku, Anatomie des sogenannten angeborenen Koloboms des gelben Flecks in normal grossen sowie im mikrophtalmischen Auge mit Parasitenbefund in der Netzhaut. ˇ Cesk. Parasitol. 6, 9 57. Jewell, M.L., Frenkel, J.K., Johnson, K.M., Reed, V., Ruiz, A., 1972. Development of Toxoplasma oocysts in neotropical felidae. Am. J. Trop. Med. Hyg. 21, 512 517. Jı´ra, J., Kozojed, V., 1970. Toxoplasmosis 1908-1967. Gustav Fischer Verlag, Stuttgart, pp. 1 464. Jı´ra, J., Kozojed, V., 1983. Toxoplasmosis 1968-1975. Gustav Fischer Verlag, Stuttgart, pp. 1 394. Jones, J.L., Kruszon-Moran, D., Sanders-Lewis, K., Wilson, M., 2007. Toxoplasma gondii infection in the United States, 1999 2004, decline from the prior decade. Am. J. Trop. Med. Hyg. 77, 405 410. Kean, B.H., Kimball, A.C., Christenson, W.N., 1969. An epidemic of acute toxoplasmosis. J. Am. Med. Assoc. 208, 1002 1004. Kotula, A.W., Dubey, J.P., Sharar, A.K., Andrew, C.D., Shen, S.K., Lindsay, D.S., 1991. Effect of freezing on infectivity of Toxoplasma gondii tissue cysts in pork. J. Food Prot. 54, 687 690. Lainson, R., 1958. Observations on the development and nature of pseudocysts and cysts of Toxoplasma gondii. Trans. R. Soc. Trop. Med. Hyg. 52, 396 407. Levaditi, C., Schoen, R., Sanchis Bayarri, V., 1928. L’ence´phalo-mye´lite toxoplasmique chronique du lapin et de la souris. C.R. Soc. Biol. 99, 37 40.

17

Luft, B.J., Conley, F.K., Remington, J.S., Laverdiere, M., Wagner, K.F., Levine, J.F., et al., 1983. Outbreak of central-nervous-system toxoplasmosis in Western Europe and North America. Lancet I, 781 784. Mateus-Pinilla, N.E., Dubey, J.P., Choromanski, L., Weigel, R.M., 1999. A field trial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T. gondii exposure for swine. J. Parasitol. 85, 855 860. Mello, U., 1910. Un cas de toxoplasmose du chien observe´ a` Turin (2). Bull. Soc. Pathol. Exot. Fil. 3, 359 363. Miller, N.L., Frenkel, J.K., Dubey, J.P., 1972. Oral infections with Toxoplasma cysts and oocysts in felines, other mammals, and in birds. J. Parasitol. 58, 928 937. Miller, M.A., Miller, W.A., Conrad, P.A., James, E.R., Melli, A.C., Leutenegger, C.M., et al., 2008. Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters. Int. J. Parasitol. 38, 1319 1328. Munday, B.L., 1972. Serological evidence of Toxoplasma infection in isolated groups of sheep. Res. Vet. Sci. 13, 100 102. Naot, Y., Remington, J.S., 1980. An enzyme-linked immunosorbent assay for detection of IgM antibodies to Toxoplasma gondii: use for diagnosis of acute acquired toxoplasmosis. J. Infect. Dis. 142, 757 766. Nicolle, C., Manceaux, L., 1908. Sur une infection a` corps de Leishman (ou organismes voisins) du gondi. C.R. Seances Acad. Sci. 147, 763 766. Nicolle, C., Manceaux, L., 1909. Sur un protozoaire nouveau du gondi. C.R. Seances Acad. Sci. 148, 369 372. Overdulve, J.P., 1970. The identity of Toxoplasma Nicolle & Manceaux, 1909 with Isospora Schneider, 1881 (I). Proc. K. Ned. Akad. Wet. C 73, 129 151. Paige, B.H., Cowen, D., Wolf, A., 1942. Toxoplasmic encephalomyelitis. V. Further observations of infantile toxoplasmosis; intrauterine inception of the disease; visceral manifestations. Am. J. Dis. Child. 63, 474 514. Perkins, E.S., 1961. Uveitis and Toxoplasmosis. J. & A. Churchill Ltd., London, pp. 1 142. Piekarski, G., Pelster, B., Witte, H.M., 1971. Endopolygenie bei Toxoplasma gondii. Z. Parasitenk. 36, 122 130. Pinkerton, H., Henderson, R.G., 1941. Adult toxoplasmosis. A previously unrecognized disease entity simulating the typhus-spotted fever group. J. Am. Med. Assoc. 116, 807 814. Pinkerton, H., Weinman, D., 1940. Toxoplasma infection in man. Arch. Pathol. 30, 374 392. Polomoshnov, A.P., 1979. Definitive hosts of Toxoplasma. In: Problems of Natural Nidality of Diseases. Institute of Zoology, Kazakh Academy of Sciences, Alma Ata, No. 10, pp. 68 72. [In Russian].

Toxoplasma Gondii

18

1. The history and life cycle of Toxoplasma gondii

Rawal, B.D., 1959. Toxoplasmosis. A dye-test on sera from vegetarians and meat eaters in Bombay. Trans. R. Soc. Trop. Med. Hyg. 53, 61 63. Remington, J.S., 1969. The present status of the IgM fluorescent antibody technique in the diagnosis of congenital toxoplasmosis. J. Pediatr. 75, 1116 1124. Remington, J.S., Miller, M.J., Brownlee, I.E., 1968. IgM antibodies in acute toxoplasmosis. II. Prevalence and significance in acquired cases. J. Lab. Clin. Med. 71, 855 866. Remington, J.S., McLeod, R., Thulliez, P., Desmonts, G., 2001. Toxoplasmosis. In: Remington, J.S., Klein, J. (Eds.), Infectious Diseases of the Fetus and Newborn Infant, fifth ed. W.B. Saunders, Philadelphia, PA, pp. 205 346. Remington, J.S., McLeod, R., Wilson, C.B., Desmonts, G., 2011. Toxoplasmosis. In: Remington, J.S., Klein, J. (Eds.), Infectious Diseases of the Fetus and Newborn Infant, seventh ed. W.B. Saunders, Philadelphia, PA, pp. 918 1041. Sabin, A.B., 1941. Toxoplasmic encephalitis in children. J. Am. Med. Assoc. 116, 801 807. Sabin, A.B., 1942. Toxoplasmosis. A recently recognized disease of human beings. Adv. Pediatr. 1, 1 53. Sabin, A.B., Feldman, H.A., 1948. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoon parasite (Toxoplasma). Science 108, 660 663. Sabin, A.B., Feldman, H.A., 1949. Persistence of placentally transmitted toxoplasmic antibodies in normal children in relation to diagnosis of congenital toxoplasmosis. Pediatrics 4, 660 664. Sabin, A.B., Warren, J., 1942. Therapeutic effectiveness of certain sulfonamide on infection by an intracellular protozoon (Toxoplasma). Proc. Soc. Exp. Biol. Med. 51, 19 23. Sanger, V.L., Cole, C.R., 1955. Toxoplasmosis. VI. Isolation of Toxoplasma from milk, placentas, and newborn pigs of asymptomatic carrier sow. Am. J. Vet. Res. 16, 536 539. Sheffield, H.G., 1970. Schizogony in Toxoplasma gondii: an electron microscope study. Proc. Helminthol. Soc. Wash. 37, 237 242. Sheffield, H.G., Melton, M.L., 1968. The fine structure and reproduction of Toxoplasma gondii. J. Parasitol. 54, 209 226. Sheffield, H.G., Melton, M.L., 1969. Toxoplasma gondii: transmission through feces in absence of Toxocara cati eggs. Science 164, 431 432. Sheffield, H.G., Melton, M.L., 1970. Toxoplasma gondii: the oocyst, sporozoite, and infection of cultured cells. Science 167, 892 893. Siim, J.C., 1956. Toxoplasmosis acquisita lymphonodosa; clinical and pathological aspects. Ann. N.Y. Acad. Sci. 64, 185 206. Silveira, C., Belfort, R., Burnier, M., Nussenblatt, R., 1988. Acquired toxoplasmic infection as the cause of

toxoplasmic retinochoroiditis in families. Am. J. Ophthalmol. 106, 362 364. Silveira, C., Belfort, R., Muccioli, C., Abreu, M.T., Martins, M.C., Victora, C., et al., 2001. A follow-up study of Toxoplasma gondii infection in Southern Brazil. Am. J. Ophthalmol. 131, 351 354. Speer, C.A., Dubey, J.P., 2005. Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites. Int. J. Parasitol. 35, 193 206. Speer, C.A., Clark, S., Dubey, J.P., 1998. Ultrastructure of the oocysts, sporocysts and sporozoites of Toxoplasma gondii. J. Parasitol. 84, 505 512. Splendore, A., 1908. Un nuovo protozoa parassita de’ conigli. incontrato nelle lesioni anatomiche d’une malattia che ricorda in molti punti il Kala-azar dell’ uomo. Nota preliminare pel. Rev. Soc. Sci. Sao Paulo 3, 109 112. Splendor, A., 2009. A new protozoan parasite in rabbits. Int. J. Parasitol. 39, 861 862. Teutsch, S.M., Juranek, D.D., Sulzer, A., Dubey, J.P., Sikes, R.K., 1979. Epidemic toxoplasmosis associated with infected cats. N. Engl. J. Med. 300, 695 699. Thalhammer, O., 1973. Prevention of congenital toxoplasmosis. Neuropadiatrie 4, 233 237. Thalhammer, O., 1978. Prevention of congenital infections. In: Perinatal Medicine, Sixth European Congress. Perinatal Medicine, Vienna, pp. 44 51. Thomas, N.J., Cole, R.A., 1996. The risk of disease and threats to the wild population. Endangered Species Update (Conservation and Management of the Southern Sea Otter Special Issue) 13, 23 27. Thomas, N.J., Dubey, J.P., Lindsay, D.S., Cole, R.A., Meteyer, C.U., 2007. Protozoal meningoencephalitis in sea otters (Enhydra lutris): a histopathological and immunohistochemical study of naturally occurring cases. J. Comp. Pathol. 137, 102 121. Torres, C.M., 1927. Morphologie d’un nouveau parasite de l’homme, Encephalitozoon chagasi, N. sp., observe dans un cas de meningo-encephalo-myelite congenitale avec myosite et myocardite. C.R. Soc. Biol. 97, 1787 1790. Wallace, G.D., 1969. Serologic and epidemiologic observations on toxoplasmosis on three Pacific Atolls. Am. J. Epidemiol. 90, 103 111. Wanko, T., Jacobs, L., Gavin, M.A., 1962. Electron microscope study of Toxoplasma cysts in mouse brain. J. Protozool. 9, 235 242. Weigel, R.M., Dubey, J.P., Siegel, A.M., Kitron, U.D., Mannelli, A., Mitchell, M.A., et al., 1995. Risk factors for transmission of Toxoplasma gondii on swine farms in Illinois. J. Parasitol. 81, 736 741. Weiland, G., Ku¨hn, D., 1970. Experimentelle ToxoplasmaInfektionen bei der Katze. II. Entwicklungsstadien des

Toxoplasma Gondii

Further reading

Parasiten im Darm. Berl. Mu¨nch. Tiera¨rztl. Wochenschr. 83, 128 132. Weinman, D., Chandler, A.H., 1954. Toxoplasmosis in swine and rodents. Reciprocal oral infection and potential human hazard. Proc. Soc. Exp. Biol. Med. 87, 211 216. Weiss, L.M., Dubey, J.P., 2009. Toxoplasmosis: a history of clinical observations. Int. J. Parasitol. 39, 895 901. Wilder, H.C., 1952. Toxoplasma chorioretinitis in adults. AMA Arch. Ophthalmol. 48, 127 136. Wilkins, M.F., O’Connell, E., 1983. Effect on lambing percentgage of vaccinating ewes with pathogenesis and pathologic anatomy of coloboma of the macula lutea in an eye of normal dimensions and in a microphthalmic eye with parasites in the retina. N.Z. Vet. J. 31, 181 182. Witte, H.M., Piekarski, G., 1970. Die OocystenAusscheidung bei experimentell infizierten Katzen in Abha¨ngigkeit vom Toxoplasma-Stamm. Z. Parasitenk. 33, 358 360. Wolf, A., Cowen, D., 1937. Granulomatous encephalomyelitis due to an encephalitozoon (encephalitozoic encephalomyelitis). A new protozoan disease of man. Bull. Neur. Inst. N.Y. 6, 306 371. Wolf, A., Cowen, D., 1938. Granulomatous encephalomyelitis due to a protozoan (Toxoplasma or Encephalitozoon). II. Identification of a case from the literature. Bull. Neur. Inst. N.Y. 7, 266 290.

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Wolf, A., Cowen, D., Paige, B., 1939a. Human toxoplasmosis: occurrence in infants as an encephalomyelitis verification by transmission to animals. Science 89, 226 227. Wolf, A., Cowen, D., Paige, B.H., 1939b. Toxoplasmic encephalomyelitis. III. A new case of granulomatous encephalomyelitis due to a protozoon. Am. J. Pathol. 15, 657 694. Wolf, A., Cowen, D., Paige, B.H., 1940. Toxoplasmic encephalomyelitis. IV. Experimental transmission of the infection to animals from a human infant. J. Exp. Med. 71, 187 214. Zulpo, D.L., Same, A.S., dos Santos, J.R., Sasse, J.P., Martin, T.A., Minutti, A.F., et al., 2018. Toxoplasma gondii: a study of oocyst re-shedding in domestic cats. Vet. Parasitol. 17 20, 2018.

Further reading Dubey, J.P., 1998. Refinement of pepsin digestion method for isolation of Toxoplasma gondii from infected tissues. Vet. Parasitol. 74, 75 77. Olafson, P., Monux, W.S., 1942. Toxoplasma infection in animals. Cornell Vet. 32, 176 190.

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C H A P T E R

2 The ultrastructure of Toxoplasma gondii David J.P. Ferguson1 and Jean-Franc¸ois Dubremetz2 1

Nuffield Department of Clinical Laboratory Science, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; Department of Biological and Medical Sciences, Faculty of Health and Life Science, Oxford Brookes University, Oxford, United Kingdom 2UMR CNRS 5235, University of Montpellier, Montpellier, France

2.1 Invasive stage ultrastructure and genesis

extensively studied stage in the T. gondii life cycle because of the ease with which large numbers can be obtained both in vivo and in vitro. These invasive stages are crescentshaped cells (2 3 7 µm approximately) with a slightly more pointed anterior end (the anterior being defined by the direction of motility) (Fig. 2.1A). They comprise a unique cytoskeleton, secretory organelles (rhoptries, micronemes, and dense granules), endosymbiotic-derived organelles (mitochondrion and apicoplast), eukaryotic universal organelles [nucleus, endoplasmic reticulum (ER), Golgi apparatus, and ribosomes], and specific structures [acidocalcisomes and plant-like vacuoles (PLVs)] all enclosed by a complex membranous structure termed the pellicle. The cytoskeleton comprises the following:

This chapter reviews the electron microscopic data on Toxoplasma gondii and its life cycle stages. Corresponding light microscopy of these stages can be found in Chapter 1, The history and life cycle of Toxoplasma gondii.

2.1.1 Basic ultrastructural morphology There are four invasive forms of T. gondii: the tachyzoite, bradyzoite, merozoite, and sporozoite. Tachyzoites and bradyzoites are associated with the intermediate host and merozoites and sporozoites with the definitive host. Tachyzoites and merozoites are responsible for the expansion of the population within a host, while the bradyzoites and sporozoites are capable of environmental transmission to new hosts. All of the infectious stages have the same basic morphology with only minor variations. The standard features will be described in this section and are based mainly on observations of tachyzoites. The tachyzoite is the most Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00002-5

• Two apical rings located beneath the plasma membrane at the apical tip of the parasite. They are uncharacterized at the molecular level but both are made of a thin ring of electron dense material, the upper one is 160 nm, the posterior one is 200 nm in diameter.

21

© 2020 Elsevier Ltd. All rights reserved.

FIGURE 2.1 Toxoplasma gondii tachyzoite ultrastructure. (Part A) Sagittal section of an intravacuolar tachyzoite: A, apicoplast; ac, acidocalcisome; C, conoid; DG, dense granule; ER, endoplasmic reticulum; G, Golgi body; HCN, host cell nucleus; MN, micronemes; Mi, mitochondria; N, nucleus; NU, nucleolus; PV, parasitophorous vacuole; R, rhoptry; tvn, tubulo-vesicular network. Bar is 1 µm. (Part B) Enlargement of the Golgi area showing the apicoplast (A) surrounded by four membranes (arrows). Bar is 0.5 µm. (Part C) Thin sections of whole tachyzoites showing a large empty vacuole: PLV/ VAC. The conditions used to prepare the cells were the same as the ones used for Fig. 2 of Miranda et al. (2010). A, apicoplast; G, Golgi; N, nucleus. Bar is 200 nm. (Part D) and (Part E) Immunogold electron microscopy labeling with anti-TgVP1 antibody (1:100) of a large, empty vacuole. The antibody used was an affinity purified rabbit antiserum generated against the peptide: SGKNEYGMSEDDPRN. The conditions used to prepare the cells were the same as the ones used for Fig. 1 of Miranda et al. (2010). Bar is 200 nm. Source: (C, D, and E) Courtesy Wandy Beatty, Microbiology Imaging Facility, Washington University.

2.1 Invasive stage ultrastructure and genesis

• The conoid is a hollow, truncated cone consisting of fibers wound into a spiral, such as a compressed spring, 400 nm in diameter at the base and 250 nm high, made of tubulin organized in a unique fashion, consisting of asymmetrical filaments of about nine protofilaments, very different from typical microtubules (Hu et al., 2002b). • Two polar rings encircle the top of the resting conoid (Fig. 2.2A). The outer ring consists of dense material covering the anterior rim of the inner membrane complex (IMC, discussed later). The inner ring is formed of material which anchors the 22 subpellicular microtubules that extend underneath the IMC for approximately twothirds of the body length (Nichols and Chiappino, 1987). These microtubules are classic 22 nm diameter hollow tubes, comprising 13 protofilaments made of tubulin (Hu et al., 2002b). • A pair of adjacent intraconoidal microtubules is also found, extending for a short distance (less than 1 µm) into the apical cytoplasm and ending anteriorly next to an apical vesicle of 40 nm that adheres to the plasma membrane (Hu et al., 2002b). The pellicle is a distinctive membrane complex that encloses the infectious stages. It consists of an outer unit membrane (plasmalemma) that completely encloses the organism and an inner layer of two closely applied unit membranes found at a fixed distance (approximately 15 nm) from the plasmalemma. The IMC consists of fused plates formed from flattened vesicles derived from the ER Golgi system (Vivier and Petitprez, 1969). The inner layer is interrupted by circular apertures at the anterior end (outer polar ring), where the conoid protrudes, and at the posterior end. The organization of the IMC has been essentially unraveled by electron microscopy (EM) freeze fracture (Morrissette et al., 1997; Porchet and Torpier, 1977). It is made of an

23

apical plate which is a single, truncated cone approximately 1 µm high, on which six longitudinal rows of rectangular plates are attached. The rows end at the posterior end of the tachyzoite by triangular plates joined in a turban-like fashion. The rows can extend straight or be twisted helicoidally. The protoplasmic faces on both sides of the IMC are covered with lines of intramembranous particles (IMPs), with 22 lines of higher density corresponding to the underlying subpellicular microtubules (Fig. 2.2C). IMPs have a 32 nm longitudinal periodicity and are lined approximately 30 nm apart (Morrissette et al., 1997). The organization of IMPs in the apical plate is distinct from that in the other plates, suggesting a distinct molecular structure in this apical area. An additional organized structure associated with the inner side of the IMC has been described by negative staining after detergent extraction, as a network of 8 10 nm filaments containing two novel proteins with extended coiled coiled domains that may play a role in determining cell shape (Mann and Beckers, 2001). The precise correlation between this network and the IMP alignments has not been defined. The pellicle has an additional adaptation termed the micropore, which is located in the apical half of the cell normally just anterior to the nucleus. The single micropore consists of a circular (approximately 150 nm in diameter) invagination of the plasmalemma through a break in the IMC (some recent studies indicate that more than one micropore may be present). The latter infolds to form an electron dense collar around the invagination (Fig. 2.2B). These structures are present throughout the development and increase in number during endopolygeny and gametogony. They are thought to act as a cytostome-like structure with extensions of the invaginated plasmalemma budding off resulting in the uptake of material (Nichols et al., 1994) (Fig. 2.2B). This process has been clearly shown to be important in the malaria

Toxoplasma Gondii

(A)

(B)

(C)

FIGURE 2.2 Details of bradyzoite (A, B) and tachyzoite (C) ultrastructure. (Part A) Apical area of a bradyzoite showing the two apical rings (a1, a2), the two polar rings (p1, p2) above and around the conoid. C, conoid; MN, micronemes; R, rhoptry. Lower picture: uptake of PV vesicular material through the micropore. Bar is 0.1 µm. (Part B) Upper picture: micropore showing the invagination of the zoite plasmalemma (arrow) through an opening and indentation (arrowhead) of the imc. Lower picture: uptake of PV vesicular material through the micropore (arrow). Bar is 0.1 µm. (Part C) Freeze fracture image of the pellicle of a tachyzoite. The three successive membranes are shown: Pe, protoplasmic face of the plasmalemma; Em, exoplasmic face of the external layer of the IMC; Pi, protoplasmic face of the inner layer of the IMC. Bar is 0.2 µm. imc, Inner membrane complex. Source: (C) From Morrissette, N.S., Murray, J.M., Roos, D.S., 1997. Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J. Cell Sci. 110 (Pt 1), 35 42, with permission.

2.1 Invasive stage ultrastructure and genesis

parasite, where the micropore is responsible for the ingestion the erythrocyte hemoglobin (Aikawa et al., 1966). Three distinct secretory organelles have been identified, which can vary in numbers and shape between the invasive stages (discussed later) (Figs. 2.1A and 2.2A). First are small rod-shaped micronemes (250 3 50 nm), located in the most apical area of the parasite, behind the conoid. They are homogeneously electron dense. Second are the rhoptries, organized as a group of elongated, club-shaped organelles that extend from within the conoid toward the nucleus. They show a long, narrow neck up to 2.5 µm long and a sac-like body about 0.25 3 1 µm in the posterior area. The contents are electron dense, except in the widened part, where the structure can be either labyrinthine or electron dense in appearance depending on the specific stage. A third type of organelle, found throughout the cell but mostly in the posterior part of the parasite, are spherical-shaped (0.3 µm diameter) structures with electron dense contents, which have been termed the dense granules. Immunoelectron microscopy has played an important role in our understanding of the functions of these organelles. With the development of antibodies to specific proteins, it has been possible to begin to identify proteins specifically located in the different organelles. It has been possible to identify proteins (MIC proteins) that are only present in the micronemes or proteins located in the dense granules (GRA proteins). Indeed, in the case of the rhoptries, it has been possible to identify proteins not just located in the rhoptries but to differentiate between those located in the bulbous region (the ROP proteins) and those specific to the neck region (the RON proteins) (Bradley et al., 2005). The nucleus occupies a central or basal location depending on the invasive stage (discussed later). It is often flattened on the upper side, where the Golgi apparatus is located. It contains a central nucleolus and small clumps

25

of electron dense heterochromatin scattered throughout the nucleoplasm. The nuclear envelope has numerous nuclear pores and is covered on its external side with ribosomes, except on the upper face, where the Golgi apparatus is located (Figs. 2.1A and B and 2.23C). The nuclear envelope is in continuity with sheets of rough ER (rER), which extend into the cytoplasm of the tachyzoite. On the upper surface of the nucleus a layer of clear vesicles of 70 nm diameter, some of which can be seen budding from the nuclear envelope, is topped by three to four flat Golgi cisternae, on top of which numerous vesicles of various contents and size can be observed (Figs. 2.1A and B and 2.23B and C). Using certain preparative techniques, one or two vesicles of c. 200 nm containing one or several electron dense droplets or crystals of various sizes in a clear background are found near the nucleus or in the posterior part of tachyzoites (Fig. 2.1A). These have been termed the acidocalcisomes and the dark content is believed to be a calcium phosphate crystal (Moreno and Zhong, 1996). In addition, under other preparative techniques, a vesicular compartment termed the plant-like vacuole can be seen (Miranda et al., 2010) (Fig. 2.1C). This compartment contains single membrane bounded vesicles of diverse size and appearance and is occupied by a less electron dense material than that present in the cytosol. The PLV has been demonstrated to have immunoreactivity to antisera to proton pyrophosphatase (TgVP1) as well as antisera to cathepsin L-like enzyme (TgCPL) and an aquaporin water-channel (TgAQP1) (Miranda et al., 2010) (Fig. 2.1D and E). Several mitochondrial profiles of 0.5 µM width and various lengths can usually be observed at various locations above and below the nucleus (Fig. 2.1A and B). These represent sections through a single-branched and elongated mitochondrion. They show the typical apicomplexan structure, with bulbous cristae.

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2. The ultrastructure of Toxoplasma gondii

Above the Golgi is the apicoplast (Fig. 2.1B and C). This organelle, limited by multiple membranes, has been identified morphologically since the early 1960s (Ogino and Yoneda, 1966; Sheffield and Melton, 1968; Vivier and Petitprez, 1969) but was only recently shown to be a typical plastid (Kohler et al., 1997). Since it appears to be a feature of all members of the Apicomplexa, with the exception of Cryptosporidium spp., the term apicoplast was proposed. In the infectious stage, it is relatively uniform in shape, up to 500 nm in diameter, bounded by possibly four membranes and filled with granular and filamentous content, in which ribosomes can be observed. The origin of the four membranes is still a matter of debate but could result from a secondary phagocytosis (Kohler et al., 1997). It has also been proposed that the four-membrane structure could result from the extensive invagination of the inner membrane of a doublemembraned organelle (Kohler, 2005).

2.1.2 Comparison of the invasive stages The infectious stages consisting of the tachyzoite, bradyzoite, merozoite, and sporozoite differ from each other in the number of the apical organelles, the shape and electron density of the rhoptries, the location of the nucleus, and the presence or absence of polysaccharide granules. The nucleus is more centrally located in the tachyzoite (Fig. 2.1A) and merozoite (Fig. 2.11B) and more basally located in the bradyzoite (Fig. 2.23B) and sporozoite (Fig. 2.21B). An additional cytoplasmic structure, not described previously, is the polysaccharide granule. The polysaccharide granules are ovoid structures (250 180 nm) of variable electron density located in both the apical and basal cytoplasms. They contain an unusual form of carbohydrate, which is biochemically more similar to plant amylopectin than animal

glycogen (Coppin et al., 2005). These granules are rarely found in tachyzoites or merozoites but are present in large numbers in bradyzoites and sporozoites (Figs. 2.21B and 2.23B). The granules appear to represent a stored energy source, which would be consistent with a possible requirement for long-term survival of the bradyzoites and sporozoites or the extra energy needed during transmission between hosts. The most marked variations are in the apical organelles (see review by Dubey et al., 1998). There are relatively few micronemes in the tachyzoite and merozoites, but these are more numerous in the bradyzoite and sporozoite. In the case of the dense granules, these are numerous (5 12) in the tachyzoite and sporozoites, with fewer in the bradyzoite and very few (2 3) in the merozoite. In the case of the rhoptries, there are differences in the number, shape, and electron density between stages. The number of rhoptries is relatively similar (5 12) for the tachyzoite, bradyzoite, and sporozoite with fewer in the merozoite (2 4). The shape of the rhoptries in the tachyzoite and sporozoites appears to have an elongated swelling with a labyrinthine appearance in comparison to the more bulbous and electron dense swelling in the merozoites and bradyzoites. These differences are summarized in Table 2.1.

2.1.3 Host cell invasion Observations on T. gondii invasion have been performed in cell cultures and on red blood cells (which appear to be a possible, although unusual, abortive host cell (HC) for this parasite) (Michel et al., 1979). Invasion is operated by a moving junction, which has the same morphological features as the one described for Plasmodium knowlesi (Aikawa et al., 1981), both in thin section and in freeze fracture. Interestingly, T. gondii makes the

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2.1 Invasive stage ultrastructure and genesis

TABLE 2.1 Summary of the morphological differences between stages of Toxoplasma gondii. Rhoptries Life cycle stage

Nucleus

Micronemes

Number

Appearance

Dense granules

Polysaccharide granules

Tachyzoite

Central

Few

5 12

Labyrinthine

Numerous

Few

Bradyzoite

Basal

Numerous

5 10

Solid

Numerous

Numerous

Merozoite

Central

Few

3 5

Solid

Few

Absent

Sporozoite

Basal

Numerous

5 10

Labyrinthine

Numerous

Numerous

same junction with nucleated cells and red blood cells (Porchet-Hennere and Torpier, 1983). It is a very close apposition of the parasite and host plasma membrane (Figs. 2.3B and 2.4B), with thickening of the host side and an accumulation of rhomboidally organized IMPs on the protoplasmic face of the host plasma membrane (Fig. 2.3C). This forms a very tight junction, which excludes small electron dense tracers such as ruthenium red. The molecular organization of the moving junction is still not fully elucidated, but data have shown that it involves proteins derived from the rhoptry neck in association with the microneme protein AMA1 (Alexander et al., 2005; Lebrun et al., 2005). Microneme exocytosis has never been clearly visualized, although it is thought to occur both during gliding motility and invasion. What has been shown is the accumulation of alignment of small clear vesicles inside the conoid in conditions of chemically triggered microneme exocytosis, as if these dense rod-like organelles gave rise to these small vesicles before or after exocytosis (Carruthers and Sibley, 1999). The docking site for microneme exocytosis is not known. Rhoptry exocytosis is easily documented upon invasion, as an apical opening in continuity with the parasite plasma membrane, facing the developing parasitophorous vacuole membrane (PVM) (Nichols et al., 1983). Freeze fracture shows an open pit in the PVM at that location

suggesting continuity between rhoptry contents and PVM or even HC cytoplasm (Fig. 2.3A). The role of the apical vesicle and of the apical rosette of IMPs located at the rhoptry exocytosis site have never been elucidated, but the rhoptries open precisely at this location and the IMP rosette disappears, just as in trichocyst exocytosis in Paramecium spp. (Beisson et al., 1976). At very early stages of invasion, when the moving junction forms, small vesicles can be seen budding from the developing vacuole or laying in the HC cytoplasm (Figs. 2.3B and 2.4A). At this stage, empty rhoptries are already observed. Therefore these vesicles correspond to the physiological counterpart of the vacuoles, which are the product of frustrated rhoptry exocytosis in the HC cytoplasm when invasion is blocked by cytochalasin D (Hakansson et al., 2001). The membrane of the developing vacuole is completely devoid of IMPs (Fig. 2.4A) (Dubremetz et al., 1993), reflecting the selective exclusion of the intramembranous HC proteins at the moving junction. However, it will acquire IMPs during the first hour of development (Porchet-Hennere and Torpier, 1983), likely due to parasite contribution, especially from dense granule protein translocation in the PVM (Dubremetz et al., 1993). Progression of the moving junction along the zoite is sometimes, but not always, correlated with parasite constriction.

Toxoplasma Gondii

FIGURE 2.3 Host cell invasion in vitro. (Part A) Serial section though the apical area of a tachyzoite at an early stage of Hela cell invasion. The moving junction is covering the apex of the tachyzoite (arrow); an empty rhoptry has exocytosed its contents in the neighboring HC cytoplasm as small vesicles (v). eR, empty rhoptry. Bar is 0.1 µm. (Part B) Freeze fracture image of the apical area of an invading tachyzoite at a stage corresponding to part (A). The typical structure of the moving junction in the protoplasmic face (Pv) of the HC plasmalemma (which will turn into the parasitophorous vacuole membrane) is visible (arrows), below the parasite apical exoplasmic plasmalemmal face. Bar is 0.1 µm. (Part C) Freeze fracture image of the apex of an invading tachyzoite at a similar stage of invasion as part (B), but corresponding to the complementary fracture faces, showing the pit (arrow) in the parasitophorous vacuole membrane (Ev, exoplasmic face) covering the site of rhoptry exocytosis in the tachyzoite plasmalemma. Pe, protoplasmic face. Bar is 0.2 µm. HC, Host cell.

2.1 Invasive stage ultrastructure and genesis

29

(A)

(B)

FIGURE 2.4 Host cell invasion in vitro. (Part A) Freeze fracture of an invading tachyzoite showing the parasitophorous membrane (Ev), a clump of membrane whorls that may correspond to material exocytosed from the rhoptries (*), and the plasmalemma of the tachyzoite. HC, host cell; Pe, protoplasmic face. Bar is 0.5 µm. (Part B) Section through an invading tachyzoite showing the moving junction (arrows) and the continuity and the difference in electron density between the HCM and the PVM. N, nucleus. Bar is 0.2 µm. HCM, Host cell plasmalemma; PVM, parasitophorous vacuole membrane.

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2. The ultrastructure of Toxoplasma gondii

2.1.4 Parasitophorous vacuole, intracellular development

2.1.5 Endodyogeny

Within minutes after closure of the parasitophorous vacuole (PV), the posterior part of the parasite invaginates and the tubulovesicular network (TVN) starts developing in this invagination (Sibley et al., 1995). The origin of the TVN is not fully understood. It contains dense granule proteins that are exocytosed from the anterior end of the parasite; this exocytosis begins before the completion of the invasion process (Dubremetz et al., 1993). But the origin of the tubular material itself, which is likely made of phospholipids, has never been elucidated; what is known is that the GRA2 protein is required to organize this network (Mercier et al., 2002), and that these tubules are in direct continuity with the PVM, although these two structures contain distinct dense granulesderived proteins (Cesbron-Delauw, 1994). Immediately after invasion, HC mitochondria and ER surround the PV and persist throughout the intracellular development (Fig. 2.8A and B). The ER is devoid of ribosomes on the side facing the vacuole. The distance between these organelles and the PV is highly conserved and is about 12 and 18 nm for mitochondria and ER, respectively (Sinai et al., 1997). The PVMassociated mitochondria may look normal but sometimes show morphological changes, with the cristae becoming larger and irregular in shape and the stroma becoming electron dense. The PV described previously is formed by actively invading parasites and is characterized by the absence of the fusion of the HC lysosomes, thus protecting the parasite during intracellular development (Jones and Hirsch, 1972; Jones et al., 1972). This probably relates to the exclusion of the HC intramembranous proteins from the PVM during invasion. In contrast, parasites within vacuoles formed by HC phagocytosis exhibit lysosome fusion and are broken down in typical phagolysosomes (Jones and Hirsch, 1972).

The tachyzoite is unique in its ability to undergo indefinite proliferation by a distinctive process termed endodyogeny, which involves parasite growth and division to form two daughters. Despite grossly resembling binary fission, endodyogeny is a highly complex event, related to the structural complexity of the formation of polarized daughters. In addition, in the tachyzoite, contrasting with the canonical asexual division mode of most Apicomplexa and even the coccidian stages of T. gondii, the apical complex and invasion-related organelles of the mother persist until the end of the endodyogeny process. Although both events occur simultaneously, we will describe the mitosis and daughter tachyzoite genesis successively. 2.1.5.1 Mitosis There have been few descriptions of T. gondii mitosis at the ultrastructural level and what has been observed can be interpreted by comparison with more detailed studies in related Apicomplexa, especially Eimeria spp. (Dubremetz, 1973). One unique feature of apicomplexan mitosis is the retention of an intact nuclear membrane throughout the process of division. Coccidian-type centrioles (150 nm diameter) consist of nine short tubules (100 nm long) centered on a central tubule. Centrosomes, or spindle pole bodies, are made of two centrioles oriented in parallel (Fig. 2.5B). Centrosomes are always found associated with centrocones, or mitotic spindle poles, usually on the apical side of the nucleus. The earliest stage of mitosis is a transnuclear funnel containing fibrous material, corresponding to an invagination of the nuclear envelope opened on both sides toward the cytoplasm (Fig. 2.5A). The mitotic spindle most likely polymerizes in this funnel which then opens in the nucleoplasm in its middle part, whereas the poles give rise to the centrocones. These latter are at first subspherical

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2.1 Invasive stage ultrastructure and genesis

31

(A)

(B)

(C)

FIGURE 2.5 Tachyzoite endodyogeny. (Part A) Early stage of mitosis: the mitotic spindle elongates into a cytoplasmic funnel through the nucleus (arrow), between the centrioles (Ce). G, Golgi body; Mi, mitochondria. Bar is 0.5 µm. (Part B) Early stage of daughter zoite genesis where a dense fiber (arrow) extends between the centrosome (Ce, centriole) and the newly formed conoid (C). The apical part of the IMC and subpellicular microtubules has started developing. Bar is 0.2 µm. (Part C) Centrocone (ct) budding off the nuclear envelope (Ne) between a centriole (Ce) and three kinetochores (arrows) in an early stage of daughter zoite formation. R, rhoptry. Bar is 0.2 µm. imc, Inner membrane complex.

invaginations of the nuclear envelope opened toward the centrosomes and through which the spindle microtubules extend. The intranuclear spindle is usually very short and transient and has rarely been described. What occurs most likely is that the kinetochores are separated immediately after the funnel opening and assemble on the nucleoplasmic side of the centrocones. Indeed, in Coccidia, caryokinesis does

not depend on mitotic spindle elongation. Centrocones soon become conical evaginations of the nuclear envelope, opened on the centrosomes and covered on the nucleoplasmic side with a layer of multilayered structures corresponding to the kinetochores (Fig. 2.5C). What is specific to this stage is that each round of mitosis occurs simultaneously with the development of two daughter individuals.

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2. The ultrastructure of Toxoplasma gondii

2.1.5.2 Zoite biogenesis Soon after the centrosomes separate and centrocones are formed, the future apical complex of each daughter tachyzoite starts to develop adjacent to each centrosome. The details of this biogenesis have not been studied as thoroughly as in Eimeria spp. (Dubremetz, 1975), but follow the same scheme (Hu et al., 2002a; Vivier, 1970; Vivier and Petitprez, 1969; Francia et al., 2012). A very early stage of development shows a bent fiber originating between the pair of centrioles and joining an area where the conoid is being assembled (Fig. 2.5B). The IMC and underlying subpellicular microtubules array appears to form around the conoid and, in a coordinated manner, starts to grow posteriorly (Figs. 2.5C and 2.6A). This occurs within the mother cell cytoplasm rather than in association with the mother cell plasmalemma that is the characteristic of daughter formation in classic schizogony undergone by most apicomplexans. Early stages are short, flattened cones above the centrocones (Fig. 2.6A), which later elongate into the grossly cylindrical shape that will eventually surround the mature organism (Fig. 2.6B). The Golgi apparatus divides concomitantly with spindle formation with each newly formed dictyosome being found on the upper nuclear envelope, near each centrocone (Pelletier et al., 2002) (Figs. 2.5A and 2.6A). The apicoplast elongates and appears to divide during daughter formation with a portion entering each daughter. Rhoptry precursors are observed at this time as heterogeneous, irregularly shaped vesicles of about 0.3 µm located near the dictyosomes, inside the IMC (Fig. 2.6A). As development proceeds, the nucleus becomes U-shaped and the developing inner complex elongates and engulfs the daughter nuclei (Fig. 2.6B), while additional organelles (rhoptry precursors and then micronemes) are formed anterior to the dictyosome. The rhoptry contents condense to eventually acquire their mature labyrinthine appearance, while the rhoptry ducts appear and elongate toward the conoid. As the daughters

grow, the IMC of the mother cell breaks down along with the anterior organelles. The fully formed daughters fill much of the mother cell cytoplasm and their IMC comes in contact with the mother cell plasmalemma to form the daughter pellicle (Fig. 2.7A and B). This is initiated at the anterior end and results in the daughters remaining connected via a small portion of residual cytoplasm before finally separating. Repeated rounds of division lead to accumulation of tachyzoites within the vacuole, which may adopt a typical rosette appearance when grown in flat cell such as human foreskin fibroblasts (Fig. 2.8A). In certain cases, each of the daughters, while remaining attached by their posterior ends, can undergo new cycles of endodyogeny (Fig. 2.8B). There is evidence for the synchronized division of the tachyzoites within a vacuole (Fig. 2.8A and B). In quantitative studies, it was observed that this was more common in vivo for avirulent parasites compared to virulent ones (Ferguson and Hutchison, 1981). This was only observed for tachyzoites and not seen during division of bradyzoites in tissue cysts. Endodyogeny is the exclusive form of asexual division undergone within the intermediate host (during tachyzoite and bradyzoite formation) and differs from the processes undergone in the definitive host (merozoite formation) or within the oocyst (sporozoite formation) (see later sections).

2.2 Coccidian development in the definitive host 2.2.1 Host parasite relationship Coccidian development is limited to the epithelial cells of the small intestine of the cat (the definitive host). In all stages undergoing coccidian development, the parasites are located within a tight-fitting, thick-walled PV (Fig. 2.9A) (Ferguson, 2004; Ferguson et al., 1974). At higher

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2.2 Coccidian development in the definitive host

33

(A)

(B)

FIGURE 2.6 Tachyzoite endodyogeny. (Part A) Early stage of endodyogeny showing two developing daughters (arrows), with early rhoptries. The Golgi body has divided. Only one nuclear pole (ct, Ce) is in the section plane. A, apicoplast; Ce, centrioles; ct, centrocone; G, Golgi body; imc, inner membrane complex; Mi, mitochondria; N, nucleus; R, rhoptry. Bar is 0.5 µm. (Part B) Later stage of endodyogeny where the daughter nuclei (N) have entered the developing zoites. Bar is 1 µm.

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2. The ultrastructure of Toxoplasma gondii

(A)

(B)

FIGURE 2.7

Tachyzoite endodyogeny. (Part A) Early budding stage where one of the daughter tachyzoites is protruding out of the mother cell by getting wrapped in the mother plasmalemma (arrow). A, apicoplast; G, Golgi body; N, nucleus; R, rhoptry. Bar is 0.5 µm. (Part B) Late stage of daughter budding at a stage where the remnants of the mother cell apical complex are still visible (arrow), while the two daughters are almost completely formed. Bar is 0.5 µm.

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2.2 Coccidian development in the definitive host

35

(A)

(B)

FIGURE 2.8 Intracellular rosette of tachyzoites. (Part A) Typical figure of intracellular tachyzoite multiplication in adherent cells grown in vitro, where divisions occur in one single plane. The vacuole and tubulovesicular network (tvn) surround the parasites, all of which are in an early stage of endodyogeny (arrows). Host cell mitochondria (arrowheads) surround the parasitophorous vacuole membrane. HCN, host cell nucleus. Bar is 1 µm. (Part B) Repeated endodyogeny showing the synchronized initiation of a new round of daughter formation (arrowheads), while the original daughters are still connected at the posterior end (arrow). HCN, host cell nucleus. Bar is 1 µm.

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2. The ultrastructure of Toxoplasma gondii

(A)

(B)

(C)

(D)

(E)

(F)

FIGURE 2.9 Toxoplasma gondii developing in enterocytes of the cat intestine. (Part A) Early developmental stage located in a thick walled, tight fitting parasitophorous vacuole (PV). N, nucleus. Bar is 1 µm. (Part B) Enlargement showing the laminated structure of the electron dense membrane limiting the parasitophorous vacuole (PVM). Note a conical structure protruding into the membrane (arrow). Bar is 100 nm. (Part C) Tangential section through the membrane of the parasitophorous vacuole illustrating the circular shape of the conical protrusion into the parasitophorous vacuole membrane (arrows). Bar is 100 nm. (Part D) Detail in which the membrane of the vacuole can be resolved into three unit membranes (arrowheads). Bar is 100 nm. (Part E) Mid stage schizont with a number of nuclei (N) and a centrally located elongated apicoplast (A). Bar is 1 µm. (Part F) Detail showing the double membranes enclosing the mitochondrion (Mi) and nucleus (N) compared to the multiple membranes enclosing the apicoplast (A). Bar is 0.5 µm.

Toxoplasma Gondii

2.2 Coccidian development in the definitive host

power the wall has a laminated appearance, which in certain areas can be seen to consist of three closely applied unit membranes (Fig. 2.9B and D). In addition, there are a number of conical-shaped dense structures impinging on the luminal surface of the PV (Ferguson, 2004) which, in certain cases, appeared to connect the surface of the parasite to the PVM (Fig. 2.9C and D). In contrast to the host/parasite relationship of the tachyzoite, there was no evidence of formation of the tubular structure within the PV or the congregation of the HC mitochondrion or strands of rER around the periphery of the PV (Fig. 2.9A). These structural differences are correlated with the lack of expression of the majority of dense granule proteins. Of the GRAs 1 8 and NTPase identified in the tachyzoite, only GRA7 and NTPase are expressed by the gut stages (Ferguson, 2004; Ferguson et al., 1999a,b). This laminated, thick-walled PV is similar to that observed for certain Isospora species (Ferguson et al., 1980) to which T. gondii is closely related but differs from those of the genus Eimeria, which are limited by a singleunit membrane (Ferguson et al., 1976).

2.2.2 Asexual development During coccidian development, only a single asexual process has been observed, which has unique structural features and has been termed endopolygeny (Piekarski et al., 1971). This term had been used previously to describe an abnormal type of development observed for the tachyzoite (Vivier, 1970). However, the abnormal tachyzoite development described did not represent an internal budding process. Therefore because of the accuracy of the description and its usage over the years, it would appear appropriate to retain the term for the description of the asexual multiplication of the coccidian stages. In studies of coccidian development of both types 2 and 3 strains of

37

T. gondii occurring between 4 and 10 days postinfection (PI), only a single process was observed, although there were marked variations in the number of daughters formed. The process involved growth of the parasite and repeated nuclear divisions (Fig. 2.9E) employing an eccentric intranuclear spindle, as described during endodyogeny. There is also a marked increase in the size of the mitochondria that are located predominantly around the periphery. In addition, it was possible to observe multiple profiles of the apicoplast (limited by four membranes) but these were more centrally located and, from immunocytochemistry, appeared to consist of a single-branched structure (Ferguson and Hutchison, 1981). These three organelles could be differentiated by their ultrastructural features (Fig. 2.9F). The number of nuclear divisions varied between parasites which had a direct effect on the number of daughters formed. It is the presence of this proliferative phase prior to daughter formation that distinguishes endopolygeny from endodyogeny. It is not clear how the number of nuclear divisions is controlled but it does not appear to relate to parasite size or a given number of nuclear divisions, since these can vary markedly between parasites (Fig. 2.10A and C). The trigger for the end of the proliferative phase and the initiation of the differentiation phase (daughter formation) is unclear. It is at the end of the proliferative phase that the elongated apicoplast divides simultaneously into a number of fragments equal to the number of nuclei. Daughter formation can occur in parasites containing between 4 and 20 nuclei and is initiated during or just after the final nuclear division (Fig. 2.10A and B). The first evidence of the initiation of daughter formation was the appearance of a conical structure formed by a number of flattened vesicles each with underlying longitudinally running microtubules and with the conoid in the apex (Fig. 2.10B). The initiation of daughter formation is synchronized with all daughters forming at the same

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(A)

(C)

2. The ultrastructure of Toxoplasma gondii

FIGURE 2.10 Early stages of

(B)

endopolygeny. (Part A) Small schizont with few nuclei (N) but showing the initiation of daughter formation (D). Bar is 1 µM. (Part B) Detail of a schizont showing the plate-like structures of the inner membrane complex representing the initiation of daughter formation (arrows). Ce, centriole; N, nucleus; NP, nuclear pole; G, Golgi body. Bar is 0.5 µm. (Part C) Low power of a large schizont with a number of nuclei (N) showing the formation of a larger number of daughters (D). Bar is 1 µm. (Part D) Detail showing the posterior growth on the inner membrane complex of the daughter to partially enclose the apicoplast (A) and nucleus (N). Note the anlagen of the rhoptry (R) and the conoid (C) in the apex of the daughter. Bar is 0.5 µm.

(D)

time (Fig. 2.10A and C). The mechanism of daughter formation is similar to that observed for the two daughters formed during endodyogeny of the tachyzoite or bradyzoite. Since this occurs at a multinucleated stage, numerous daughters are formed, thus the appropriateness of the term endopolygeny. The simultaneous formation of a large number of daughters requires an extremely wellcoordinated process to ensure that all daughters receive a full complement of organelles and are therefore viable. As daughter formation progresses by the posterior growth of the IMC, it encloses a nucleus, apicoplast, and mitochondrion (Fig. 2.10D). In the apical cytoplasm, one or two electron dense spherical structures representing the nascent rhoptries

and a number of cigar-shaped micronemes appear (Fig. 2.10D). The merozoites have relatively few dense granules and these appear to form late in daughter development. This posterior growth continues until the merozoite is fully formed and contains the full complement of organelles. In the apical cytoplasm, there is maturation of the rhoptries with the development of the duct leading to the conoid. In contrast to the tachyzoite and bradyzoites, the bulbous end of the rhoptry remains spherical. At this point the daughters fill the mother cell cytoplasm but are still enclosed in the schizont plasmalemma. The final stage is the invagination of the mother cell plasmalemma, starting at the anterior of the daughter and progressing posteriorly to form the outer membrane of the

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2.2 Coccidian development in the definitive host

pellicle of each daughter (Fig. 2.11A). A single micropore is formed in the pellicle just anterior to the nucleus. The merozoites often remain attached to a small amount of residual cytoplasm at the posterior end. These bananashaped daughters can be seen forming fan-like structures (Fig. 2.11B and C). The merozoites are released from the HC into the lumen, where they can reinvade enterocytes. However, this process has not been observed by EM. Unlike many Eimeria species, there does not appear to be the distinct sequential generations of schizogony, which differ from each other in

(A)

39

their size and number of daughters formed. However, in studies of the early stages of infection (1 3 days), additional asexual processes have been described (Speer and Dubey, 2005). It has been reported that certain developing parasites have a similar host/parasite relationship and undergo endodyogeny and repeated endodyogeny (type B schizonts), while others appeared intermediate (type C schizonts). The type B schizonts have a similar relationship to that described for parasites invading the small intestine of the intermediate host (Dubey, 1997; Speer and Dubey, 1998). These stages appear to be rare and could represent examples, where FIGURE 2.11 Late stages of endopolygeny. (Part A) Late schizont showing the daughters filling the mother cell cytoplasm and the invagination of the plasmalemma around the daughters (arrows). N, nucleus; R, rhoptry; MN, microneme. Bar is 1 µm. (Part B) Mature schizont with fully formed merozoites with the characteristic apical organelles. N, nucleus; C, conoid; R, rhoptry; DG, dense granule. Bar is 1 µm. (Part C) Scanning electron micrograph of a fracture through the epithelial cells of a villus. A number of small Tr and two mature schizonts with crecentic-shaped merozoites (arrows) can be seen. Bar is 2 µm. Tr, Trophozoites.

(B)

(C)

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40

2. The ultrastructure of Toxoplasma gondii

the initial invading bradyzoite failed to undergo conversion to coccidian development but underwent conversion to tachyzoite development as seen in the intermediate host. It is known that the cat can act as an intermediate host as well as the definitive host.

2.2.3 Sexual development After an unknown number of asexual cycles, certain merozoites on entering a new enterocyte can develop into either male (microgametocyte) or female (macrogametocyte) gametocytes. In microgametogony, this results in the formation of multiple (15 30) male (microgametes) and, in macrogametogony, the formation of a single female (macrogamete). The trigger for the conversion from asexual to sexual development is unknown. Nor is it known what is responsible for deciding whether an invading merozoite develops into either a microgametocyte or macrogametocyte. The initiation of gametocyte formation appears to be less controlled in T. gondii than other species of Coccidia. In the majority of Eimeria spp., there are a fixed number of asexual cycles followed by the vast majority of merozoites simultaneously develop into sexual stages. In T. gondii, there does not appear to be a distinct conversion with a mixture of both asexual and sexual stages observed throughout enteric development. There were no ultrastructural features that could identify the merozoite that would develop into sexual stages nor were there any differences in the host/parasite relationship or PV. On entering the HC the merozoite becomes more spherical and loses the majority of its apical organelles, such as the rhoptries and dense granules, although the conoid and a few micronemes remain. This stage (trophozoite) begins to grow and there appears to be an increase in the size of the mitochondrion/mitochondria, which are located around the periphery.

2.2.3.1 Microgametogony and the microgamete There are relatively few descriptions of microgametogony (Colley and Zaman, 1970; Dubey et al., 1998; Ferguson et al., 1974; Pelster and Piekarski, 1971). Initially it is impossible to differentiate between the proliferative phase of endopolygeny and microgametogony with both processes involving continued growth and repeated nuclear divisions. It has been reported that the earliest stage allowing differentiation between schizogony and microgametogony is based on the difference in the distribution of the nuclear chromatin (Ferguson et al., 1974). During schizogony the electron dense heterochromatin remains dispersed throughout the nuclei (Fig. 2.9E), whereas in the later stages of microgametogony, the heterochromatin condenses into electron dense masses at the periphery of the nucleus (Fig. 2.12A). In microgametogony the nuclei move to the periphery of the cell with two centrioles and a dense plaque (perforatorium) located between the nuclei and the plasmalemma (Fig. 2.12A). The centrioles become the basal bodies for the developing flagella, which begin to grow by protruding into the PV (Fig. 2.12B). Interestingly, although the centrioles differ from metazoan centrioles, the flagella have the typical nine peripheral duplet tubules with the two central microtubules. As this flagellar growth occurs, the chromatin condensation continues at the side of the nucleus closest to the centrioles with the other part of the nucleus having a more electron lucent appearance. In addition, a mitochondrion is located adjacent to each nucleus (Fig. 2.12B). There is no significant development in the apicoplast during this process (Ferguson et al., 2005). Microgamete development continues with flagellar growth and protrusion of a portion of cytoplasm containing the basal bodies, the electron dense portion of the nucleus, and a mitochondrion into the lumen of the PV (Fig. 2.12B D). As this occurs, there is division

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2.2 Coccidian development in the definitive host

(A)

(B)

(C)

(D)

41

FIGURE 2.12 Electron micrographs of various stages in the process of microgametogony. (Part A) Mid stage microgametocyte showing the peripherally located nuclei with areas of condensed chromatin. Centriole (Ce) and the plate-like perforatorium (P) can be seen located between the nuclei (N) and the plasmalemma. Bar is 1 µm. (Part B) Detail showing the protrusion the flagella (F) plus the dense portion of the nucleus and a mitochondrion (Mi) into the PV. Bar is 0.5 µm. (Part C) Late stage showing a number of microgametes forming in the PV, while still attached to the mother cell (arrows). N, nucleus; F, flagellum. Bar is 1 µm. (Part D) Detail from part (C) showing elongating nucleus (N) and Mi of the microgamete still connected to the mother cell (arrows). Bar is 0.5 µm. Toxoplasma Gondii

42

(A)

2. The ultrastructure of Toxoplasma gondii

FIGURE 2.13 The structure of the mature microgamete. (Part A) Longitudinal TEM section through a microgamete showing the dense nucleus (N) and the anterior mitochondrion (Mi) and the basal bodies of the two flagella (F). Bar is 1 µm. (Part B) SEM of a microgamete illustrating the nucleus (N) and the two very long posteriorly pointing flagella (F). Bar is 1 µm.

(B)

of the nucleus with the electron dense portion separating from the electron lucent portion. The electron dense portion enters the developing microgamete and the lucent portion remains within the mother cell as a residual nucleus. The microgametocyte of T. gondii produces relatively few (15 30) microgametes (Fig. 2.12C). The immature microgametes are still attached to the mother cell by a narrow cytoplasmic isthmus (Fig. 2.12C and D). Maturation continues with each microgamete becoming elongated in appearance and consisting of an electron dense nucleus with a mitochondrion located between the nucleus and the basal bodies, from which the two very long flagella run toward the posterior (Fig. 2.13A and B). In addition, there is an electron dense plate termed the perforatorium in the apex and four longitudinally running microtubules (Ferguson et al., 1974). Once fully formed, the microgametes detach from the microgametocyte leaving a large residual cytoplasmic body.

2.2.3.2 Macrogametogony and the macrogamete The development of the macrogametocyte has been described in a few studies (Colley and Zaman, 1970; Ferguson et al., 1975; Pelster and Piekarski, 1972). It is associated with the growth of the trophozoite and the appearance of a large nucleus with dispersed chromatin and a large nucleolus but no nuclear division. As the macrogametocyte grows, there is a marked increase in the size of the peripherally located mitochondrion and the centrally located apicoplast (Fig. 2.14A). In addition, a number of Golgi bodies are distributed throughout the cytoplasm. The first distinct organelle of macrogametogony is the appearance of flocculent material condensed within dilatations of the rER (Fig. 2.14B and D). This material represents the initiation of formation of the wall-forming body type 2 (WFB2) socalled because of their role in the formation of

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2.2 Coccidian development in the definitive host

(A)

43

(B)

(C)

(D)

FIGURE 2.14 Various stages in the development of the macrogametocyte. (Part A) Early macrogametocyte characterized by the central nucleus (N) with a large nucleolus (Nu). The cytoplasm contains a number of profiles of an enlarged mitochondrion (Mi) and an enlarged Golgi body (G). A few PG and lipid droplets (L) were present in the cytoplasm. Bar is 1 µm. (Part B) Mid stage macrogametocyte showing increasing numbers of PG and lipid droplets (L) and the appearance of wall forming bodies type 1 (W1) and type 2 (W2) in the cytoplasm and an increase in size of the apicoplast (A). N, nucleus. Bar is 1 µm. (Part C) Mature macrogamete showing the centrally located nucleus (N) with adjacent apicoplast (A). The cytoplasm contains numerous wall forming bodies of type 1 (W1) and a few type 2 (W2) plus numerous PG and lipid droplets (L). Bar is 2 µm. (Part D) Detail of the cytoplasm of a mature macrogamete showing the numerous dense granules representing the wall forming bodies type 1 (W1). The wall forming body type 2 (W2) is located within the rough endoplasmic reticulum. PG, polysaccharide granule; L, lipid droplet. Bar is 0.5 µm. PG, Polysaccharide granules.

the oocyst walls (discussed later). A Golgi body is often associated with the membrane of ER surrounding the WFB2. As maturation continues, there is an increase in size and number of the WFB2 and a number of electron dense membrane bound granules appear to form

from vesicles produced by the Golgi bodies (Fig. 2.14C). These were of various sizes and were termed the wall-forming body type 1 (WFB1) (Fig. 2.14D). However, it has been possible using immunoelectron microscopy to identify two populations of membrane bound

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2. The ultrastructure of Toxoplasma gondii

electron dense granules (Ferguson et al., 2000). One population, which appears to be involved in the formation of the outer veil, is termed the veil-forming bodies (VFB). These were originally termed WFB type 1a, but, with the identification of similar granules in the macrogametocyte of Eimeria maxima, it was proposed that VFB may be a more appropriate term (Ferguson et al., 2003). The WFB1 appear slightly larger than the VFB. As the VFBs and WFBs are being synthesized, there is also the synthesis of numerous polysaccharide granules and lipid droplets and an expansion of the apicoplast (Fig. 2.14C). When fully developed, the macrogametocyte can be considered to be a mature macrogamete (Fig. 2.14C). This is not a sharp division but one of convenience to differentiate the developing stage from the mature gamete.

2.2.4 Oocyst wall formation The oocyst wall is a multilayer structure, which is extremely resistant to physical and chemical insults. As such, it is fundamental to the survival of the parasite. Without this wall, the parasite could not survive in the external environment for the extended periods required for transmission between hosts by fecal contamination. The oocyst wall is a complex structure consisting of distinct layers (Ferguson et al., 1975; Speer et al., 1998). The oocyst wall is synthesized, while the macrogamete is still within the HC. In reviewing these data with reference to later observations for both T. gondii and Eimeria spp., the wall can be divided into three zones. The first is the formation of a loose outer veil consisting of two-thirds membranes [termed layer 1 3 (Ferguson et al., 1975)], which is formed by the fusion of the VFB with the macrogamete plasmalemma and release of their contents (Ferguson et al., 2000). This occurs during the maturation of the macrogamete. This is followed by the triggered

secretion of the WFB1, which occurs simultaneously in the mature macrogamete to form the outer layer of the oocyst wall [termed layer 4 (Ferguson et al., 1975)] (Fig. 2.15A). This initially forms a thick layer that undergoes polymerization to form a 30 70 nm electron dense layer. Finally, the contents of the WFB2 are released and coalesce to form the electron lucent inner layer of the oocyst wall [termed layer 5 (Ferguson et al., 1975)] (Fig. 2.15B and D). The cytoplasmic mass loses the WFBs during oocyst wall formation and is characterized by a central electron lucent nucleus and cytoplasm packed with polysaccharide granules and lipid droplets (Fig. 2.15B). This process is identical to that described for the closely related genus Eimeria (Ferguson et al., 2003). For correct formation of the oocyst wall, there is a requirement for tight control and sequential secretion of the VFB and the WFB1 and 2. From the available data for the Coccidia, it would be most accurate to consider the outer veil as part of the early development as it is lost by the time oocysts are released with the feces. The oocyst proper can be considered as a double-layered structure (reviewed by Belli et al., 2006). The outer electron dense layer is thinner in the T. gondii oocyst (Fig. 2.15D) than those of Eimeria spp. (Belli et al., 2006). The formation and polymerization of the inner layer has a dramatic effect on the ability to process the oocyst for ultrastructural examination. To date, no technique has been developed, which will allow the oocysts of T. gondii or any other coccidian oocyst to be examined by EM. Over the past 30 years, numerous attempts, using many electron microscopic fixatives and embedding protocols, have resulted in failure. The two layers provide different structural and chemical protection. The outer layer contains mostly proteins and carbohydrate and appears to provide structural strength. In contrast the inner layer has high lipid content and appears to provide the protection from chemical insult by its impervious nature (even to EM

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2.2 Coccidian development in the definitive host

(A)

45

(B)

(C)

(D)

FIGURE 2.15 Early stages of oocyst formation. (Part A) Early stage of oocyst wall showing the formation of the outer veil (V) and partial formation of the outer layer of the oocyst wall (arrows). Note this is associated with the loss of the VFB and the WFB1 from the macrogamete cytoplasm, while the WFB2 (W2) remains. L, lipid droplet; PG, polysaccharide granule; N, nucleus. Bar is 1 µm. (Part B) Newly released oocyst showing the outer veil (V) and fully formed OW enclosing a cytoplasmic mass containing PG and lipid droplets (L). Bar is 1 µm. (Part C) SEM showing a number of microgametes (Mi) apparently attach to a macrogamete/oocyst (Ma) with two adjacent merozoites (Me). Bar is 1 µm. (Part D) Detail of the oocyst wall consisting of the outer veil (V) plus the thin electron dense outer layer (O) and the thicker inner layer (I) which separates from the plasmalemma (P) of the cytoplasmic mass. Bar is 100 nm. OW, Oocyst wall; PG, polysaccharide granule.

reagents). Work on the properties of the oocyst is continuing in the closely related genus Eimeria (Belli et al., 2006).

2.2.5 Fertilization It would appear logical that, if sexual development takes place, there will be fusion

between a microgamete and a macrogamete to form a fertilized zygote. However, this process has never been visualized. It could be expected that the mature microgametes and macrogametes are released from the HCs and fertilization takes place in the lumen. Indeed, macrogametes/oocysts with attached microgametes have been observed on rare occasions (Ferguson, 2002) (Fig. 2.15C). However, it has

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2. The ultrastructure of Toxoplasma gondii

been described previously, oocyst wall formation is initiated prior to release of the macrogamete from the HC. An additional anomaly in T. gondii is the formation of very few microgametocytes producing relatively few microgametes in relation to the number of macrogametes. It is a universal feature of plants and animals that there is a vast excess of male gamete formation because of the importance of ensuring maximum fertilization of the female gametes. That fertilization can occur has been proven from the identification of cross fertilized parasites (Pfefferkorn and Pfefferkorn, 1980). However, T. gondii, unlike most other metazoan, is normally haploid and whether this will affect the necessity for fertilization is open to question (Ferguson, 2002).

2.2.6 Oocyst and extracellular sporulation The oocyst is the only stage of T. gondii that is capable of undergoing extracellular development—all other development processes can only occur within viable HCs. The oocysts are excreted in an unsporulated form with a single undifferentiated cytoplasmic mass: the primary sporoblast (Fig. 2.16A). In the external environment, asexual development (sporulation) occurs, which finally results in the formation of two sporocysts, each of which contains four sporozoites. Initial attempts to study this process were unsuccessful because of our inability to process the oocyst for ultrastructural examination. It was only possible to overcome this problem by developing a technique that involved freezing and cryosectioning of the oocysts prior to processing for EM (BirchAndersen et al., 1976). The aim was to fracture the oocyst wall without destroying the cytoplasmic mass within. This technique was inefficient with destruction of a large proportion of oocysts. However, a few oocysts remained intact and these were used to examine the

ultrastructural changes associated with sporulation. Due to the difficulties, these studies have been limited to a series of papers on the sporulation of Eimeria brunetti (Ferguson et al., 1978a,b) as model for the genera Eimeria and T. gondii (Ferguson et al., 1979a,b,c). The quality of the ultrastructural observations is limited due to the technical difficulties, but the developmental process could be followed. The original central cytoplasmic mass termed the primary sporoblast was similar to that of the macrogamete (Figs. 2.15B and 2.16A). The cytoplasm containing a single large nucleus plus numerous polysaccharide and lipid droplets admixed with mitochondria and rER is enclosed by a unit membrane (Fig. 2.16A). In T. gondii the nucleus underwent two rounds of division giving rise to four nuclei (Fig. 2.16B). The cytoplasmic mass then underwent elongation and became limited by two additional membranes. This was followed by cytokinesis of the cytoplasmic mass with the formation of centrally located infoldings of the limiting membranes (Fig. 2.16C), which finally fused, thus dividing the primary sporoblast into two spherical secondary sporoblasts, each with two nuclei (Fig. 2.16D). This process is summarized diagrammatically in Fig. 2.17. As each secondary sporoblast develops, it becomes more elongated or cigar-shaped and develops into the sporocyst, which is characterized by the formation of the sporocyst wall (Fig. 2.18A, D, and E). In T. gondii the wall of the sporocyst appears to be formed by material secreted from the cytoplasm that condenses on one of the limiting membranes. It forms a distinctive structure comprising four plates (Fig. 2.18 D and E) joined by specialized sutures with an overlaying thin layer of electron dense material (Figs. 2.18B and 2.19A). This wall has various banded striations, which could be consistent with organized repetitive protein structures which probably provide structural strength and increase resistance to external insult (Fig. 2.19B).

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2.2 Coccidian development in the definitive host

(A)

(B)

(C)

(D)

47

FIGURE 2.16 Early oocyst sporulation in the external environment. (Part A) Section through an unsporulated oocyst (0 h) showing the central nucleus (N) with cytoplasm containing a Golgi body (G), mitochondria (Mi) and a number of PG and lipid droplets (L). Bar is 1 µm. (Part B) Early stage in sporulation with the cytoplasmic mass containing a number of nuclei (N). PG, polysaccharide granules; L, lipid droplets. Bar is 1 µm. (Part C) Section through an oocyst in which the cytoplasmic mass has started to divide (arrows) to form the two secondary sporoblasts. Note the two nuclei (N) in one of the forming sporoblasts. PG, polysaccharide granules. Bar is 1 µm. (Part D) Section through the two secondary sporoblasts. N, nucleus; L, lipid droplet; PG, polysaccharide granules. Bar is 1 µm. PG, Polysaccharide granules.

Within the cytoplasm of the developing sporocyst, a nucleus was observed at either end of the elongated sporocyst with the majority of the cytoplasm containing polysaccharide granules and lipid droplets (Fig. 2.18A). It was observed that the anlagen of two daughters formed adjacent to the plasmalemma above each nucleus at either end of the sporocyst (Fig. 2.18C). The process of daughter formation was similar to that observed during endodyogeny with the nucleus appearing to divide during the posterior growth of the IMC of each daughter with two daughters forming at either end of the sporocyst (Fig. 2.18C). This inner membrane growth continued until the daughters were fully formed

and enclosed a nucleus, apicoplast and mitochondrion and the apical organelles (micronemes, rhoptries, and dense granules). This formation of the daughters adjacent to the sporocyst plasmalemma differs from the internal formation associated with endodyogeny or endopolygeny. In this situation, it was observed that the plasmalemma invaginated with the growth of the IMC to form the sporozoite pellicle and in this respect it is similar to classical schizogony. This resulted in the formation of four daughters (two from each end). A small residual cytoplasmic mass remains within each sporocyst. The process of sporulation is represented diagrammatically in Fig. 2.20.

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2. The ultrastructure of Toxoplasma gondii

FIGURE 2.17 A diagrammatic representation of the changes observed during the development of the zygote and formation of the sporoblasts (A) primary sporoblast; (B) four nuclei stage; (C) cytokinesis; (D) secondary sporoblasts.

2.2.7 Excystation There have been few ultrastructural studies on the process of excystation (Christie et al., 1978; Ferguson et al., 1979d; Speer et al., 1998). In certain studies the oocyst wall was broken mechanically by grinding, although it has been reported that reasonable excystation can occur without this process (Speer et al., 1998). Excystation is stimulated by incubation in a mixture of trypsin and bile salts (sodium taurocholate). This excystation fluid appears to act on the sporocyst wall causing increased tension, which results in an infolding of the edges of the plates along the suture lines (Fig. 2.21A and B). At the sutures, there is a separation of the inner aspect of the inner layer of the sporocyst wall, which initially remained attached at the outer edge (Fig. 2.21C). This connection eventually ruptures and with it the outer membrane of the sporocyst

wall (Fig. 2.21D). There appears to be rapid separation of the plates and infolding to form scroll-like structures that allow the sporozoites to escape (Fig. 2.21E).

2.3 Development in the intermediate host 2.3.1 Tachyzoite development When an intermediate host is infected by ingestion of tissue cysts or oocysts, the bradyzoites and sporozoites are released into the lumen of the small intestine. These invade the enterocyte or intraepithelial lymphocytes of the small intestine or pass through into the lamina propria and invade cells there. The process of invasion has not been observed but is likely to be similar to that described previously

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2.3 Development in the intermediate host

(A)

FIGURE 2.18 Development of the sporocyst with sporozoite formation. (Part A) Early development of the sporocyst showing the elongated appearance with a nucleus (N) located at either end, and the cytoplasm containing PG and lipid droplets (L). Bar is 1 µm. (Part B) Cross section through the sporocyst wall at the junction between plates showing the outer (O) and inner (I) layers. There is a swelling of the plates of the inner layer at the junction where they are joined by an IS of material. Bar is 100 nm. (Part C) Enlargement of part of a sporoblast showing the nucleus (N) and the two dense plaques (arrows) representing the initiation of daughter formation. Bar is 1 µm. (Part D) Advanced stage of SP formation showing the nucleus becoming enclosed by the inner membrane complex of the daughters. The junction between the four plates of the sporocyst wall can be seen (arrows). Bar is 1 µm. (Part E) Scanning electron micrograph illustrating the raised junctions between the plates (arrows). Bar is 1 µm. IS, Intermediate strip; PG, polysaccharide granules.

(B)

(C)

(D)

(E)

for tachyzoites (Section 2.1.3). In either case the parasite (bradyzoite or sporozoite) defaults to tachyzoite development with formation of the characteristic PV and undergoes multiplication

by endodyogeny (see has been described Dubey (Dubey et Dubey, 1998). From

Toxoplasma Gondii

Section 1.5). This process in detail by Speer and al., 1998; Speer and in vitro studies it was

50

2. The ultrastructure of Toxoplasma gondii

FIGURE 2.19 A diagrammatic representation of a cross section (Part A) and the 3D (Part B) appearances of the sporocyst wall at the junction between the four plates which form the sporocyst. I, Inner layer; IS, intermediate strip.

FIGURE 2.20 A diagrammatic representation of the changes occurring during the development of the sporocysts and formation of the sporozoites; (A) primary sporoblast; (B) four nuclei stage; (C) cytokinesis; (D) secondary sporoblasts.

originally proposed that the sporozoite entered a HC and formed an enlarged PV, which it then left to form a second vacuole within which it underwent tachyzoite development

(Speer et al., 1995); however, this was not observed in their in vivo studies and could represent an in vitro artifact (Dubey et al., 1997). Thereafter, the tachyzoites disseminated

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2.3 Development in the intermediate host

(A)

(B)

(C)

(E)

51

(D)

FIGURE 2.21 Changes in the sporocyst associated with excystation. (Part A) Scanning electron micrograph of a sporocyst undergoing excystation showing the separation of the plates of the sporocyst wall (arrows). Bar is 1 µm. (Part B) Transmission electron micrograph through an excysting sporocyst showing separation and infolding of the plates of the sporocyst wall (arrows). The SP contain a posteriorly located N and numerous MN and PG. C, conoid. Bar is 1 µm. (Part C) Early stage in excystation showing inward curling (arrow) of the sporocyst wall at the junction of the plates of the inner layer (I) resulting in a separation of the plates and the IS. O, outer layer. Bar is 100 nm. (Part D) More advance stage of excystation showing separation of the inner plates and rupture of the outer layer (arrow). Bar is 200 nm. (Part E) Example of the continued curling of the plates of the sporocyst wall to form tightly wound scroll-like structures. Bar is 100 nm. IS, Intermediate strip; MN, micronemes; PG, polysaccharide granules; SP, sporozoites.

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systemically via the vascular system to all organs of the body. In various organs they undergo proliferation by endodyogeny in many cell types and were present initially in large numbers in the lungs and spleen but by 6 10 days had invaded all organs, including the brain. However, in immune competent mice (genetically resistant to Toxoplasma), the number of lesions and tachyzoites peaked at about 12 days and by 21 days it was difficult to identify tachyzoites in any organ, including the brain, even using immunocytochemistry.

2.3.2 Stage conversion: tachyzoite to bradyzoite There appears to be very marked tissue tropism in relation to the organs where the majority of tissue cysts are formed. The two tissues where the majority of tissue cysts are observed are the striated muscles, including the heart and the central nervous system. However, this can vary between species. For example, the majority of tissue cysts are located in the musculature in pigs (Dubey, 1986), but predominantly in the brains of mice. This again contrasts with the in vitro situation where almost any cell type can act as a HC during stage conversion. This again emphasizes the need for caution when extrapolating from in vitro results. Stage conversion was examined in detail in mice. At 12 15 days postoral infection, lesions were observed in the brain which consisted of numbers of parasites undergoing tachyzoite development admixed with parasites forming early tissue cysts (Fig. 2.22A). It was observed that only a small subpopulation of the tachyzoites underwent conversion. The lesions consisted of numbers of extracellular tachyzoites plus a few intracellular organisms located in typical tachyzoite-like PVs (Fig. 2.22B and D). These could often be identified as inflammatory cells, which formed part of the lesion. However, it was

also possible to identify a number of early tissue cysts (Fig. 2.22C and E). A number of cysts were seen within the lesion and indeed it was possible to observe two cysts forming within the one HC. These could be differentiated from tachyzoitelike vacuoles on the distinctive structure of the PV and PMV enclosing parasites that could be identified at the 1 2 cell stage (Fig. 2.22E). The distinctive PV appears to form at the time of invasion. This is also consistent with the immunocytochemical results using the stage-specific antibodies (SAG1 and ENO2 for tachyzoites and BAG1 and ENO1 for bradyzoites). The PVs of early tissue cyst were characterized by their tight fit and being limited by a membrane with numerous irregular, shallow invaginations (Fig. 2.22E). These vacuoles lacked the tubular network but possessed a thin layer of amorphous material. In addition, there was no congregating of the HC mitochondrion or rER around the vacuole. When examined by immunoelectron microscopy, it was found that the material beneath the membrane reacted positively to the cyst wall protein recognized by the antibody CC2 and to antibody to CST1 (Tomita et al., 2013). It was reported that a small subpopulation of the tachyzoites from peritoneal exudates contained lucent cytoplasmic granules which were positive with CC2 (Gross et al., 1996). It is interesting to speculate that there is a subpopulation of tachyzoites which, on reaching the correct environment, may be preprogramed and are able to initiate tissue cyst formation directly. These tissue cysts continue to enlarge over the next three weeks with the bradyzoites dividing by endodyogeny (Fig. 2.23A). Initially, a large number of the parasites are undergoing endodyogeny, but the proportion of dividing organism reduces during the first 28 days PI and from 3 months onward very few dividing parasites are seen (Ferguson and Hutchison, 1987a). During this early development the cyst enlarges and the depth of the invaginations increases and a more distinct

Toxoplasma Gondii

(A)

(B)

(C)

(D)

(E)

FIGURE 2.22 Early stages of development in the mouse brain. (Part A) Low magnification of a section through the brain of a mouse at 15 days postinfection showing lesion undergoing stage conversion. Note the formation of a number of early tissue cysts (Cy) and the group of tachyzoite-like organisms (T). Bar is 5 µm. (Part B) Part of a cell in the brain showing a tachyzoite (T) within a typical tachyzoite parasitophorous vacuole with its TN. Bar is 1 µm. (Part C) Example of a very early tissue cyst containing two parasites contained within a tight fitting vacuole (PV). N, nucleus. Bar is 1 µm. (Part D) Detail from part (B) showing the strand of rER associated with the parasitophorous vacuole membrane characteristic of a T-containing vacuole. Bar is 100 nm. (Part E) Detail from part (C) showing the undulating parasitophorous vacuole membrane (arrows) and the absence of HC organelles associated with the membrane. HC, host cell. Bar is 100 nm. HC, Host cell; rER, rough endoplasmic reticulum; TN, tubular network.

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2. The ultrastructure of Toxoplasma gondii

(A)

(B)

(C)

(D)

(E)

FIGURE 2.23 Mature tissue cysts and bradyzoites in mouse brain. (Part A) Part of the periphery of an early tissue cyst (21 days postinfection) showing the CW and the thin rim of HC cytoplasm. The cyst contains a number of bradyzoites (Br) and electron lucent organisms undergoing endodyogeny (arrows). Bar is 1 µm. (Part B) Longitudinal sectioned bradyzoite showing the posteriorly located nucleus, numerous PG plus DG, rhoptries (R), MN, and conoid (C). Bar is 1 µm. (Part C) Detail of the region just anterior to the nucleus of the bradyzoite in part B showing the Golgi body (G) and the apicoplast (A). PG, polysaccharide granule. Bar is 100 nm. (Part D) Low power of a mature tissue cyst (1 year postinfection) showing a large number of bradyzoites (Br) enclosed by a CW retained within a HC. Bar is 10 µm. (Part E) Detail from the periphery of the cyst in part (D) showing the CW with deep invaginations of the limiting membrane of the cyst into the underlying granular material. Note the HC can be definitively identified as a neuron because of the presence of synapses (S). Bar is 100 nm. CW, Cyst wall; DG, dense granules; HC, host cell; MN; micronemes; PG, polysaccharide granule.

underlying amorphous layer forms (Fig. 2.23E). Within the early tissue cyst the bradyzoites still have similar ultrastructural features to the tachyzoites, particularly rhoptries, which have a

labyrinthine appearance (Ferguson and Hutchison, 1987b). This shows that although the structure is that of a tissue cyst and the expression of marker molecules (BAG1, ENO1,

Toxoplasma Gondii

2.3 Development in the intermediate host

and LDH2) are those of bradyzoites, the specific ultrastructural features lag behind. It was often 21 28 days before typical bradyzoites could be identified. In addition, many of the dividing parasites had more electron lucent cytoplasm with few organelles (Fig. 2.23A).

2.3.3 Structure of the tissue cyst and bradyzoite The structure of the mature tissue cyst observed from 3 to 24 months PI (approximately the life span of the mouse) remained relatively unchanged (Fig. 2.23D). The first important observation was that throughout this period the tissue cysts were retained within a viable HC (Fig. 2.23D and E). It was originally thought that the mature cysts were extracellular; however, on ultrastructural examination, a thin rim of HC cytoplasm could be observed enclosing the tissue cysts (Ferguson and Hutchison, 1987b). This may explain the lack of an immune response to the tissue cysts; they are masked by the HC. With the limited host cytoplasm available it is difficult to identify the cell type; however, in the majority of cases, the HCs could be identified as neurons because of the defining presence of synapses (Fig. 2.23E). It is not possible to identify the other HCs, although their features are consistent with neurons. There are variations in the thickness of the cyst wall between tissue cysts with some showing deep invaginations of the limiting membrane forming a complex network of interconnecting channels all embedded in the homogeneous granular material. There is also some vesicle formation on the inner aspect (Fig. 2.23E) (Ferguson and Hutchison, 1987a). In the mature cysts the bradyzoites appeared more elongated than the tachyzoites with a posteriorly located nucleus (Fig. 2.23B). There were numerous micronemes and few dense granules, although this was variable. The rhoptries had more bulbous ends and

55

were electron dense. The major difference was the presence of numerous polysaccharide granules (Fig. 2.23B and D). The majority of tissue cysts appear as a single structure but it is possible to find small groups of tissue cysts of different sizes. It has been suggested, from the very early studies (Lainson, 1958), that this could represent daughter cyst formation resulting from the escape of individual bradyzoites to form new cysts. However, extensive immunocytochemical and ultrastructural studies fail to find supporting evidence. It is possible to observe the formation of groups of cysts from as early as 14 days PI (Fig. 2.22A). In addition, when examined by EM, the cysts, although of different sizes, appear to be of similar maturity. The bradyzoites in all the cysts appeared as mature organisms with no evidence of the feature described previously for immature cysts. In addition, given the immunological response of the host to exposed parasite antigens, it would be expected that any escaped bradyzoite would invoke an inflammatory response (see Section 2.3.5).

2.3.4 Inflammatory changes in the brains of infected mice Toxoplasma infection of the brain was associated with inflammatory changes in around the meninges and certain blood vessels within the brain (Ferguson et al., 1991). During the acute phase, many of the inflammatory cells were lymphocytes and monocytes with a few polymorphic leukocytes. In chronically infected mice, with numerous tissue cysts in the brain, there was the continuous presence of inflammatory cells which cuff the small blood vessels within the neuropil and the vessels of the meninges (Fig. 2.24A, B, and C). However, it should be noted that these inflammatory cells show no tropism toward the tissue cysts (Fig. 2.24B). In chronic infections (3 24 months PI), the majority of inflammatory

Toxoplasma Gondii

56

2. The ultrastructure of Toxoplasma gondii

(A)

(B)

(C)

FIGURE 2.24 Inflammatory cell infiltration of chronically infected mouse brain. (Part A) Small blood vessel from the brain of a chronically infected mouse showing a number of monocytes (M) and plasma cells (P) cuffing the vessel and also a plasma cell in the neuropil. Bar is 10 µm. (Part B) Light micrograph of the brain of a chronically infected mouse showing numerous inflammatory cells within the meninges and cuffing the blood vessels (arrows). Note the tissue cyst (Cy) invokes no reaction. Bar is 20 µm. (Part C) Electron micrograph of a large vessel close to the meninges showing the large number of cuffing monocytes (M) and plasma cells (P). Bar is 10 µm.

cells were plasma cells or monocytes/macrophages (Fig. 2.24C). In addition, it is possible to observe numerous plasma cells around the small vessels and free in the neuropil (Fig. 2.24A).

2.3.5 Cyst rupture in immune competent hosts One of the clinical problems in immunocompromised hosts was recrudescence of the infection resulting in stage conversion back to

actively proliferating and tissue-destructive tachyzoites. To examine the situation in an immune competent host, the brains of immune competent, chronically infected mice, were examined. It was found that, indeed, a very small percentage of tissue cysts were rupturing at any given time during chronic infections (Ferguson et al., 1989). The initial change appeared to be death of the HC. With the exposure of the parasite antigens in the cyst wall there was evidence for a rapid and massive cell mediated immune response involving

Toxoplasma Gondii

2.3 Development in the intermediate host

numerous inflammatory cells (monocytes and even neutrophils). These were observed around the still apparently intact cyst (Fig. 2.25A). With the rupture of the cyst wall there was further

(A)

57

influx of macrophages into the cyst (Fig. 2.25C). The macrophages phagocytosed the bradyzoites where heterophagosomes were formed, resulting in destruction of the parasites (Fig. 2.25B).

(B)

(C)

FIGURE 2.25 Tissue cyst rupture in an immune competent mouse brain. (Part A) Low-power image of a tissue cyst with an intact CW but with loss of the host cell. Note the number of inflammatory cells, monocytes (M), surrounding the cyst. Bar is 10 µm. (Part B) Detail from the tissue cyst in part (C) showing a macrophage with a phagocytic vacuole containing degenerating bradyzoites (Br). Bar is 1 µm. (Part C) Section through a ruptured tissue cyst in an immunocompetent host showing the fractured CW partially enclosing the bradyzoites (Br). Note the numerous macrophages (M) surrounding and invading into the tissue cyst and phagocytizing the bradyzoites. Bar is 5 µm. CW, Cyst wall.

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2. The ultrastructure of Toxoplasma gondii

This resulted in the formation of small inflammatory lesions (microglia nodules) with some evidence of HC apoptosis, but limited host tissue damage. The bradyzoites appeared to be destroyed before they could undergo replication or stage conversion to tachyzoites. These observations were confirmed by immunocytochemical examination of the lesions.

unpublished), the process of cyst formation from the time the parasite entered the HC was similar to that observed in vivo. However, other studies suggest that there may be some tachyzoite-like development before the vacuole takes on the features of the tissue cyst (Soete et al., 1994).

References

2.3.6 Development in vitro It is often stated that T. gondii is very easy to culture and this has made it very popular as a molecular model. However, it needs to be emphasized that normally only the tachyzoite and tachyzoite development occurs in cell cultures. It is possible to trigger tissue cyst formation in vitro under stress conditions and recent data suggests that it is also possible to reproduce development undergone by the coccidian stages in vitro (see Chapter 18: Bradyzoite and sexual stage development, for a review of the development of bradyzoites and coccidian stages in vitro). 2.3.6.1 Tachyzoite development in vitro In vitro, tachyzoites undergo similar development to that described previously (Sections 1.4 and 1.5) irrespective of the type of HC used. The host/parasite relationship and the proliferation by endodyogeny and repeated endodyogeny are identical to that described previously. 2.3.6.2 Bradyzoite development in vitro This was first described in astrocytes in the 1980s (Jones et al., 1986) and techniques for inducing this stage conversion were identified in the 1990s. It was observed that factors (pH changes, oxygen tension) that induce stress in the culture system appear to stimulate conversion to cyst formation. Unlike the in vivo situation where there is marked selection of the type of HC (neurons, muscle cells), in vitro almost any cell type could act as a HC for cyst formation. In certain in vitro studies (Ferguson,

Aikawa, M., Hepler, P.K., Huff, C.G., Sprinz, H., 1966. The feeding mechanism of avian malarial parasites. J. Cell Biol. 28, 355 373. Aikawa, M., Miller, L.H., Rabbege, J.R., Epstein, N., 1981. Freeze-fracture study on the erythrocyte membrane during malarial parasite invasion. J. Cell Biol. 91, 55 62. Alexander, D.L., Mital, J., Ward, G.E., Bradley, P.J., Boothroyd, J.C., 2005. Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog. 1 (2), e17. Beisson, J., Lefort-Tran, M., Pouphile, M., Rossignol, M., Satir, B., 1976. Genetic analysis of membrane differentiation in Paramecium. Freeze-fracture study of the trichocyst cycle in wild-type and mutant strains. J. Cell Biol. 69, 126 143. Belli, S.I., Smith, N.C., Ferguson, D.J.P., 2006. The coccidian oocysts a tough nut to crack. Trends Parasitol. 22 (9), 416 423. Birch-Andersen, A., Ferguson, D.J., Pontefract, R.D., 1976. A technique for obtaining thin sections of coccidian oocysts. Acta Pathol. Microbiol. Scand. [B] 84, 235 239. Bradley, P.J., Ward, C., Cheng, S.J., Alexander, D.L., Coller, S., Coombs, G.H., et al., 2005. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii. J. Biol. Chem. 280, 34245 34258. Carruthers, V.B., Sibley, L.D., 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol. Microbiol. 31, 421 428. Cesbron-Delauw, M.F., 1994. Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitol. Today 10, 293 296. Christie, E., Pappas, P.W., Dubey, J.P., 1978. Ultrastructure of excystment of Toxoplasma gondii oocysts. J. Protozool. 25, 438 443. Colley, F.C., Zaman, V., 1970. Observations on the endogenous stages of Toxoplasma gondii in the cat ileum. II. Electron microscope study. Southeast Asian J. Trop. Med. Public Health 1, 456 480.

Toxoplasma Gondii

References

Coppin, A., Varre, J.S., Lienard, L., Dauvillee, D., Guerardel, Y., Soyer-Gobillard, M.O., et al., 2005. Evolution of plant-like crystalline storage polysaccharide in the protozoan parasite Toxoplasma gondii argues for a red alga ancestry. J. Mol. Evol. 60, 257 267. Dubey, J.P., 1986. A review of toxoplasmosis in pigs. Vet. Parasitol. 19, 181 223. Dubey, J.P., 1997. Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii. J. Eukaryot. Microbiol. 44, 592 602. Dubey, J.P., Speer, C.A., Shen, S.K., Kwok, O.C., Blixt, J.A., 1997. Oocyst-induced murine toxoplasmosis: life cycle, pathogenicity and stage conversion in mice fed Toxoplasma gondii oocysts. J. Parasitol. 83, 870 882. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structures of Toxoplasma gondii tachyzoites, bradyzoites and sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267 299. Dubremetz, J.F., 1973. Etude ultrastructurale de la mitose schizogonique chez la coccidie Eimeria necatrix (Johnson 1930). J. Ultrastruct. Res. 42, 354 376. Dubremetz, J.F., 1975. La gene`se des Me´rozoı¨tes chez la coccidie Eimeria necatrix. Etude Utrastructurale. J. Protozool. 22, 71 84. Dubremetz, J.F., Achbarou, A., Bermudes, D., Joiner, K.A., 1993. Kinetics and pattern of organelle exocytosis during Toxoplasma gondii/host-cell interaction. Parasitol. Res. 79, 402 408. Ferguson, D., 2002. Toxoplasma gondii and sex: essential or optional extra? Trends Parasitol. 18, 351. Ferguson, D.J., 2004. Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. Int. J. Parasitol. 34, 347 360. Ferguson, D.J., Hutchison, W.M., 1981. Comparison of the development of avirulent and virulent strains of Toxoplasma gondii in the peritoneal exudate of mice. Ann. Trop. Med. Parasitol. 75, 539 546. Ferguson, D.J., Hutchison, W.M., 1987a. The host-parasite relationship of Toxoplasma gondii in the brains of chronically infected mice. Virchows Arch. A Pathol. Anat. Histopathol. 411, 39 43. Ferguson, D.J., Hutchison, W.M., 1987b. An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitol. Res. 73, 483 491. Ferguson, D.J., Hutchison, W.M., Dunachie, J.F., Siim, J.C., 1974. Ultrastructural study of early stages of asexual multiplication and microgametogony of Toxoplasma gondii in the small intestine of the cat. Acta Pathol. Microbiol. Scand [B] Microbiol. Immunol. 82, 167 181.

59

Ferguson, D.J., Hutchison, W.M., Siim, J.C., 1975. The ultrastructural development of the macrogamete and formation of the oocyst wall of Toxoplasma gondii. Acta Pathol. Microbiol. Scand. [B] 83, 491 505. Ferguson, D.J., Birch-Andersen, A., Hutchison, W.M., Siim, J.C., 1976. Ultrastructural studies on the endogenous development of Eimeria brunetti. Acta Pathol. Microbiol. Scand. [B] 84B, 401 413. Ferguson, D.J., Birch-Andersen, A., Hutchison, W.M., Siim, J.C., 1978a. Light and electron microscopy on the sporulation of the oocysts of Eimeria brunetti. I. Development of the zygote and formation of the sporoblasts. Acta Pathol. Microbiol. Scand. [B] 86, 1 11. Ferguson, D.J., Birch-Andersen, A., Hutchison, W.M., Siim, J.C., 1978b. Light and electron microscopy on the sporulation of the oocysts of Eimeria brunetti. II. Development into the sporocyst and formation of the sporozoite. Acta Pathol. Microbiol. Scand. [B] 86, 13 24. Ferguson, D.J., Birch-Andersen, A., Siim, J.C., Hutchison, W.M., 1979a. Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. I. Development of the zygote and formation of the sporoblasts. Acta Pathol. Microbiol. Scand. [B] 87B, 171 181. Ferguson, D.J., Birch-Andersen, A., Siim, J.C., Hutchison, W.M., 1979b. Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. II. Formation of the sporocyst and structure of the sporocyst wall. Acta Pathol. Microbiol. Scand. [B] 87B, 183 190. Ferguson, D.J., Birch-Andersen, A., Siim, J.C., Hutchison, W.M., 1979c. Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. III. Formation of the sporozoites within the sporocysts. Acta Pathol. Microbiol. Scand. [B] 87, 253 260. Ferguson, D.J., Birch-Andersen, A., Siim, J.C., Hutchison, W.M., 1979d. An ultrastructural study on the excystation of the sporozoites of Toxoplasma gondii. Acta Pathol. Microbiol. Scand. [B] 87, 277 283. Ferguson, D.J., Birch-Andersen, A., Hutchinson, W.M., Siim, J.C., 1980. Ultrastructural observations showing enteric multiplication of Cystoisospora (Isospora) felis by endodyogeny. Z. Parasitenkd. 63, 289 291. Ferguson, D.J., Hutchison, W.M., Pettersen, E., 1989. Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study. Parasitol. Res. 75, 599 603. Ferguson, D.J., Graham, D.I., Hutchison, W.M., 1991. Pathological changes in the brains of mice infected with Toxoplasma gondii: a histological, immunocytochemical and ultrastructural study. Int. J. Exp. Pathol. 72, 463 474. Ferguson, D.J., Cesbron-Delauw, M.F., Dubremetz, J.F., Sibley, L.D., Joiner, K.A., Wright, S., 1999a. The expression and distribution of dense granule proteins in the

Toxoplasma Gondii

60

2. The ultrastructure of Toxoplasma gondii

enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Exp. Parasitol. 91, 203 211. Ferguson, D.J., Jacobs, D., Saman, E., Dubremetz, J.F., Wright, S.E., 1999b. In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii. Parasitology 119 (Pt 3), 259 265. Ferguson, D.J., Brecht, S., Soldati, D., 2000. The microneme protein MIC4, or an MIC4-like protein, is expressed within the macrogamete and associated with oocyst wall formation in Toxoplasma gondii. Int. J. Parasitol. 30, 1203 1209. Ferguson, D.J., Belli, S.I., Smith, N.C., Wallach, M.G., 2003. The development of the macrogamete and oocyst wall in Eimeria maxima: immuno-light and electron microscopy. Int. J. Parasitol. 33, 1329 1340. Ferguson, D.J., Henriquez, F.L., Kirisits, M.J., Muench, S.P., Prigge, S.T., Rice, D.W., et al., 2005. Maternal inheritance and stage-specific variation of the apicoplast in Toxoplasma gondii during development in the intermediate and definitive host. Eukaryot. Cell 4, 814 826. Francia, M.E., Jordan, C.N., Patel, J.D., Sheiner, L., Demerly, J.L., Fellows, J.D., et al., 2012. Cell division in apicomplexan parasites is organized by a homolog of the striated rootlet fiber of algal flagella. PLoS Biol. 10 (12), e1001444. Gross, U., Bohne, W., Soete, M., Dubremetz, J.F., 1996. Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii. Parasitol. Today 12, 30 33. Hakansson, S., Charron, A.J., Sibley, L.D., 2001. Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole. EMBO J. 20, 3132 3144. Hu, K., Mann, T., Striepen, B., Beckers, C.J., Roos, D.S., Murray, J.M., 2002a. Daughter cell assembly in the protozoan parasite Toxoplasma gondii. Mol. Biol. Cell 13, 593 606. Hu, K., Roos, D.S., Murray, J.M., 2002b. A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J. Cell Biol. 156, 1039 1050. Jones, T.C., Hirsch, J.G., 1972. The interaction between Toxoplasma gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J. Exp. Med. 136, 1173 1194. Jones, T.C., Yeh, S., Hirsch, J.G., 1972. The interaction between Toxoplasma gondii and mammalian cells. I. Mechanism of entry and intracellular fate of the parasite. J. Exp. Med. 136, 1157 1172. Jones, T.C., Bienz, K.A., Erb, P., 1986. In vitro cultivation of Toxoplasma gondii cysts in astrocytes in the presence of gamma interferon. Infect. Immun. 51, 147 156.

Kohler, S., 2005. Multi-membrane-bound structures of Apicomplexa: I. The architecture of the Toxoplasma gondii apicoplast. Parasitol. Res. 96, 258 272. Kohler, S., Delwiche, C.F., Denny, P.W., Tilney, L.G., Webster, P., Wilson, R.J., et al., 1997. A plastid of probable green algal origin in apicomplexan parasites. Science 275, 1485 1489. Lainson, R., 1958. Observations on the development and nature of pseudocysts and cysts of Toxoplasma gondii. Trans. R. Soc. Trop. Med. Hyg. 52, 396 407. Lebrun, M., Michelin, A., El Hajj, H., Poncet, J., Bradley, P. J., Vial, H., et al., 2005. The rhoptry neck protein RON4 relocalizes at the moving junction during Toxoplasma gondii invasion. Cell. Microbiol. 7, 1823 1833. Mann, T., Beckers, C., 2001. Characterization of the subpellicular network, a filamentous membrane skeletal component in the parasite Toxoplasma gondii. Mol. Biochem. Parasitol. 115, 257 268. Mercier, C., Dubremetz, J.F., Rauscher, B., Lecordier, L., Sibley, L.D., Cesbron-Delauw, M.F., 2002. Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. Mol. Biol. Cell 13, 2397 2409. Michel, R., Schupp, K., Raether, W., Bierther, F.W., 1979. Formation of a close junction during invasion of erythrocytes by Toxoplasma gondii in vitro. Int. J. Parasitol. 10, 309 313. Miranda, K., Pace, D.A., Cintron, R., Rodrigues, J.C., Fang, J., Smith, A., et al., 2010. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Mol. Microbiol. 76 (6), 1358 1375. Moreno, S.N.J., Zhong, L., 1996. Acidocalcisomes in Toxoplasma gondii tachyzoites. Biochem. J. 313, 655 659. Morrissette, N.S., Murray, J.M., Roos, D.S., 1997. Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J. Cell Sci. 110 (Pt 1), 35 42. Nichols, B.A., Chiappino, M.L., 1987. Cytoskeleton of Toxoplasma gondii. J. Protozool. 34, 217 226. Nichols, B.A., Chiappino, M.L., O’Connor, G.R., 1983. Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J. Ultrastruct. Res. 83, 85 98. Nichols, B.A., Chiappino, M.L., Pavesio, C.E., 1994. Endocytosis at the micropore of Toxoplasma gondii. Parasitol. Res. 80, 91 98. Ogino, N., Yoneda, C., 1966. The fine structure and mode of division of Toxoplasma gondii. Arch. Ophthalmol. 75, 218 227. Pelletier, L., Stern, C.A., Pypaert, M., Sheff, D., Ngo, H.M., Roper, N., et al., 2002. Golgi biogenesis in Toxoplasma gondii. Nature 418, 548 552.

Toxoplasma Gondii

References

Pelster, B., Piekarski, G., 1971. Electron microscopical studies on the microgametogeny of Toxoplasma gondii. Z. Parasitenkd. 37, 267 277. Pelster, B., Piekarski, G., 1972. Ultrastructure of the macrogametes in Toxoplasma gondii. Z. Parasitenkd. 39, 225 232. Pfefferkorn, L.C., Pfefferkorn, E.R., 1980. Toxoplasma gondii: genetic recombination between drug resistant mutants. Exp. Parasitol. 50, 305 316. Piekarski, G., Pelster, B., Witte, H.M., 1971. Endopolygeny in Toxoplasma gondii. Z. Parasitenkd. 36, 122 130. Porchet, E., Torpier, G., 1977. Etude du germe infectieux de Sarcocystis tenella et Toxoplasma gondii par la technique du cryodecapage. Z. Parasitenkd. 54, 101 124. Porchet-Hennere, E., Torpier, G., 1983. Relations entre Toxoplasma et sa cellule-hoˆte. Protistologica 19, 357 370. Sheffield, H.G., Melton, M.L., 1968. The fine structure and reproduction of Toxoplasma gondii. J. Parasitol. 54, 209 226. Sibley, L.D., Niesman, I.R., Parmley, S.F., Cesbron-Delauw, M.F., 1995. Regulated secretion of multi-lamellar vesicles leads to formation of a tubulovesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J. Cell Sci. 108, 1669 1677. Sinai, A.P., Webster, P., Joiner, K.A., 1997. Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J. Cell. Sci. 110 (Pt 17), 2117 2128. Soete, M., Camus, D., Dubremetz, J.F., 1994. Experimental induction of bradyzoite-specific antigen expression and

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cyst formation by the RH strain of Toxoplasma gondii in vitro. Exp. Parasitol. 78, 361 370. Speer, C.A., Dubey, J.P., 1998. Ultrastructure of early stages of infections in mice fed Toxoplasma gondii oocysts. Parasitology 116 (Pt 1), 35 42. Speer, C.A., Dubey, J.P., 2005. Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites. Int. J. Parasitol. 35, 193 206. Speer, C.A., Tilley, M., Temple, M.E., Blixt, J.A., Dubey, J. P., White, M.W., 1995. Sporozoites of Toxoplasma gondii lack dense-granule protein GRA3 and form a unique parasitophorous vacuole. Mol. Biochem. Parasitol. 75, 75 86. Speer, C.A., Clark, S., Dubey, J.P., 1998. Ultrastructure of the oocysts, sporocysts and sporozoites of Toxoplasma gondii. J. Parasitol. 84, 505 512. Tomita, T., Bzik, D.J., Ma, Y.F., Fox, B.A., Markillie, L.M., Taylor, R.C., et al., 2013. The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence. PLoS Pathog. 9 (12), e1003823. Vivier, E., 1970. Observations nouvelles sur la reproduction asexue´e de Toxoplasma gondii et conside´rations sur la notion d’endogene`se. C. R. Acad. Sci. Paris 271, 2123 2126. Vivier, E., Petitprez, A., 1969. Le complexe membranaire et son e´volution lors de l’e´laboration des individus-fils de Toxoplasma gondii. J. Cell. Biol. 43, 329 342.

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C H A P T E R

3 Molecular epidemiology and population structure of Toxoplasma gondii Marie-Laure Darde´1,2, Aure´lien Mercier1,2, Chunlei Su3, Asis Khan4 and Michael E. Grigg4 1

INSERM, Univ. Limoges, CHU Limoges, UMR 1094, Tropical Neuroepidemiology, Institute of Epidemiology and Tropical Neurology, Limoges, France 2Centre National de Re´fe´rence Toxoplasmose/ Toxoplasma Biological Resource Center, CHU Limoges, Limoges, France 3Department of Microbiology, The University of Tennessee, Knoxville, TN, United States 4Laboratory of Parasitic Diseases, Molecular Parasitology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States

3.1 Introduction

one locus was enough to determine the strain affiliation to one of the three lineages. However, even in these first studies, a few strains exhibited genetic profiles that do not fit into the three main clonal lineages. They mainly originated from wild areas either in North America (Howe and Sibley, 1995) or in South America (Darde´, 1996; Darde´ et al., 1992), suggesting that a different population structure might exist outside the domestic environment of Europe and the United States. Some of these “exotic” strains were isolated from human patients with severe forms of Toxoplasma infections, reinforcing the hypothesis of a role of the infecting strain in clinical expression of toxoplasmosis (Darde´, 1996). Since then, the use of multilocus genotyping, a continuous progress in the development of

The earliest studies on genetic diversity of Toxoplasma gondii were published nearly 30 years ago using the genetic tools that were then available, that is, multilocus enzyme electrophoresis (MLEE) (Darde´ et al., 1988, 1992) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) on a few genetic markers (Sibley and Boothroyd, 1992). The available strains were also limited, regarding both their number and their geographical origins. This has led to a simple vision of a clonal population structure of T. gondii, essentially limited to three clonal lineages, namely, types I
III (Howe and Sibley, 1995). Consequently, it was inadequately suggested that genetic analysis of just Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00003-7

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© 2020 Elsevier Ltd. All rights reserved.

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3. Molecular epidemiology and population structure of Toxoplasma gondii

new genotyping tools, and a considerable effort to isolate strains across different continents, originating from various biotopes (wild, domestic, or anthropized) and from a variety of host species has made it possible to describe a much more complex population structure. The whole-genome sequencing (WGS) added a new dimension to our understanding of the genetic architecture and global population genetic structure. The current thinking on the molecular epidemiology of the parasite includes sexual recombination events that shape the parasite population differently depending on the environment, the host biology and behavior, and on the influence of exchanges promoted by human activity. At the same time the virulence mechanisms of the different strains in the mouse model are increasingly understood. However, the knowledge on the role of the T. gondii strain types in human toxoplasmosis is progressing more slowly because of the limitations in the possibilities of isolating the parasite from humans, of obtaining clinical data, and the multiple host factors that can influence the consequences of the infection.

3.2 Genetic markers

2004, 2006), multilocus DNA sequencing (Fraza˜o-Teixeira et al., 2011; Khan et al., 2006, 2007, 2011a,b; Lehmann et al., 2000; Miller et al., 2008a,b; Pan et al., 2012). Serotyping is a different approach for strain typing and for population genetic study, based on the use of peptides derived from polymorphic sites of the genes coding for T. gondii antigens including GRA6 and GRA7 (Kong et al., 2003). Currently, the most widely used genotyping methods for T. gondii genetic diversity studies are multilocus genotyping using PCR-RFLP, MS markers, and less frequently, multilocus sequence-based typing (MLST). In any case, genotyping methods should imperatively rely on multilocus markers as they can capture genetic diversity and genetic recombination with good resolution. Genotyping methods based on a single locus analysis are no longer admitted. The number of loci is a matter of debate, but it may be considered that a minimum of five markers, each located on a different chromosome, is acceptable. These markers applied to a large population of isolates allowed the definition of genetic clusters that were further refined by WGS (Lorenzi et al., 2016).

3.2.1 Microsatellites

Numerous genotyping techniques and markers have been developed to distinguish T. gondii isolates. They are suitable for the studies of molecular epidemiology and population genetics of the parasite. These techniques include MLEE (Darde´ et al., 1992), mobile genetic elements PCR (Terry et al., 2001), random-amplified polymorphic DNA PCR (Ferreira Ade et al., 2004; Guo and Johnson, 1995), PCR-RFLP (Cristina et al., 1995; Ferreira et al., 2006; Grigg et al., 2001a,b; Howe and Sibley, 1995; Khan et al., 2005a; Sibley and Boothroyd, 1992; Su et al., 2006), microsatellite (MS) analysis (Ajzenberg et al., 2002a, 2004, 2010; Blackston et al., 2001; Lehmann et al.,

MS sequences are short nucleotide tandem repeats of two to six nucleotides that occur 2
20 times. They are hypervariable due to fast accumulation of length polymorphisms by intraallelic polymerase slippage on MS sequence during replication. Several sets of MS markers have been used in different studies with 5
15 markers (Ajzenberg et al., 2002a, 2010; Blackston et al., 2001; Lehmann et al., 2004, 2006; Sreekumar et al., 2003). Currently, a multiplex PCR for 15 MSs allowing multilocus analysis of isolates following a single PCR amplification provides a simple method with high resolution in genotyping T. gondii strains (Ajzenberg et al., 2010) (Table 3.1). In general

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3.2 Genetic markers

TABLE 3.1 Microsatellite markers and polymerase chain reaction (PCR) primers used for the 15 microsatellite (MS) multiplex PCR assay. Marker MS TUB2

Chromosome IX

Primer sequencesa

Repeat motif

Size range (bp)

0

(F) 5 6-FAM-GTCCGGGTGTTCCTACAAAA 3

[TG/AC]n

0

287
291

(R) 50 TTGGCCAAAGACGAAGTTGT 30 W35

II

[TC/AG]n [TG/AC]n

(F) 50 HEX-GGTTCACTGGATCTTCTCCAA 30 0

(R) 5 AATGAACGTCGCTTGTTTCC 3 TgM-A

X

[TG/AC]n

242
248

0

(F) 50 HEX-GGCGTCGACATGAGTTTCTC 30

203
211

(R) 50 TGGGCATGTAAATGTAGAGATG 30 B18

VIIa

[TG/AC]n

(F) 50 6-FAM-TGGTCTTCACCCTTTCATCC 30 0

156
170

0

(R) 5 AGGGATAAGTTTCTTCACAACGA 3 B17

XII

[TC/AG]n

(F) 50 HEX-AACAGACACCCGATGCCTAC 30

334
366

(R) 50 GGCAACAGGAGGTAGAGGAG 30 M33

IV

[TC/AG]n

(F) 50 6-FAM- TACGCTTCGCATTGTACCAG 30 0

(R) 5 TCTTTTCTCCCCTTCGCTCT 3 IV.1

IV

[TG/AC]n

165
173

0

(F) 50 HEX-GAAGTTCGGCCTGTTCCTC 30

272
282

(R) 50 TCTGCCTGGAAAAGGAAAGA 30 XI.1

XI

[TG/AC]n

(F) 50 6-FAM-GCGTGTGACGAGTTCTGAAA 30 0

354
362

0

(R) 5 AAGTCCCCTGAAAAGCCAAT 3 M48

Ia

[TA/AT]n

(F) 50 6-FAM-AACATGTCGCGTAAGATTCG 30

209
243

(R) 50 CTCTTCACTGAGCGCCTTTC 30 M102

VIIa

[TA/AT]n

(F) 50 NED-CAGTCCAGGCATACCTCACC 30 0

(R) 5 CAATCCCAAAATCCCAAACC 3 N60

Ib

[TA/AT]n

164
196

0

(F) 50 NED-GAATCGTCGAGGTGCTATCC 30

132
157

(R) 50 AACGGTTGACCTGTGCGAGT 30 N82

XII

[TA/AT]n

(F) 50 HEX-TGCGTGCTTGTCAGAGTTC 30 0

105
145

0

(R) 5 GCGTCCTTGACATGCACAT 3 AA

VIII

[TA/AT]n

(F) 50 NED-GATGTCCGGTCAATTTTGCT 30

251
332

(R) 50 GACGGGAAGGACAGAAACAC 30 N61

VIIb

[TA/AT]n

(F) 50 6-FAM-ATCGGCGGTGGTTGTAGAT 30 0

79
123

0

(R) 5 CCTGATGTTGATGTAAGGATGC 3 N83

X

[TA/AT]n

(F) 50 6-FAM-ATGGGTGAACAGCGTAGACA 30 0

(R) 5 GCAGGACGAAGAGGATGAGA 3

306
338

0

a In each pair of primers, the forward one was 50 -end labeled with flurorescein: 6-carboxyfluorescein (6-FAM) for MS TUB2, XI.1, B18, N83, N61, M33, and M48; hexachlorofluorescein (HEX) for MS TgM-A, B17, N82, W35, and IV.1; 2,70 ,80 -benzo-50 -fluoro-20 ,4,7-trichloro-5-carboxyfluorescein (NED) for AA, N60, and M102.

F, Forward primer; MS, microsatellite; R, reverse primer. Adapted from Ajzenberg, D., Collinet, F., Mercier, A., Vignoles, P., Darde´, M.-L., 2010. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. J. Clin. Microbiol. 48, 4641
4645. https://doi.org/10.1128/JCM.01152-10.

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the mutation rate for MSs is 1022
1025 per locus per replication, which is several orders of magnitude faster than that of single nucleotide polymorphisms (SNPs) (Goldstein and Schlotterer, 1999). This high rate of mutation has been considered as a limitation to the use of these makers in epidemiological studies as they can be prone to homoplasy, that is, the number of repeats can expand and contract by strand slippage during replication. However, the mutation rate is varying according to the position of the markers in the target organism’s genome and the nature of the nucleotide repeats. In the case of the 15 MS multiplex PCR the homoplasy has been minimized by the selection of variable and numerous MS markers located on 11 different chromosomes (Table 3.1). The eight MS markers with a repeat motif consisting of [TG/AC]n or [TC/AG]n (TUB2, W35, TgM-A, B18, B17, M33, IV.1, and XI.1) have a lower mutability, allowing a typing level, that is the ability of markers to distinguish the major clonal lineages from atypical strains. The higher mutability of the seven MS markers with a repeat motif consisting of [TA/ AT]n (M48, M102, N60, N82, AA, N61, and N83) make these markers ideal as fingerprinting markers for distinguishing closely related isolates or analyzing the intratype population structure. For instance, they allowed finding a spatial structure population among strains belonging to the type II lineage in France or in Europe (Ajzenberg et al., 2015; Verma et al., 2015). The Simpson’s index of diversity, tested on 369 type II isolates, was found to be 0.999, that is, reaching nearly the maximum of 1.0 (Ajzenberg et al., 2010). The high degree of polymorphism provided by MS markers and their more rapid mutation rate is making this technique especially informative for molecular epidemiological studies, for analyzing recent evolutionary events, and for individual identification of T. gondii isolates during outbreak investigation (Demar et al., 2007; Vielmo et al., 2019).

Apart from the risk of homoplasy, the limitations of this assay are the availability of an automated sequencer and the sensitivity of the method, estimated to be between 50 and 100 T. gondii genome equivalents per 5 μL of DNA sample (see Table 3.2).

3.2.2 Polymerase chain reaction restriction fragment length polymorphism Multilocus PCR-RFLP typing of T. gondii has been widely used for population genetic studies due to its ease of use and cost effectiveness (Cristina et al., 1995; Ferreira et al., 2006; Grigg et al., 2001a,b; Howe and Sibley, 1995; Khan et al., 2005a; Pena et al., 2008; Shwab et al., 2014; Sibley and Boothroyd, 1992; Su et al., 2006, 2010). This method relies on endonucleases to recognize SNPs among DNA sequences, digest PCR products, and reveal distinct DNA banding patterns by agarose gel electrophoresis. The mutation rate for SNP in T. gondii is not known but expected to be close to the rate of 1029
10210 per nucleotide position per replication for eukaryotes in general (Goldstein and Schlotterer, 1999), making it an excellent tool to study distantly related parasite strains. Sibley and Boothroyd (1992) were the first to use PCR-RFLP markers in studying T. gondii genetic diversity and its association with virulence in mice. When analyzing 28 strains from a variety of host on five continents using three PCR-RFLP markers, they demonstrated that the 10 virulent strains have an identical genotype, whereas the 18 avirulent strains were moderately polymorphic, suggesting that virulent strains originated from a single lineage. Since then, many sets of PCR-RFLP markers were developed and applied to distinguish T. gondii strains collected from a variety of hosts in different regions. Here we have no intention to make an exhaustive list of all published articles on this subject but to include a few

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3.2 Genetic markers

TABLE 3.2 Advantages and limits of the main techniques for Toxoplasma strain genotyping. Technique

Number of loci

Technique references

Advantages

Limits

• High sensitivity (estimated at $ 10 Toxoplasma gondii genome equivalents per PCR) • Useful for clonal lineages versus atypical strains distinction • Well-defined designation scheme for atypical or unique strains

• Rely on nested PCR that need caution during manipulation to avoid DNA contamination • Need two successive PCR runs (initial step of multiplex PCR amplification) • Poor intratype distinction or individual strains identification

Multiplex 10 multilocus PCRRFLP

Su et al. (2010)

Multiplex PCR of microsatellite markers

15

Ajzenberg • High discrimination power et al. (2010) • Useful for clonal lineages versus atypical strains distinction and intratype distinction • Distinction between natural type I strain and RH strain (PCR positive control) in case of contamination suspicion

• Low sensitivity (estimated to be between 50 and 100 T. gondii genome equivalents per PCR) • Homoplasy in highly polymorphic fingerprint markers • No definite designation scheme for atypical or unique strains

Whole-genome sequencing

Whole genome

Lorenzi • Very high discrimination power et al. (2016) • Precise identification of recombination/admixture patterns

• Expensive (although the cost is rapidly dropping) • Cumbersome and lengthy for routine use • Still only used for fine genomic exploration (identification of specific genes or recombination patterns) rather than routine strain characterization

PCR, Polymerase chain reaction; RFLP, restriction fragment length polymorphism. Adapted from Galal, L., Ajzenberg, D., Hamidovi´c, A., Durieux, M.-F., Darde´, M.-L., Mercier, A., 2018. Toxoplasma and Africa: one parasite, two opposite population structures. Trends Parasitol. 34, 140
154. https://doi.org/10.1016/j.pt.2017.10.010; Galal, L., Schares, G., Stragier, C., Vignoles, P., Brouat, C., Cuny, T., et al., 2019,. Diversity of Toxoplasma gondii strains shaped by commensal communities of small mammals. Int. J. Parasitol. https://doi.org/10.1016/j.ijpara.2018.11.004.

representatives in the references (Cristina et al., 1995; Ferreira et al., 2006; Grigg et al., 2001a,b; Howe and Sibley, 1995; Khan et al., 2005a; Pena et al., 2008; Su et al., 2010). By analyzing 106 strains from a variety of hosts (including human) from North America and Europe using five PCR-RFLP markers, Howe and Sibley (1995) revealed the dominance of three clonal lineages (types I
III) of T. gondii and concluded that the parasite was clonal with the type II lineage accounted for 50% of isolates, and the population diversity was very limited in the regions.

The main limitation of the multilocus PCRRFLP typing method is its relative low resolution in identifying individual T. gondii strains (Table 3.2). In addition, the PCR-RFLP markers initially developed to genotype T. gondii were based on SNPs that differentiate archetypal North American and European clonal strains types I
III (Su et al., 2006). While these PCRRFLP markers will differentiate atypical genotypes that contain unique combinations of the types I
III alleles, they fail to detect unique polymorphisms present within each locus that atypical strains of T. gondii possess (Khan et al., 2007).

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3. Molecular epidemiology and population structure of Toxoplasma gondii

3.2.3 Multilocus DNA sequence typing

3.2.4 Serotyping

Although multilocus MS and PCR-RFLP typing methods are simple, rapid, and costeffective ways to genotype pathogens, they do not capture the true extent of polymorphism present within each locus. Because of the limitations of MS and PCR-RFLP typing, as well as the high resolution and decreased cost for sequencing, multilocus DNA sequence typing (either based on introns in genes or partial sequencing of housekeeping genes) has been increasingly applied to study genetic diversity of T. gondii strains, particularly those that are atypical (Fraza˜o-Teixeira et al., 2011; Grigg et al., 2001a,b; Khan et al., 2007, 2011a,b; Lehmann et al., 2000; Miller et al., 2008a,b; Pan et al., 2012). SAG1, SAG2, SAG3, SAG4, BTUB, c22-8, c29-2, L358, PK1, Apico, GRA6, GRA7, and B1 (Dubey et al., 2008a; Grigg and Boothroyd, 2001; Su et al., 2006) are the most widely used sequence-typing markers that provide highly sensitive genotyping. These sequence markers were developed as nested PCRs that can detect as low as 5
10 parasites in cerebral spinal fluid or ocular fluids (Grigg et al., 2001a,b; Khan et al., 2005a). However, most current studies use only a limited number of loci to investigate a small collection of strains, limiting the information from these studies so that genetic relatedness among different genotypes of T. gondii cannot be assessed. To overcome this limitation, Su et al. (2012) genotyped 138 atypical strains by DNA sequencing of four introns presumed to be under neutral selection from three different genes, including UPRT, EF, and HP that comprised 1775 bp. Cluster analysis using neighbor-net network, structure, and principle coordinate analysis (PCA) organized the 138 unique PCR-RFLP genotypes into 15 distinct haplogroups, constituting six major clades of T. gondii.

While PCR-based methodologies have been used successfully to genotype strains infecting people and animals, it is not always possible to obtain sufficient parasite DNA from infected tissues or blood samples to attempt molecular typing. Samples from humans also tend to be biased, as they are largely from people who exhibit symptomatic disease and likely do not capture the true diversity of strains that cause asymptomatic, chronic infections. Toxoplasma induces a wide spectrum of species-specific diseases ranging from lymphadenopathy, chorioretinitis, ileitis, encephalitis, congenital disease as well as behavioral modifications, and numerous studies have demonstrated clinical disease is, at least partially dependent, on strain type (Boothroyd and Grigg, 2002; de-laTorre et al., 2013; Grigg et al., 2001a,b; Hutson et al., 2015; McLeod et al., 2012; Shobab et al., 2013). Since the vast majority of human hosts harbor relatively benign infections, little is known about the strains that cause asymptomatic infection. To address this knowledge gap, that is, whether benign versus symptomatic patients are infected with different strain types of Toxoplasma, a variety of serological typing assays have been developed that employ strain-specific polymorphic epitopes arrayed on microchips or coupled to carrier proteins in order to capture strain-specific antibodies induced during parasite infection (Grigg, 2007; Kong et al., 2003; Maksimov et al., 2012a,b; Sousa et al., 2008). These methodologies are distinct from commercially available diagnostic proteins or lysate preparations that identify Toxoplasma infection. The commercial assays work independent of parasite genotype to serologically diagnose all Toxoplasma infections. The serotyping methodology obviates the necessity to isolate parasite DNA or actual organisms and relies on the detection of strain-

Toxoplasma Gondii

3.2 Genetic markers

specific antibodies circulating in infected animal and human sera. Because a significant proportion of antibodies induced during natural infection are restricted to highly immunogenic, polymorphic epitopes present in immunodominant surface proteins or secreted antigens, that is, SAG2A (SRS34A), GRA3, GRA5, GRA6, and GRA7, a detectable number of antibodies against Toxoplasma are epitope-specific and clear serotypes exist (Kong et al., 2003; Maksimov et al., 2012a,b; Sousa et al., 2008). This focus of strong immunity against polymorphic epitopes, coupled with the unusually limited number of alleles possessed by different T. gondii strains (especially in North America and Europe), has permitted the development of these diagnostic strain-typing methodologies. Using only a limited number of polymorphic peptides, either presented by a carrier molecule or in a microarray format, it is possible to pull out diagnostic strain-specific antibodies present in infected people and animals to effectively “serotype” the major strains of T. gondii known to cause disease globally (Kong et al., 2003; Nowakowska et al., 2006). Using just a few microliters of human or animal sera, the serotyping assay is run as a titration and is capable of quantifying the detection of epitopes that discriminate between infections caused by type II from all other T. gondii strains, referred collectively as “Not Exclusively II” or NE-II (McLeod et al., 2012). However, within the NE-II group, distinct serotypes exist and these can be used to differentiate between infections caused by type I/III from type X (also referred to as haplogroup 12, or HG12) from other nonarchetypal strains that encompass the total genetic diversity within the species (i.e., not type I, II, III, or X) (Grigg, 2007). As few as five polymorphic peptides, derived from the diagnostic GRA6 and GRA7 genes, are sufficient to make this distinction.

69

Multiple studies have since been performed using different suites of polymorphic peptides derived from surface and secreted proteins in an attempt to increase the number of polymorphic peptides available, and these studies have independently confirmed that the original peptide epitopes discovered on GRA6 and GRA7 remain the most effective (Maksimov et al., 2012a,b; Sousa et al., 2008). Interestingly, the GRA6 epitope, determined to be the immunodominant B-cell epitope (Kong et al., 2003), is also an immunodominant T-cell epitope (Blanchard et al., 2008). While the serotyping assay has worked remarkably well in regions of the world where archetypal strains dominate T. gondii population genetics (e.g., North America and Europe) (Contopoulos-Ioannidis et al., 2015; Kong et al., 2003; McLeod et al., 2012; Morisset et al., 2008; Nowakowska et al., 2006; Shobab et al., 2013), it has been suggested that the assay would fail in regions such as Brazil, where strain genetic diversity is substantial (Ajzenberg, 2012). This logic, however, belies the fundamental basis for the success of the assay. The assay utilizes polymorphic peptide epitopes identified in highly immunogenic genes that are strainvariable. Hence, it is the 10
12 amino acid epitope, not the underlying allele or strain genetic type that determines the specificity of the assay. For example, the strain responsible for causing a human toxoplasmosis outbreak in St. Isabel do Ivai, Brazil, serotyped as I/III. Sequencing of the isolate from the outbreak established that it was a nonarchetypal strain and that it possessed a unique allele at GRA6. However, the 12 amino acid epitope was identical to the epitope found in type I or III strains, so not surprisingly, sera from the outbreak patients had a I/III serotype (Vaudaux et al., 2010). Hence, reactivity does not predict genotype but rather epitope. The same was

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3. Molecular epidemiology and population structure of Toxoplasma gondii

true for patients infected with type X (HG12). Here, the GRA6 epitope contains both polymorphisms that distinguish II from I/III, so it possesses dual reactivity, and serotypes as II 5 I/III, and this is diagnostic in its own right (Grigg, 2007; McLeod et al., 2012). Furthermore, nonarchetypal strains that possess different epitopes at these immunodominant sites induce antibodies during natural infection that either fail to react or cross-react with nonpolymorphic amino acids present within each strain-typing epitope. So it is the serologic “signature” that is identified across multiple peptides used in the assay that is diagnostic, and this may point to the likelihood of infection with an atypical parasite genotype. In effect the epitopes act as markers in the genome for association studies, rather than for strain genotyping. The utility of the assay is perhaps best illustrated for its ability to associate specific serotypes with disease cohorts (see Section 3.7) (McLeod et al., 2012; Hutson et al., 2015). In patients presenting with a more serious form of ocular toxoplasmosis (OT), a novel “NR” (for nonreactive) serotype has been found associated with patient cohorts in Germany, the United States, and Colombia (de-la-Torre et al., 2013; Kong et al., 2003; Shobab et al., 2013). Patients with this serotype fail to react with the strain-typing peptides but possess high anti
Toxoplasma antibody titers. Whether this reflects a failure of the individual to mount an appropriate immune response to control the severity of the infection or is the result of infection with a nonarchetypal parasite genotype that possesses different epitopes at the diagnostic peptides is unclear. Strains amplified from some of the infected patients were identified to be nonarchetypal, and the NR serotype was determined to be predictive for identifying patients at increased risk of developing disease, which underscores the utility of serotyping to manage disease.

The assay is not without its limitations, however. First, it is essential to control for natural infection antibody titers, as patients with waning titers may produce a false-negative reactivity at the peptide typing epitope, so lack of reactivity may simply reflect a low concentration of strain-specific antibodies that fail to reach the threshold necessary to produce a positive result. Second, failure to react to a straintyping epitope may be real, so it is essential to identify additional polymorphic epitopes in patients who are infected with strains that produce the NR serotype to extend the resolution of the assay. Lastly, as the assay transitions to more sensitive light-based luciferase immunoprecipitation system or fluorochrome-based systems that can be multiplexed to increase sensitivity, the generation of additional epitopes will resolve the utility of the assay substantially. What is clear is that the assay has provided a new tool to assess the extent of strain heterogeneity infecting humans and animals in different geographic locations and to address or predict serotypes that associate with different clinical disease. Ultimately, this will provide important information for the establishment of healthcare guidelines to reduce communities and/or susceptible population groups that are more at risk for exposure to toxoplasmosis.

3.2.5 Whole-genome sequencing In the last decade, genotyping by sequencing fueled the transition from population genetics to population genomics after the introduction of massively parallel DNA sequencing technologies or next-generation sequencing (NGS). While genotyping a standardized panel of MSs or RFLP-based markers was widely and successfully used to genotype T. gondii, WGS has revolutionized our understanding of the genetic architecture and global population genetic structure (Lorenzi et al., 2016; Minot et al., 2012). Currently, more than a hundred

Toxoplasma Gondii

3.2 Genetic markers

whole-genome sequences representing 16 different haplogroups of T. gondii are deposited into the Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) where the data are freely available and can be used for comparative genomic analyses. Although most of the recent WGS was conducted based on second-generation sequencing platforms such as Illumina HiSeq or MiSeq (Lorenzi et al., 2016; Minot et al., 2012), initial wholegenome sequences (10 3 coverage) of the type I strain GT-1, type II strain ME49, and type III strain VEG were completed using a random shotgun approach at TIGR (currently the J. Craig Venter Institute, United States). The whole-genome shotgun sequences (available in ToxoDB: www.toxodb.org) (Kissinger et al., 2003) and the composite genome map from the types I
III strains of T. gondii (Khan et al., 2005a; Sibley, 2009) were used to define T. gondii’s 14 chromosomes and total genetic size of 592cM with an average map unit of B104 kb/ cM (Khan et al., 2005b). This genome map provides a framework for pursuing comparative population genetic studies, particularly as the reference ME49 genome is a high confidence assembly that can reliably be used to map short reads. As such, reads generated from NGS are facilitating comparative population genomic analyses, including investigating structural variation, copy number variation, and recombination among different T. gondii strains at whole-genome resolution. Recently, 62 strains selected from the 15 distinct haplogroups comprising 138 unique genotypes were subjected to WGS and ref-mapped against ME49 (Lorenzi et al., 2016). Wholegenome comparison using the reference genome identified a total of 802,764 SNPs. Interestingly, neighbor-net network, admixture, and PCA showed a similar structure and haplogroup pattern as that depicted from the low-resolution PCR-RFLP and intron sequencing analyses (Fig. 3.1A) (Lorenzi et al., 2016). In

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contrast, comparative WGS carried out on a subset of 26 strains of distinct and shared ancestry across multiple lineages produced a model of extensive recombination and recent expansion of distinct admixture groups (Minot et al., 2012). To test this model, Lorenzi et al. (2016) investigated the genome-wide inheritance patterns using chromosome painting to depict local admixture across strains. Analogous to the previous observations (Minot et al., 2012), chromosome painting also showed the inheritance of shared haploblocks across strains indicating extensive genetic recombination among strains of different clades (Fig. 3.1B) (Lorenzi et al., 2016). However, three haploblocks in particular (ChrIa, right end of ChrXI, and parts of ChrXII) were shown to be highly conserved suggesting that these haploblocks were being retained because they conferred some selective advantage (Khan et al., 2007, 2011b; Minot et al., 2012). Recently, Zhang et al. (2017) developed a software suite known as PopNet to detect these regions of shared ancestry and chromosome paint to visualize such genetic admixture patterns among related strains using a Markov clustering approach. Chromosome painting analysis using PopNet was conducted using genome-wide SNPs from 24 isolates of diverse haplogroups of T. gondii (Fig. 3.1C). Consistent with previous studies (Lorenzi et al., 2016; Minot et al., 2012), PopNet also showed a mosaic structure across chromosomes of diverse haplogroups, which led to the conclusion that extensive recombination has shaped the current population structure of T. gondii (Zhang et al., 2017). Importantly, because it was an “all-against-all” comparison, it was able to visualize the genomic position and number of distinct genetic ancestries that make up the highly successful type II clonal lineage (Fig. 3.1C). Thus NGS is providing fascinating new insights into the population genomics of T. gondii and establishing how genetic hybridization has dramatically impacted the

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FIGURE 3.1 Population genetic structure and chromosome painting of Toxoplasma gondii. (A) Neighbor-net analysis of 62 T. gondii based on genome-wide SNPs, which were identified by reference mapping using Me49 as a reference strain (Lorenzi et al., 2016). (B) Chromosome painting using shared inheritance of ancestral blocks across 62 T. gondii strains (Lorenzi et al., 2016). (C) PopNet representation of shared inheritance of ancestral blocks depicted the recombination pattern among types I
II and haplogroup 12 strains of T. gondii (Zhang et al., 2017).

long-term evolution and genetic relatedness of the current suite of T. gondii strains circulating globally. Although generalized use of WGS is still hindered by some basic challenges including cost, bioinformatics analysis, interpretation of the data, and data storage infrastructure, the resolution of the data provided by WGS now allows us to develop methods to detect parasite genetic factors that control adaptive processes using population-based studies. Genome-wide association studies (GWAS) have been conducted using Plasmodium isolates to detect candidate loci responsible for antimalarial drug selection (Cheeseman et al., 2012; Miotto et al., 2015; Volkman et al., 2012) and loci under balancing selection due to host immune pressure (Weedall and Conway, 2010). Recently, WGSs of two Chinese 1 strains that differed significantly in

mouse virulence were analyzed with the intent to identify genotype
phenotype associations (Cheng et al., 2015). Analysis of the WGS data identified significant variation in a number of effector molecules, suggesting that this technique shows promise, but no validation experiments were rigorously pursued. However, this first approach suggests that a meta-analysis approaches specific genetic loci that are associated with a specific phenotype, such as virulence. NGS-based metagenomic approaches likewise offer the possibility of profiling entire microbial communities associated with clinical disease. Each of the different typing methods discussed above has its advantage and disadvantage. Though the whole-genome sequence provides the highest resolution in identifying T. gondii strains, it is hindered by the

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requirements of isolating the parasites, expanding the parasites in cell culture, purify them, and extract DNA for sequencing. The cost to sequence a 65 Mb T. gondii genome is still a factor to consider. In addition, DNA samples extracted from tissue samples of clinical toxoplasmosis are not suitable for WGS due to low concentration of T. gondii DNA. In these cases, PCR-based typing methods (MS, PCR-RFLP, and MLST) are still the better ways to identify the parasites. Given that none of the currently employed PCR-based typing methods have markers to cover all 14 chromosomes, and these markers may not necessarily capture all diversity in the population, additional markers may be selected based on whole-genome sequences from a diverse set of over 60 T. gondii strains (Lorenzi et al., 2016).

3.2.6 Correspondence between haplogroups, polymerase chain reaction restriction fragment length polymorphism, and microsatellite genotype designation Currently, there is no standard for genotype designation. Given that different methods (MSs, PCR-RFLP, sequencing, etc.) are used to type a variety of isolates, and each method has its own scheme of classification, genotype designation has been confusing. Among these methods, the conventional designation of genotypes assumed the clonal population structure with the three dominant lineages, namely, types I
III (also known as types 1, 2, and 3). The ToxoDB PCR-RFLP genotype naming system applies to isolates typed by 10 genetic markers, including SAG1, SAG2 (50 -30 SAG2 and alt.SAG2), SAG3, BTUB, GRA6, L358, c22-8, c29-2, PK1, and Apico (Su et al., 2010). These markers can further divide the type II lineage into “type II clonal” and “type II variant.” The type II clonal has a type II allele at locus Apico, whereas the type II variant has a type I allele.

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The designation of clonal lineages characterized by the 15 MS markers is in agreement with other techniques for the three archetypal lineages and often refers to their geographic origins for other clonal lineages (e.g., Chinese 1, Africa 1, Caribbean 1, 2, etc.). With these MS markers, the name “unique genotype” or “atypical” is attributed to strains belonging to none of the clonal lineages defined by this genotyping method. The haplogroup naming system is based on DNA sequencing of five introns, including UPRT, MIC, BTUB, HP, and EF (Khan et al., 2007, 2011b). To clarify the correspondence between the three typing methods, 956 strains were genotyped by both PCR-RFLP and MS markers, and genotyping was further refined for 138 of them using intron sequencing. Clustering methods were used to condense the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major ancestral populations or clades from A to F (Su et al., 2012). WGS performed on 62 representative strains confirmed this clustering, with a few exceptions (notably, identification of a 16th haplogroup) (Lorenzi et al., 2016). Correspondence between WGS (Lorenzi et al., 2016), MLST (Su et al., 2012), PCR-RFLP (Su et al., 2012; www.toxodb.org), and MS genotypes (Ajzenberg et al., 2010) together with the main geographical distribution of the genotypes is presented in Table 3.3. This correspondence is easily observed for strains belonging to the different clonal lineages, allowing deducing from a MS or PCR-RFLP genotype, the belonging of the isolate to a particular haplogroup or clade. For instance, the conventional types I, II, and III lineages are grouped into clades A, D, and C, respectively, and correspond to PCR-RFLP genotype #1 (type II clonal, belonging to haplogroup 2), genotype #2 (type III, belonging to haplogroup 3), genotype #3 (type II variant, haplogroup 2), and genotype #10 (type I, part of haplogroup 1). It

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3. Molecular epidemiology and population structure of Toxoplasma gondii

TABLE 3.3 Correspondence of Toxoplasma gondii genotypes between whole-genome sequencing, multilocus sequencebased typing (MLST), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), or 15 microsatellite (MS) markers and their main geographical origin. Clade and haplogroups defined by whole-genome sequencing (Lorenzi et al., 2016)

Haplogroups defined by MLST (Su et al., 2012)

ToxoDB PCR-RFLP Genotype # (www. toxodb.org; Su et al., 2012)

Groups defined by microsatellite typing using 15 MSs (Ajzenberg et al., 2010)

Main geographical origin

A1

/

91

Atypical

South America

38

/

South America

10

Type I

Asia, South America

17

Atypical

South America

27; 35; 55

Atypical

Central America; South America

/

139

III/II

North America

2

127

II/III

North America

1; 3

Type II

Europe; North America, North Africa

128

II

South America

129

Atypical

South America

2

Type III

Worldwide

73

Atypical

North America

12

Caribbean 2

South America, Carribean

133

Type III

North America

72

Type III

North America

2; 7

II; III

North America; Central America

13

Caribbean 1

Caribbean

25

Caribbean 3

South America

26

Atypical

South America

31

Caribbean 2

South America

50

Type III

Central America

79

Atypical

South America

83

III variant

Caribbean

1

/ D2

/

C3

/

a

3

/

a

(Continued)

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3.2 Genetic markers

TABLE 3.3 (Continued) Clade and haplogroups defined by whole-genome sequencing (Lorenzi et al., 2016)

B4

Haplogroups defined by MLST (Su et al., 2012)

4

B8

/

A15

5

F5 / A6

6

/

A7

B8 /

7

8

ToxoDB PCR-RFLP Genotype # (www. toxodb.org; Su et al., 2012)

Groups defined by microsatellite typing using 15 MSs (Ajzenberg et al., 2010)

Main geographical origin

90

Atypical

North America

115; 118; 125; 130

Atypical

North America; Caribbean; South America

140

Type III

Central America

141

Caribbean 3

Caribbean

11

Atypical

South America

17

Atypical

South America

119; 104

Atypical

South America

47

Atypical

South America

17

Atypical

South America

76; 92; 93; 99; 107; 108; 124; 126

Atypical

South America; Africa

106

/

South America

52

Atypical

Central America

60; 95; 98; 193

Amazonian

South America

22; 37; 65; 100

Atypical

South America

6

Africa 1

Africa

80; 42; 85

Atypical

South America

33; 41; 51; 56; 70; 82; 84; 105

Atypical

South America

17

/

South America

86

Africa 1

South America

77

Atypical

South America

28

Atypical

North America

19

Atypical

South America

32; 40; 59; 64; 69; 71; 75; 121

Atypical

South America; North America

53

Atypical

South America

94

Atypical

South America (Continued)

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3. Molecular epidemiology and population structure of Toxoplasma gondii

TABLE 3.3 (Continued) Clade and haplogroups defined by whole-genome sequencing (Lorenzi et al., 2016)

Haplogroups defined by MLST (Su et al., 2012)

ToxoDB PCR-RFLP Genotype # (www. toxodb.org; Su et al., 2012)

Groups defined by microsatellite typing using 15 MSs (Ajzenberg et al., 2010)

Main geographical origin

B8

9

21

Atypical

South America

8

Atypical

North America

14

Atypical

South America

45; 46; 67; 114; 116; 120; 138

Atypical

South America

78

Caribbean 1

South America

123

III/II

South America

60; 97; 194

Amazonian

South America

/

96

Atypical

South America

B4

34

Caribbean 1

South America

4; 5

HG12

North America

39

HG12

North America

49

Atypical

Caribbean

74

II/III or variant B18

North America

Chinese 1

Asia

C9

/

F10

D12

10

12

/

D13

a

13

9

137

Africa 4

Africa

14

203

Africa 3

Africa

36; 88

Atypical

South America

61

Atypical

South America

/

63

Atypical

South America

B8

111

Atypical

South America

/

134

III variant TUB2

South America

23; 44; 63; 81; 101; 109; 135

Atypical

Central America; South America

136

/

South America

197

Atypical Guiana

South America

66

Atypical

North America

/ A14 / A15

D11

15

a

11

A15

/

43

/

Central America

/

/

20

Africa 4

Africa; Asia (Continued)

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3.3 Evolutionary history

TABLE 3.3 (Continued) Clade and haplogroups defined by whole-genome sequencing (Lorenzi et al., 2016)

Haplogroups defined by MLST (Su et al., 2012)

ToxoDB PCR-RFLP Genotype # (www. toxodb.org; Su et al., 2012)

Groups defined by microsatellite typing using 15 MSs (Ajzenberg et al., 2010)

Main geographical origin

/

/

54

III

North America

/

/

112

/

Caribbean

/

/

29

/

South America

/

/

132

/

Africa

/

/

16

/

Central America

/

/

102

/

Central America

/

/

62

/ / / / / E16

/

/

South America

a

/

South America

a

/

South America

57

/

30

a

/

/

South America

a

/

South America

a

131

/

North America

15; 17; 202

HG16

South America, France (human)

48

/

122

/ b

16

a

Grouping did not correspond between Network and Structure analyses (Su et al., 2012). Haplogroup not well defined in Su et al. (2012), individualized using microsatellites markers and whole-genome sequencing (Lorenzi et al., 2016). /: haplogroup not defined. Genotype matching between techniques was not always possible, as haplogroups were not defined for all of them. Bold genotypes correspond to those defined by all techniques. Adapted from Galal, L., Hamidovi´c, A., Darde´, M.L., Mercier, M., 2019. Diversity of Toxoplasma gondii strains at the global level and its determinants. Food Waterborne Parasitol. e00052. b

is more uncertain for strains originating from areas, such as South American countries, where sexual recombination shuffles alleles of the different markers. However, more and more studies are jointly using RFLP and MS genotyping techniques (Aubert et al., 2010; Feitosa et al., 2017; Santos et al., 2018; Stajner et al., 2013; Vielmo et al., 2019) or comparing them (Chaichan et al., 2017; Galal et al., 2018). This may allow in the future a better homogenization between clustering by these different techniques, associated with the increasing use of the WGS.

3.3 Evolutionary history T. gondii is a generalist parasite, whose clade of cyst-forming coccidian parasites also includes closely related apicomplexan parasites Hammondia spp., Neospora spp., and Sarcocystis spp. Although these tissue-cyst forming coccidian parasites evolved from a recent common ancestor over 400 million years (Fig. 3.2A), they differ from each other in their host range, life cycle, and modes of transmission (Lorenzi et al., 2016). Currently, the understanding of the genetic basis behind these differences is

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3. Molecular epidemiology and population structure of Toxoplasma gondii

FIGURE 3.2 Comparative genome analysis of closely related tissue-cyst forming coccidian parasites (Lorenzi et al., 2016). (A) Phylogenetic relationship between apicomplexan parasites based on conserved DEAD box helicase. (B) Circos plot representation of synteny map among closely related tissue-cyst forming coccidian.

still in its infancy. The dates of divergence of these different species are quite controversial and difficult to estimate due to the lack of knowledge on the phylogeny of this group. Phylogeny of some Apicomplexa (Eimeria tenella, Sarcocystis tenella, Sarcocystis neurona, Cystoisospora belli, Hammondia hammondi, Neospora caninum, and T. gondii) was performed with the regions of the small subunit (SSU) and ITS1 of ribosomal DNA. This phylogeny does not solve the relationship between H. hammondi, N. caninum, and T. gondii and shows trifurcation for these three parasites. The node corresponding to this trifurcation was estimated at 12 million years using an average substitution rate of the SSU region calculated from several taxa (Su et al., 2003). Another phylogeny including a larger number of coccidial species and based on 18S ribosomal RNA sequences shows that T. gondii is closer to H. hammondi than to N. caninum (Morrison et al., 2004). With an average 18S rRNA substitution rate of 1% per 100 million years, the most recent common ancestor (TMRCA) of N. caninum and T. gondii was estimated at 11.3 million years. However, N. caninum, H. hammondi, and T. gondii seem very similar on this phylogeny with fewer nucleotide differences

between these three species of different genera than between some species of the same genus, such as two Besnoitia species (Morrison et al., 2004). In summary the appearance of the species T. gondii is estimated at about 11 million years based on the previous dating, but the separation of the three species N. caninum, H. hammondi, and T. gondii do not seem very clear because the sequences used in this study have few polymorphisms to distinguish the three parasites (Morrison, 2005). Another study comparing the two complete genomes of N. caninum and T. gondii, applying molecular clock theory, estimated the divergence between these two species at about 28 million years with a confidence interval between 21.7 and 42.7 million years (Reid et al., 2012). Only a few chromosomal rearrangements and net gain/loss of genetic information have been observed between T. gondii, Neospora, and Hammondia based on WGS (Fig. 3.2B) (Lorenzi et al., 2016). For instance, these three parasites have similar genome sizes (62
65 Mb), though Sarcocystis has a much larger genome (B127 Mb). All four genomes have similar genetic architectures, indicated by similar percentages of GC content and number of predicted coding sequences (B7000 to 8000).

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3.3 Evolutionary history

The most abundant predicted coding sequences are serine/threonine (S/T) kinases, RNAbinding proteins, PP2C-type S/T phosphatases, calcium-binding motifs (EF-hands), plant-like AP2 transcription factors and a family of surface antigens (SRA) known as SRS family (Lorenzi et al., 2016). Interestingly, there is a significant expansion of SRS family in Neospora (227 NcSRSs genes and 52 NcSRSs pseudogenes) that are tandemly arrayed in multigene clusters throughout the genome (Reid et al., 2012). In addition, there are significant differences in diversification of these genes between and within parasite species. Importance of these highly abundant and tandemly arrayed proteins, particularly S/T kinases and SRS family (known as secreted pathogenesis determinants or SPDs), has been documented for the regulation of the host immune response as a means of mediating parasite virulence (Behnke et al., 2011; Hunter and Sibley, 2012; Jung et al., 2004; Reese et al., 2011; Saeij et al., 2006; Taylor et al., 2006; White et al., 2014). Summarily, these SPDs evolved separately and independently in these closely related tissue-cyst forming coccidian parasites, leading to differences in host adaptation and modes of transmission. Studies have documented that genetic recombination has significance influence in shaping the population genetic structure of T. gondii (Boyle et al., 2006; Khan et al., 2007; Lorenzi et al., 2016; Minot et al., 2012). Initially, analysis of recent genealogy of types I
III utilizing expressed sequence tag sequencing based on 4324 genome-wide SNPs showed that a single genetic cross can lead to the evolution and expansion of a clonal population of T. gondii (Boyle et al., 2006). Subsequently, STRUCTURE analysis of 46 representative T. gondii isolates by sequencing eight introns from five unlinked loci showed that current T. gondii populations are derived from genetic recombination of four ancestral lineages (Khan et al., 2007). The first ancestral lineage was previously denoted as the “α” strain which crossed with an ancestral type II strain (type

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II1) and gave rise to the modern-day type I strains (Boyle et al., 2006; Khan et al., 2007). This ancestral lineage most closely resembles the current haplogroup six strains, including GPHT, BOF, FOU, and TgCatBr2. The second ancestral lineage is most closely related to circulating type II strains PTG, DEG, and PIH. The third ancestral linage was previously denoted as the “β” strain which crossed with an ancestral type II strain (type II3) and gave rise to the circulating type 3 strains (Boyle et al., 2006; Khan et al., 2007). This third ancestral lineage is closely related to haplogroup 9 strains P89 and TgCatBr3 (Boyle et al., 2006; Khan et al., 2007). The fourth ancestral lineage most closely resembles current haplogroup 4 strains such as MAS, TgCatBr18, and TgCatBr25 (Khan et al., 2007). Intron sequences also revealed that HG12 was evolved through a recombination between type II and a unique parental lineage designated as “γ” (Khan et al., 2011a). Thus initial genealogy analysis of these genotypes based on very few SNPs and genetic markers was interpreted as an indication that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture (Su et al., 2012). However, highresolution genome-wide SNPs data using WGS of several members from each haplogroup, chromosome painting, and mapping of shared ancestry (Fig. 3.1B and C) showed extensive mosaic genetic architecture across diverse haplogroups, leading to the conclusion that the most current isolates of T. gondii evolved through extensive recombination between lineages (Lorenzi et al., 2016; Minot et al., 2012). Moreover, genome-wide comparisons between highly conserved versus nonconserved regions showed that T. gondii’s unusual population genetic structure has been shaped by the inheritance of these conserved blocks that encode gene cassettes referred to as SPDs (Lorenzi et al., 2016). These conserved SPD haploblocks appear to have introgressed from a single lineage into multiple diverse lineages and have been shown to encode highly

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polymorphic, secreted effector proteins that appear to confer enhanced transmission, host range potential, pathogenicity, and overall fitness. The geographical origin of the common ancestor of the current parasite lineages is still being debated with two main opposing hypotheses. In 2006 a study (Lehmann et al., 2006) based mainly on MSs calculated the genetic diversity of different populations using strains isolated from chickens in different parts of the world. A total of 275 isolates and seven loci were included. The highest diversities have been found in South America. A network representation of haplotypes showed a separation between Eurasian and South American strains but also a close relationship between certain genotypes present in several geographical regions suggesting a recent removal of a single genotype with good migration capacity. A Bayesian statistical model allowing the strains to be grouped without a priori on their geographical origin also showed that they formed four populations: two confined to South and Central America, one present in Europe, Asia, Africa, and North America but apparently absent from South America, and one cosmopolitan. An Fst-based analysis measuring the divergence between each population and a theoretical “ancestral” population showed that South American populations had smaller Fst, suggesting that they had less diverged from the common “ancestral” population than the others (Lehmann et al., 2006). These data led to the conclusion that the place of origin of T. gondii was South America, where only wild Felids in low abundance could serve as definitive hosts for the parasite, before the introduction of the domestic cat in the 16th century. This scenario proposes at least two migrations from South America. A first migration toward Eurasia could have been achieved through bird migrations. However, bird migration between North America and Eurasia is a rare event that alone is not sufficient to explain the very recent spread of multi-continental genotypes on a

global scale and the low proportion of genotypes implicated. The alternative hypothesis suggests a role for human activities in the dispersal of T. gondii between the European and American continents, through maritime trade. It started in the 16th century with the slave trade and continued in the 18th and 19th centuries with the transport of agricultural products from South and Central America. This may have allowed some introductions of strains, leading to the emergence of a globally widespread population in Europe, Asia, Africa, North America where there is an abundance of domestic cats, in contrast to South America. It has been proposed that the geographical isolation of South America may have led to the division of two ancestral allele lines. The lower frequency of recombinants in North America and Eurasia could then be explained by a shorter time to produce these recombinants compared to South America, which would be the origin of this parasite, with a high selfreplication rate (Lehmann et al., 2006). These hypotheses were then contradicted in another study (Khan et al., 2007), which assumed that South American strains would have come from North America and would have been introduced into South America with their definitive hosts. The felids have indeed diversified when they entered in South America (O’Brien et al., 2008; O’Brien and Johnson, 2007). This study used intron sequencing and did not make any conclusion about the origin itself. But they hypothesized that a separation would have occurred between the North American and South American strains 1 million years ago when T. gondii entered in South America, which contradicts the scenario of Lehmann et al. (2006). However, this study focused on a small number of strains (46) mainly from North and South America. Europe was poorly represented and Asia and Africa were not included in the analysis, unlike the work of Lehmann et al. (2006). A third, more recent study tried to clarify these hypotheses (Bertranpetit et al., 2017).

Toxoplasma Gondii

3.3 Evolutionary history

Thus in 2017, the work of Bertranpetit et al. aimed to propose a phylogeographic origin of the current population of T. gondii strains, by adapting a methodology already applied to Plasmodium falciparum (Tanabe et al., 2010). A global collection of 168 T. gondii isolates collected from 13 populations representing five continents was sequenced for five gene fragments: GRA6, GRA7, SAG3, UPRT1, and UPRT7 (140 SNPs from 3153 bp per isolate). Phylogeny based on maximum likelihood methods with an estimate of the age of TMRCA and geostatistical analyses were performed to infer the hypothetical origin of T. gondii. Bertranpetit et al. (2017) show that the current strains of the parasite probably evolved from a South American ancestor about 1.5 million years ago

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and were able to reconstruct the global spread of the subsequent pathogen (Fig. 3.3). This emergence would be much more recent than the appearance of the ancestral form of T. gondii estimated about 11 million years ago (Morrison et al., 2004) and therefore subsequent to the arrival of the felids in this part of the world (O’Brien et al., 2008; O’Brien and Johnson, 2007). The authors propose that the ancestral lineage of T. gondii was introduced into South America with felids. The evolution of oral infectivity of tissue cysts through carnivorism and the diversification of felids in this region of the world would have allowed the emergence of a new strain with a much more effective transmission capacity than the ancestral line, allowing it to supplant it and have a pandemic distribution.

FIGURE 3.3 Historical spread of Toxoplasma gondii inferred from five molecular markers (Bertranpetit et al., 2017). Haplotype diversity within populations (gray circles) is correlated to the geographic distance from various origins, indicated using color shading (see legend). Strong negative correlations (red shading) indicate substantial loss of diversity inferred from the corresponding location. The best supported origin (plain dot) corresponds to strongest loss of diversity (i.e., lowest correlation), indicated in inset. Plain black lines indicate the corresponding shortest path from the origin to the various populations.

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3. Molecular epidemiology and population structure of Toxoplasma gondii

3.4 Global diversity and population structure Genetic diversity and population structure are strongly influenced by the intensity of sexual recombination and evolutionary selection of the recombinants as further demonstrated by WGS (Lorenzi et al., 2016). Reproduction without genetic recombination leads to a clonal population. With such population structure, it is expected to see identical genotypes over large geographic areas and at interval of many years. If a background level of genetic recombination is coupled with clonal expansion of a few genotypes, an epidemic population structure is expected. In this scenario a number of uncommon multilocus genotypes coexist with an overrepresentation of a few major genotypes. Random mating without selective expansion of any progeny will lead to a panmictic population structure. These different population structures are largely influenced both by parasitic and host factors and by environmental factors and human activities. The overall population consisted of clusters of highly abundant, overrepresented clonal genotypes along with more diverse groups that may be derived from genetic crosses (Lorenzi et al., 2016; Su et al., 2012). The distribution of genotypes showed a strong geographic separation, with widespread clonal genotypes in North hemisphere, in Africa and in Asia, and highly diverse genotypes in South America.

3.4.1 Geographical distribution Numerous studies have been conducted in diverse countries for analyzing T. gondii genotypes of isolates circulating in diverse countries and hosts. The studies are most often carried out in free-range chickens, as sentinels of the environmental contamination with T. gondii oocysts (Lehmann et al., 2006). Genotypes infecting wild species or other domestic

animals are less frequently studied. Although epidemiological studies presenting genotypes infecting humans may be biased due to travel or to consumption of imported food, they usually reflect genotypes circulating in animals and environment (Ajzenberg et al., 2002b). The analysis of 1457 T. gondii samples collected worldwide identified 189 genotypes based on the 10 PCR-RFLP markers (Shwab et al., 2014) Genotype distribution based on these PCRRFLP studies in different geographical regions is summarized in Fig. 3.4. 3.4.1.1 Europe In Europe, type II is largely predominant in published studies, from the extreme North (the Artic archipelago of Svalbarg, Finland) (Jokelainen et al., 2011; Jokelainen and Nylund, 2012; Prestrud et al., 2008) to the Mediterranean countries (Italy, Portugal, Greece). However, although this has to be confirmed by more studies, there may be a gradient in the prevalence of this type from North to South, type II being more prevalent in Northern and Western Europe, than in Southern countries, closer to Africa or Middle East. In France, it is found in more than 90% of human congenital toxoplasmosis but also in nearly all isolates originating from a large variety of animals (Ajzenberg et al., 2002a; Aubert et al., 2010; Darde´, 2008; Dume`tre et al., 2006; Halos et al., 2010; Richomme et al., 2009). It was also predominant in Germany (Herrmann et al., 2012a,b, 2013), Belgium (De Craeye et al., 2011), Switzerland (Berger-Schoch et al., 2011; Frey et al., 2012), or in Poland (Nowakowska et al., 2006). Isolated cases due to type II were reported in Serbia (Djurkovi´c-Djakovi´c et al., 2006), in Italy (Chessa et al., 2014), in Czech Republic (Machaˇcova´ et al., 2016; Slany et al., 2016), or in Romania (Costache et al., 2013). In Southern countries, although type II is still present, type III seems to be more frequently isolated (Dubey et al., 2006; Klun et al., 2017; Messaritakis et al., 2008; Vilares et al., 2014). Genotypes

Toxoplasma Gondii

3.4 Global diversity and population structure

FIGURE 3.4

83

Geographical distribution of Toxoplasma gondii genotypes (Shwab et al., 2014).

different from clonal lineages type II or III and natural recombinant isolates were occasionally detected in wild animals suggesting a higher diversity than usually described (Burrells et al., 2013; Calero-Bernal et al., 2015; Herrmann et al., 2010, 2012a,b; Markovi´c et al., 2014). 3.4.1.2 Africa Several genetic studies on T. gondii in Africa also suggested clonal population structure with a few dominant lineages. Galal et al. (2018), in a recent literature review, have summarized studies on the genotypic diversity of African strains of T. gondii published until May 2017. To this literature review, two recent

articles on South African genotypes of T. gondii can also be added (Luka´sˇ ova´ et al., 2018; Shwab et al., 2018). In these three articles, only strains genotyped by multilocus analysis (RFLP markers or MSs) were included. They originated from humans or from domestic animals. Out of 381 strains, approximately 94% belonged to five predominant clonal lineages: type II, type III, Africa 1 (haplogroup 6), Africa 3 (haplogroup 14), and a group of strains genetically very close to strains belonging to haplogroup 13. This clearly shows the clonal structure of T. gondii’s genetic diversity in African domestic environments. Country mapping of African genotypes of T. gondii has

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3. Molecular epidemiology and population structure of Toxoplasma gondii

revealed a contrasting spatial structure of parasite diversity across the continent. In North and East Africa (as far as South Africa), type II appears to be the predominant lineage, while Africa 1 or Africa 3 is almost absent. On the other hand, Africa 1 is largely spread in tropical Africa, while type II is much rarer there. type III, considered as a worldwide genotype, is also present on the African continent. The determinants of this structure remain to be clarified. It is worth noticing that the Africa 1 genotype is similar to ToxoDB genotype #6 (type BrI) which has been frequently identified in Brazil, South America (Pena et al., 2008). Given the geographical diversity of Africa, these data need to be completed by a broader sampling of T. gondii in order to better understand its population structure. In addition, the genetic diversity of strains circulating in the wild in Africa remains unknown. 3.4.1.3 Asia A recent review of geographical distribution of T. gondii genotypes from 390 isolates or DNA extracts in Asia revealed 36 genotypes (Chaichan et al., 2017). There is a clear association of genotypes with geographical areas. Overall, PCR-RFLP genotype #9 (Chinese 1, haplogroup 13) is the most frequently identified lineage in Southeast Asia, followed by genotype #10 (type I, haplogroup 1) in the same region. Toward northwest Asia, genotypes #1 and #3 (collectively type II, haplogroup 2) and #3 (type III, haplogroup 3) become the dominant lineages. Along the coastal line of south Asia to east Africa, genotype #20 is more frequently identified. Overall, available data indicated relatively low genetic diversity of T. gondii in Asia. It is suggested that, such genetic structure of T. gondii in Asia is the consequence of the arisen and expansion of agriculture, trade exchanges, and human migrations (Chaichan et al., 2017; Shwab et al., 2018). However, most of the continent remains underexplored. Except for China, genotypic

data are only available for 11 Asian countries. A high diversity of host species in the tropical part of Asia can be associated with greater genetic diversity as observed in other tropical areas (Mercier et al., 2011). In fact, the highest proportion of atypical strains is found in tropical South Asia (Malaysia, Myanmar, Sri Lanka). Further studies are needed in unexplored Asian countries to better understand the genetic structure of the T. gondii population on this continent (Chaichan et al., 2017). 3.4.1.4 Australia Data on T. gondii diversity in Australia are limited. Genotype #1 (type II) strain was reported from a domestic dog and a seal (AlQassab et al., 2009; Donahoe et al., 2014). Genotype #3 (type II variant) was also isolated from a common wombat, a peach-faced lovebird, and seven domestic cats in Australia (Brennan et al., 2016; Cooper et al., 2015; Donahoe et al., 2015). These data suggest that the type II lineage is common in Australia. Different genotypes were isolated from Australian wildlife (a wombat, a wallaby, two woylies, a mouse, a meerkat, and eight kangaroo samples) and from domestic animals (one horse, one cat, and two goats) (Parameswaran et al., 2010). These genotypes were characterized by atypical alleles that were interpreted as a result of genetic drift following the isolation of archetypal strains (type I, II, or III) imported into Australia during early European settlement. This possible genetic drift was further confirmed by sequencing of only three loci (B1, SAG2, and SAG3) performed on isolates from 16 macropods (Pan et al., 2012).The observed allelic diversity suggested that most genotypes exist as minor variants of established archetypal lineages by the accumulation of new mutations. Recombination of these variants was responsible for the diversification of strains on this continent. The presence of multiple infections in these macropods will favor recombination in feral cats, enhancing diversity

Toxoplasma Gondii

3.4 Global diversity and population structure

(Pan et al., 2012). Overall, the number of T. gondii isolates studied in Australia is still very limited, and more studies are needed to have a better view of T. gondii genetic diversity and population structure. 3.4.1.5 North America Genetic studies of T. gondii in North America revealed higher diversity than that in Europe, Africa, and Asia (Shwab et al., 2014). However, a few types including genotypes #1 and #3 (collectively type II), genotypes #4 and #5 (collectively type 12, HG12), and genotype #2 (type III) are dominant in this region. Genotype #10 (type I) is rarely encountered. Genotype #5 (type 12, HG12) is identified as the major genotype in wildlife in North America (Dubey et al., 2011b; Jiang et al., 2018; Khan et al., 2011a). It includes previously reported type A and X strains from sea otters (Miller et al., 2004; Sundar et al., 2008). Comparison of T. gondii isolates from domestic animals and wildlife indicates a lower genetic diversity in the former, with the dominance of genotypes #1, #2, and #3 in domestic animals (Jiang et al., 2018). This suggests the partition of domestic versus sylvatic transmission routes for T. gondii. Recent study of 67 DNA samples from human toxoplasmosis revealed the dominance of type II parasites, followed by significant proportion of the parasites with the atypical genotypes of South America origins, indicating that T. gondii in human toxoplasmosis from the United States are more diverse than that from Europe (Pomares et al., 2018). 3.4.1.6 Central and South America After the initial discovery of clonal population structure of T. gondii in Europe and North America (Darde´ et al., 1992; Howe and Sibley, 1995; Sibley and Boothroyd, 1992), it was speculated that this is a general rule applies worldwide. However, a decade later, several studies on T. gondii diversity in South America revealed distinct genotypes and higher genetic

85

diversity than expected (Ajzenberg et al., 2004; Ferreira et al., 2006; Khan et al., 2006; Lehmann et al., 2004, 2006). These findings challenged the dogma of clonal population structure and stimulated a wave of population genetics studies on T. gondii. Up to date, numerous studies have confirmed the high genetic and genotypic diversity among T. gondii strains circulating in Central and South America. However, the genetic diversity and population structure are not homogeneous in this subcontinent. Genotyping analysis of T. gondii from Central America (Guatemala, Nicaragua, Costa Rica), Caribbean (Grenada, St. Kitts), and North and Western part of South America (Venezuela, Colombia, Peru, and Chile) showed a moderate diversity, with the presence of the type II lineage, the high frequency of type III (genotype #2), and related genotypes belonging to haplogroup 3 (Chikweto et al., 2017; Dubey et al., 2013, 2016; Hamilton et al., 2017; Rajendran et al., 2012; Su et al., 2012). These haplogroup 3
related genotypes were designated as Caribbean genotypes in the MS naming system (Mercier et al., 2011). In Brazil a large number of isolates from a variety of animals from different areas were intensively studied revealing a high genotypic diversity. Pena et al. (2008) genotyped 125 isolates from chickens, dogs, and cats in Brazil and identified 48 genotypes with 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates from different hosts and locations were designated as types BrI, BrII, BrIII, and BrIV, and they were considered relatively common lineages in Brazil. In a more recent study of 385 T. gondii samples in Brazil, 106 genotypes were identified, but there is no clear dominance of any genotype (Shwab et al., 2014). For a global comparison, Brazilian isolates were found in different haplogroups including 6, 14, 4, 8, 9, and 15, indicating their diversity (Su et al., 2012). However, the types II and III isolates are infrequently encountered in this part of the continent (More´ et al., 2012). The genetic

Toxoplasma Gondii

86

3. Molecular epidemiology and population structure of Toxoplasma gondii

diversity is high in the Amazonian strains observed in the rainforest of French Guiana (Ajzenberg et al., 2004; Mercier et al., 2011); some of them were clustered in the haplogroups 5 and 10 (Su et al., 2012). New genotypes are continuously identified in Brazil, particularly from wild animals (Vitaliano et al., 2014) or previously not studied region (Feitosa et al., 2017). In a recent report, 37 Argentinean samples were divided into 21 multilocus PCR-RFLP genotypes, indicating high genetic diversity in Argentina (Bernstein et al., 2018). Among the 37 samples, five new genotypes were identified. Most samples grouped to genotype #2 (type III). In addition, the genotypes #1 and #3 (type II) were also identified. These results suggest a unique population structure with combination of atypical genotypes and the common types II and III lineages in Argentina.

2.

3.

3.4.2 Factors affecting transmission and genetic exchange Toxoplasma gondii is a heterogamous parasite with sexual and asexual life stages in definitive and intermediate hosts, respectively. However, population structure studies reveal a high proportion of clonality in some parts of the world, suggesting a restricted use of this sexual cycle. The limited genetic exchange in a given population may result of an upstream inhibition of recombination or a downstream elimination of recombinant genotypes by natural selection (Tibayrenc and Ayala, 2002). Both biological and environmental factors influence the proportion of genetic exchanges. 3.4.2.1 Biological factors Several of the biological properties of T. gondii may account for the limited genetic exchanges: 1. Horizontal transmission through carnivory among intermediate hosts: T. gondii can

4.

5.

effectively bypass the sexual stage by cycling, presumably indefinitely, among intermediate hosts (Grigg and Sundar, 2009; Su et al., 2003). Vertical, transplacental, transmission: There is evidence that, in some host species, T. gondii may be serially vertically transmitted (Hide et al., 2009; Innes et al., 2009; Miller et al., 2008a,b; Williams et al., 2005). This would lead to a local clonal expansion of the parasite. Self-fertilization or self-mating: Successful mating of male and female gametes originating from a single parasite clone (i.e., with identical genetic background) has been demonstrated experimentally (Cornelissen and Overdulve, 1985; Pfefferkorn et al., 1977). This results in a clonal expansion via sex and meiosis (Wendte et al., 2010). The sexual stage infection in the cat is relatively transient, and it is thus likely that the majority of infections involve only a single T. gondii isolate derived from a single prey source. This means that self-fertilization or “selfing” would be common and would limit the flow of genes between strains (Howe and Sibley, 1995). This has also been suggested as being the main factor in the emergence of epidemic clones (Wendte et al., 2010). Parthenogenesis: Many macrogametes of the parasite remain unfertilized but are capable of forming oocysts in the small intestine of cats by parthenogenesis (Ferguson, 2002). Genetic factors: The presence of monomorphic ChrIa and other genetic factors significantly correlated with greater oocysts production in domestic cats, which may confer an advantage for transmission in anthropized environment. Hence, preference presence of monomorphic ChrIa for greater transmission in domestic cats may lead to reduce diversity (Khan et al., 2014).

Toxoplasma Gondii

3.4 Global diversity and population structure

6. Immunity against reinfection: The presence of only one strain in a prey species is a key to the transmission dynamics of the parasite, conditioning clonal expansion both by selfing and by carnivory transmission. Multiple infections may be due either to simultaneous ingestion of different preys or of an oocyst sample with different genotypes or to reinfection. Toxoplasma is classically known to induce a strong immune response both in intermediate and in definitive hosts, preventing infection by a new isolate. In murine models, reinfection was possible only when the prime and the challenge strains belonged to two different types (Dao et al., 2001; Elbez-Rubinstein et al., 2009). In a context of a highly predominant type circulating in the environment, such as type II in Europe, reinfection should be the exception and infection with a single isolate the rule, reinforcing the clonal structure. A mixed infection with two different isolates both belonging to type II has only been described once, with highly polymorphic MS markers (Ajzenberg et al., 2002a). Ingestion by a cat of such a prey infected by two different isolates belonging to the same type will not result in the emergence of a new clonal type. Mixed infections, possibly resulting from reinfections, were more often described in countries where numerous different genotypes are already circulating and where recombination events occur more frequently (Dubey et al., 2007, 2009; Pan et al., 2012). 7. Fitness: The expansion of types II and III was suggested to be facilitated by their ability to effectively outcompete other genotypes (Grigg and Sundar, 2009; Sibley and Ajioka, 2008). These superfit strains were supposed to possess the right mix of alleles for key proteins such as ROP or GRA proteins (Boothroyd, 2009). Some

87

recombinant genotypes resulting from a genetic out-crossing in a definitive host may be unable to develop in some intermediate hosts. However, until now, this has not been demonstrated. The notion of fitness was also hypothesized to explain the higher diversity of T. gondii strains paralleling the host range diversity (see below influence of environmental factors) (Mercier et al., 2011). 3.4.2.2 Dynamics of transmission between different environments or hosts The use of an ecological approach for T. gondii strain epidemiology is still rarely performed (Galal et al., 2019). For analyzing a phenomenon, which can occur over a relatively short period, the rapidly evolving MSs are the best suited markers. MSs were able to show the interpenetration of strains from anthropized and from wild areas in French Guiana, with the possibility of hybrid strains, or the influence of human activities on clustering of T. gondii population (Mercier et al., 2011). Genetic studies using PCR-RFLP markers and DNA sequencing were also essential for understanding transmission networks for T. gondii in Californian marine mammals. The same strains were found in Californian sea otters, in the terrestrial mammals, notably in wild felid hosts, from the adjacent coastal areas, as well as in a filter-feeding invertebrate collected near the shoreline. This study provided evidence for a mechanism through which this terrestrial parasite could infiltrate the marine environment via land-to-sea run-off and bioconcentration of oocysts in prey species of sea otters and other marine mammals (Miller et al., 2008a,b). Further discussion of the dynamics of transmission in the environment is found in the next section about the environmental and human factors influencing genetic population structure of T. gondii strains.

Toxoplasma Gondii

88

3. Molecular epidemiology and population structure of Toxoplasma gondii

3.4.2.3 Environmental and human factors The analysis of a genetic population structure must consider the opportunities for transmission between hosts in different environments. For T. gondii, important factors to take into account are the felid population (density, diversity, hunting areas, and dietary intake), the richness of prey species, and the possibility of circulation of these species. The variations in the dynamics of T. gondii transmission in an urban
rural
nonanthropized gradient, in temperate and in tropical environments, have been reviewed in Gilot-Fromont et al. (2012). They have consequences in terms of T. gondii genetic population structure. In domestic areas, only a limited number of host species such as cats, a few meat-producing animals, and peridomestic mammals and birds are involved in T. gondii domestic cycle. Among many genotypes, the three clonal lineages seemed to be most successfully adapted to these domestic hosts (Lehmann et al., 2003). They may have diverged about 10,000 years ago, which coincides with the domestication of companion and agricultural animals in the Fertile Crescent region. In Europe or North America, intensive breeding of a narrow range of domestic meat-producing animals together with cat domestication offered a major niche to types II and III (Shwab et al., 2018). In rural areas, farm buildings which shelter both cats and small intermediate hosts (small peridomestic rodents and birds) are reservoirs of infection, from which transmission of clonal types can radiate to the surrounding wild environment (Jiang et al., 2018), leading to an impoverishment of genetic diversity even in wildlife. In such a context (anthropized environment), for example, in Europe, strains found in the neighboring wildlife are usually similar to strains found in domestic animals (Aubert et al., 2010; De Craeye et al., 2011; Halos et al., 2010). Human activities lead to an impoverishment of diversity limiting recombination and thus gene flow in T. gondii (Jiang et al., 2018; Mercier et al., 2011).

Remote tropical areas, such as the rainforest, are species rich habitats that harbor many species of birds and mammals, including a large number of felids. This could sustain a greater diversity of parasite genotypes in order to colonize the maximum of ecological niches (Ajzenberg et al., 2004; Mercier et al., 2011). The different behavior of wild felids compared to that of domestic cats (larger hunting areas and dietary intake) and the number of possible preys influence hybridization patterns and gene flow of the parasite and thus its genetic structure of populations (Galal et al., 2019). Definitive hosts are more frequently infected by multiple T. gondii genotypes, which then cross and recombine before transmission to a new intermediate hosts (Gilot-Fromont et al., 2012; Pan et al., 2012). The possibility of reinfection by different strains may be another source of increasing diversity (ElbezRubinstein et al., 2009; Jensen et al., 2015). All these ecological factors lead to a high genetic diversity. For instance, in the French Guiana rainforest (Amazonian forest), one of the most important hotspots of diversity with at least 183 mammal species, including 8 of 39 known wild felid species, and 718 bird species, “wild” strains exhibited a remarkably higher genetic diversity than strains from the adjacent anthropized environment (Mercier et al., 2011). Besides, strains from the anthropized environment clustered into a few widespread lineages from the Caribbean region, whereas the “wild” population of strains does not exhibit any clear genetic clustering structure nor any linkage disequilibrium, supporting the hypothesis of an important mixing in this natural, undisturbed ecosystem. An intermediate situation may be present in countries, such as the United States or Canada, where large territories are still nonanthropized. Here, a genotypic diversity of T. gondii in the wild animals is present, coexisting with clonal lineages in anthropized areas (Dubey et al., 2008a, 2011b; Wendte et al., 2011). At the

Toxoplasma Gondii

3.4 Global diversity and population structure

confluence between both environments, wild animals may penetrate in anthropized areas and domestic animals are exposed to the wild through wild game, soil, or running water. The consequences of this interpenetration in terms of T. gondii genotypes are diverse: (1) detection of T. gondii strains with “hybrid” genotypes between the “wild” population and the anthropized population reflecting genetic exchanges, (2) strains from the wild environment found in domestic animals, or (3), on the opposite, strains from the anthropized environment found in wild animals. This was shown in the French Guianan context (Mercier et al., 2011), where 89% of the territory is covered by equatorial forest and where 80% of the human population is concentrated in the remaining 10% of urban/anthropized territory. In this context the “Amazonian” strains from the forest, considered more virulent for humans, represent a real risk for the population when they enter or exchange genes with the anthropized environment. We can also detect the influence of wild strains in North America by the presence of diverse genotypes in domestic animals (Dubey et al., 2008b, 2011a; Jiang et al., 2018). While human activities with urbanization, fragmentation of landscape, deforested areas, farming, domestication of cats and other animals modify T. gondii ecology by clustering the parasite population, in the meantime (Mercier et al., 2011), transportation of these strains through large distances by commercial exchange and transportation of animals since human trade on an intercontinental scale leads to expansion of clonal lineages. This mechanism was proposed as an explanation for the large distribution of the same lineages across countries or continents such as types II and III, or Africa 1/BRI (same clonal type from haplogroup 6, respectively, described by MS and RFLP markers in Africa and Brazil) (Chaichan et al., 2017; Galal et al., 2018; Lehmann et al., 2006; Mercier et al., 2011; Shwab et al., 2018). This phenomenon has been amplified since the

89

16th century with the expansion of world trade, particularly through maritime transport as a potential vector for the spread of cats, and more particularly brown rats, black rats, and domestic mice (Galal et al., 2019). Since the slave trade and the colonial period, European ships could have allowed the spread of types II and III strains from European ports to ports in the other regions involved in these exchanges (Aplin et al., 2011; Bonhomme et al., 2011; Galal et al., 2018). The same is true for the Africa 1 strains between West and Central Africa and Brazil (Lehmann et al., 2006; Mercier, 2010). These invasive rodents, in addition to being probably the most relevant reservoirs within the domestic cycle of T. gondii (Dubey et al., 1995; Hejlı´cek et al., 1997), would in this way be the presumed vectors of intercontinental migration for some strains. Galal et al. (2018, 2019) also propose that these small mammal species, due to their variable adaptations to different parasite lines (Murillo-Leo´n et al., 2019) may cause more in-depth changes in the genetic structure of Toxoplasma populations in the areas newly colonized by these species (Galal et al., 2018, 2019). Hassan et al. (2018) have thus experimentally demonstrated highly contrasted responses for the same type I strain in different subspecies of Mus musculus: M. m. musculus and M. m. castaneus appear resistant, whereas M. m. domesticus dies within days of infection (Hassan et al., 2018). In contrast, these three subspecies, infected with the African lineage Africa 1 or with most South American strains, all develop fatal toxoplasmosis. These observations lead to suggest that the house mouse (M. m. domesticus) could have spread to northern Africa over several millennia due to the quasiabsence in this region of T. gondii Africa 1 strain (Galal et al., 2018). On the other hand, considering the virulence expressed in domestic mice (M. m. domesticus) by Africa 1, the fact that this lineage is widespread in the tropical regions of West and Central Africa could have constituted a barrier

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90

3. Molecular epidemiology and population structure of Toxoplasma gondii

to invasion by this subspecies of mice more recently introduced in this region during the colonial period (Dalecky et al., 2015). Conversely, when this invasive host species establishes and proliferates, this phenomenon could lead to the decline of this parasitic lineage (Galal et al., 2019). In addition, the existence in this same region of some species of native small commensal mammals that are able to be competent reservoirs of the Africa 1 lineage, such as the Cricetomys gambianus (Galal et al., 2019), or potential reservoir, such as Mastomys natalensis that survives infection by a type I lineage belonging to the same clade as Africa 1 (Fujii et al., 1983), would allow an increased infection spectrum for the parasite. This would give Africa 1 a selective advantage over other less mouse-virulent strains (Khan et al., 2009). Indeed, only strains capable of chronically infecting local species of small mammals will be able to be transmitted to cats and spread into the environment (Lilue et al., 2013). These mechanisms could therefore shape the structure of Toxoplasma populations on a larger scale worldwide, as host specific profiles in terms of genetic susceptibility and resistance to different strains of T. gondii would determine the transmission capacities and persistence of parasitic strains (Galal et al., 2019). Regarding the natural environmental factors that can affect the migration of strains of T. gondii on a large scale, the great diversity of host species of the parasite augurs well for a multitude of pathways for its migration. Among these hosts, a role of migratory birds in the global expansion of T. gondii has been mentioned in the literature (Can et al., 2014; Karakavuk et al., 2018; Lehmann et al., 2006; Prestrud et al., 2007, 2008). Given the quantitative importance of bird migration flows over millions of years, the hypothesis of the role of migratory birds is highly plausible (Galal et al., 2019). Karakavuk et al. (2018) have thus tried to explain the presence in Turkey of strains belonging to the Africa 1 lineage (Do¨s¸ kaya

et al., 2013) by testing the hypothesis of migratory birds without however being able to demonstrate it. However, although North and South America are linked by large migratory bird flows, both subcontinents have highly divergent populations of T. gondii, suggesting a more secondary role of this pathway in the spread of the parasite (Lehmann et al., 2006). Concerning natural migrations via terrestrial hosts of T. gondii, their role seems to be less important in the intercontinental spread of strains over a reduced time scale, with more impact at a local or regional level.

3.5 Outbreak investigations Toxoplasma infection in immunocompetent adults is not typically associated with serious disease sequelae and usually presents with mild flulike symptoms before resolving into an asymptomatic, life-long infection. In fact the majority of seropositive people have no history of infection and have no idea when they were infected. Therefore it is significant, from a molecular and epidemiological perspective, when point-source outbreaks or foci of infection are identified. Indeed, outbreaks have contributed significantly to our understanding of the pathogenesis and underlying genetic factors associated with clinical disease, and they offer relevant insight into how the parasite spreads in nature and how to manage or prevent the transmission of disease. A recent, systematic review of 437 reported human toxoplasmosis outbreaks found that the vast majority of those associated with high numbers of infected, symptomatic, individuals were the result of ingesting oocysts contaminating water and soil samples (Meireles et al., 2015). It is likely that this number underestimates truth, and that many more outbreaks have occurred, but these go unreported because they fail to cause sufficient disease to be noticed. Table 3.4 lists a collection of 15 large outbreaks that have occurred during the

Toxoplasma Gondii

TABLE 3.4 Human toxoplasmosis outbreaks. Route of transmission

Stage

People symptomatic

Isolate

Date Symptoms

Reference

Sa˜o Jose´ dos Campos, Brazil

Unknown

Unknown Unknown

99




1966 Febrile lymphadenopathy

Magaldi et al. (1969)

Alabama, United States

Cat feces

Oocyst

30

10




1976 Chorioretinitis, fever, neurologic deficits

Stagno et al. (1980)

Atlanta, United States

Inhalation

Oocyst

86

37




1977 Febrile reaction, lymphadenopathy

Teutsch et al. (1979)

Butte County, United States

Goat’s milk

Cyst

24

10




1978 Retinitis

Sacks, et al. (1982)

Panama Canal

Water

Oocyst

98

39




1979 Fever, lymphadenopathy, myalgia

Benenson et al. (1982), Sulzer et al. (1986)

Victoria, Canada

Water

Oocyst

2894
7718

110

Atypical 1995 Retinitis, lymphadenopathy

Bowie et al. (1997), Burnett et al. (1998)

Sa˜o Paulo, Brazil

Water

Oocyst

Unknown

113




Gattas et al. (2000)

Santa Isabel, Brazil

Water

Oocyst

426

155

Atypical 2001 Fever, lymphadenitis, myalgia

De Moura et al. (2006), Vaudaux et al. (2010)

Izmir, Turkey

Cat feces

Oocyst

1797

171




Doganci et al. (2006)

Patam, Suriname

Water

Oocyst

33

11

Atypical 2003 Fever, myalgia, death

Demar et al. (2007)

Coimbatore, India

Water

Oocyst

Unknown

248




2004 Chorioretinitis

Palanisamy et al. (2006), Balasundaram et al. (2010)

Ouro Preto do Oeste, Brazil

Water

Oocyst

Unknown

78




2011 Febrile lymphadenopathy, myalgia

Santana et al. (2015)

Ponta de Pedras, Brazil

Ac¸ai juice

Oocyst

270

73




2013 Febrile lymphadenopathy, myalgia

Unpublished

Sa˜o Marcos, Brazil

Undercooked meat

Cyst

1000

154




2015 Febrile lymphadenopathy, myalgia

Unpublished

Santa Maria, Brazil

Water

Oocyst

1116

460




2018 Fever, retinitis, lymphadenopathy

Unpublished

Place of outbreak

People exposed

1999 Lymphadenopathy

2002 Fever, myalgia, lymphadenopathy

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3. Molecular epidemiology and population structure of Toxoplasma gondii

past half century. In the 14 outbreaks where the stage of infection was known, all but two were the result of infection by the oocyst stage. Whether this reflects the degree to which infection is occurring by oocysts in nature, or reflects a difference in the pathogenic potential of acquired infection by different transmission routes, that is, ingestion of oocysts versus tissue cysts, is enigmatic. In support of the second scenario, oocyst-derived infections using the VEG strain in mice are known to produce more virulent disease than infection by tissue cysts (Dubey and Frenkel, 1973). Until recently, it was generally accepted that undercooked meat from Toxoplasma infected food animals was the major source of acquired infection (Jones et al., 2009). However, with the development of new serological tests capable of distinguishing the parasite stage initiating infection, several studies have suggested that the majority of acquired infections are by ingestion of oocysts, rather than tissue cysts (Boyer et al., 2011; Hill et al., 2011; Santana et al., 2015). Support for this prevailing perspective is corroborated by natural outbreaks in marine mammals that are only infected by the oocyst stage (Conrad et al., 2005). These animals exhibit high seroprevalence rates indicating that oocyst transmission is not insignificant and may possibly be dominant. The unanswered question, however, is why the oocyst-derived outbreaks in Table 3.4 were associated with more virulent disease? One possible explanation is that parasite genotype plays a major role in determining the severity of disease (Boothroyd and Grigg, 2002). Alternatively, the fecundity of infection, which determines the concentration or load of infectious particles and/or the route of transfer (aerosolization, soil, and water), may also influence inoculation size or infection competency. A detailed overview of the outbreaks listed in Table 3.4 thus provides relevant insight into the possible mechanisms at play that likely influence the size and severity of the various outbreaks below.

A large-scale epidemic of toxoplasmosis observed in 1966 in Brazil occurred over a 2month period, from late March to mid-May, with 99 individuals developing illness including fever, lymphadenopathy, headaches, and myocardial involvement in a school population of 500 students at the University of Sa˜o Jose´ dos Campos in Sa˜o Paulo state (Magaldi et al., 1969; Magaldi et al., 1967). The etiological agent was presumed to be Toxoplasma as all patients possessed high Sabin
Feldman dye test titers (in excess of 4000) and a significant percentage of the students tested 6 months later saw an increase in their anti
Toxoplasma titers. No systematic epidemiological investigation was performed for what has ostensibly been regarded as the first-reported, large-scale outbreak of clinical toxoplasmosis in people. An outbreak in 1976 involving 10 individuals from a family in Alabama, United States, was the first study to epidemiologically link the ingestion of oocysts from cat feces that contaminated soil with clinical disease in people (Stagno et al., 1980). It also described a high incidence of associated eye disease, with 3 of 10 infected children developing chorioretinitis. In 1977 another outbreak at a riding stable in Atlanta, United States, conclusively linked the ingestion of oocysts with human disease (Teutsch et al., 1979). In all, 37 individuals came down with toxoplasmosis, the result of inhalation of dust contaminated with oocysts defecated by barn cats that got aerosolized during an equestrian event. In May 1999 113 students at a university campus in Brazil presented with lymphadenopathy and acute toxoplasmosis (Gattas et al., 2000). The outbreak was linked to the university campus cafeteria, and no new cases were reported after a 2-μm filter was installed to remove particulate matter, including Toxoplasma oocysts from the water supply. Although it could not be ruled out that this was a point-source outbreak from eating undercooked meat, the sheer size of the outbreak, and the presence of a significant feral cat community on campus suggested that oocyst

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contamination was the more likely source of infection. Likewise, another outbreak in Izmir, Turkey, in September 2002 identified 171 acutely infected students at a local boarding school (population of 1797 students) that experienced cervical lymphadenopathy, low-grade fever, and flulike symptoms, with the majority of cases restricted to junior-grade students that ate out of a particular dining hall (Doganci et al., 2006). The investigators concluded that the likely source of the outbreak was oocysts from a feral cat colony in close proximity to the dining hall that serviced the junior-grade students, as food served in all dining halls was from the same source, and only a very limited number of students using the other dining halls were symptomatic and IgM positive. The first demonstration that transmission of acquired toxoplasmosis can occur through the ingestion of raw milk occurred in 1978 in Butte County, United States. A family cluster of 10 cases of acute toxoplasmosis was epidemiologically linked to the consumption of raw goat’s milk (Sacks et al., 1982). In Parana´, Brazil, in 1993, a child that was exclusively breast-fed by a mother acutely infected after eating mutton during an outbreak established that transmission of Toxoplasma infection is also possible by the consumption of raw human milk from an acutely infected mother (Bonametti et al., 1997). Waterborne transmission has also been implicated as the source for several massacquired toxoplasmosis outbreaks that occurred after water supplies were contaminated with oocysts shed in cat feces (Benenson et al., 1982; Bowie et al., 1997; Burnett et al., 1998; de Moura et al., 2006). In Panama a 1979 outbreak of febrile toxoplasmosis afflicting 35 US Army soldiers occurred after drinking unfiltered water from a jungle water source during a routine training mission. Importantly, afflicted troops had treated the jungle water with iodine tablets prior to consumption, which was not an effective treatment option to preclude infection (Benenson et al., 1982;

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Sulzer et al., 1986). The largest recorded waterborne outbreak of clinical toxoplasmosis occurred in Victoria, Canada, in March 1995. As many as 7718 people were infected, and more than 100 individuals experienced overt clinical disease (Bowie et al., 1997; Eng et al., 1999). The source of the outbreak was a small, public reservoir that was unfiltered and only treated its water by chlorination. Transmission of the outbreak strain was linked to oocysts from wild cougars nearby the reservoir (Aramini et al., 1998; Aramini et al., 1999), and one isolate recovered from a cougar in the vicinity of the reservoir was shown to be acutely virulent in a mouse infection model (Khan et al., 2007). The incidence of eye involvement was quite high, 20 out of 97 (21%) symptomatic individuals examined developed toxoplasmic retinochoroiditis (Burnett et al., 1998), which is significantly greater than reported frequencies of 1%
2% prevalence for ocular disease among infected individuals in North America (Holland, 2003). It was this increased incidence of finding acute toxoplasmic retinitis in a local practice that identified the outbreak (Burnett et al., 1998). Whether the high incidence of eye involvement simply reflected a preselection bias because only symptomatic individuals were examined, or was the result of a parasite genotype that is more capable of causing eye disease, is not known. But frequencies of eye disease associated with infection in the range of 19%
23% have been recorded throughout Brazil, and this higher prevalence is thought to be due, at least in part, to parasite genotype (Bahia-Oliveira et al., 2003; Commodaro et al., 2016; Glasner et al., 1992; Silveira et al., 2001). Another outbreak was identified after a striking increase in the incidence of acquired OT occurred over a 5 month period (from September 2004 to January 2005) in Coimbatore, India (Palanisamy et al., 2006). A total of 402 serum samples screened for acute Toxoplasma infection in an ophthalmology

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clinic from January 2001 until February 2005 noted a dramatic increase in the frequency of eye lesions and seroconversion consistent with OT in September 2004. In all, 248 individuals were identified over a short period, the majority of which possessed high IgM titers. The available evidence suggested that a reservoir that supplied only chemically treated, unfiltered water to Coimbatore City was the likely source, as its catchment was infested with domestic and feral cats, and the increased incidence of infection was reported shortly after the rainfall season (Balasundaram et al., 2010; Holland, 2010). All of these studies point to the importance of filtering public water supplies to reduce the human health risk of infection by Toxoplasma oocysts that are deposited in the environment by feral and wild cat populations. With new reagents available to serotype, perform high-resolution genotyping and to integrate epidemiological datasets, it is increasingly possible to identify the source and strain of parasite that causes the epidemic, with the ultimate goal to determine why some outbreaks have, for example, increased eye disease rate or greater morbidity. A large waterborne outbreak of clinical toxoplasmosis occurred in Santa Isabel do Ivai, Parana´ state, Brazil, in 2001 (de Moura et al., 2006). Between October and December 2001, at least 426 individuals were identified as acutely infected out of 2884 people tested serologically. The outbreak was epidemiologically linked to a cistern that served as the town’s water supply, and it was remarkable for its high prevalence of symptomatic, systemic disease with associated eye involvement. T. gondii isolates were recovered from roof-top water tanks, a cat caught at the cistern (Dubey et al., 2004), and from chickens collected in the surrounding environment immediately following the outbreak event (Vaudaux et al., 2010). The original study isolated Toxoplasma from the water source and genotyped the isolate using a single genetic marker (de Moura et al., 2006). A follow-up

study utilized PCR-DNA sequencing at multiple genotyping markers to establish that the three isolates linked to the outbreak source were epidemic clones of a single genotype, and it also found that the outbreak clone infected 4/11 chickens that foraged nearby the outbreak (Vaudaux et al., 2010). Supporting the molecular findings, a dominant serotype identical to the serotype recovered from mice infected with the outbreak clone was identified among human patients, providing compelling evidence that serotyping is a useful epidemiologic tool to identify individuals infected with an outbreak agent (Vaudaux et al., 2010). A MSbased typing scheme was then applied to determine the precise molecular genotype of the T. gondii isolates associated with the human waterborne outbreak in Brazil (Wendte et al., 2010). When the MS markers were applied to the same set of isolates, the two strains isolated from the water cistern were confirmed to be identical, but genetically different from the majority of the chicken strains found in the neighboring environment, except for one chicken isolate. The higher level of resolution provided by the MS typing provided increased confidence in the conclusion that self-mating within a cat had caused the epidemic expansion and transmission of sufficient oocysts from a single parasite clone to cause the outbreak and reshape the local population genetic structure (Wendte et al., 2010). Another outbreak of human toxoplasmosis infecting 11 patients in a small village of 33 inhabitants in Suriname also used MS genotyping to demonstrate that all five patients from whom parasites were isolated had been infected with the same, previously undiscovered genotype (Demar et al., 2007). Similarly, MS markers were used to analyze two fatal outbreaks of toxoplasmosis in an outdoor captive breeding colony of squirrel monkeys (Saimiri sciureus) that occurred in 2001 and 2006 at the Institut Pasteur in French Guiana (Carme et al., 2009a). The 2001 and 2006 outbreaks were

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shown to be due to two different T. gondii strains. The 2001 strain exhibited a type 2 genotype, whereas the 2006 strain was atypical. In 2006 the outbreak consisted of two successive episodes, 3 weeks apart. The second event leading to the death of 20 squirrel monkeys was believed to be related to direct contamination by tachyzoites of bronchopulmonary origin from dying monkeys from the first event. All samples collected during both episodes of the 2006 outbreak had the same multilocus genotype using 12 MS markers, providing compelling evidence that the same T. gondii strain was responsible for both outbreak episodes. Within the last decade, an additional four outbreaks have occurred in Brazil, which remain largely unpublished. No systematic studies have yet been performed on these outbreaks to track their source, the transmission dynamics, or the molecular origins of the strains associated with these outbreaks, and such studies will no doubt be important to perform in order to evaluate why these outbreaks were associated with clinical disease. One of the outbreaks, in Ouro Preto do Oeste, produced 78 symptomatic infections in 2011 and was linked to oocyst contamination of a water supply. A recently developed serological test capable of identifying antibodies restricted to the CCp5A sporozoite antigen suggested that infection in these individuals was by oocysts (Santana et al., 2015). Another point-source outbreak that occurred in May 2013 in Ponta de Pedras, Brazil was associated with febrile lymphadenopathy in 73 individuals out of 270 people exposed after consuming Ac¸ai juice using unfiltered water that was likely contaminated with oocysts. In 2015, over 1000 individuals were thought to be exposed to Toxoplasma after eating undercooked pork meat from a single distributor over a 2 month period in Sa˜o Marcos, Brazil. In total, 154 individuals were symptomatic. Lastly, a large outbreak in 2018 was identified in Santa Maria, State of Rio Grande do Sul, Brazil. Between March and

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April 2018, a dramatic increase in cases of febrile syndrome identified 1116 cases, of which 460 individuals had confirmed laboratory diagnoses of acute toxoplasmosis. The outbreak was associated with eye disease in 14% of confirmed cases and is thought to be due to drinking unfiltered water contaminated with oocysts from a public water source. As samples from these outbreaks become available, it will now be possible to apply the nascent tools described herein to better infer the strain genotypes as well as the molecular and epidemiologic basis for the size, extent, and source of parasites in these outbreaks to put them into context with the other well-studied outbreaks.

3.6 Toxoplasma genotype and biological characteristics Parasite strain
specific difference in mouse virulence is the most notable phenotypic marker, which has been well defined for three canonical clonal types I
III strains of T. gondii. Type I strains are lethal in all strains of laboratory mice with inoculation of less than 10 tachyzoites (LD100 5 1), referred to as accurately virulent; by contrast, types II and III are considered intermediate to nonvirulent (LD100 $ 103) (Sibley and Boothroyd, 1992). Interestingly, like type I strains most of the South American divergent haplogroups (4
10) are also highly virulent in mice model (Grigg and Suzuki, 2003; Khan et al., 2007). Although types II and III are generally considered as nonvirulent in mice, progressive deterioration and death of mice, notably with neurological symptoms, can occur a few weeks or months after inoculation (Darde´ et al., 1988). Oral infection, which is the natural route of infection, also showed parasite strain
specific differences in mouse pathogenicity. Oral infection with type II strains produced more severe ileitis than type III strains in mouse model (Liesenfeld, 2002).

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The higher virulence of type I in mice compared to type II or III has been correlated with in vitro biological properties: type I displays enhanced migration under soft agarose plates, as well as enhanced transmigration across polarized epithelia or across extracellular matrix. They also show a higher rate of ex vivo penetration of lamina propria and submucosa (Barragan and Sibley, 2002, 2003). This ability to cross epithelial barriers rapidly and reach the bloodstream within hours postinfection might be an important predeterminant of parasite dissemination in vivo in susceptible host species. In cell culture, type I grows faster than type II or III and convert from tachyzoite to bradyzoite much slower rate than type II strains (Soeˆte et al., 1993). The higher growth rate of type I parasites either due to a higher reinvasion rate, or higher migration distance or to a shorter doubling time may also explain the higher tissue burden observed in mice infected with virulent strains (Saeij et al., 2005). Linkage mapping analysis in experimental segregating population such as F1 progeny of an experimental cross is a powerful classical genetic method to dissect the genetic loci of complex traits such as virulence. Quantitative trait locus (QTL) mapping has been successfully applied using multiple genetic crosses to identify the virulence loci and has identified remarkably a small number of polymorphic genes including a rhoptry S/T protein kinase ROP18 (Saeij et al., 2006; Taylor et al., 2006) and a polymorphic pseudokinase ROP5 (Behnke et al., 2011; Reese et al., 2011). In addition to QTL mapping a biochemical approach to detect binding partners of ROP5 by affinity purification (TAP) tagging and mass spectrometry identified another kinase ROP17 (Etheridge et al., 2014), which is together with ROP18 and in association with ROP5 phosphorylates immunity-related GTPases to control acute virulence in mice model. Thus mouse virulence phenotype of T. gondii is a multilocus trait, and the same finding was

observed in naturally recombinant isolates. Among the five recombinant (II X III) genotypes identified in an oocyst sample, two were of low virulence in mice, one showed an intermediate virulence (LD50 $ 102 but ,104 tachyzoites) or high (LD50 , 102 tachyzoites) virulence phenotype in mice, and two were highly virulent (LD50 , 102 tachyzoites) in mice (Herrmann et al., 2012a,b). Although in vitro studies demonstrate different intrinsic properties of the different strains, the expression of this virulence in a given host species is a more complex trait, which depends on several host and parasite characteristics. The definition of T. gondii virulence with respect to mouse infection leads to much ambiguity when defining virulence factors, since other hosts and especially man may behave quite differently from mice (Dubremetz and Lebrun, 2012). The most divergent strains, Amazonian strains, which have virulence traits in mice but also in immunocompetent humans, are useful for fundamental studies of pathogenicity. Over the last decade, several studies have documented that after invading the host cells, T. gondii actively reprograms the gene expression profile of the host cells by thwarting the host cell transcription machinery, including energy metabolism, immune responses, and signaling (Blader et al., 2001). Interestingly, T. gondii infection controls the host cell signaling pathways in a parasite strain specific manner: types I and III strains sustain the host signal transducer and activator of transcription 3 (STAT3) and STAT6 activity, whereas type II strains cannot maintain activation of STAT3 and STAT6. Mapping of virulence QTLs using II X III F1 progeny identified a single ROP kinase known as ROP16 (Saeij et al., 2006). It has been proposed that ROP16 can subvert the IL-12 response and inhibit the nuclear factor kappa B (NF-KB). Similarly, strain specific differences in NF-KB activation and IL-12 production were observe in murine-derived macrophages and peritoneal exudate cell. In

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3.7 Toxoplasma gondii genotype and human disease

comparison to type I strains, type II strains induce higher level of NF-KB activation and IL12 production. QTL mapping using type II X III F1 progeny identified GRA15, which is a secreted dense granule protein (Rosowski et al., 2011). Comparative analysis of genomewide gene expression profile differences identified another ROP kinase: ROP38, which inhibits the host cell transcription (Peixoto et al., 2010). Recent studies showed that the combination of polymorphic alleles of virulent genes rhoptry protein 18 (ROP18) and ROP5 is strongly associated with T. gondii virulence in laboratory mice; therefore these two genes may serve as genetic markers to predict virulence of the parasite strains (Shwab et al., 2016). Consistent with other innate immune sensing system, T. gondii infection actives inflammasome response in mice, primary human monocytes, and THP-1 cells in parasite strain specific manner through activation of NLRP1 and 3 by recruiting caspase-1/11 (Brahmer et al., 2012; Cirelli et al., 2014; Ewald et al., 2014; Gorfu et al., 2014; Gov et al., 2017; Topalian et al., 2012). Murine bone marrow
derived macrophages infected with types II and 11 T. gondii strains rapidly release cleaved interleukin 21β (IL-1β) with no pyroptosis. Conversely, mice infected with T. gondii produce substantial quantities of IL-18, which reduces parasite replication and promotes murine survival (Gorfu et al., 2014). It has been also well documented that GRA15 is the parasite factor that is partially responsible for strains specific differences in activation of inflammasome by differentially inducing NF-KB signaling pathway (Gorfu et al., 2014; Gov et al., 2017). Although there is parasite strain specific differences in activation of inflammasome in murine model and THP-1 cells, a survey of T. gondii strains from distinct genetic background revealed that all strains are capable of inducing NLRP1 variant-dependent rapid pyroptosis (Cirelli et al., 2014) in rat model. Forward and reverse genetic analysis

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including knockdown and overexpression identified Nlrp1 gene plays a pivotal role in T. gondii induced pyroptosis, inhibition of parasite growth, and induction of IL-1β/IL-18 (Cirelli et al., 2014).

3.7 Toxoplasma gondii genotype and human disease 3.7.1 Circumstances of isolation and genetic typing The circumstances during which T. gondii strains can be isolated from human cases of toxoplasmosis are relatively rare. They necessitate the retrieval of tachyzoites from pathological samples collected for diagnosis purposes (blood, amniotic fluid, bronchoalveolar lavage fluid, aqueous humor, etc.) or of cysts in tissues collected via biopsy or necropsy. Isolates from human toxoplasmosis come predominantly from cases of congenital toxoplasmosis or from immunodeficient patients and much less frequently from symptomatic acquired toxoplasmosis in immunocompetent patients. As a diagnostic assay, strain isolation via mouse inoculation is not easy to perform and tend to be abandoned in favor of PCR-based assays, which are more sensitive and standardized, give faster results, and bypass the need of animal care facilities. The sensitivity of the PCR-based methods theoretically allows direct analysis of the parasite genotype from primary clinical sample. This would enable genotyping to be performed on small quantities of pathological products such as ocular fluids or on formaldehyde fixed tissues. In reality the low number of parasites present in some samples and the frequent presence of PCR inhibitors often led to negative results with single-copy genotyping markers. Increasing the sensitivity of genotyping is theoretically possible with nested PCR assays, but this procedure greatly amplifies the risk of

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DNA contamination. Genetic typing of clinical samples must be easy to perform and suitable for screening of a large number of isolates. As for molecular epidemiology studies, the most widely techniques currently used for genotyping T. gondii in human toxoplasmosis rely on PCR-RFLP and MS markers (Ajzenberg et al., 2010; Su et al., 2006). Strain isolation or direct typing on pathological products can only be performed during symptomatic toxoplasmosis. To interpret properly the influence of T. gondii genotype on the different clinical aspects of human toxoplasmosis, we should also genotype the vast majority of human infections that are asymptomatic. Although limited in its resolution (most often II vs non-II serotypes), the noninvasive method of serological typing can provide interesting information on strains infecting asymptomatic patients. A serotyping study based on GRA5 and GRA6 polymorphism showed a homogeneous distribution of serotype II in Europe and of serotypes I and III in South America, whatever the clinical presentation of the infection, that is, symptomatic or asymptomatic (Morisset et al., 2008). This suggests that the vast majority of asymptomatic infected patients harbor the strains that circulate in their geographic environment. For instance, it is untrue to claim that human infections are more frequently due to type II; it is just an artifact due to the more frequent isolation of the parasite in European patients. African patients are infected by African strains (Ajzenberg et al., 2009), and genotypes found in southeastern Brazilian or Chinese patients overlapped those circulating in animals from the same area (Silva et al., 2014; Wang et al., 2013). As a consequence of the spatial distribution of strains, finding in a clinical sample a genotype uncommon in a given country should prompt an epidemiological investigation (travel outside the country, geographical origin of the patient, consumption of imported food) (Pomares et al., 2011, 2018).

3.7.2 Congenital toxoplasmosis Congenital toxoplasmosis is the main source of T. gondii isolation in humans from samples of amniotic fluid, placenta, cord blood, and tissues of aborted fetuses. Amniotic fluids or even placentas are available mainly in countries, such as France, where a systematic prenatal screening of congenital toxoplasmosis is performed. In this case, nearly all the isolates responsible for congenital cases of Toxoplasma infection, including the majority of asymptomatic cases at birth, can be submitted for genotyping. Otherwise, strain isolation comes mainly from the more severe cases of symptomatic congenital toxoplasmosis, introducing a clinical bias. The largest series of congenital cases with genotyping data has been conducted in a multicenter study in France (Ajzenberg et al., 2002b). Typing performed on all the strains consecutively isolated in congenital cases in French laboratories revealed that they almost all belonged to type II. Type II isolates were found in all the different aspects of congenital disease from lethal infection and severe neuroocular involvement in early maternal infections to isolated chorioretinitis or latent toxoplasmosis in late maternal infections. Though type II strains can sometimes prove highly pathogenic to the human fetus after a third-trimester infection (Kieffer et al., 2011), the severity of fetal infection caused by type II strains is clearly linked to the period of gestation at which the mother becomes infected with a decreasing risk of clinical disease when gestational age increases. The question is to know whether non-type II strains are more virulent for the fetus than type II strains. According to the data available, type III strains seem to behave roughly like type II strains in congenital toxoplasmosis and true type I strains are until now too rarely isolated to understand how they are interacting with the human host (Ajzenberg et al., 2010).

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3.7 Toxoplasma gondii genotype and human disease

However, there is a growing body of evidence to link some atypical strains to a higher burden in congenital toxoplasmosis (Delhaes et al., 2010; Demar et al., 2007; Elbez-Rubinstein et al., 2009). This should probably be nuanced according to the nature of the so-called atypical genotypes. For example, an “atypical” strain circulating in the Pacific Islands from French Polynesia has caused latent congenital toxoplasmosis after early maternal infection (Yera et al., 2014).The higher pathogenicity of some atypical strains has been suggested to explain the more severe ocular disease in congenitally infected children in Brazil when compared to those infected in Europe (Gilbert et al., 2008). The diversity of strains in the Brazilian environment is also present in humans and could explain the higher burden of the disease in infants congenitally infected with some mousevirulent T. gondii strains in Brazil. Unfortunately, genotyping studies from human congenital cases are still limited in this area to correlate clinical findings to specific genotypes (Carneiro et al., 2013; Ferreira et al., 2011; Silva et al., 2014). The analysis of 24 strains isolated from congenital cases revealed the genetic diversity of T. gondii isolates from newborns in southeastern Brazil (Carneiro et al., 2013; Silva et al., 2014), with a total of 14 different genotypes (mostly the clonal Brazilian types BRII and BRIII, and the ToxoDB genotypes #108 and #206). Of these 24 isolates completely genotyped, 20 originated from newborns with retinochoroiditis and there was no association with a specific genotype. In fact, reports of atypical strains or strains different from type II or III in congenital toxoplasmosis are scarce, but most of these cases were severe even after late maternal infection during pregnancy (Ajzenberg et al., 2002b; Delhaes et al., 2010; Demar et al., 2007; Do¨s¸ kaya et al., 2013; Elbez-Rubinstein et al., 2009; Pardini et al., 2019). Furthermore, the fact that atypical strains are able to reinfect women with a past infection to T. gondii is another clue to suggest

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that these strains with a different genetic background trigger an inappropriate immune response in the host that increases the risk of severe damages for the fetus (Elbez-Rubinstein et al., 2009). Because isolation of strains is a difficult task from clinical samples, a partial answer to the question of the higher pathogenicity of non-type II strains versus type II strains may come from serotyping instead of genotyping even though serotyping assays have limitations. In a study conducted in congenitally infected infants in the United States with a serotyping assay, severe disease and eye severity at birth were more common in infants with non-type II serotypes than in those with type II serotypes (McLeod et al., 2012). In the same cohort of US congenital cases, different anatomical patterns of hydrocephaly were associated with type II versus not-exclusively type II serotypes, serotype II pattern giving more frequently rise to aqueductal obstruction (Hutson et al., 2015). The non-type II serotypes could belong to the HG12 that is endemic in the United States (Khan et al., 2011a; Pomares et al., 2018). While severity of disease is influenced by trimester in which infection is acquired by the mother and likely by the genetic background of strains, other factors including genetic predisposition may contribute. Studies suggest that polymorphisms in genes affecting immune response, including HLA (Mack et al., 1999) or NALP1 (Witola et al., 2011), or in genes playing a role in developmental processes, such as ABCA4 and COL2A1 (Jamieson et al., 2008), could influence clinical outcome in congenital toxoplasmosis.

3.7.3 Postnatally acquired toxoplasmosis in immunocompetent patients 3.7.3.1 Ocular toxoplasmosis The influence of T. gondii genotype in OT has largely been suggested by epidemiological

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data showing a significantly higher prevalence and severity of OT in South America compared to Europe and by experimental OT model demonstrating a different immune response with type II or South American strains (Arantes et al., 2015; Pfaff et al., 2014). For example, 0.6% of the general population of Alabama and Maryland in the United States had retinochoroidal scars consistent with T. gondii infection (Holland, 2003) compared to 6% in Armenia, Colombia, to 21% in Erechim, Southern Brazil (Pfaff et al., 2014). A higher incidence of OT was also found in West African patients indirectly suggesting that strains from this region of Africa may also have a higher tropism for retinal tissue (Gilbert et al., 1995; Saad et al., 2018). There are very few studies reporting the direct genotyping analysis of ocular fluid samples due to the scarcity of these samples. These low-volume samples (usually ,200 μL) require invasive diagnostic procedures, and most of the collected volume is dedicated for the biological diagnosis of OT. Isolation of strains from ocular samples in mice or by cell culture is usually never attempted because of these volume limitations. When PCR-based assays are performed for detecting T. gondii, the extracted DNA may be used for direct genotyping analyses, but the amount of parasitic DNA is often too low to permit amplification of single-copy genetic markers leading to unsuccessful or incomplete typing results. Moreover, genotyping analyses performed directly on aqueous and vitreous humor samples may lead to a bias toward an underrepresentation of the common cases of OT, because these ocular fluid samples are usually collected when the clinical presentation is severe and/or atypical (Subauste et al., 2011). To overcome these limitations the serotyping technique seems interesting at least to differentiate serotype II from nonserotype II in OT (Morisset et al., 2008; Shobab et al., 2013). Interestingly, patients with OT are more prone to present a NR serotype (see Section 2.4).

The available multilocus genotyping data of OT in Brazil were mainly collected in the southern states of the country where the prevalence of the disease is known to be extremely high. Genotypes were retrieved directly from patients’ samples in the city of Erechim, Rio Grande do Sul state (Khan et al., 2006) and in diverse cities of Sao˜ Paulo state (Ferreira et al., 2011), whereas one strain was collected indirectly from environmental samples implicated as the source of a large waterborne toxoplasmosis outbreak that caused a high prevalence of eye involvement in Santa Isabel do Ivai, Parana´ state (de Moura et al., 2006; Silveira et al., 2015; Vaudaux et al., 2010). It is hard to draw any definite conclusion from these studies because the number of cases is very limited, but some unexpected findings deserve attention. The genetic diversity of T. gondii strains in OT appears limited when compared to the genetic data of strains collected in animals in Brazil. In Sao˜ Paulo state for example, it is striking to see that only one genotype (ToxoDB genotype # 65) was collected from seven patients with OT (Ferreira et al., 2011), whereas in the same state of Sao˜ Paulo, 20 different genotypes were collected from 46 cats (Pena et al., 2008). The ToxoDB genotype #65 seems to be common in Brazil since it has also been described in immunosuppressed patients with cerebral toxoplasmosis (Ferreira et al., 2011) and in animals elsewhere in this country (Pena et al., 2008). Similarly, most of the 11 patients from Erechim were infected by only one genotype and the strain involved in the outbreak of Santa Isabel do Ivai had the BrI genotype, which is considered as a major clonal lineage in Brazil, because it has been sampled in numerous animals from different areas. Interestingly, the BrI genotype is equivalent to the Africa 1 genotype found in cases of OT in African patients (Fekkar et al., 2011) reinforcing the hypothesis of retinal tropism of this genotype associated with a higher incidence of OT in Africa (Gilbert et al., 1995). All these

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data suggest that, among the numerous genotypes circulating in Brazil, only a few ones may be responsible for the high burden of OT in Brazil and are common in the environment. A major argument for the high virulence of strains collected in patients from Erechim and Sao˜ Paulo state is indirectly highlighted by the fact that they were not characterized from ocular fluid samples but from peripheral blood (Ferreira et al., 2011; Khan et al., 2006). This prolonged parasitemia in ocular disease in Brazil has been confirmed by direct microscopic observation of tachyzoites in blood samples (Silveira et al., 2011). Studies in the United States and Europe have reported contradictory results. In France and in several European countries, OT involves predominantly type II strains (Fekkar et al., 2011; Morisset et al., 2008). This result is not surprising since type II strains are the most common strains in the European environment. In Germany, a direct genotyping on a limited sample of ocular fluids detected type II in five cases of OT (Herrmann et al., 2014), but a serotyping study found a higher prevalence of non-type II serotype (Shobab et al., 2013). However, the geographical origin of the patients was not available. According to one survey that investigated the genetic background of strains causing atypical and/or severe OT in the United States, most cases in immunocompetent patients were not due to the common type II or HG12 strains but involved atypical genotypes (Grigg et al., 2001a,b). Because epidemiological information is lacking, it is not possible to know if these atypical strains were collected in patients originating from South America where such atypical genotypes are common. It is however possible that these atypical genotypes circulate in the United States and may be more virulent than type II strains. Of note, in Canada, the largest outbreak of toxoplasmosis, with a high rate of OT, ever reported was likely associated to an atypical strain shed by a cougar in one of

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two main reservoirs of municipal drinking water serving the Greater Victoria area (Bowie et al., 1997; Burnett et al., 1998; Dubey et al., 2008a). 3.7.3.2 Disseminated toxoplasmosis Atypical strains with highly divergent genotypes have been involved in the rare cases of disseminated toxoplasmosis observed in otherwise healthy people. The disease is characterized by high and prolonged fever associated with lung involvement which may be lifethreatening and need intensive care management. The parasitic DNA is usually genotyped from peripheral blood or bronchoalveolar lavage fluid samples. Most of these cases have been described in French Guiana (Carme et al., 2002, 2009b; Demar et al., 2007, 2012; Groh et al., 2012), but some cases may occur in other Amazonian countries (Nunura et al., 2010) or elsewhere such as in Europe (De SalvadorGuilloue¨t et al., 2006; Pomares et al., 2011), or in Africa (Gachet et al., 2018). In French Guiana, this severe infection is acquired after consumption of wild game or drinking unfiltered water from the Amazonian rainforest of French Guiana. In Europe, these cases are thought to be acquired after consumption of imported meat, especially horsemeat, from South America. In French Guiana, the responsible strains were the highly divergent “Amazonian” strains, quite different from the strains circulating in the coastal area of this French South American department (Mercier et al., 2011). WGS of some of these highly diverse Amazonian strains shows their belonging to the ancestral clade F, with no clear clustering into well-defined haplogroups (Lorenzi et al., 2016). Although the determinants of strainspecific differences in virulence for human infection remain unknown (Niedelman et al., 2012), it should be noted that these strains possess type I alleles for ROP18 locus of T. gondii, associated with virulence in mice. However, an

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outbreak in Suriname showed that the same atypical strain could lead to a broad spectrum of clinical manifestations ranging from mild illness to life-threatening disease (Demar et al., 2007). Clinical manifestations are also milder in children (Blaizot et al., 2018). These data strongly suggest that the host status and the inoculum size of T. gondii related to different dietary practices or hygiene habits is of paramount importance to explain the severity of symptoms in toxoplasmosis.

3.7.4 Postnatally acquired toxoplasmosis in immunocompromised patients In AIDS patients the most frequent clinical presentation of toxoplasmosis is encephalitis. The material for isolating and genotyping strains is rarely available in these patients, because cerebral biopsies are almost never performed and PCR assays usually test negative in blood samples. In patients with severe toxoplasmosis whose immunosuppression is not caused by HIV infection, such as those who undergo allogeneic hematopoietic stem-cell transplantation, T. gondii is often detected in blood and BAL samples and therefore could be genotyped, but the incidence of this opportunistic infection in those patients is much lower than in AIDS patients. Immunosuppressed patients usually reactivate the strain they asymptomatically acquired years before when they were immunocompetent. It is then expected that the genetic background of strains in these patients reflects the one observed in the general population in a given area. For example, an immunocompromised patient with a past immunity to T. gondii and who have always lived in France will have a high probability to reactivate a type II strain because type II strains account for .95% of strains in the French environment. In other areas with a higher diversity of strains the overrepresentation of a given genotype in

immunocompromised patients is likely to reflect a disproportionately high infection with this genotype in meat-producing animals and environment of this area (Wang et al., 2013). The largest collection of multilocus T. gondii genotypes with epidemiological, clinical, and outcome data in immunocompromised people was obtained from a French multicenter study (Ajzenberg et al., 2009). The genotype of T. gondii strains was strongly linked to the presumed geographical origin of infection. Type II strains were predominant in patients that acquired the infection in Europe, whereas non-type II strains were more commonly recovered from patients with infections acquired outside Europe. Two main genotypes were recovered from patients who acquired the infection in several sub-Saharan African countries (the clonal lineage Africa 1) and in the French West Indies (genotype Caribbean 1), common genotypes in these areas. The distribution of T. gondii genotypes (type II vs non-type II) was not significantly different between patients with AIDS and non-HIV patients nor was it significantly different for different sites of infection and outcome. These data suggested that the genotype of the strain does not play a major role in the pathophysiology and severity of toxoplasmosis in immunocompromised patients. This conclusion should be reevaluated in the context of the diversity of South American strains. In Brazil (Sao Paulo state), AIDS patients with focal cerebral infection were mainly infected with genotype ToxoDB#65, a common genotype in this Brazilian area (Ferreira et al., 2011), whereas those who died of a disseminated infection were infected with other strains (Bastos da Silva et al., 2016).

3.8 Conclusion and perspective on Toxoplasma genotype and human disease The role of the strain in the severity of human toxoplasmosis is likely to be a reality but is difficult to assess because the scenario of

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References

pathogenicity is complex and involves several factors such as genetic background of the strain, inoculum dose, suppression, or immaturity of the host immune system and genetic predisposition. Because of their association with a high burden of ocular disease in Brazil and their ability to cause life-threatening symptoms in otherwise healthy people in the Amazonian forest, the atypical highly divergent strains circulating in tropical South America appear to be more pathogenic for humans than those circulating elsewhere in the world. It has been predicted that super infection may drive the high genetic diversity in South American strains by increasing the probability of presence of two different genotypes in an intermediate host. As the drivers of pathogenicity in humans are still poorly understood, it remains unclear whether the higher pathogenicity of South American strains is associated with true strain-specific differences in virulence, super infection ability, or lack of adaptive host response. In the post
genomic era, hundreds of GWAS have been conducted in human population to identify the loci associated with human diseases. Drug resistance loci in P. falciparum parasites have been discovered successfully using GWAS and QTL. Recently, Orjuela-Sa´nchez et al. (2010) demonstrated an assessment of the feasibility of GWAS to identify the genetic basis of Plasmodium vivax pathogenicity (Orjuela-Sa´nchez et al., 2010). A recent metagenomics study of an HIV-positive patient with diffuse brain lesions identified T. gondii as the infectious agent responsible (Hu et al., 2018). Genomic innovation of long reads sequencing including PacBio and Oxford Nanopore sequencing (MinION) is the paradigm shifting methodology to construct the whole genome of pathogens from metagenomic data set (Conlan et al., 2014). Thus these studies and technologies provide the proof of principle that it should soon be possible to sequence the entire genome of T. gondii within complex samples utilizing such NGS-based

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metagenomics approaches. Performed on a large number of well-documented clinical cases, this approach may contribute to our understanding of strain pathogenicity in humans.

Acknowledgments We would like to acknowledge Lokman Galal and Azra Hamidovic for their contribution to the hypotheses on parasitic diversity and to the production of some tables.

References Ajzenberg, D., 2012. High burden of congenital toxoplasmosis in the United States: the strain hypothesis? Clin. Infect. Dis. 54, 1606
1607. Available from: https://doi. org/10.1093/cid/cis264. Ajzenberg, D., Ban˜uls, A.L., Tibayrenc, M., Darde´, M.L., 2002a. Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups. Int. J. Parasitol. 32, 27
38. Ajzenberg, D., Cogne´, N., Paris, L., Bessie`res, M.-H., Thulliez, P., Filisetti, D., et al., 2002b. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J. Infect. Dis. 186, 684
689. Available from: https://doi.org/10.1086/342663. Ajzenberg, D., Ban˜uls, A.L., Su, C., Dume`tre, A., Demar, M., Carme, B., et al., 2004. Genetic diversity, clonality and sexuality in Toxoplasma gondii. Int. J. Parasitol. 34, 1185
1196. Available from: https://doi.org/10.1016/j. ijpara.2004.06.007. Ajzenberg, D., Yera, H., Marty, P., Paris, L., Dalle, F., Menotti, J., et al., 2009. Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings. J. Infect. Dis. 199, 1155
1167. Ajzenberg, D., Collinet, F., Mercier, A., Vignoles, P., Darde´, M.-L., 2010. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. J. Clin. Microbiol. 48, 4641
4645. Available from: https://doi.org/10.1128/JCM.01152-10. Ajzenberg, D., Collinet, F., Aubert, D., Villena, I., Darde´, M.-L., French ToxoBs network group, et al., 2015. The rural-urban effect on spatial genetic structure of type II Toxoplasma gondii strains involved in human congenital toxoplasmosis, France, 2002
2009. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 36, 511
516. Available from: https://doi.org/10.1016/j.meegid. 2015.08.025.

Toxoplasma Gondii

104

3. Molecular epidemiology and population structure of Toxoplasma gondii

Al-Qassab, S., Reichel, M.P., Su, C., Jenkins, D., Hall, C., Windsor, P.A., et al., 2009. Isolation of Toxoplasma gondii from the brain of a dog in Australia and its biological and molecular characterization. Vet. Parasitol. 164, 335
339. Available from: https://doi.org/10.1016/j. vetpar.2009.05.019. Aplin, K.P., Suzuki, H., Chinen, A.A., Chesser, R.T., ten Have, J., Donnellan, S.C., et al., 2011. Multiple geographic origins of commensalism and complex dispersal history of black rats. PLoS One 6, e26357. Available from: https://doi.org/10.1371/journal. pone.0026357. Aramini, J.J., Stephen, C., Dubey, J.P., 1998. Toxoplasma gondii in Vancouver Island cougars (Felis concolor vancouverensis): serology and oocyst shedding. J. Parasitol. 84, 438
440. Aramini, J.J., Stephen, C., Dubey, J.P., Engelstoft, C., Schwantje, H., Ribble, C.S., 1999. Potential contamination of drinking water with Toxoplasma gondii oocysts. Epidemiol. Infect. 122, 305
315. Arantes, T.E.F., Silveira, C., Holland, G.N., Muccioli, C., Yu, F., Jones, J.L., et al., 2015. Ocular involvement following postnatally acquired Toxoplasma gondii infection in Southern Brazil: a 28-year experience. Am. J. Ophthalmol. 159, 1002
1012. e2. Available from: https://doi.org/10.1016/j.ajo.2015.02.015. Aubert, D., Ajzenberg, D., Richomme, C., Gilot-Fromont, E., Terrier, M.E., de Gevigney, C., et al., 2010. Molecular and biological characteristics of Toxoplasma gondii isolates from wildlife in France. Vet. Parasitol. 171, 346
349. Available from: https://doi.org/10.1016/j. vetpar.2010.03.033. Bahia-Oliveira, L.M., Jones, J.L., Azevedo-Silva, J., Alves, C. C., Orefice, F., Addiss, D.G., 2003. Highly endemic, waterborne toxoplasmosis in north Rio de Janeiro state, Brazil. Emerg. Infect. Dis. 9, 55
62. Balasundaram, M.B., Andavar, R., Palaniswamy, M., Venkatapathy, N., 2010. Outbreak of acquired ocular toxoplasmosis involving 248 patients. Arch. Ophthalmol. 128, 28
32. Barragan, A., Sibley, L.D., 2002. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. J. Exp. Med. 195, 1625
1633. Barragan, A., Sibley, L.D., 2003. Migration of Toxoplasma gondii across biological barriers. Trends Microbiol. 11, 426
430. Bastos da Silva, I., Batista, T.P., Martines, R.B., Kanamura, C.T., Ferreira, I.M.R., Vidal, J.E., et al., 2016. Genotyping of Toxoplasma gondii: DNA extraction from formalinfixed paraffin-embedded autopsy tissues from AIDS patients who died by severe disseminated toxoplasmosis. Exp. Parasitol. 165, 16
21. Available from: https:// doi.org/10.1016/j.exppara.2016.03.004.

Behnke, M.S., Khan, A., Wootton, J.C., Dubey, J.P., Tang, K., Sibley, L.D., 2011. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc. Natl. Acad. Sci. U.S. A. 108, 9631
9636. Available from: https://doi.org/ 10.1073/pnas.1015338108. Benenson, M.W., Takafuji, E.T., Lemon, S.M., Greenup, R. L., Sulzer, A.J., 1982. Oocyst-transmitted toxoplasmosis associated with ingestion of contaminated water. N. Engl. J. Med. 307, 666
669. Berger-Schoch, A.E., Herrmann, D.C., Schares, G., Mu¨ller, N., Bernet, D., Gottstein, B., et al., 2011. Prevalence and genotypes of Toxoplasma gondii in feline faeces (oocysts) and meat from sheep, cattle and pigs in Switzerland. Vet. Parasitol. 177, 290
297. Available from: https:// doi.org/10.1016/j.vetpar.2010.11.046. Bernstein, M., Pardini, L., More´, G., Unzaga, J.M., Su, C., Venturini, M.C., 2018. Population structure of Toxoplasma gondii in Argentina. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 65, 72
79. Available from: https://doi.org/10.1016/j.meegid.2018. 07.018. Bertranpetit, E., Jombart, T., Paradis, E., Pena, H., Dubey, J., Su, C., et al., 2017. Phylogeography of Toxoplasma gondii points to a South American origin. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 48, 150
155. Available from: https://doi.org/10.1016/j. meegid.2016.12.020. Blackston, C.R., Dubey, J.P., Dotson, E., Su, C., Thulliez, P., Sibley, D., et al., 2001. High-resolution typing of Toxoplasma gondii using microsatellite loci. J. Parasitol. 87, 1472
1475. Available from: https://doi.org/ 10.1645/0022-3395(2001)087[1472:HRTOTG]2.0.CO;2. Blader, I., Manger, I.D., Boothroyd, J.C., 2001. Microarray analysis reveals previously unknown changes in Toxoplasma gondii infected human cells. J. Biol. Chem. 276, 24223
24231. Blaizot, R., Nabet, C., Blanchet, D., Martin, E., Mercier, A., Darde´, M.-L., et al., 2018. Pediatric Amazonian Toxoplasmosis caused by atypical strains in French Guiana, 2002
2017. Pediatr. Infect. Dis. J. Available from: https://doi.org/10.1097/INF.0000000000002130. Blanchard, N., Gonzalez, F., Schaeffer, M., Joncker, N.T., Cheng, T., Shastri, A.J., et al., 2008. Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum. Nat. Immunol. 9, 937
944. Available from: https://doi.org/10.1038/ni.1629. Bonametti, A.M., Passos Jdo, N., da Silva, E.M., Bortoliero, A.L., 1997. [Outbreak of acute toxoplasmosis transmitted thru the ingestion of ovine raw meat]. Rev. Soc. Bras. Med. Trop. 30, 21
25 [Article in Portuguese].

Toxoplasma Gondii

References

Bonhomme, F., Orth, A., Cucchi, T., Rajabi-Maham, H., Catalan, J., Boursot, P., et al., 2011. Genetic differentiation of the house mouse around the Mediterranean basin: matrilineal footprints of early and late colonization. Proc. R. Soc. Lond. B: Biol. Sci. 278, 1034
1043. Available from: https://doi.org/10.1098/rspb.2010. 1228. Boothroyd, J.C., 2009. Expansion of host range as a driving force in the evolution of Toxoplasma. Mem. Inst. Oswaldo Cruz 104, 179
184. Boothroyd, J.C., Grigg, M.E., 2002. Population biology of Toxoplasma gondii and its relevance to human infection: do different strains cause different disease? Curr. Opin. Microbiol. 5, 438
442. Bowie, W.R., King, A.S., Werker, D.H., Isaac-Renton, J.L., Bell, A., Eng, S.B., et al., 1997. Outbreak of toxoplasmosis associated with municipal drinking water. The BC Toxoplasma Investigation Team. Lancet Lond. Engl. 350, 173
177. Boyer, K., Hill, D., Mui, E., Wroblewski, K., Karrison, T., Dubey, J.P., et al., 2011. Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America. Clin. Infect. Dis. 53, 1081
1089. Boyle, J.P., Rajasekar, B., Saeij, J.P.J., Ajioka, J.W., Berriman, M., Paulsen, I., et al., 2006. Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103, 10514
10519. Available from: https://doi.org/10.1073/pnas.0510319103. Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P., et al., 2012. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455
2465. Available from: https://doi.org/10.1056/NEJMoa1200694. Brennan, A., Donahoe, S.L., Beatty, J.A., Belov, K., Lindsay, S., Briscoe, K.A., et al., 2016. Comparison of genotypes of Toxoplasma gondii in domestic cats from Australia with latent infection or clinical toxoplasmosis. Vet. Parasitol. 228, 13
16. Available from: https://doi.org/ 10.1016/j.vetpar.2016.06.008. Burnett, A.J., Shortt, S.G., Isaac-Renton, J., King, A., Werker, D., Bowie, W.R., 1998. Multiple cases of acquired toxoplasmosis retinitis presenting in an outbreak. Ophthalmology 105, 1032
1037. Available from: https://doi.org/10.1016/S0161-6420(98)960043. Burrells, A., Bartley, P.M., Zimmer, I.A., Roy, S., Kitchener, A.C., Meredith, A., et al., 2013. Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian carnivores in the UK. Parasitology 140, 1768
1776. Available from: https://doi.org/10.1017/ S0031182013001169.

105

Calero-Bernal, R., Saugar, J.M., Frontera, E., Pe´rez-Martı´n, J. E., Habela, M.A., Serrano, F.J., et al., 2015. Prevalence and genotype identification of Toxoplasma gondii in wild animals from southwestern Spain. J. Wildl. Dis. 51, 233
238. Available from: https://doi.org/10.7589/2013-09-233. ¨ zdemir, H.G., Can, H., Do¨s¸ kaya, M., Ajzenberg, D., O ˙ S.G., et al., 2014. Genetic characterization Caner, A., Iz, of Toxoplasma gondii isolates and toxoplasmosis sero˙ prevalence in stray cats of Izmir, Turkey. PLoS One 9, e104930. Available from: https://doi.org/10.1371/journal.pone.0104930. Carme, B., Bissuel, F., Ajzenberg, D., Bouyne, R., Aznar, C., Demar, M., et al., 2002. Severe acquired toxoplasmosis in immunocompetent adult patients in French Guiana. J. Clin. Microbiol. 40, 4037
4044. Carme, B., Ajzenberg, D., Demar, M., Simon, S., Darde´, M. L., Maubert, B., et al., 2009a. Outbreaks of toxoplasmosis in a captive breeding colony of squirrel monkeys. Vet. Parasitol. 163, 132
135. Available from: https:// doi.org/10.1016/j.vetpar.2009.04.004. Carme, B., Demar, M., Ajzenberg, D., Darde´, M.L., 2009b. Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana. Emerg. Infect. Dis. 15, 656
658. Available from: https://doi.org/10.3201/ eid1504.081306. Carneiro, A.C.A.V., Andrade, G.M., Costa, J.G.L., Pinheiro, B.V., Vasconcelos-Santos, D.V., Ferreira, A.M., et al., 2013. Genetic characterization of Toxoplasma gondii revealed highly diverse genotypes for isolates from newborns with congenital toxoplasmosis in southeastern Brazil. J. Clin. Microbiol. 51, 901
907. Available from: https://doi.org/10.1128/JCM.02502-12. Chaichan, P., Mercier, A., Galal, L., Mahittikorn, A., Ariey, F., Morand, S., et al., 2017. Geographical distribution of Toxoplasma gondii genotypes in Asia: a link with neighboring continents. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 53, 227
238. Available from: https://doi.org/10.1016/j.meegid.2017.06.002. Cheeseman, I.H., Miller, B.A., Nair, S., Nkhoma, S., Tan, A., Tan, J.C., et al., 2012. A major genome region underlying artemisinin resistance in malaria. Science 336, 79
82. Available from: https://doi.org/10.1126/ science.1215966. Cheng, W., Liu, F., Li, M., Hu, X., Chen, H., Pappoe, F., et al., 2015. Variation detection based on nextgeneration sequencing of type Chinese 1 strains of Toxoplasma gondii with different virulence from China. BMC Genomics 16, 888. Chessa, G., Chisu, V., Porcu, R., Masala, G., 2014. Molecular characterization of Toxoplasma gondii type II in sheep abortion in Sardinia, Italy. Parasite Paris Fr. 21, 6. Available from: https://doi.org/10.1051/parasite/ 2014007.

Toxoplasma Gondii

106

3. Molecular epidemiology and population structure of Toxoplasma gondii

Chikweto, A., Sharma, R.N., Tiwari, K.P., Verma, S.K., Calero-Bernal, R., Jiang, T., et al., 2017. Isolation and RFLP genotyping of Toxoplasma gondii in free-range chickens (Gallus domesticus) in Grenada, West Indies, revealed widespread and dominance of clonal type III parasites. J. Parasitol. 103, 52
55. Available from: https://doi.org/10.1645/15-945. Cirelli, K.M., Gorfu, G., Hassan, M.A., Printz, M., Crown, D., Leppla, S.H., et al., 2014. Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii. PLoS Pathog. 10, e1003927. Available from: https://doi.org/10.1371/journal.ppat.1003927. Commodaro, A.G., Chiasson, M., Sundar, N., Rizzo, L.V., Belfort Jr., R., Grigg, M.E., 2016. Elevated Toxoplasma gondii infection rates for retinas from Eye Banks, Southern Brazil. Emerg. Infect. Dis. 22, 691
693. Conlan, S., Thomas, P.J., Deming, C., Park, M., Lau, A.F., Dekker, J.P., et al., 2014. Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae. Sci. Transl. Med. 6, 254ra126. Available from: https://doi.org/10.1126/ scitranslmed.3009845. Conrad, P.A., Miller, M.A., Kreuder, C., James, E.R., Mazet, J., Dabritz, H., et al., 2005. Transmission of Toxoplasma: clues from the study of sea otters as sentinels of Toxoplasma gondii flow into the marine environment. Int. J. Parasitol. 35, 1155
1168. Contopoulos-Ioannidis, D., Wheeler, K.M., Ramirez, R., Press, C., Mui, E., Zhou, Y., et al., 2015. Clustering of Toxoplasma gondii infections within families of congenitally infected infants. Clin. Infect. Dis. 61, 1815
1824. Available from: https://doi.org/10.1093/cid/civ721. ˇ Cooper, M.K., Slapeta, J., Donahoe, S.L., Phalen, D.N., 2015. Toxoplasmosis in a pet peach-faced lovebird (Agapornis roseicollis). Korean J. Parasitol. 53, 749
753. Available from: https://doi.org/10.3347/kjp.2015.53.6.749. Cornelissen, A.W., Overdulve, J.P., 1985. Sex determination and sex differentiation in coccidia: gametogony and oocyst production after monoclonal infection of cats with free-living and intermediate host stages of Isospora (Toxoplasma) gondii. Parasitology 90 (Pt 1), 35
44. Costache, C.A., Colosi, H.A., Blaga, L., Gyo¨rke, A., Pa¸stiu, A.I., Colosi, I.A., et al., 2013. First isolation and genetic characterization of a Toxoplasma gondii strain from a symptomatic human case of congenital toxoplasmosis in Romania. Parasite Paris Fr. 20, 11. Available from: https://doi.org/10.1051/parasite/2013011. Cristina, N., Darde´, M.L., Boudin, C., Tavernier, G., PestreAlexandre, M., Ambroise-Thomas, P., 1995. A DNA fingerprinting method for individual characterization of Toxoplasma gondii strains: combination with isoenzymatic characters for determination of linkage groups. Parasitol. Res. 81, 32
37.

Dalecky, A., Baˆ, K., Piry, S., Lippens, C., Diagne, C.A., Kane, M., et al., 2015. Range expansion of the invasive house mouse Mus musculus domesticus in Senegal, West Africa: a synthesis of trapping data over three decades, 1983
2014. Mammal Rev. 45, 176
190. Available from: https://doi.org/10.1111/mam.12043. Dao, A., Fortier, B., Soete, M., Plenat, F., Dubremetz, J.F., 2001. Successful reinfection of chronically infected mice by a different Toxoplasma gondii genotype. Int. J. Parasitol. 31, 63
65. Darde´, M.L., 1996. Biodiversity in Toxoplasma gondii. Curr. Top. Microbiol. Immunol. 219, 27
41. Darde´, M.L., 2008. Toxoplasma gondii, “new” genotypes and virulence. Parasite Paris Fr. 15, 366
371. Available from: https://doi.org/10.1051/parasite/ 2008153366. Darde´, M.L., Bouteille, B., Pestre-Alexandre, M., 1988. Isoenzymic characterization of seven strains of Toxoplasma gondii by isoelectrofocusing in polyacrylamide gels. Am. J. Trop. Med. Hyg. 39, 551
558. Darde´, M.L., Bouteille, B., Pestre-Alexandre, M., 1992. Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications. J. Parasitol. 78, 786
794. De Craeye, S., Speybroeck, N., Ajzenberg, D., Darde´, M.L., Collinet, F., Tavernier, P., et al., 2011. Toxoplasma gondii and Neospora caninum in wildlife: common parasites in Belgian foxes and Cervidae? Vet. Parasitol. 178, 64
69. Available from: https://doi.org/10.1016/j.vetpar.2010. 12.016. Delhaes, L., Ajzenberg, D., Sicot, B., Bourgeot, P., Darde´, M.-L., Dei-Cas, E., et al., 2010. Severe congenital toxoplasmosis due to a Toxoplasma gondii strain with an atypical genotype: case report and review. Prenat. Diagn. 30, 902
905. Available from: https://doi.org/ 10.1002/pd.2563. Demar, M., Ajzenberg, D., Maubon, D., Djossou, F., Panchoe, D., Punwasi, W., et al., 2007. Fatal outbreak of human toxoplasmosis along the Maroni River: epidemiological, clinical, and parasitological aspects. Clin. Infect. Dis. 45, e88
e95. Available from: https://doi. org/10.1086/521246. Demar, M., Hommel, D., Djossou, F., Peneau, C., Boukhari, R., Louvel, D., et al., 2012. Acute toxoplasmoses in immunocompetent patients hospitalized in an intensive care unit in French Guiana. Clin. Microbiol. Infect. 18, E221
E231. Available from: https://doi.org/10.1111/ j.1469-0691.2011.03648.x. de Moura, L., Bahia-Oliveira, L.M.G., Wada, M.Y., Jones, J. L., Tuboi, S.H., Carmo, E.H., et al., 2006. Waterborne toxoplasmosis, Brazil, from field to gene. Emerg. Infect. Dis. 12, 326
329. Available from: https://doi.org/ 10.3201/eid1202.041115.

Toxoplasma Gondii

References

De Salvador-Guilloue¨t, F., Ajzenberg, D., Chaillou-Opitz, S., Saint-Paul, M.-C., Dunais, B., Dellamonica, P., et al., 2006. Severe pneumonia during primary infection with an atypical strain of Toxoplasma gondii in an immunocompetent young man. J. Infect. 53, e47
e50. Available from: https://doi.org/10.1016/j.jinf.2005.10.026. Djurkovi´c-Djakovi´c, O., Klun, I., Khan, A., Nikoli´c, A., Knezevi´c-Usaj, S., Bobi´c, B., et al., 2006. A human origin type II strain of Toxoplasma gondii causing severe encephalitis in mice. Microbes Infect. 8, 2206
2212. Available from: https://doi.org/10.1016/j.micinf.2006. 04.016. Doganci, L., Tanyuksel, M., Araz, E.R., Besirbellioglu, B.A., Erdem, U., Ozoguz, C.A., et al., 2006. A probable outbreak of toxoplasmosis among boarding school students in Turkey. Clin. Microbiol. Infect. 12, 672
674. Donahoe, S.L., Rose, K., Slapeta, J., 2014. Multisystemic toxoplasmosis associated with a type II-like Toxoplasma gondii strain in a New Zealand fur seal (Arctocephalus forsteri) from New South Wales, Australia. Vet. Parasitol. 205, 347
353. Available from: https://doi. org/10.1016/j.vetpar.2014.07.022. ˇ Donahoe, S.L., Slapeta, J., Knowles, G., Obendorf, D., Peck, S., Phalen, D.N., 2015. Clinical and pathological features of toxoplasmosis in free-ranging common wombats (Vombatus ursinus) with multilocus genotyping of Toxoplasma gondii type II-like strains. Parasitol. Int. 64, 148
153. Available from: https://doi.org/10.1016/j. parint.2014.11.008. ˘ Do¨s¸ kaya, M., Caner, A., Ajzenberg, D., Degirmenci, A., Darde´, M.-L., Can, H., et al., 2013. Isolation of Toxoplasma gondii strains similar to Africa 1 genotype in Turkey. Parasitol. Int. 62, 471
474. Available from: https://doi.org/10.1016/j.parint.2013.06.008. Dubey, J.P., Frenkel, J.K., 1973. Experimental toxoplasma infection in mice with strains producing oocysts. J. Parasitol. 59, 505
512. Dubey, J.P., Weigel, R.M., Siegel, A.M., Thulliez, P., Kitron, U.D., Mitchell, M.A., et al., 1995. Sources and reservoirs of Toxoplasma gondii infection on 47 swine farms in Illinois. J. Parasitol. 81, 723
729. Dubey, J.P., Navarro, I.T., Sreekumar, C., Dahl, E., Freire, R.L., Kawabata, H.H., et al., 2004. Toxoplasma gondii infections in cats from Parana, Brazil: seroprevalence, tissue distribution, and biologic and genetic characterization of isolates. J. Parasitol. 90, 721
726. Dubey, J.P., Vianna, M.C.B., Sousa, S., Canada, N., Meireles, S., Correia da Costa, J.M., et al., 2006. Characterization of Toxoplasma gondii isolates in freerange chickens from Portugal. J. Parasitol. 92, 184
186. Available from: https://doi.org/10.1645/GE-652R.1. Dubey, J.P., Lo´pez-Torres, H.Y., Sundar, N., Velmurugan, G.V., Ajzenberg, D., Kwok, O.C.H., et al., 2007. Mouse-

107

virulent Toxoplasma gondii isolated from feral cats on Mona Island, Puerto Rico. J. Parasitol. 93, 1365
1369. Available from: https://doi.org/10.1645/GE-1409.1. Dubey, J.P., Quirk, T., Pittt, J.A., Sundar, N., Velmurugan, G.V., Kwok, O.C.H., et al., 2008a. Isolation and genetic characterization of Toxoplasma gondii from raccoons (Procyon lotor), cats (Felis domesticus), striped skunk (Mephitis mephitis), black bear (Ursus americanus), and cougar (Puma concolor) from Canada. J. Parasitol. 94, 42
45. Available from: https://doi.org/10.1645/GE1349.1. Dubey, J.P., Sundar, N., Hill, D., Velmurugan, G.V., Bandini, L.A., Kwok, O.C.H., et al., 2008b. High prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption in the USA. Int. J. Parasitol. 38, 999
1006. Available from: https://doi.org/10.1016/j.ijpara.2007. 11.012. Dubey, J.P., Moura, L., Majumdar, D., Sundar, N., Velmurugan, G.V., Kwok, O.C.H., et al., 2009. Isolation and characterization of viable Toxoplasma gondii isolates revealed possible high frequency of mixed infection in feral cats (Felis domesticus) from St Kitts, West Indies. Parasitology 136, 589
594. Available from: https://doi. org/10.1017/S0031182009006015. Dubey, J.P., Rajendran, C., Ferreira, L.R., Martins, J., Kwok, O.C.H., Hill, D.E., et al., 2011a. High prevalence and genotypes of Toxoplasma gondii isolated from goats, from a retail meat store, destined for human consumption in the USA. Int. J. Parasitol. 41, 827
833. Available from: https://doi.org/10.1016/j.ijpara.2011.03.006. Dubey, J.P., Velmurugan, G.V., Rajendran, C., Yabsley, M. J., Thomas, N.J., Beckmen, K.B., et al., 2011b. Genetic characterisation of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type. Int. J. Parasitol. 41, 1139
1147. Available from: https://doi.org/10.1016/j. ijpara.2011.06.005. Dubey, J.P., Tiwari, K., Chikweto, A., Deallie, C., Sharma, R., Thomas, D., et al., 2013. Isolation and RFLP genotyping of Toxoplasma gondii from the domestic dogs (Canis familiaris) from Grenada, West Indies revealed high genetic variability. Vet. Parasitol. 197, 623
626. Available from: https://doi.org/10.1016/j. vetpar.2013.07.029. Dubey, J.P., Verma, S.K., Villena, I., Aubert, D., Geers, R., Su, C., et al., 2016. Toxoplasmosis in the Caribbean islands: literature review, seroprevalence in pregnant women in ten countries, isolation of viable Toxoplasma gondii from dogs from St. Kitts, West Indies with report of new T. gondii genetic types. Parasitol. Res. 115, 1627
1634. Available from: https://doi.org/10.1007/ s00436-015-4900-6.

Toxoplasma Gondii

108

3. Molecular epidemiology and population structure of Toxoplasma gondii

Dubremetz, J.F., Lebrun, M., 2012. Virulence factors of Toxoplasma gondii. Microbes Infect. 14, 1403
1410. Dume`tre, A., Ajzenberg, D., Rozette, L., Mercier, A., Darde´, M.-L., 2006. Toxoplasma gondii infection in sheep from Haute-Vienne, France: seroprevalence and isolate genotyping by microsatellite analysis. Vet. Parasitol. 142, 376
379. Available from: https://doi.org/10.1016/j. vetpar.2006.07.005. Elbez-Rubinstein, A., Ajzenberg, D., Darde´, M.-L., Cohen, R., Dume`tre, A., Yera, H., et al., 2009. Congenital toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J. Infect. Dis. 199, 280
285. Available from: https://doi.org/10.1086/595793. Eng, S.B., Werker, D.H., King, A.S., Marion, S.A., Bell, A., Issac-Renton, J.L., et al., 1999. Computer-generated dot maps as an epidemiologic tool: investigating an outbreak of toxoplasmosis. Emerg. Infect. Dis. 5, 815
819. Etheridge, R.D., Alaganan, A., Tang, K., Lou, H.J., Turk, B. E., Sibley, L.D., 2014. The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice. Cell Host Microbe 15, 537
550. Available from: https://doi. org/10.1016/j.chom.2014.04.002. Ewald, S.E., Chavarria-Smith, J., Boothroyd, J.C., 2014. NLRP1 is an inflammasome sensor for Toxoplasma gondii. Infect. Immun. 82, 460
468. Available from: https://doi.org/10.1128/IAI.01170-13. Feitosa, T.F., Ribeiro Vilela, V.L., de Almeida-Neto, J.L., Dos Santos, A., de Morais, D.F., Alves, B.F., et al., 2017. High genetic diversity in Toxoplasma gondii isolates from pigs at slaughterhouses in Paraı´ba state, northeastern Brazil: circulation of new genotypes and Brazilian clonal lineages. Vet. Parasitol. 244, 76
80. Available from: https://doi.org/10.1016/j.vetpar.2017.07.017. Fekkar, A., Ajzenberg, D., Bodaghi, B., Touafek, F., Le Hoang, P., Delmas, J., et al., 2011. Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France. J. Clin. Microbiol. 49, 1513
1517. Available from: https://doi.org/10.1128/JCM.02196-10. Ferguson, D.J.P., 2002. Toxoplasma gondii and sex: essential or optional extra? Trends Parasitol. 18, 355
359. Ferreira, I.M.R., Vidal, J.E., de Mattos Cde, C., de Mattos, L. C., Qu, D., Su, C., et al., 2011. Toxoplasma gondii isolates: multilocus RFLP-PCR genotyping from human patients in Sao Paulo State, Brazil identified distinct genotypes. Exp. Parasitol. 129, 190
195. Available from: https:// doi.org/10.1016/j.exppara.2011.06.002. Ferreira Ade, M., Vitor, R.W., Carneiro, A.C., Brandao, G. P., Melo, M.N., 2004. Genetic variability of Brazilian Toxoplasma gondii strains detected by random amplified polymorphic DNA-polymerase chain reaction (RAPD-

PCR) and simple sequence repeat anchored-PCR (SSRPCR). Infect. Genet. Evol. 4, 131
142. Ferreira Ade, M., Vitor, R.W., Gazzinelli, R.T., Melo, M.N., 2006. Genetic analysis of natural recombinant Brazilian Toxoplasma gondii strains by multilocus PCR-RFLP. Infect. Genet. Evol. 6, 22
31. Fraza˜o-Teixeira, E., Sundar, N., Dubey, J.P., Grigg, M.E., de Oliveira, F.C.R., 2011. Multi-locus DNA sequencing of Toxoplasma gondii isolated from Brazilian pigs identifies genetically divergent strains. Vet. Parasitol. 175, 33
39. Frey, C., Berger-Schoch, A., Herrmann, D., Schares, G., Mu¨ller, N., Bernet, D., et al., 2012. Incidence and genotypes of Toxoplasma gondii in the muscle of sheep, cattle, pigs as well as in cat feces in Switzerland. Schweiz. Arch. Tierheilkd. 154, 251
255. Available from: https://doi.org/10.1024/0036-7281/a000341 [Article in German]. Fujii, H., Kamiyama, T., Hagiwara, T., 1983. Species and strain differences in sensitivity to Toxoplasma infection among laboratory rodents. Jpn. J. Med. Sci. Biol. 36, 343
346. Gachet, B., Elbaz, A., Boucher, A., Robineau, O., Fre´alle, E., Ajana, F., et al., 2018. Acute toxoplasmosis in an immunocompetent traveller to Senegal. J. Travel Med. 25. Available from: https://doi.org/10.1093/jtm/tay086. Galal, L., Ajzenberg, D., Hamidovi´c, A., Durieux, M.-F., Darde´, M.-L., Mercier, A., 2018. Toxoplasma and Africa: one parasite, two opposite population structures. Trends Parasitol. 34, 140
154. Available from: https:// doi.org/10.1016/j.pt.2017.10.010. Galal, L., Schares, G., Stragier, C., Vignoles, P., Brouat, C., Cuny, T., et al., 2019. Diversity of Toxoplasma gondii strains shaped by commensal communities of small mammals. Int. J. Parasitol 49, 267
275. Available from: https://doi.org/10.1016/j.fawpar.2019.e00052. Galal, L., Hamidovi´c, A., Darde´, M.L., Mercier, M., 2019. Diversity of Toxoplasma gondii strains at the global level and its determinants. Food Waterborne Parasitol e00052. Gattas, V.L., Nunes, E.M., Soares, A.L.B., Pires, M.A., Pinto, P.L.S., de Andrade, H.F., 2000. Acute toxoplasmose outbreak at campus of the University of Sao Paulo related to food or water oocyst contamination. In: Paper Presented at International Conference on Emerging Infectious Diseases, Atlanta, GA. Gilbert, R.E., Stanford, M.R., Jackson, H., Holliman, R.E., Sanders, M.D., 1995. Incidence of acute symptomatic Toxoplasma retinochoroiditis in south London according to country of birth. BMJ 310, 1037
1040. Gilbert, R.E., Freeman, K., Lago, E.G., Bahia-Oliveira, L.M. G., Tan, H.K., Wallon, M., et al., 2008. Ocular sequelae of congenital toxoplasmosis in Brazil compared with Europe. PLoS Negl. Trop. Dis. 2, e277. Available from: https://doi.org/10.1371/journal.pntd.0000277.

Toxoplasma Gondii

References

Gilot-Fromont, E., Le´lu, M., Darde´, M.-L., Richomme, C., Aubert, D., Afonso, E., et al., 2012. The life cycle of Toxoplasma gondii in the natural environment. In: Djakovi´c, O.D. (Ed.), Toxoplasmosis. IntechOpen, Rijeka. Available from: https://doi.org/10.5772/48233. Glasner, P.D., Silveira, C., Kruszon-Moran, D., Martins, M. C., Burnier Jr., M., Silveira, S., et al., 1992. An unusually high prevalence of ocular toxoplasmosis in Southern Brazil. Am. J. Ophthalmol. 114, 136
144. Goldstein, D.B., Schlotterer, C., 1999. Microsatellites: Evolution and Applications, Microsatellites and Other Simple Sequences: Genomic Context and Mutational Mechanisms. Oxford University Press, Oxford. Gorfu, G., Cirelli, K.M., Melo, M.B., Mayer-Barber, K., Crown, D., Koller, B.H., et al., 2014. Dual role for inflammasome sensors NLRP1 and NLRP3 in murine resistance to Toxoplasma gondii. mBio 5. Available from: https://doi.org/10.1128/mBio.01117-13. Gov, L., Schneider, C.A., Lima, T.S., Pandori, W., Lodoen, M.B., 2017. NLRP3 and potassium efflux drive rapid IL1beta release from primary human monocytes during Toxoplasma gondii infection. J. Immunol. 199, 2855
2864. Available from: https://doi.org/10.4049/ jimmunol.1700245. Grigg, M.E., 2007. The biology of Toxoplasma gondii. In: Soldata, D. (Ed.), Population Genetics, Sex and the Emergence of Clonal Lines of Toxoplasma gondii. Horizon Press. Grigg, M.E., Boothroyd, J.C., 2001. Rapid identification of virulent type I strains of the protozoan parasite Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis of the B1 gene. J. Clin. Microbiol. 39, 398
400. Grigg, M.E., Sundar, N., 2009. Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics. Int. J. Parasitol. 39, 925
933. Available from: https://doi.org/10.1016/j. ijpara.2009.02.005. Grigg, M.E., Suzuki, Y., 2003. Sexual recombination and clonal evolution of virulence in Toxoplasma. Microbes Infect. 5, 685
690. Grigg, M.E., Ganatra, J., Boothroyd, J.C., Margolis, T.P., 2001a. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J. Infect. Dis. 184, 633
639. Grigg, M.L.E., Bonnefoy, S., Hehl, A.B., Suzuki, Y., Boothroyd, J.C., 2001b. Success and virulence in Toxoplasma as the result of sexual recombination between two distinct ancestries. Science 294, 161
165. Available from: https://doi.org/10.1126/science. 1061888. Groh, M., Faussart, A., Villena, I., Ajzenberg, D., Carme, B., Demar, M., et al., 2012. Acute lung, heart, liver, and

109

pancreatic involvements with hyponatremia and retinochoroiditis in a 33-year-old French Guianan patient. PLoS Negl. Trop. Dis. 6, e1802. Available from: https:// doi.org/10.1371/journal.pntd.0001802. Guo, Z.G., Johnson, A.M., 1995. Genetic characterization of Toxoplasma gondii strains by random amplified polymorphic DNA polymerase chain reaction. Parasitology 111, 127
132. Halos, L., The´bault, A., Aubert, D., Thomas, M., Perret, C., Geers, R., et al., 2010. An innovative survey underlining the significant level of contamination by Toxoplasma gondii of ovine meat consumed in France. Int. J. Parasitol. 40, 193
200. Available from: https://doi.org/ 10.1016/j.ijpara.2009.06.009. Hamilton, C.M., Kelly, P.J., Boey, K., Corey, T.M., Huynh, H., Metzler, D., et al., 2017. Predominance of atypical genotypes of Toxoplasma gondii in free-roaming chickens in St. Kitts, West Indies. Parasit. Vectors 10, 104. Available from: https://doi.org/10.1186/s13071-017-2019-6. Hassan, M.A., Olijnik, A.-A., Frickel, E.-M., Saeij, J.P., 2018. Clonal and atypical Toxoplasma strain differences in virulence vary with mouse sub-species. Int. J. Parasitol. 49, 63
70. Available from: https://doi.org/10.1016/j. ijpara.2018.08.007. Hejlı´cek, K., Litera´k, I., Nezval, J., 1997. Toxoplasmosis in wild mammals from the Czech Republic. J. Wildl. Dis. 33, 480
485. Available from: https://doi.org/10.7589/ 0090-3558-33.3.480. Herrmann, D.C., Pantchev, N., Vrhovec, M.G., Barutzki, D., Wilking, H., Fro¨hlich, A., et al., 2010. Atypical Toxoplasma gondii genotypes identified in oocysts shed by cats in Germany. Int. J. Parasitol. 40, 285
292. Available from: https://doi.org/10.1016/j.ijpara.2009. 08.001. Herrmann, D.C., Ba¨rwald, A., Maksimov, A., Pantchev, N., Vrhovec, M.G., Conraths, F.J., et al., 2012a. Toxoplasma gondii sexual cross in a single naturally infected feline host: generation of highly mouse-virulent and avirulent clones, genotypically different from clonal types I, II and III. Vet. Res. 43, 39. Available from: https://doi. org/10.1186/1297-9716-43-39. Herrmann, D.C., Maksimov, P., Maksimov, A., Sutor, A., Schwarz, S., Jaschke, W., et al., 2012b. Toxoplasma gondii in foxes and rodents from the German Federal States of Brandenburg and Saxony-Anhalt: seroprevalence and genotypes. Vet. Parasitol. 185, 78
85. Available from: https://doi.org/10.1016/j.vetpar.2011.10.030. Herrmann, D.C., Wibbelt, G., Go¨tz, M., Conraths, F.J., Schares, G., 2013. Genetic characterisation of Toxoplasma gondii isolates from European beavers (Castor fiber) and European wildcats (Felis silvestris silvestris). Vet. Parasitol. 191, 108
111. Available from: https://doi. org/10.1016/j.vetpar.2012.08.026.

Toxoplasma Gondii

110

3. Molecular epidemiology and population structure of Toxoplasma gondii

Herrmann, D.C., Maksimov, P., Hotop, A., Groß, U., Da¨ubener, W., Liesenfeld, O., et al., 2014. Genotyping of samples from German patients with ocular, cerebral and systemic toxoplasmosis reveals a predominance of Toxoplasma gondii type II. Int. J. Med. Microbiol. IJMM 304, 911
916. Available from: https://doi.org/10.1016/ j.ijmm.2014.06.008. Hide, G., Morley, E.K., Hughes, J.M., Gerwash, O., Elmahaishi, M.S., Elmahaishi, K.H., et al., 2009. Evidence for high levels of vertical transmission in Toxoplasma gondii. Parasitology 136, 1877
1885. Available from: https://doi.org/10.1017/ S0031182009990941. Hill, D., Coss, C., Dubey, J.P., Wroblewski, K., Sautter, M., Hosten, T., et al., 2011. Identification of a sporozoitespecific antigen from Toxoplasma gondii. J. Parasitol. 97, 328
337. Holland, G.N., 2003. Ocular toxoplasmosis: a global reassessment. Part I: Epidemiology and course of disease. Am. J. Ophthalmol. 136, 973
988. Holland, G.N., 2010. An epidemic of toxoplasmosis: lessons from Coimbatore, India. Arch. Ophthalmol. 128, 126
128. Howe, D.K., Sibley, L.D., 1995. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J. Infect. Dis. 172, 1561
1566. Hu, Z., Weng, X., Xu, C., Lin, Y., Cheng, C., Wei, H., et al., 2018. Metagenomic next-generation sequencing as a diagnostic tool for toxoplasmic encephalitis. Ann. Clin. Microbiol. Antimicrob. 17, 45. Available from: https:// doi.org/10.1186/s12941-018-0298-1. Hunter, C.A., Sibley, L.D., 2012. Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat. Rev. Microbiol. 10, 766
778. Hutson, S.L., Wheeler, K.M., McLone, D., Frim, D., Penn, R., Swisher, C.N., et al., 2015. Patterns of hydrocephalus caused by congenital Toxoplasma gondii infection associate with parasite genetics. Clin. Infect. Dis. 61, 1831
1834. Available from: https://doi.org/10.1093/ cid/civ720. Innes, E.A., Bartley, P.M., Buxton, D., Katzer, F., 2009. Ovine toxoplasmosis. Parasitology 136, 1887
1894. Available from: https://doi.org/10.1017/ S0031182009991636. Jamieson, S.E., de Roubaix, L.-A., Cortina-Borja, M., Tan, H.K., Mui, E.J., Cordell, H.J., et al., 2008. Genetic and epigenetic factors at COL2A1 and ABCA4 influence clinical outcome in congenital toxoplasmosis. PLoS One 3, e2285. Available from: https://doi.org/10.1371/journal.pone.0002285. Jensen, K.D.C., Camejo, A., Melo, M.B., Cordeiro, C., Julien, L., Grotenbreg, G.M., et al., 2015. Toxoplasma gondii superinfection and virulence during secondary infection

correlate with the exact ROP5/ROP18 allelic combination. mBio 6, e02280. Available from: https://doi.org/ 10.1128/mBio.02280-14. Jiang, T., Shwab, E.K., Martin, R., Gerhold, R., Rosenthal, B., Dubey, J.P., et al., 2018. A partition of Toxoplasma gondii genotypes across spatial gradients and among host species, and decreased parasite diversity towards areas of human settlement in North America. Int. J. Parasitol. 48, 611
619. Available from: https://doi.org/ 10.1016/j.ijpara.2018.01.008. Jokelainen, P., Nylund, M., 2012. Acute fatal toxoplasmosis in three Eurasian red squirrels (Sciurus vulgaris) caused by genotype II of Toxoplasma gondii. J. Wildl. Dis. 48, 454
457. Available from: https://doi.org/10.7589/ 0090-3558-48.2.454. Jokelainen, P., Isomursu, M., Na¨reaho, A., Oksanen, A., 2011. Natural Toxoplasma gondii infections in European brown hares and mountain hares in Finland: proportional mortality rate, antibody prevalence, and genetic characterization. J. Wildl. Dis. 47, 154
163. Available from: https://doi.org/10.7589/ 0090-3558-47.1.154. Jones, J.L., Dargelas, V., Roberts, J., Press, C., Remington, J. S., Montoya, J.G., 2009. Risk factors for Toxoplasma gondii infection in the United States. Clin. Infect. Dis. 49, 878
884. Jung, C., Lee, C.Y., Grigg, M.E., 2004. The SRS superfamily of Toxoplasma surface proteins. Int. J. Parasitol. 34, 285
296. Available from: https://doi.org/10.1016/j. ijpara.2003.12.004. Karakavuk, M., Aldemir, D., Mercier, A., Atalay S¸ ahar, E., Can, H., Murat, J.-B., et al., 2018. Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey. PLoS One 13, e0196159. Available from: https://doi.org/10.1371/ journal.pone.0196159. Khan, A., Su, C., German, M., Storch, G.A., Clifford, D.B., Sibley, L.D., 2005a. Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J Clin Microbiol 43, 5881
5887. Available from: https://doi.org/10.1128/ jcm.43.12.5881-5887.2005. Khan, A., Taylor, S., Su, C., Mackey, A.J., Boyle, J., Cole, R. H., et al., 2005b. Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii. Nucleic Acids Res. 33, 2980
2992. Khan, A., Jordan, C., Muccioli, C., Vallochi, A.L., Rizzo, L.V., Belfort, R., et al., 2006. Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil. Emerg. Infect. Dis. 12, 942
949.

Toxoplasma Gondii

References

Khan, A., Fux, B., Su, C., Dubey, J.P., Darde, M.L., Ajioka, J.W., et al., 2007. Recent transcontinental sweep of Toxoplasma gondii driven by a single monomorphic chromosome. Proc. Natl. Acad. Sci. U.S.A. 104, 14872
14877. Available from: https://doi.org/10.1073/ pnas.0702356104. Khan, A., Taylor, S., Ajioka, J.W., Rosenthal, B.M., Sibley, L.D., 2009. Selection at a single locus leads to widespread expansion of Toxoplasma gondii lineages that are virulent in mice. PLoS Genet. 5, e1000404. Available from: https://doi.org/10.1371/journal.pgen.1000404. Khan, A., Dubey, J.P., Su, C., Ajioka, J.W., Rosenthal, B.M., Sibley, L.D., 2011a. Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America. Int. J. Parasitol. 41, 645
655. Available from: https://doi.org/10.1016/j. ijpara.2011.01.005. Khan, A., Miller, N., Roos, D.S., Dubey, J.P., Ajzenberg, D., Darde´, M.L., et al., 2011b. A monomorphic haplotype of chromosome Ia is associated with widespread success in clonal and nonclonal populations of Toxoplasma gondii. mBio 2, e00228
e00211. Available from: https:// doi.org/10.1128/mBio.00228-11. Khan, A., Ajzenberg, D., Mercier, A., Demar, M., Simon, S., Darde´, M.L., et al., 2014. Geographic separation of domestic and wild strains of Toxoplasma gondii in French Guiana correlates with a monomorphic version of chromosome1a. PLoS Negl. Trop. Dis. 8, e3182. Available from: https://doi.org/10.1371/journal. pntd.0003182. Kieffer, F., Rigourd, V., Ikounga, P., Bessieres, B., Magny, J.-F., Thulliez, P., 2011. Disseminated congenital Toxoplasma infection with a type II strain. Pediatr. Infect. Dis. J. 30, 813
815. Available from: https://doi. org/10.1097/INF.0b013e31821b8dfe. Kissinger, J.C., Gajria, B., Li, L., Paulsen, I.T., Roos, D.S., 2003. ToxoDB: accessing the Toxoplasma gondii genome. Nucleic Acids Res. 31, 234
236. Klun, I., Uzelac, A., Villena, I., Mercier, A., Bobi´c, B., Nikoli´c, A., et al., 2017. The first isolation and molecular characterization of Toxoplasma gondii from horses in Serbia. Parasit. Vectors 10, 167. Available from: https:// doi.org/10.1186/s13071-017-2104-x. Kong, J.-T., Grigg, M.E., Uyetake, L., Parmley, S., Boothroyd, J.C., 2003. Serotyping of Toxoplasma gondii infections in humans using synthetic peptides. J. Infect. Dis. 187, 1484
1495. Available from: https://doi.org/ 10.1086/374647. Lehmann, T., Blackstone, C.R., Parmley, S.F., Remington, J. S., Dubey, J.P., 2000. Strain typing of Toxoplasma gondii: comparison of antigen-coding and housekeeping genes. J. Parasitol. 86, 960
971.

111

Lehmann, T., Graham, D.H., Dahl, E., Sreekumar, C., Launer, F., Corn, J.L., et al., 2003. Transmission dynamics of Toxoplasma gondii on a pig farm. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 3, 135
141. Lehmann, T., Graham, D.H., Dahl, E.R., Bahia-Oliveira, L. M.G., Gennari, S.M., Dubey, J.P., 2004. Variation in the structure of Toxoplasma gondii and the roles of selfing, drift, and epistatic selection in maintaining linkage disequilibria. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 4, 107
114. Available from: https://doi.org/10.1016/j.meegid.2004.01.007. Lehmann, T., Marcet, P.L., Graham, D.H., Dahl, E.R., Dubey, J.P., 2006. Globalization and the population structure of Toxoplasma gondii. Proc. Natl. Acad. Sci. U. S.A. 103, 11423
11428. Available from: https://doi. org/10.1073/pnas.0601438103. Liesenfeld, O., 2002. Oral infection of C57BL/6 mice with Toxoplasma gondii: a new model of inflammatory bowel disease? J. Infect. Dis. 185, S96
S101. Lilue, J., Mu¨ller, U.B., Steinfeldt, T., Howard, J.C., 2013. Reciprocal virulence and resistance polymorphism in the relationship between Toxoplasma gondii and the house mouse. eLife 2, e01298. Available from: https:// doi.org/10.7554/eLife.01298. Lorenzi, H., Khan, A., Behnke, M.S., Namasivayam, S., Swapna, L.S., Hadjithomas, M., et al., 2016. Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes. Nat. Commun. 7, 10147. Available from: https://doi.org/10.1038/ncomms10147. Luka´sˇ ova´, R., Kobe´dova´, K., Halajian, A., Ba´rtova´, E., Murat, J.-B., Rampedi, K.M., et al., 2018. Molecular detection of Toxoplasma gondii and Neospora caninum in birds from South Africa. Acta Trop. 178, 93
96. Available from: https://doi.org/10.1016/j. actatropica.2017.10.029. ˇ ´ kovska´, A., Sedla´k, K., Machaˇcova´, T., Ajzenberg, D., Za Ba´rtova´, E., 2016. Toxoplasma gondii and Neospora caninum in wild small mammals: seroprevalence, DNA detection and genotyping. Vet. Parasitol. 223, 88
90. Available from: https://doi.org/10.1016/j. vetpar.2016.04.018. Mack, D.G., Johnson, J.J., Roberts, F., Roberts, C.W., Estes, R.G., David, C., et al., 1999. HLA-class II genes modify outcome of Toxoplasma gondii infection. Int. J. Parasitol. 29, 1351
1358. Magaldi, C., Elkis, H., Pattoli, D., de Queiroz, J.C., Coscina, A.L., Ferreira, J.M., 1967. [Outbreak of toxoplasmosis in a Paulist seminary in Braganza (Sao Paulo state). Clinical, serological and epidemiological aspects]. Rev. Saude Publ. 1, 141
171 [Article in Portuguese].

Toxoplasma Gondii

112

3. Molecular epidemiology and population structure of Toxoplasma gondii

Magaldi, C., Elkis, H., Pattoli, D., Coscina, A.L., 1969. Epidemic of toxoplasmosis at a university in Sao-Josedos Campos, S.P. Brazil. 1. Clinical and serologic data. Rev. Latinoam. Microbiol. Parasitol. (Mex.) 11, 5
13. Maksimov, P., Zerweck, J., Maksimov, A., Hotop, A., Gross, U., Pleyer, U., et al., 2012a. Peptide microarray analysis of in silico-predicted epitopes for serological diagnosis of Toxoplasma gondii infection in humans. Clin. Vaccine Immunol. 19, 865
874. Available from: https://doi.org/10.1128/CVI.00119-12. Maksimov, P., Zerweck, J., Maksimov, A., Hotop, A., Gross, U., Spekker, K., et al., 2012b. Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptidemicroarray. PLoS One 7, e34212. Available from: https://doi.org/10.1371/journal.pone.0034212. Markovi´c, M., Ivovi´c, V., Stajner, T., Djoki´c, V., Klun, I., Bobi´c, B., et al., 2014. Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia. Comp. Immunol. Microbiol. Infect. Dis. 37, 173
179. Available from: https://doi.org/10.1016/j. cimid.2014.03.001. McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981
2009). Clin. Infect. Dis. 54, 1595
1605. Available from: https://doi.org/10.1093/cid/cis258. Meireles, L.R., Ekman, C.C., Andrade Jr., H.F., Luna, E.J., 2015. Human toxoplasmosis outbreaks and the agent infecting form. findings from a systematic review. Rev. Inst. Med. Trop. Sao Paulo 57, 369
376. Mercier, A., 2010. Approche e´cologique, e´pide´miologique et ge´ne´tique de la biodiversite´ de Toxoplasma gondii en zone tropicale humide: exemples du Gabon et de la Guyane Franc¸aise. Limoges Univ. Mercier, A., Ajzenberg, D., Devillard, S., Demar, M.P., de Thoisy, B., Bonnabau, H., et al., 2011. Human impact on genetic diversity of Toxoplasma gondii: example of the anthropized environment from French Guiana. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 11, 1378
1387. Available from: https://doi.org/ 10.1016/j.meegid.2011.05.003. Messaritakis, I., Detsika, M., Koliou, M., Sifakis, S., Antoniou, M., 2008. Prevalent genotypes of Toxoplasma gondii in pregnant women and patients from Crete and Cyprus. Am. J. Trop. Med. Hyg. 79, 205
209. Miller, M.A., Grigg, M.E., Kreuder, C., James, E.R., Melli, A.C., Crosbie, P.R., et al., 2004. An unusual genotype of Toxoplasma gondii is common in California sea otters (Enhydra lutris nereis) and is a cause of mortality. Int. J. Parasitol. 34, 275
284.

Miller, M.A., Conrad, P., James, E.R., Packham, A., ToyChoutka, S., Murray, M.J., et al., 2008a. Transplacental toxoplasmosis in a wild southern sea otter (Enhydra lutris nereis). Vet. Parasitol. 153, 12
18. Available from: https://doi.org/10.1016/j.vetpar.2008.01.015. Miller, M.A., Miller, W.A., Conrad, P.A., James, E.R., Melli, A.C., Leutenegger, C.M., et al., 2008b. Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters. Int. J. Parasitol. 38, 1319
1328. Available from: https://doi.org/10.1016/j.ijpara.2008.02.005. Minot, S., Melo, M.B., Li, F., Lu, D., Niedelman, W., Levine, S. S., et al., 2012. Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity. Proc. Natl. Acad. Sci. U.S.A. 109, 13458
13463. Available from: https://doi.org/10.1073/pnas.1117047109. Miotto, O., Amato, R., Ashley, E.A., MacInnis, B., AlmagroGarcia, J., Amaratunga, C., et al., 2015. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat. Genet. 47, 226
234. More´, G., Maksimov, P., Pardini, L., Herrmann, D.C., Bacigalupe, D., Maksimov, A., et al., 2012. Toxoplasma gondii infection in sentinel and free-range chickens from Argentina. Vet. Parasitol. 184, 116
121. Available from: https://doi.org/10.1016/j.vetpar.2011.09.012. Morisset, S., Peyron, F., Lobry, J.R., Garweg, J., Ferrandiz, J., Musset, K., et al., 2008. Serotyping of Toxoplasma gondii: striking homogeneous pattern between symptomatic and asymptomatic infections within Europe and South America. Microbes Infect. 10, 742
747. Available from: https://doi.org/10.1016/j.micinf.2008.04.001. Morrison, D.A., 2005. How old are the extant lineages of Toxoplasma gondii? Parassitologia 47, 205
214. Morrison, D.A., Bornstein, S., Thebo, P., Wernery, U., Kinne, J., Mattsson, J.G., 2004. The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa). Int. J. Parasitol. 34, 501
514. Available from: https://doi.org/10.1016/j.ijpara.2003.11.006. Murillo-Leo´n, M., Mu¨ller, U.B., Zimmermann, I., Singh, S., Widdershooven, P., Campos, C., et al., 2019. Molecular mechanism for the control of virulent Toxoplasma gondii infections in wild-derived mice. Nat. Commun. 10, 1233. Available from: https://doi.org/10.1038/s41467019-09200-2. Niedelman, W., Gold, D.A., Rosowski, E.E., Sprokholt, J.K., Lim, D., Farid Arenas, A., et al., 2012. The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferongamma response. PLoS Pathog. 8, e1002784. Available from: https://doi.org/10.1371/journal.ppat.1002784.

Toxoplasma Gondii

References

Nowakowska, D., Colo´n, I., Remington, J.S., Grigg, M., Golab, E., Wilczynski, J., et al., 2006. Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological testing of samples from infants in Poland diagnosed with congenital toxoplasmosis. J. Clin. Microbiol. 44, 1382
1389. Available from: https://doi. org/10.1128/JCM.44.4.1382-1389.2006. Nunura, J., Va´squez, T., Endo, S., Salazar, D., Rodriguez, A., Pereyra, S., et al., 2010. Disseminated toxoplasmosis in an immunocompetent patient from Peruvian Amazon. Rev. Inst. Med. Trop. Sao Paulo 52, 107
110. O’Brien, S.J., Johnson, W.E., 2007. The evolution of cats. Genomic paw prints in the DNA of the world’s wild cats have clarified the cat family tree and uncovered several remarkable migrations in their past. Sci. Am. 297, 68
75. O’Brien, S.J., Johnson, W., Driscoll, C., Pontius, J., PeconSlattery, J., Menotti-Raymond, M., 2008. State of cat genomics. Trends Genet. TIG 24, 268
279. Available from: https://doi.org/10.1016/j.tig.2008.03.004. Orjuela-Sa´nchez, P., Karunaweera, N.D., da Silva-Nunes, M., da Silva, N.S., Scopel, K.K.G., Gonc¸alves, R.M., et al., 2010. Single-nucleotide polymorphism, linkage disequilibrium and geographic structure in the malaria parasite Plasmodium vivax: prospects for genome-wide association studies. BMC Genet. 11, 65. Available from: https://doi.org/10.1186/1471-2156-11-65. Palanisamy, M., Madhavan, B., Balasundaram, M.B., Andavar, R., Venkatapathy, N., 2006. Outbreak of ocular toxoplasmosis in Coimbatore, India. Indian J. Ophthalmol. 54, 129
131. Pan, S., Thompson, R.C.A., Grigg, M.E., Sundar, N., Smith, A., Lymbery, A.J., 2012. Western Australian marsupials are multiply infected with genetically diverse strains of Toxoplasma gondii. PLoS One 7, e45147. Available from: https://doi.org/10.1371/journal.pone.0045147. Parameswaran, N., Thompson, R.C.A., Sundar, N., Pan, S., Johnson, M., Smith, N.C., et al., 2010. Non-archetypal Type II-like and atypical strains of Toxoplasma gondii infecting marsupials of Australia. Int. J. Parasitol. 40, 635
640. Available from: https://doi.org/10.1016/j. ijpara.2010.02.008. Pardini, L., Bernstein, M., Carral, L.A., Kaufer, F.J., Dellarupe, A., Gos, M.L., et al., 2019. Congenital human toxoplasmosis caused by non-clonal Toxoplasma gondii genotypes in Argentina. Parasitol. Int. 68, 48
52. Available from: https://doi.org/10.1016/j. parint.2018.10.002. Peixoto, L., Chen, F., Harb, O.S., Davis, P.H., Beiting, D.P., Brownback, C.S., Ouloguem, D., Roos, DS., 2010. Integrative genomic approaches highlight a family of

113

parasite-specific kinases that regulate host responses. Cell Host Microbe 8, 208
218. Available from: https:// doi.org/10.1016/j.chom.2010.07.004. Pena, H.F.J., Gennari, S.M., Dubey, J.P., Su, C., 2008. Population structure and mouse-virulence of Toxoplasma gondii in Brazil. Int. J. Parasitol. 38, 561
569. Available from: https://doi.org/10.1016/j.ijpara.2007. 09.004. Pfaff, A.W., de-la-Torre, A., Rochet, E., Brunet, J., Sabou, M., Sauer, A., et al., 2014. New clinical and experimental insights into Old World and neotropical ocular toxoplasmosis. Int. J. Parasitol. 44, 99
107. Available from: https://doi.org/10.1016/j.ijpara.2013.09.007. Pfefferkorn, E.R., Pfefferkorn, L.C., Colby, E.D., 1977. Development of gametes and oocysts in cats fed cysts derived from cloned trophozoites of Toxoplasma gondii. J. Parasitol. 63, 158
159. Pomares, C., Ajzenberg, D., Bornard, L., Bernardin, G., Hasseine, L., Darde, M.-L., et al., 2011. Toxoplasmosis and horse meat, France. Emerg. Infect. Dis. 17, 1327
1328. Available from: https://doi.org/10.3201/ eid1707.101642. Pomares, C., Devillard, S., Holmes, T.H., Olariu, T.R., Press, C.J., Ramirez, R., et al., 2018. Genetic characterization of Toxoplasma gondii DNA samples isolated from humans living in North America: an unexpected high prevalence of atypical genotypes. J. Infect. Dis. 218, 1783
1791. Available from: https://doi.org/10.1093/ infdis/jiy375. Prestrud, K.W., Asbakk, K., Fuglei, E., Mørk, T., Stien, A., Ropstad, E., et al., 2007. Serosurvey for Toxoplasma gondii in arctic foxes and possible sources of infection in the high Arctic of Svalbard. Vet. Parasitol. 150, 6
12. Available from: https://doi.org/10.1016/j.vetpar.2007.09.006. Prestrud, K.W., Asbakk, K., Mørk, T., Fuglei, E., Tryland, M., Su, C., 2008. Direct high-resolution genotyping of Toxoplasma gondii in arctic foxes (Vulpes lagopus) in the remote arctic Svalbard archipelago reveals widespread clonal Type II lineage. Vet. Parasitol. 158, 121
128. Available from: https://doi.org/10.1016/j. vetpar.2008.08.020. Rajendran, C., Su, C., Dubey, J.P., 2012. Molecular genotyping of Toxoplasma gondii from Central and South America revealed high diversity within and between populations. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 12, 359
368. Available from: https://doi.org/10.1016/j.meegid.2011.12.010. Reese, M.L., Zeiner, G.M., Saeij, J.P., Boothroyd, J.C., Boyle, J.P., 2011. Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence. Proc. Natl. Acad. Sci. U.S.A. 108, 9625
9630.

Toxoplasma Gondii

114

3. Molecular epidemiology and population structure of Toxoplasma gondii

Reid, A.J., Vermont, S.J., Cotton, J.A., Harris, D., HillCawthorne, G.A., Ko¨nen-Waisman, S., et al., 2012. Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: coccidia differing in host range and transmission strategy. PLoS Pathog. 8, e1002567. Available from: https://doi.org/ 10.1371/journal.ppat.1002567. Richomme, C., Aubert, D., Gilot-Fromont, E., Ajzenberg, D., Mercier, A., Ducrot, C., et al., 2009. Genetic characterization of Toxoplasma gondii from wild boar (Sus scrofa) in France. Vet. Parasitol. 164, 296
300. Available from: https://doi.org/10.1016/j.vetpar.2009.06.014. Rosowski, E.E., Lu, D., Julien, L., Rodda, L., Gaiser, R.A., Jensen, K.D., et al., 2011. Strain-specific activation of the NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208, 195
212. Saad, S., Delbarre, M., Saad, R., Berguiga, M., Benisty, D., Marechal, M., et al., 2018. Failure of systemic oral doxycycline in preventing ocular toxoplasmic retinochoroiditis in French military personnel. J. R. Army Med. Corps 164, 122
123. Available from: https://doi.org/10.1136/ jramc-2017-000854. Sacks, J.J., Roberto, R.R., Brooks, N.F., 1982. Toxoplasmosis infection associated with raw goat’s milk. JAMA 248, 1728
1732. Saeij, J.P., Boyle, J.P., Grigg, M.E., Arrizabalaga, G., Boothroyd, J.C., 2005. Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains. Infect. Immun. 73, 695
702. Saeij, J.P.J., Boyle, J.P., Coller, S., Taylor, S., Sibley, L.D., Brooke-Powell, E.T., et al., 2006. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314, 1780
1783. Santana, S.S., Gebrim, L.C., Carvalho, F.R., Barros, H.S., Barros, P.C., Pajuaba, A.C., et al., 2015. CCp5A protein from Toxoplasma gondii as a serological marker of oocyst-driven infections in humans and domestic animals. Front. Microbiol. 6, 1305. Santos, S.V., Pena, H.F.J., Talebi, M.G., Teixeira, R.H.F., Kanamura, C.T., Diaz-Delgado, J., et al., 2018. Fatal toxoplasmosis in a southern muriqui (Brachyteles arachnoides) from Sa˜o Paulo state, Brazil: pathological, immunohistochemical, and molecular characterization. J. Med. Primatol. 47, 124
127. Available from: https:// doi.org/10.1111/jmp.12326. Shobab, L., Pleyer, U., Johnsen, J., Metzner, S., James, E.R., Torun, N., et al., 2013. Toxoplasma serotype is associated with development of ocular toxoplasmosis. J. Infect. Dis. 208, 1520
1528. Available from: https://doi.org/ 10.1093/infdis/jit313.

Shwab, E.K., Zhu, X.-Q., Majumdar, D., Pena, H.F.J., Gennari, S.M., Dubey, J.P., et al., 2014. Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping. Parasitology 141, 453
461. Available from: https://doi.org/10.1017/ S0031182013001844. Shwab, E.K., Jiang, T., Pena, H.F.J., Gennari, S.M., Dubey, J. P., Su, C., 2016. The ROP18 and ROP5 gene allele types are highly predictive of virulence in mice across globally distributed strains of Toxoplasma gondii. Int. J. Parasitol. 46, 141
146. Available from: https://doi.org/ 10.1016/j.ijpara.2015.10.005. Shwab, E.K., Saraf, P., Zhu, X.-Q., Zhou, D.-H., McFerrin, B.M., Ajzenberg, D., et al., 2018. Human impact on the diversity and virulence of the ubiquitous zoonotic parasite Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A.115, E6956
E6963 Available from: https://doi.org/10.1073/ pnas.1722202115. Sibley, L.D., 2009. Development of forward genetics in Toxoplasma gondii. Int. J. Parasitol. 39, 915
924. Sibley, L.D., Ajioka, J.W., 2008. Population structure of Toxoplasma gondii: clonal expansion driven by infrequent recombination and selective sweeps. Annu. Rev. Microbiol. 62, 329
351. Available from: https://doi. org/10.1146/annurev.micro.62.081307.162925. Sibley, L.D., Boothroyd, J.C., 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 359, 82
85. Available from: https://doi.org/ 10.1038/359082a0. Silva, L.A., Andrade, R.O., Carneiro, A.C.A.V., Vitor, R.W. A., 2014. Overlapping Toxoplasma gondii genotypes circulating in domestic animals and humans in Southeastern Brazil. PLoS One 9, e90237. Available from: https://doi.org/10.1371/journal.pone.0090237. Silveira, C., Belfort Jr., R., Muccioli, C., Abreu, M.T., Martins, M.C., Victora, C., et al., 2001. A follow-up study of Toxoplasma gondii infection in southern Brazil. Am. J. Ophthalmol. 131, 351
354. Silveira, C., Vallochi, A.L., Rodrigues da Silva, U., Muccioli, C., Holland, G.N., Nussenblatt, R.B., et al., 2011. Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis. Br. J. Ophthalmol. 95, 396
400. Available from: https://doi. org/10.1136/bjo.2008.148205. Silveira, C., Muccioli, C., Holland, G.N., Jones, J.L., Yu, F., de Paulo, A., et al., 2015. Ocular involvement following an epidemic of Toxoplasma gondii infection in Santa Isabel do Ivaı´, Brazil. Am. J. Ophthalmol. 159, 1013
1021. e3. Available from: https://doi.org/ 10.1016/j.ajo.2015.02.017. Slany, M., Reslova, N., Babak, V., Lorencova, A., 2016. Molecular characterization of Toxoplasma gondii in pork

Toxoplasma Gondii

References

meat from different production systems in the Czech Republic. Int. J. Food Microbiol. 238, 252
255. Available from: https://doi.org/10.1016/j.ijfoodmicro. 2016.09.020. Soeˆ te, M., Fortier, B., Camus, D., Dubremetz, J.F., 1993. Toxoplasma gondii: kinetics of bradyzoitetachyzoite interconversion in vitro. Exp. Parasitol. 76, 259
264. Sousa, S., Ajzenberg, D., Vilanova, M., Costa, J., Darde, M. L., 2008. Use of GRA6-derived synthetic polymorphic peptides in an immunoenzymatic assay to serotype Toxoplasma gondii in human serum samples collected from three continents. Clin. Vaccine Immunol. 15, 1380
1386. Available from: https://doi.org/10.1128/ CVI.00186-08. Sreekumar, C., Graham, D.H., Dahl, E., Lehmann, T., Raman, M., Bhalerao, D.P., et al., 2003. Genotyping of Toxoplasma gondii isolates from chickens from India. Vet. Parasitol. 118, 187
194. Stagno, S., Dykes, A.C., Amos, C.S., Head, R.A., Juranek, D. D., Walls, K., 1980. An outbreak of toxoplasmosis linked to cats. Pediatrics 65, 706
712. Stajner, T., Vasiljevi´c, Z., Vuji´c, D., Markovi´c, M., Risti´c, G., Mi´ci´c, D., et al., 2013. Atypical strain of Toxoplasma gondii causing fatal reactivation after hematopoietic stem cell transplantion in a patient with an underlying immunological deficiency. J. Clin. Microbiol. 51, 2686
2690. Available from: https://doi.org/10.1128/ JCM.01077-13. Su, C., Evans, D., Cole, R.H., Kissinger, J.C., Ajioka, J.W., Sibley, L.D., 2003. Recent expansion of Toxoplasma through enhanced oral transmission. Science 299, 414
416. Available from: https://doi.org/10.1126/ science.1078035. Su, C., Zhang, X., Dubey, J.P., 2006. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. Int. J. Parasitol. 36, 841
848. Available from: https://doi.org/10.1016/j.ijpara.2006.03.003. Su, C., Shwab, E.K., Zhou, P., Zhu, X.Q., Dubey, J.P., 2010. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology 137, 1
11. Available from: https://doi. org/10.1017/S0031182009991065. Su, C., Khan, A., Zhou, P., Majumdar, D., Ajzenberg, D., Darde´, M.-L., et al., 2012. Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proc. Natl. Acad. Sci. U.S.A. 109, 5844
5849. Available from: https://doi.org/10.1073/pnas.1203190109.

115

Subauste, C.S., Ajzenberg, D., Kijlstra, A., 2011. Review of the series “Disease of the year 2011: toxoplasmosis” pathophysiology of toxoplasmosis. Ocul. Immunol. Inflamm. 19, 297
306. Available from: https://doi.org/ 10.3109/09273948.2010.605198. Sulzer, A.J., Franco, E.L., Takafuji, E., Benenson, M., Walls, K.W., Greenup, R.L., 1986. An oocyst-transmitted outbreak of toxoplasmosis: patterns of immunoglobulin G and M over one year. Am. J. Trop. Med. Hyg. 35, 290
296. Sundar, N., Cole, R.A., Thomas, N.J., Majumdar, D., Dubey, J.P., Su, C., 2008. Genetic diversity among sea otter isolates of Toxoplasma gondii. Vet. Parasitol. 151, 125
132. Tanabe, K., Mita, T., Jombart, T., Eriksson, A., Horibe, S., Palacpac, N., et al., 2010. Plasmodium falciparum accompanied the human expansion out of Africa. Curr. Biol. CB 20, 1283
1289. Available from: https://doi.org/ 10.1016/j.cub.2010.05.053. Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., et al., 2006. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314, 1776
1780. de-la-Torre, A., Sauer, A., Pfaff, A.W., Bourcier, T., Brunet, J., Speeg-Schatz, C., et al., 2013. Severe South American ocular toxoplasmosis is associated with decreased Ifnγ/Il-17a and increased Il-6/Il-13 intraocular levels. PLoS Negl. Trop. Dis. 7, e2541. Available from: https:// doi.org/10.1371/journal.pntd.0002541. Terry, R.S., Smith, J.E., Duncanson, P., Hide, G., 2001. MGE-PCR: a novel approach to the analysis of Toxoplasma gondii strain differentiation using mobile genetic elements. Int. J. Parasitol. 31, 155
161. Teutsch, S.M., Juranek, D.D., Sulzer, A., Dubey, J.P., Sikes, R.K., 1979. Epidemic toxoplasmosis associated with infected cats. N. Engl. J. Med. 300, 695
699. Tibayrenc, M., Ayala, F.J., 2002. The clonal theory of parasitic protozoa: 12 years on. Trends Parasitol. 18, 405
410. Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., McDermott, D.F., et al., 2012. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443
2454. Available from: https://doi.org/10.1056/NEJMoa1200690. Vaudaux, J.D., Muccioli, C., James, E.R., Silveira, C., Magargal, S.L., Jung, C., et al., 2010. Identification of an atypical strain of Toxoplasma gondii as the cause of a waterborne outbreak of toxoplasmosis in Santa Isabel do Ivai, Brazil. J. Infect. Dis. 202, 1226
1233. Available from: https://doi.org/10.1086/656397.

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116

3. Molecular epidemiology and population structure of Toxoplasma gondii

Verma, S.K., Ajzenberg, D., Rivera-Sanchez, A., Su, C., Dubey, J.P., 2015. Genetic characterization of Toxoplasma gondii isolates from Portugal, Austria and Israel reveals higher genetic variability within the type II lineage. Parasitology 142, 948
957. Available from: https://doi. org/10.1017/S0031182015000050. Vielmo, A., Pena, H.F.J., Panziera, W., Bianchi, R.M., De Lorenzo, C., Oliveira, S., et al., 2019. Outbreak of toxoplasmosis in a flock of domestic chickens (Gallus gallus domesticus) and guinea fowl (Numida meleagris). Parasitol. Res. 118, 991
997. Available from: https:// doi.org/10.1007/s00436-019-06233-w. Vilares, A., Gargate´, M.J., Ferreira, I., Martins, S., Ju´lio, C., Waap, H., et al., 2014. Isolation and molecular characterization of Toxoplasma gondii isolated from pigeons and stray cats in Lisbon, Portugal. Vet. Parasitol. 205, 506
511. Available from: https://doi.org/10.1016/j. vetpar.2014.08.006. Vitaliano, S.N., Soares, H.S., Minervino, A.H.H., Santos, A. L.Q., Werther, K., Marvulo, M.F.V., et al., 2014. Genetic characterization of Toxoplasma gondii from Brazilian wildlife revealed abundant new genotypes. Int. J. Parasitol. Parasites Wildl. 3, 276
283. Available from: https://doi.org/10.1016/j.ijppaw.2014.09.003. Volkman, S.K., Neafsey, D.E., Schaffner, S.F., Park, D.J., Wirth, D.F., 2012. Harnessing genomics and genome biology to understand malaria biology. Nat. Rev. Genet. 13, 315
328. Available from: https://doi.org/10.1038/nrg3187. Wang, L., Chen, H., Liu, D., Huo, X., Gao, J., Song, X., et al., 2013. Genotypes and mouse virulence of Toxoplasma gondii isolates from animals and humans in China. PLoS One 8, e53483. Available from: https://doi. org/10.1371/journal.pone.0053483. Weedall, G.D., Conway, D.J., 2010. Detecting signatures of balancing selection to identify targets of anti-parasite immunity. Trends Parasitol. 26, 363
369. Available from: https://doi.org/10.1016/j.pt.2010.04.002.

Wendte, J.M., Miller, M.A., Lambourn, D.M., Magargal, S. L., Jessup, D.A., Grigg, M.E., 2010. Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii. PLoS Genet. 6, e1001261. Available from: https://doi.org/10.1371/journal.pgen.1001261. Wendte, J.M., Gibson, A.K., Grigg, M.E., 2011. Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife. Vet. Parasitol. 182, 96
111. Available from: https://doi.org/10.1016/j. vetpar.2011.07.018. White, M.W., Radke, J.R., Radke, J.B., 2014. Toxoplasma development—turn the switch on or off? Cell. Microbiol. 16, 466
472. Available from: https://doi. org/10.1111/cmi.12267. Williams, R.H., Morley, E.K., Hughes, J.M., Duncanson, P., Terry, R.S., Smith, J.E., et al., 2005. High levels of congenital transmission of Toxoplasma gondii in longitudinal and cross-sectional studies on sheep farms provides evidence of vertical transmission in ovine hosts. Parasitology 130, 301
307. Witola, W.H., Mui, E., Hargrave, A., Liu, S., Hypolite, M., Montpetit, A., et al., 2011. NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells. Infect. Immun. 79, 756
766. Available from: https://doi.org/10.1128/IAI.00898-10. Yera, H., Ajzenberg, D., Lesle, F., Eyrolle-Guignot, D., Besnard, M., Baud, A., et al., 2014. New description of Toxoplasma gondii genotypes from French Polynesia. Acta Trop. 134, 10
12. Available from: https://doi. org/10.1016/j.actatropica.2014.02.011. Zhang, J., Khan, A., Kennard, A., Grigg, M.E., Parkinson, J., 2017. PopNet: a Markov clustering approach to study population genetic structure. Mol. Biol. Evol. 34, 1799
1811. Available from: https://doi.org/10.1093/ molbev/msx110.

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C H A P T E R

4 Human Toxoplasma infection Rima McLeod1, William Cohen1, Samantha Dovgin1, Lauren Finkelstein1 and Kenneth M. Boyer2 1

The University of Chicago, Chicago, IL, United States 2Rush University Medical Center, Chicago, IL, United States

4.1 Clinical manifestations and course 4.1.1 Introduction and history Toxoplasmosis refers to the disease caused by Toxoplasma gondii (Remington et al., 2011; Delair et al., 2011; McLeod et al., 2012; Dubey et al., 2012c). As reviewed in Chapter 1, The history and life cycle of Toxoplasma gondii, the first record of a human case ascribed to infection with T. gondii was Janku’s report of a child with hydrocephalus in 1923 (Janku, 1923). In 1939 Wolf et al. (1939) described a case of toxoplasmic encephalitis. Systemic symptoms of infection with T. gondii were first reported in adults in 1940 (Pinkerton and Weinman, 1940). Sabin reported the first case of encephalitis due to T. gondii in infants (Sabin, 1941) and, with Feldman, developed a serologic test, the Sabin Feldman dye test, to diagnose infection (Sabin and Feldman, 1948). Lymphadenopathy was recognized as a symptom in older children and adults by Siim (1951) and Gard and Magnusson (1951). Encephalitis due to T. gondii in immune-compromised patients was first

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00004-9

reported from patients with Hodgkin’s disease during immunosuppressive treatment (Flament-Durand et al., 1967) and shortly after by Vietzke et al. (1968). Hogan recognized T. gondii as a cause of ocular infection, retinitis, and scarring (Hogan et al., 1957) and O’Connor and Perkins confirmed this could be effectively treated with pyrimethamine and sulfadiazine (O’Connor, 1974; Perkins et al., 1956). Several authors and groups described different aspects of congenital infections noting symptomatic congenital infections at birth in persons living in Europe and North America and/or late, progressive neurological and ophthalmologic manifestations in untreated children, even in those with subclinical infections at birth, and improved outcomes with prompt diagnosis and initiation of treatment prenatally as well as during infancy (Frenkel and Friedlander, 1951; Eichenwald, 1957, 1960; Couvreur and Desmonts, 1962, 1964; Kimball et al., 1971; Saxon et al., 1973; Alford et al., 1974; Thalhammer, 1975; Couvreur et al., 1976; Remington and Desmonts, 1976; Stagno et al., 1977; Desmonts and Remington, 1980;

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Wilson et al., 1980a,b; Aspo¨ck and Pollak, 1982; Desmonts, 1982; Koppe and Kloosterman, 1982; Couvreur et al., 1955, 1979, 1984; Desmonts and Couvreur, 1979, 1984; McLeod et al., 1985, 1992, 2000, 2006a, 2009, 2012; Moncada and Montoya, 2012; Morin et al., 2012; Koppe et al., 1986; Daffos et al., 1988; Hohlfeld et al., 1989, 1994; Forestier, 1991; McAuley et al., 1994; Swisher et al., 1994; Roizen et al., 1995, 2006; Mets et al., 1996; Patel et al., 1996; Thulliez, 2001a; Beghetto et al., 2003; Romand et al., 2004; Boyer et al., 2005, 2011; Kieffer et al., 2008; Phan et al., 2008a,b; Weiss and Dubey, 2009; Cortina-Borja et al., 2010; Noble et al., 2010; Villena et al., 2010; Peyron et al., 2011; Remington et al., 2011; Ajzenberg, 2012; Andrade et al., 2012; Dubey et al., 2012b; Olariu et al., 2011; Wallon et al., 2013). Manifestations of congenital infection may be prevented or reduced and there is clear benefit from early treatment as demonstrated by several large studies (see Section 4.3). Also, such treatment appears to be effective for infections with parasites with differing genotypes in the United States (McLeod et al., 2012). T. gondii can cause neurological and ophthalmologic disease when acquired postnatally in those without apparent immune compromise, as well as for patients with lymphomas and other immune compromise (Townsend et al., 1975; Couvreur and Thulliez, 1996). This postnatal infection has since been recognized as the most common infectious cause of retinitis and uveitis in the world (Silveira et al., 1988; Glasner et al., 1992; Kortbeek et al., 2009; Delair et al., 2011). Acute acquired toxoplasmosis causing a mononucleosis-like illness during postnatally acquired infection in persons without known immune compromise was reported by Remington et al. (1962). T. gondii infections causing destruction and/or inflammation of the heart, pericardium, skeletal muscle, lung, skin (dermatomyositis), as well as brain and eye, were reported subsequently (Palma et al., 1984; Pollock, 1979; Montoya et al., 1997;

Cunningham, 1982; Paspalaki et al., 2001; Prado et al., 1978; Greenlee et al., 1975; McCabe et al., 1987; Montoya and Remington, 2008). Clinicians in Brazil (Silveira et al., 1988, 2001, 2002; Glasner et al., 1992; Carme et al., 2002, 2009; de Amorim Garcia et al., 2004; Lehmann et al., 2006; Gilbert et al., 2008; Vasconcelos-Santos et al., 2009; VasconcelosSantos and Queiroz Andrade, 2011; Vasconcelos-Santos, 2012; Vaudaux et al., 2010; Ajzenberg, 2012; Andrade et al., 2012; reviewed in Dubey et al., 2012c; Lavinsky et al., 2012; Ore´fice et al., 2012) and in tropical areas such as Panama and regions with Amazon tributaries (Darde et al., 1998; Carme et al., 2002, 2009; Demar et al., 2007, 2012) noted severe, frequent, and recurrent eye and other substantial disease manifestations, including pneumonias requiring respiratory support, and death, with acute acquired toxoplasmosis (Demar et al., 2007, 2012). It appears that the genotype of the infecting strain of T. gondii might sometimes in humans contribute significantly to differences in virulence of parasites as it does in other animals (see Chapter 3: Molecular epidemiology and population structure of Toxoplasma gondii, for a discussion of genotypes) (Sibley and Boothroyd, 1992; Grigg et al., 2001; Vallochi et al., 2005; Lehmann et al., 2006; Darde, 2008; Gilbert et al., 2008; Ajzenberg, 2012; Demar et al., 2012, reviewed in Dubey et al., 2012c; McLeod et al., 2012). T. gondii infections transmitted into seronegative heart transplant recipients by hearts from donors with latent T. gondii infection, in one case presenting as a brain abscess, was reported in 1979 (Ryning et al., 1979; McLeod et al., 1979). It later was confirmed in several series of cases that a heart from a seropositive donor transplanted into a seronegative recipient could lead to reactivation of latent T. gondii infection in the donor heart and present significant risk of life-threatening disease for the recipient. Pyrimethamine prophylaxis now effectively prevents disease in such patients.

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Cases with disseminated or organ damaging toxoplasmosis with each type of solid organ, bone marrow, and stem cell transplantation, and with many different kinds of immunosuppressive treatments, were noted in subsequent years (Remington, 1974; Ryning et al., 1979; Conley et al., 1981; Luft et al., 1983; Wreghitt et al., 1992, 1995; Singer et al., 1993; Slavin et al., 1994; Bretagne et al., 1995; Aubert et al., 1996; Renoult et al., 1997; Sing et al., 1999; Martino et al., 2000, 2005; Abgrall et al., 2001; Montoya et al., 2001, 2010; Botterel et al., 2002; Mele et al., 2002; Lassoued et al., 2007; Matsuo et al., 2007; Derouin et al., 2008; Bautista et al., 2012; Bories et al., 2012; Busemann et al., 2012; Caselli et al., 2012; Ferna`ndez-Sabe´ et al., 2012; Osthoff et al., 2012; Strabelli et al., 2012; Vaughan and Wenzel, 2012). One of the earliest documented cases of toxoplasmic encephalitis in persons with AIDS occurred in a homosexual man in the late 1970s, who lived in Chicago but had traveled to Haiti. He also developed Pneumocystis pneumonia and tuberculosis and his HIV infection was recognized later. He was one of the first patients in the US AIDS epidemic (Levin et al., 1983), before HIV was identified as a cause of the concomitant opportunistic infections in AIDS. It soon became clear that toxoplasmic encephalitis was the most common of the central nervous system (CNS) infections that were presenting manifestations during the HIV epidemic, particularly in areas of higher seroprevalence of T. gondii infection (Luft et al., 1983; Post et al., 1983; Luft and Remington, 1984, 1992; Levy et al., 1986, 1988, 1990; Haverkos et al., 1987; Kovacs, 1995; Leport et al., 1996; Abgrall et al., 2001; Furco et al., 2008; Arslan et al., 2012; Basavaprabhu et al., 2012; GuevaraSilva et al., 2012; Kim et al., 2012). Carefully designed and implemented prenatal serologic screening programs to diagnose primary T. gondii infections acquired in gestation were mandated by law in France in 1972 (Desmonts and Couvreur, 1974a,b; Daffos

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et al., 1988). This was combined with an approach for diagnosis and treatment of the infection in utero, including diminishing intervals to each month for obtaining serum samples for gestational screening, and continuous treatment with pyrimethamine and sulfadiazine with leucovorin when congenital infection was likely. There has been an almost complete eradication of severe symptomatic congenital toxoplasmosis in France since the implementation of these programs (Kieffer et al., 2008; Cortina-Borja et al., 2010; McLeod et al., 2009; Peyron et al., 2011; Stillwaggon et al., 2011; Wallon et al., 2013). Serotype II predominance in France was documented, indicating that the analyses of outcomes applied to serotype II infections (Ajzenberg, 2012; Darde, 2008; Peyron et al., 2011). An alternative approach was developed in Austria (Aspo¨ck and Pollak, 1982) and Germany (Hotop et al., 2012), where any acutely infected pregnant woman receives pyrimethamine, sulfadiazine, and leucovorin beginning at the 18th week of gestation or beginning after that time if the infection is acquired later in gestation. A European group, EMSCOT, analyzed pooled data over time from many countries that had different approaches (Dunn et al., 1999; Naessens et al., 1999; Gilbert et al., 1999). There is an improvement of neurological findings at birth with shorter intervals between diagnosis and treatment in gestation (Cortina-Borja et al., 2010; SYROCOT, 2007). The EMSCOT SYROCOT studies included evaluations that were combined from centers in Europe. There were considerable variations in their standard of care, diagnostic measures, treatments, and timing of implementation of treatment. In the analyses, when data from these differing centers were pooled, these varied methods did not allow the investigators to identify significant effects on neurological signs and symptoms when durations from diagnosis to initiation of varied treatments were greater than 4 weeks or to identify effectiveness of treatment in reducing

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retinal disease. Effectiveness of treatment in preventing subsequent retinal disease could be identified, however, when data from the more uniform individual centers were analyzed separately (Kieffer et al., 2008). Studies of additional modifications to try to identify whether alternative, abbreviated treatment durations might be effective, but less costly, are currently ongoing in France. Feldman, Eichenwald, Stagno, Alford, Wilson, Remington, the National Collaborative Chicago-Based, Congenital Toxoplasmosis Study (NCCCTS), and the Palo Alto Research Foundation, United States serology reference laboratory group have, in various publications, emphasized how severe congenital infection can be at birth without prenatal screening and subsequent treatment. This is often the case in regions without screening programs, such as in North America (Feldman, 1953; Eichenwald, 1957, 1960; Alford et al., 1974; Stagno et al., 1977; Wilson et al., 1980a,b; McLeod et al., 1985, 1992, 2000, 2006a, 2012, 2013a,b; McAuley et al., 1994; Montoya and Remington, 2008; Olariu et al., 2011; Remington et al., 2011; Moncada and Montoya, 2012). The mixture of parasite genetic serotypes during this time in the United States was defined by the NCCCTS with the Grigg group at the National Institutes of Health (McLeod et al., 2012). These investigators demonstrated that, in the United States, congenital toxoplasmosis is not caused predominantly by serotype II parasites as in Europe; only approximately a third of those infected in the North American NCCCTS have type II parasites. The oocyst life cycle stages were discovered in cat intestine contemporaneously by Hutchison and Frenkel (Hutchison et al., 1970; Frenkel et al., 1970; reviewed in Dubey, 1998). Proteins specific to the oocyst and sporozoite life cycle stages have been identified (Kasper and Ware, 1989; Hill et al., 2011). The frequency of unrecognized exposure to oocysts in mothers of congenitally infected infants

(referred to the NCCCTS) was studied by Hill et al. (2011) and Boyer et al. (2011). Using a serologic assay, to detect antibody to a sporozoite protein present only for less than 8 months after acquisition, family clusters and recreational riding stable, work, and waterborne epidemics were identified that were caused by unrecognized exposure to oocysts in North America. This demonstrated that education alone about avoiding risk factors in the United States, although it may be helpful in limiting infections, is not likely to be sufficient to eliminate this infection (Gollub et al., 2008; Boyer et al., 2011). A mathematical model and algorithm to predict the cost and benefit of gestational serologic screening, diagnoses, and prenatal and postnatal treatment has been developed (Stillwaggon et al., 2011) and, when applied to data from the United States, suggested that prenatal screening and in utero treatment would be cost effective (Stillwaggon et al., 2011), while another study estimated the healthcare costs of this disease (Collier et al., 2012). A newborn screening program for T. gondii infections has documented occult infections with brain and eye damage in infants born in Massachusetts and New Hampshire (Guerina et al., 1994). A small number of obstetrical practices in the United States have implemented screening and treatment for primary acquisition of T. gondii infection during gestation. The development of serologic and molecular tests for diagnosis involved many groups (Sabin and Feldman, 1948; Araujo et al., 1971; Desmonts and Couvreur, 1974a,b; Walls et al., 1977; Balsari et al., 1980; Desmonts and Remington, 1980; Naot et al., 1981a,b; Naot and Remington, 1980; Remington et al., 1985; Thulliez et al., 1986, 1989; Hedman et al., 1989, 1993; Dannemann et al., 1990; Grover et al., 1990; Stepick-Biek et al., 1990; Wong et al., 1993; Petithory et al., 1996; Pinon et al., 1996; Liesenfeld et al., 1997, 2001a,b; Montoya, 2002; Montoya et al., 2002; Romand et al., 2004; Press et al., 2005; Bricker-Hildalgo et al., 2007;

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Montoya and Remington, 2008; Remington et al., 2011; L’Ollivier et al., 2012; Pinto et al., 2012; Villard et al., 2012). This has occurred through the latter half of the previous century and continues to the present. These advances in serologic methods defined and changed the way in which physicians diagnose the various syndromes, with varying manifestations, caused by this infection (Remington et al., 1962, 2011; Greenlee et al., 1975; Prado et al., 1978; Pollock, 1979; Cunningham, 1982; Palma et al., 1984; McCabe et al., 1987; Montoya et al., 1997; Paspalaki et al., 2001; Binquet et al., 2003; Wallon et al., 2004; Garweg et al., 2005; Kodjikian et al., 2006; Montoya et al., 2010; Delair et al., 2011; Bories et al., 2012; Brownback et al., 2012; Burrowes et al., 2012; Karanis et al., 2013; McLeod et al., 2012; Toporovski et al., 2012). These serologic tests facilitated a large number and variety of seroepidemiologic studies (e.g., Desmonts et al., 1965a,b; Ambroise-Thomas et al., 1966; Benenson et al., 1982; Luft and Remington, 1984; Papoz et al., 1986; Cook et al., 2000; Jones et al., 2001, 2007, 2009; Jones and Dubey, 2012; Jones and Roberts, 2012; Alvarado-Esquivel et al., 2002, 2011; Bahia-Oliverira et al., 2003; Lindsay et al., 2003; Conrad et al., 2005; Wainwright et al., 2007; Aptouramani et al., 2012; Garabedian et al., 2012; Hill and Dubey, 2013; Mosti et al., 2012). Definition of differences in parasite genetics and serotyping for a few of these differences have been developed (Sibley and Boothroyd, 1992; Kong et al., 2003; Lehmann et al., 2006; Darde, 2008; Dubey et al., 2012a; Maksimov et al., 2012; Minot et al., 2012; Carneiro et al., 2013). Pyrimethamine (with folinic acid) and sulfadiazine can treat the infection synergistically in vitro and in animal models (Eyles and Coleman, 1955) and was effective in treating human retinochoroiditis and uveitis including in placebo-controlled studies (Perkins et al., 1956; Garin et al., 1985; Mets et al., 1996; Brezin et al., 2003; Holland et al., 2008; Delair et al.,

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2011). Folinic acid was found to rescue mammalian bone marrow from antifolate toxicity without affecting the inhibitory effect of pyrimethamine on the parasite in vitro at the concentrations achieved with current treatment regimens (Frenkel and Hitchings, 1957; McLeod et al., 1990). It was also found that pyrimethamine (with folinic acid) and sulfadiazine can effectively treat the infection in immunecompromised persons, including transplant recipients, persons with AIDS, and those with other immune suppression (Weiss et al., 1988; LePort et al., 1996; Delair et al., 2011; Ryning et al., 1979; Ruskin and Remington, 1976). In pregnant women, in an algorithm that included spiramycin in some cases and pyrimethamine and sulfadiazine in other cases, treatment appeared to block transmission to the fetus and attenuated or eliminated disease at birth (Desmonts and Couvreur, 1974a,b; Aspo¨ck and Pollak, 1982; Couvreur et al., 1988; Hohlfeld et al., 1989; Brezin et al., 2003; McLeod et al., 2006a, 2009, 2012; Kieffer et al., 2008; Cortina-Borja et al., 2010; Wallon et al., 2013; Peyron et al., 2011; Hotop et al., 2012). In infants, treatment has been shown to attenuate or eliminate signs of infection and illness (Couvreur and Desmonts, 1962; McAuley et al., 1994; McLeod et al., 2006a, 2012). Further, it was demonstrated that antimicrobial prophylaxis against recurrence of retinal disease (e.g., with TMP SMX; Silveira et al., 2002) is effective. Alternative medicines for sulfadiazine hypersensitive persons (McLeod et al., 2006b) also have been identified. A study identifying clinical findings across human lifetimes combined with characterization of parasite and host genetics is being carried out. This is proving to be a powerful tool for understanding pathogenesis and consequences of this infection and the diseases it causes. This analysis is providing fundamental insights into pathogenic mechanisms for humans (Mack et al., 1999; Jamieson et al., 2008, 2009, 2010; Lees et al., 2010; Peixoto et al., 2010; Tan et al., 2010; Cong et al., 2011a,b, 2012; Witola et al.,

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2011; Be´la et al., 2012; McLeod et al., 2012; Dutra et al., 2013). Experiments of nature in which mutations have occurred in human genes, or where diseases profoundly modify a specific immune function, also are informative, for example, toxoplasmosis in the setting of disruption of the function of CD4 T cells, CD40 ligands, or TNF-α (Karanovic et al., 2019). T. gondii infection is very common, present in 1/3 to 1/2 of humans in the world. There is regional variation in prevalence and recognition of this infection. Two examples come from France and the United States (Lykins et al., 2018a,b; El Bissati et al., 2018; Fig. 4.1A F). Other countries such as Brazil and Colombia have very high prevalence with variability between regions (Dubey et al., 2012b; Go´mezMarin et al., 2011). This infection may be asymptomatic, have presented symptoms that range from mild to severe, or possibly have symptoms not attributed to the infection. The possibility that there may be neurobehavioral consequences from chronic infection in some persons has been proposed, with findings associated with infection in animal models, transcriptome studies with neuronal cells, and with seroprevalence association studies providing a foundation raising questions about causality (Ferguson et al., 1991; Brown et al., 2005; Hermes et al., 2008; Niebuhr et al., 2008; Bech-Nielsen, 2012; Goodwin et al., 2012; Guenter et al., 2012; Horacek et al., 2012; Hamdani et al., 2013; Holub et al., 2013). The hypothesis is that treatment of underlying toxoplasmosis might provide adjunctive or even definitive therapy for these neurobehaˇ ´ vioral diseases (Torres et al., 1993; Kankova et al., 2012; Kaushik et al., 2012; Lester, 2012; Pearce et al., 2012; Petersen et al., 2012; Torrey et al., 2012; Xiao et al., 2012a,b; Flegr, 2013). The fact that the parasite resides in the brain across lifetimes has raised the possibility that it might even shape language and cultures (Lafferty, 2006; Gajewski et al., 2014). Cause and effect have not been proven for humans for any

of these associations. The well-recognized, proven, treatable, and longstanding problems that congenital and ocular infection cause, and disease in immune-compromised persons that impact on quality of life, cause deaths, morbidity, and costs for care (Stillwaggon et al., 2011; Prusa et al., 2017) provide reason enough to develop definitive, better treatments and preventive measures. These include effective application of prenatal serologic screening and treatment programs, vaccines and treatments to prevent infection and/or sequelae, which are challenges for the future.

4.1.2 Postnatally acquired infection in children and adults 4.1.2.1 Adults and older children with primary, acute acquired Toxoplasma gondii infection T. gondii can be acquired by inadvertent and unrecognized ingestion of meat that has not been cooked to “well done,” which contains encysted T. gondii (Dubey and Beattie, 1998; Dubey et al., 2012b; Fig. 4.1G). Desmonts et al. (1965a,b) documented that infections from undercooked meat in an orphanage in France could lead to infection. Ingestion of shellfish was also defined as a risk factor in the United States in epidemiologic surveys (Jones et al., 2009). Mussels can become infected when they filter seawater contaminated with oocysts from surrounding rivers. Ingestion of contaminated shellfish caused mortality in sea otters (Conrad et al., 2005; Miller et al., 2002; Shapiro et al., 2016). Other marine animals have also been infected (Sharma et al., 2018; Barbieri et al., 2016). Identification of T. gondii DNA has demonstrated contamination of water and fish residing in that water. This is a particular problem in Colombia (Bahia-Oliveira et al., 2017; Go´mez-Marin et al., 2011; Fig. 4.1). Infection can also be acquired from oocysts contaminating freshwater or food, including fruits and

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FIGURE 4.1

(Continued).

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FIGURE 4.1 Acute acquired Toxoplasma gondii infection. (A) Regional variation of T. gondii infection in the United States (El Bissati et al., 2018). (B) Regional variation of T. gondii infection in France (El Bissati et al., 2018). (C) Large data analysis based on CPT codes in patients within the United States: age of persons at first diagnosis by gender (Lykins et al., 2016). (D) Large data analysis disease manifestations (conjunctivitis was reported even though toxoplasma does not cause

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L

vegetables, and likely from inhaling oocyst contaminated dust/soil (Teutsch et al., 1979; Benenson et al., 1982; Isaac-Renton et al., 1998; Wainwright et al., 2007; Shapiro et al., 2010; Boyer et al., 2011; Hill and Dubey, 2013; Lass et al., 2012; Gallas-Lindemann et al., 2013; Dubey et al., 2012c; Fig. 4.1H). Dog fur can be contaminated with oocysts and serve as a transport vector (Frenkel and Parker, 1996) as can insects such as cockroaches and flies. Oocysts can persist in water and warm, moist soil for up to a year (Fig. 4.1H J), even one oocyst is infectious, and an acutely infected member of the cat family can excrete up to 5

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million oocysts during a period of 2 weeks after acquisition (Dubey and Beattie, 1998; Lindsay et al., 2003; Fritz et al., 2012b; Aguirre et al., 2019; Trivin˜o-Valencia et al., 2016). T. gondii can cause isolated family (Luft and Remington, 1984; Contopoulos-ioannidis et al., 2015), educational facility (dos Santos et al., 2010; Morris et al., 2012), or work (Magaldi et al., 1969) clusters of infection as well as epidemics due to multiple exposures to the same infectious source (Isaac-Renton et al., 1998; Bowie et al., 1997; Burnett et al., 1998; Dubey and Beattie, 1998; Jones et al., 2009; Jones and Dubey, 2012; Vaudaux et al., 2010; Karanis

conjunctivitis) (Lykins et al., 2016). (E) Prevalence by locale. Dark green is highest prevalence (Lykins et al., 2016). (F) Coded by medicines used to treat infection (Lykins et al., 2016). (G) Life cycle of T. gondii and modes of acquisition. (H) The definitive hosts of T. gondii are members of the Felidae family (Gerhold and Jessup, 2012). Color photos depict recently documented modes of oocyst acquisition, including contaminated mussels, drinking water and schoolyards in Brazil, the fur of dogs, unwashed fruits and vegetables, and goat cheese. 2D gel depicts a protein specific to T. gondii sporozoites (shown in red and indicated by white arrow). Table shows limits of correlation between presence of antibodies to oocysts and self-determined risk factors for oocyst exposure during pregnancy of mothers in NCCCTS, 1981 98. (I) Relationship of transmission patterns to water and soil (Go´mez-Marin et al., 2011). (J) Detection of T. gondii DNA in public drinking water (treated water) (Bahia-Oliveira et al., 2017). (K) Symptoms and signs in persons with acute acquired toxoplasmosis from a riding stable epidemic and in uninfected controls in Atlanta, GA, United States. Oocysts were the source of infection in the riding stable epidemic. Light green bars represent persons with acute acquired toxoplasmosis. Dark green bars represent uninfected controls. Percentages refer to persons with acute acquired toxoplasmosis displaying a particular symptom or sign of infection. Persons with acute acquired toxoplasmosis are more likely to exhibit symptoms, such as fever, lymphadenopathy, headache, myalgia, stiff neck, anorexia, arthralgia, rash, confusion, and hepatitis, than those uninfected. Most persons (95%) with acute acquired toxoplasmosis in this epidemic displayed a sign or symptom of infection, but only a few (7%) were correctly diagnosed by their physician. (L) Histopathology of lymph node from a person with acute toxoplasmosis. Black arrow indicates cluster of epithelioid histiocytes. Source: (H) Hill, D., Coss, C., Dubey, J.P., et al., 2011. Identification of a sporozoite-specific antigen from Toxoplasma gondii. J. Parasitol. 97 (2), 328 337 with permission; Boyer, K., Hill, D., Mui, E., et al., 2011. Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America. Clin. Infect. Dis. 53 (11), 1081 with permission. Image from Remington, J.S., McLeod, R. Toxoplasmosis. In: Braude, A.l. (Ed.) International Textbook of Medicine, Medical Microbiology and Infectious Disease, vol. II. WB Saunders, Philadelphia. 1981; 1818, with permission. Photographs in H from Estuary: ,http://www.travelmezze.com/images/estuary-abel-tasman-national-park-new-zealand1.JPG’.; Otter: ,http://carinbondar.com/wp-content/uploads/2010/11/seaotter2.jpg.; Water supply: ,http://www.sswm.info/sites/default/files/toolbox/DOLMAN%20and%20LUNDQUIST%202008%20Roof%20Water%20Harvesting %20for%20a%20low%20Impact%20Water%20Supply%20Brazil.jpg.; Schoolyard: ,http://thisismyhappiness.com/wp-content/ uploads/2011/10/Playing-soccer-with-local-kids.jpg.; Dog: ,http://images.cpcache.com/merchandise/514_400x400_NoPeel.jpg? region 5 name:FrontCentre,id:70011053,w:16.; Blueberries: ,http://www.thehomesteadgarden.com/wp-content/uploads/2012/10/ Blueberry_Cluster.jpg.; Goat: ,http://www.formaggiokitchen.com/shop/images/goat%20sampler2.jpg.; Goat cheese: ,http://upload. wikimedia.org/wikipedia/commons/a/a1/Domestic_goat_feeding_on_capeweed.jpg.. 2D gel from Hill, D., Coss, C., Dubey, J.P., et al., 2011. Identification of a sporozoite-specific antigen from Toxoplasma gondii. J. Parasitol. 97 (2), 328 337. Data in table from Teutsch, S.M., Juranek, D.D., Sulzer, A., Dubey, J.P., Sikes, R.K., 1979. Epidemic toxoplasmosis associated with infected cats. N. Engl. J. Med. 300, 695 699, with permission. (K) Data from Teutsch, S.M., Juranek, D.D., Sulzer, A., Dubey, J.P., Sikes, R.K., 1979. Epidemic toxoplasmosis associated with infected cats. N. Engl. J. Med. 300, 695 699, with permission. (L) Image from Dorfman, R.F., Remington, J.S., 1973. Value of lymph-node biopsy in the diagnosis of acute toxoplasmosis. N. Engl. J. Med. 289, 878 881, with permission.

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et al., 2013; Lass et al., 2012). In the NCCCTS the source of infection was rarely identified by mothers of congenitally infected children (Boyer et al., 2011). In the NCCCTS study 78% of the mothers whose serum was tested in the perinatal period had an antibody to an 11 kDa T. gondii sporozoite protein, present exclusively in oocyst acquired infections in the 6 8 months after infection is acquired (Boyer et al., 2011; Hill et al., 2011). Frequency and severity of disease manifestation may vary by geographic location or be associated with parasite genetics (McLeod et al., 2012). Dose, route of infection, and host genetics likely influence manifestations and outcomes for people (Fig. 4.1). Acute acquired infection in older children and adults in Europe is believed to most often be asymptomatic. Desmonts reported that B10% of mothers of congenitally infected children reported symptoms or signs. In an epidemic in the United States in Atlanta, Georgia (Teutsch et al., 1979; Dubey et al., 1981), 95% of the 37 infected persons reported symptoms including headache, myalgias, and fever, but only 7% were correctly diagnosed by their primary physician (Fig. 4.1K). These associated symptoms of headache and myalgia in patients with lymphadenopathy were emphasized in the United States (Wong et al., 2012) and Brazilian cases (reviewed in Dubey et al., 2012c). In the United States, illness and small peripheral retinal scars appear to be relatively common (B10%) in mothers of infants with T. gondii infection (Noble et al., 2010). Symptoms are, in part, associated with parasite serotype (McLeod et al., 2012). Non type II infections more often, but not exclusively, were more symptomatic. Pregnancy may also result in recrudescence of retinal disease (Andrade et al., 2012). Toxoplasmic lymphadenopathy can involve any nodes but most often involves cervical nodes which present as nontender, discrete, firm, not matted or fixed to contiguous tissues and do not suppurate. Mesenteric nodes that cause pain with fever may be mistaken for

appendicitis and a pectoral node may be mistaken for breast cancer. Lymphadenopathy may be either self-limited or associated with prolonged symptoms, such as fatigue, for more than a year. Toxoplasmosis may account for 5% of clinically significant lymphadenopathy cases (Dorfman and Remington, 1973; Luft and Remington, 1984; McCabe et al., 1987; Montoya et al., 2010; Natella et al., 2012; Rollins-Raval et al., 2012). Fever, malaise, night sweats, myalgias, sore throat, hepatosplenomegaly or small numbers of circulating, atypical lymphocytes may accompany the adenopathy. Adenopathy and symptoms usually resolve within a few months to a year. The nodes have characteristic, distinctive histopathology with epithelioid histiocytes and monocytoid cells that encroach on and blur the margins of germinal centers (Dorfman and Remington, 1973; Luft and Remington, 1984; Natella et al., 2012; RollinsRaval et al., 2012; Fig. 4.1L). Plasma dendritic cells are identified in the nodes but are not in tightly packed clusters as in certain malignancies (Rollins-Raval et al., 2012). Fine-needle aspirate of lymph nodes has been used to establish the diagnosis of toxoplasmic lymphadenopathy (Natella et al., 2012). Occasionally, acquired infection may be associated with myositis or a sepsis-like syndrome (Demar et al., 2012). Townsend et al. (1975) described toxoplasmic encephalitis, meningoencephalitis, and meningitis with 50% of the cases occurring in persons without known immune compromise. Polymyositis, dermatomyositis (Greenlee et al., 1975; Pollock, 1979; Palma et al., 1984), myocarditis, pericarditis, pancarditis (Prado et al., 1978; Cunningham, 1982; Montoya et al., 1997), pneumonia, mesenteric lymphadenopathy mimicking appendicitis, gastrointestinal symptoms (Schreiner and Liesenfeld, 2009), hepatocellular abnormalities, Guillain Barre´ syndrome, headaches, fever, and visual symptoms have all been reported with acute acquired infection (Remington et al., 1962). Couvreur and Thulliez (1996) described neurological and

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retinal disease in acute acquired infection. There is evidence in animal models that gastrointestinal involvement occurs in some instances (Schreiner and Liesenfeld, 2009; Benson et al., 2012; Mennechet et al., 2004). How frequently this infection causes these symptoms and whether they are dependent on inoculum size, life cycle stage, parasite strain, unrecognized immune deficiencies, and variability in immune responses of the person remain to be determined. Host genetics can influence toxoplasmic chorioretinitis (Jamieson et al., 2008, 2009; Dutra et al., 2013) and parasite strain, as defined by Kong et al. (2003), may be associated with eye disease in adults (Holland et al., 1996; Grigg et al., 2001). Symptoms have ranged from short and self-limited to severe symptoms with prolonged fever, chronic lymphadenopathy, fatigue and progressive, recurrent retinochoroiditis (Masur et al., 1978; Teutsch et al., 1979; Benenson et al., 1982; Luft and Remington, 1984; Silveira et al., 1988, 2001; Glasner et al., 1992; Couvreur and Thulliez, 1996; Bowie et al., 1997; Burnett et al., 1998; Delair et al., 2008; Montoya and Remington, 2008; Holland et al., 2008). Severe acute infection associated with interstitial pneumonia and death have been reported in epidemics in the Amazon region, and along its tributaries, including the Maroni River in Guyana (Carme et al., 2002, 2009; Demar et al., 2007, 2012). Other common symptoms included fever, weight loss, increased liver enzymes, lymphadenopathy, headache, rash, retinochoroiditis, myocarditis, myositis, and neurological disorders. Severe manifestations, such as pneumonia, in adults also have been described from Brazil (Leal et al., 2007; Dubey et al., 2012c). In some cases the severe clinical syndromes have been associated with infection with atypical T. gondii genotypes (Grigg et al., 2001; Vallochi et al., 2005; Khan et al., 2006; Vaudaux et al., 2010; Carneiro et al., 2013). Severe T. gondii infections reported

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from South America (Carme et al., 2002, 2009; Demar et al., 2007), with diffuse interstitial pneumonia as a key symptom, often required ventilator support and intensive care modalities and even then fatalities occurred in some cases. Severe retinal involvement also characterizes both acute acquired, chronic recurrent and congenital infections, particularly in Brazil, but also elsewhere (Silveira et al., 1988; Glasner et al., 1992; Burnett et al., 1998; Delair et al., 2008; Gilbert et al., 2008; Montoya and Remington, 2008; Vasconcelos-Santos and Queiroz Andrade, 2011; Brownback et al., 2012; Denes et al., 2013; Dubey et al., 2012c; Toporovski et al., 2012). 4.1.2.2 The special problem of primary infection during gestation Acute T. gondii infection in pregnant women does not differ from T. gondii infection in other immune-competent individuals. The infection may be asymptomatic, although cervical lymphadenopathy may occur, and chorioretinal lesions may develop. Due to an asymptomatic course, acute infection may go unnoticed, or symptoms may not be attributed to T. gondii infection. Thus the infection may be transmitted to the fetus without the pregnant woman realizing she is acutely infected. A program for serodiagnosis and treatment of primary infection during gestation and polymerase chain reaction (PCR) diagnosis of congenital toxoplasmosis in the fetus was developed in France and Belgium (Fig. 4.2). Treatment is used to prevent transmission to the fetus and to eliminate, or reduce, sequelae if transmission occurs. A separate approach has been developed and utilized in Germany and Austria (Hotop et al., 2012; Prusa et al., 2017; Wallon et al., 2013; El Bissati et al., 2018) and more recently adopted in some areas of France for infections acquired between 14 and 17 weeks gestation (Mandelbrot et al., 2018; Montoya, 2018; Peyron et al., 2019; Bobi´c et al., 2019).

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FIGURE 4.2 Primary Toxoplasma gondii infection during gestation: the French approach and data enable prenatal diagnosis of congenital infection. (A) Schematic diagram of screening algorithm for acquisition of primary Toxoplasma infection during gestation utilized in France and some US obstetrical practices. Ideally, screening would be initiated shortly before pregnancy, or even earlier in gestation than 12 weeks (i.e., 6 8 weeks gestation). (B) Top: Diagnosis of infants with

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There is now extensive literature attributing many behavioral and neurological diseases to T. gondii infections (Brown et al., 2005; Niebuhr et al., 2008; Bech-Nielsen, 2012; Fabiani et al., 2013; Goodwin et al., 2012; Guenter et al., 2012; Hamdani et al., 2013; Horacek et al., 2012; Holub et al., 2013). This grew from observations that chronic infection in rodents is associated with ongoing neurobehavioral, neuroimaging, histologic, dopaminergic, cytokine, and transcriptomic abnormalities in brain in both infected and contiguous cells (Ferguson et al., 1991; Webster et al., 2006; Hermes et al., 2008; Vyas and Sapolsky, 2010; Mitra et al., 2012). Differing animal species have varied manifestations of disease based on their genetics, for example, new and old world primates (Araujo et al., 1973) and various strains of mice. There is a literature that has ascribed causal associations between an increased seroprevalence of T. gondii with a wide variety of illnesses including seizures (Stommel et al., 2001), bipolar disease and schizophrenia (Webster et al., 2006, 2013; Henriquez et al.,

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2009; Torrey et al., 2012; Webster et al., 2013), motor vehicle accidents, slowed reaction times which become progressively worse with duration of infection, sex of offspring, curmudgeonly behavior in men and “sex-kitten” behavior in women (Flegr, 2013), suicide (Godwin, 2012; Okusaga and Postolache, 2012; Pedersen et al., 2012), memory loss, depression, gluten intolerance (Severance et al., 2012), and diminished verbal memory measured on RBANS test in young urban professionals (Torrey and Yolken, abstract, Toxoplasmosis International Meeting, The Netherlands, 2009), among others (Ferguson et al., 1991; Brown et al., 2005; Hermes et al., 2008; Niebuhr et al., 2008; Bech-Nielsen, 2012; Fabiani et al., 2013; Godwin, 2012; Goodwin et al., 2012; Guenter et al., 2012; Hamdani et al., 2013; Horacek et al., 2012; Holub et al., 2013). At present, there is no definitive proof that T. gondii causes or contributes significantly to any of these conditions, other than seizures, in humans. Proposed mechanisms include parasite production of dopamine, tyrosine hydroxylase, and the inflammatory immune response elicited by infection. Webster et al. (2013) have proposed how rat behavioral changes with a dopamine connection might be studied in

PCR in AF according to gestational age, in weeks, at maternal infection. Open bars indicate PCR sensitivity. Shaded bars indicate negative predictive values of PCR assay. 95% CI shown in parenthesis above bars. Middle: Percentage of infants undergoing amniocentesis and cases of congenital toxoplasmosis according to gestational age, in months, at maternal infection. Open bars indicate amniocentesis. Shaded bars indicate cases of congenital toxoplasmosis. Note first trimester sensitivity 5 76%. Bottom left: Correlation between concentration of Toxoplasma in AF and gestational age, in weeks, at maternal infection Shaded squares indicate severe signs of infection. Open circles indicate mild or no signs of infection. Higher concentrations of T. gondii in AF are more likely earlier in gestation and correspond to severe disease in infants. Lower concentrations of T. gondii are more likely later in gestation and correspond to mild or no disease in infants. n 5 sample size. Bottom right: Comparison of median parasite concentration in AF between infants with mild/no infection and infants with severe infection for maternal infections acquired before or after 20 weeks’ gestation. Open bars indicate infants with subclinical infection. Shaded bars represent infants with infectious sequelae. Severe infections in infants are more likely if maternal infection is acquired before 20 weeks’ gestation. AF, Amniotic fluid; CI, confidence interval. Source: Image from (A) McLeod, R., Lee, D., Boyer, K., 2013b in press. Diagnosis of Congenital Toxoplasmosis: A Practical Procedural Atlas, with permission; (B): McLeod, R., Lee, D., Boyer, K., 2013a in press. Toxoplasmosis in the Foetus and Newborn Infant; Romand, S., Chosson, M., Franck, J., et al., 2004. Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of foetal infection with Toxoplasma gondii. Am. J. Obstet. Gynecol. 190, 797 802; Wallon, M., Kieffer, F., Binquet, C., Thulliez, P., Garcia-Meric, P., Dureau, P., et al., 2011. Congenital toxoplasmosis: randomized comparison of strategies for retinochoroiditis prevention. Therapie 66 (6), 473 480, with permission.

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humans. If there is a cause-and-effect relationship between T. gondii infection and neurological and behavioral diseases, such as schizophrenia, bipolar disease, memory loss, and dementia, for some persons, it must be uncommon, at least in the first few decades after infection. This is because, in carefully studied cohorts of mothers and their congenitally infected children, there have been only rare persons with these neuropsychiatric or other diseases (McLeod, unpublished data). This indicates that if there is a cause-andeffect relationship between T. gondii infection and neurobehavioral disease, other cofactors must play a substantial role. Host genetics is one of those critical factors (Roberts et al., 2014; Karanovic et al., 2019; Ngoˆ et al., 2017). Other insights into pathogenesis, host and parasite genetics, and their interplay are also influential (see Section 4.5 and Chapter 25 "Innate Immunity to Toxoplasma gondii").

4.1.3 Congenital infection 4.1.3.1 The fetus, infant, and older child During the 1940s an understanding developed that acute acquired maternal infection resulted in congenital toxoplasmosis in the fetus and newborn infant. Holmdahl and Holmdahl (1955) found that 2 out of 23,260 children had clinical toxoplasmosis in a study performed from 1948 to 1951. In 1953 Feldman reported a series of 103 congenitally infected children. In this series, 99% had eye lesions, 63% had intracranial calcifications and 56% had psychomotor retardation (Feldman, 1953). Examples of some of these manifestations are shown in Figs. 4.3 4.5. These observations initiated an interest in congenital infection among scientists in Europe (Couvreur, 1955). A study from Austria reported frequent, similar symptoms in children with congenital toxoplasmosis (Aspo¨ck and Pollak, 1982). A study from France demonstrated that the seroprevalence

in pregnant women in Paris was 85%, with a relatively high risk of acquisition of Toxoplasma infection in seronegative women (Desmonts et al., 1965a, 1965b). The incidence of seropositivity among nonimmigrant women in Stockholm, Sweden ranged from 47.7% in 1957 to 21.1% in 1987 (Forsgren et al., 1991). Another group demonstrated that in pregnant women, seropositivity was 14% in Stockholm, Sweden and 26% in southern Sweden (Evenga˚rd et al., 2001). Seropositivity in Poland is nearly 60% and increases with age (Paul et al., 2000). Eichenwald (1960) described the presence of severe disease in a cohort of children referred to him in New York with a poor prognosis for vision, cognition, and motor function with seizures and hearing loss in two of four children who did not have symptoms at birth (Fig. 4.3A). The earlier work of Couvreur (1955) and Couvreur and Desmonts (1962, 1964) was followed by a larger study from France of 374 pregnancies (Desmonts and Couvreur, 1974a,b). Koppe 1982, 1986) and Stagno et al. (1977) described substantial ophthalmologic and neurological sequelae in untreated children with congenital toxoplasmosis, both when symptomatic and asymptomatic in the perinatal period (Desmonts and Couvreur, 1984; McLeod et al., 2009; Thulliez, 2001a; Remington et al., 2011). T. gondii in North America still often presents as a severe disease when diagnosed at birth in the absence of systematic screening programs (McLeod et al., 1990, 2006a, 2012; McAuley et al., 1994; Olariu et al., 2011). The severity of symptomatic infection as well as sequelae of infections thought to be mild or asymptomatic at birth was detailed by Eichenwald (1957). Wilson et al. (1980a) noted later neurological and ophthalmologic sequelae even for those thought to be asymptomatic at birth. Several studies have documented that prompt treatment can prevent transmission from mother to child and reduce clinical symptoms in children (Couvreur and Desmonts, 1962; Roux et al.,

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FIGURE 4.3 Toxoplasma gondii congenital infection causes disease when untreated. (A) Data from Eichenwald study of patient outcome at 4 years of age or older. n 5 sample size. (B) Data from Koppe study of visual outcome for children who were asymptomatic at birth, untreated or treated for less than a month, and evaluated at ages 6 and 20 years. Percentages are those children with retinal disease. There are negative outcomes for children with congenital toxoplasmosis who were untreated or treated for only 1 month. (C) Prevalence of various manifestations of Toxoplasma infection in children at or near birth referred to the NCCCTS (1981 2009). These include prematurity, r/o sepsis, skin rash, hepatomegaly, splenomegaly, microphthalmia, chorioretinitis, hydrocephalus, microcephalus, seizures, CNS calcifications, thrombocytopenia and anemia. (D) Images depicting manifestations of congenital toxoplasmosis. Manifestations include splenomegaly and hepatomegaly. Manifestations of the skin can include petechiae and/or blueberry muffin rash. The

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1976; Desmonts, 1982; Couvreur et al., 1984, 1993; Daffos et al., 1988; Hohlfeld et al., 1989, 1994; Forestier, 1991; Foulon et al., 1994, 1999; Gilbert et al., 2001; Thulliez, 2001a; Peyron et al., 2011; Berrebi et al., 2007; Kieffer et al., 2008; Montoya et al., 2010; Wallon et al., 2013). Congenital infection results when acute acquired T. gondii infection occurs in previously seronegative pregnant women. Women who are seropositive before conception, as a rule, do not transmit infection to the fetus. There have, however, been rare cases reported of congenital infection where the mother acquired infection 1 2 months before conception (e.g., a previously seropositive woman from Brazil but living in Switzerland transmitted to her fetus after a trip to Brazil during her pregnancy (Kodjikian et al., 2004) and a woman in Brazil with chronic infection and active retinal disease who transmitted to her fetus (Andrade et al., 2012)). Immunecompromised women (e.g., those treated with corticosteroids) may occasionally transmit a chronic infection during pregnancy resulting in congenital infection (Desmonts and Couvreur,

1984; Mitchell et al., 1990). Persons with HIV in the context of available retroviral treatment may, but in one series did not, transmit their chronic T. gondii infection to their fetus when pregnant (Dunn et al., 1996). The clinical manifestations of congenital toxoplasmosis (Fig. 4.3) vary depending on the trimester when the infection was acquired. Congenital infection of the fetus in women infected just before conception is extremely rare (Vogel et al., 1996; Remington et al., 2011) and even during the first few weeks of pregnancy, the maternal fetal transmission rate is only a small percentage. There is an inverse relationship between the rate of transmission and the severity of the infection (Desmonts and Couvreur; 1984; Remington et al., 2011; Wallon et al., 2013). Without treatment, infections acquired in the first trimester result in congenital infection in 10% 25% of fetuses (Couvreur and Desmonts, 1962, 1964; Stillwaggon et al., 2011). The rate of transmission rises to 30% 50% in those women infected during the second trimester and 60% 70% for those infected during the third trimester. These studies on risk and

infant depicted here has a blueberry muffin rash secondary to cytomegalovirus infection. In Toxoplasma infection, appearance of blueberry muffin rash appears similar. Manifestations involving the eye include chorioretinitis which can result in scarring. Involvement of the brain can manifest in hydrocephalus and/or calcifications. (E) Incidence of ophthalmologic manifestations of congenital toxoplasmosis. n, Sample size. (F) Incidence of central nervous system and retinal manifestations of congenital toxoplasmosis as documented by the New England Screening Program. CNS, Central nervous system. Source: (A) Image from McLeod, R., Kieffer, F., Sautter, M., Hosten, T., Pelloux, H., 2009. Why prevent, diagnose and treat congenital toxoplasmosis?. Mem. Inst. Oswaldo Cruz 104, 320 344. (B) Image from McLeod, R., Kieffer, F., Sautter, M., Hosten, T., Pelloux, H., 2009. Why prevent, diagnose and treat congenital toxoplasmosis?. Mem. Inst. Oswaldo Cruz 104, 320 344; based on data from Eichenwald, H.F., 1957. Congenital toxoplasmosis: a study of 150 cases. Am. J. Dis. Chld. 94, 411 412; Eichenwald, H.F., 1960. A study of congenital toxoplasmosis, with particular emphasis on clinical manifestations, sequelae and therapy. In: Siim, J. (Ed.), Human Toxoplasmosis, Munksgaard, Copenhagen, pp. 41 49; Koppe, J.G., Kloosterman, G.J., 1982. Congenital toxoplasmosis: long-term follow-up. Padiatr. Padol. 17, 171 179; Koppe, J.G., Loewer-Sieger, D.H., de Roever-Bonnet, H., 1986. Results of 20-year follow-up of congenital toxoplasmosis. Lancet 1 (8475), 254 256, with permission. (C) Image from McLeod, R., Lee, D., Boyer, K., 2013b in press. Diagnosis of Congenital Toxoplasmosis: A Practical Procedural Atlas, with permission. (D) Petechiae image from ,http://dermatology.about.com/library/blpetechiaephoto.htm. Blueberry muffin rash image from Mehta, V., Balachandran, C., Lonikar, V., 2008. Blueberry muffin baby: a pictorial differential diagnosis. Dermatol Online J. 14(2), 8. Fundus photographs from Phan, L., Kasza, K., Jalbrzikowski, J., et al., 2008a. Longitudinal study of new eye lesions in treated congenital toxoplasmosis. Ophthalmology 115, with permission. Hydrocephalus image from Swisher, C.N., Boyer, K., McLeod, R., The Toxoplasmosis Study Group, 1994. Congenital toxoplasmosis. Semin. Pediatr. Neurol., 4 25, with permission. Calcification images from Patel, D.V., Holfels, E.M., Vogel, N.P., et al., 1996. Resolution of intracranial calcifications in infants with treated congenital toxoplasmosis. Radiology 199 (2), 433 440 1996, with permission. (E) Data from Mets, M., Holfels, E., Boyer, K.M., et al., 1996. Eye manifestations of congenital toxoplasmosis. Am. J. Ophthalmol. 122, 309 324, with permission.

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FIGURE 4.4 Parasite serotype and congenital infection in the United States. (A) Allele-specific peptides. Top: The peptide sequence at the GRA6/7 locus determines parasite serotype II or NE-II (not exclusively II). Bottom: Summary of the NCCCTS. (B) NCCCTS patients with antibodies to GRA6/7 peptides to type II or type I/III parasites, or both, based on decade of birth. Right: Distribution of patients with serologic responses to parasite serotype II or NE-II. Left: Distribution of patients with serologic responses designated as serotype II, IIa, atypical, I/IIIa, or I/III. Persons with responses to both

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severity of transmission were done in areas with a predominance of type II strains and not in areas (e.g., the United States or South America) where other, differing genotypes predominate. Several studies have demonstrated that treatment of the pregnant woman with anti T. gondii medicines reduces the incidence and severity of manifestations of congenital infection (Desmonts and Couvreur, 1984; Thulliez, 1992, 2001a; Kieffer et al., 2008; McLeod et al., 2009, 2012; Cortina-Borja et al., 2010; Hotop et al., 2012; Remington et al., 2011; Stillwaggon et al., 2011; Wallon et al., 2013; Figs. 4.2 4.5). The clinical manifestations of congenital toxoplasmosis usually are most severe if infection is acquired before week 26 of gestation. In

these cases the retina and central nervous system are commonly affected with nonspecific signs, including retinochoroiditis, strabismus, blindness, epilepsy, psychomotor or mental retardation, encephalitis, microcephaly, intracranial calcification, hydrocephalus anemia, jaundice, rash, and petechiae due to thrombocytopenia (Remington et al., 2001, 2011; Fig. 4.3). Newborns infected in the third trimester may be asymptomatic at birth but, with careful examination, signs such as meningitis and retinitis are frequently noted at birth. Sequelae, such as chorioretinitis, often develop later in life without treatment (Koppe et al., 1982, 1986; Phan et al., 2008a,b; Kieffer et al., 2008; Wallon et al., 2010; Peyron et al., 2011). Desmonts and Couvreur (1984), Stagno et al. (1977), and The

serotype II and NE-II peptides are designated as IIa if the response was greater to serotype II, designated as I/IIIa if the response was greater to serotype I/III, or designated as atypical if the response was equal to serotypes II and I/III. Rx indicates the persons in the NCCCTS cohort who were diagnosed in the perinatal period. No Rx indicates persons in the NCCCTS cohort who missed being treated during the first year of life. All include Rx plus No Rx. P, P-Value. (C) Distribution of parasite serotypes. Top map shows distribution of parasite serotype in the United States by birthplace and United States region. Bottom map shows distribution of parasite serotypes by birthplace and climate. N, Sample size. (D) Prevalence of parasite serotype by country. Type II serotype toxoplasmic infections predominate in France. NE-II toxoplasmic infections predominate in Brazil. Serotypes of toxoplasmic infections are more varied in the United States. N, Sample size; P, P-value. (E) Associations between parasite serotype and demographics. Toxoplasmic infection with NE-II serotype associated with residing in rural locales, lower SES and Hispanic ethnicity. N, Sample size; P, P-value. (F) Associations between parasite serotype and treated/untreated cohorts. Serotypes are similarly distributed between treated and untreated cohorts. N, Sample size; P, P-value. (G) Associations between parasite serotype and prematurity. Toxoplasmic infection with NE-II serotype associated, but not exclusively, with prematurity. P, P-Value. (H) Associations between parasite serotype and disease severity at birth. Toxoplasmic infection with NE-II serotype is associated, but not exclusively, with severe disease at birth. NCCCTS, National Collaborative Chicago-Based, Congenital Toxoplasmosis Study; SES, socioeconomic status. Source: (B) Image from McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Toxoplasmosis Study Group. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981 2009). Clin. Infect. Dis. 54 (11), 1595 1605, with permission. (C) Image from McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Toxoplasmosis Study Group. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981 2009). Clin. Infect. Dis. 54 (11), 1595 1605, with permission. Pomares, C., Devillard, S., Holmes, T.H., Olariu, T. R., Press, C.J., Ramirez, R., et al. (2018). Genetic characterization of Toxoplasma gondii DNA samples isolated from humans living in North America: an unexpected high prevalence of atypical genotypes. J. Infect. Dis., 218(11), 1783 1791. https://doi.org/10.1093/ infdis/jiy375 (Pomares et al., 2018) identified similar diversity in parasite genotypes in the United States. (H) Images from McLeod, R., Lee, D., Boyer, K., 2013a in press. Toxoplasmosis in the Foetus and Newborn Infant; data from McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Toxoplasmosis Study Group. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981 2009). Clin. Infect. Dis. 54 (11), 1595 1605, with permission. Pattern of hydrocephalus may vary with serotype. Hutson, S.L., Wheeler, K.M., McLone, D., Frim, D., Penn, R., Swisher, C.N., et al., 2015. Patterns of hydrocephalus caused by congenital Toxoplasma gondii infection associate with parasite genetics. Clin. Infect. Dis., 61 (12), 1831 1834. https://doi.org/10.1093/cid/civ720 (Hutson et al., 2015); McLeod, R., Wheeler, K.M., Boyer, K., 2015. Reply to Wallon and Peyron. Clin. Infect. Dis., 62(6), 812 814. https://doi.org/10.1093/cid/civ1036 (McLeod et al., 2015); Wallon, M., Peyron, F., 2015. Effect of antenatal treatment on the severity of congenital toxoplasmosis. Clin. Infect. Dis., 62(6), 811 812. https://doi.org/ 10.1093/cid/civ1035 (Wallon and Peyron, 2015).

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FIGURE 4.5 Manifestations of ocular toxoplasmosis improve with treatment. (A) Fundus photographs of follow-up of vitritis. Left panel depicts severe vitritis. Middle panel depicts resolving vitritis with active lesion. Right panel depicts resolved vitritis and healed lesion. (B) Fundus photographs of follow-up of toxoplasmic lesion. Left image depicts active lesion. Right image depicts healed scar. (C) Fundus photographs of follow-up of toxoplasmic lesion. Left image depicts active retinal lesion (arrow). Right panel depicts completely resolved lesion within 1 month of initiating treatment. (D) Fundus photographs of follow-up of toxoplasmic lesion in an infant. Left panel, “near birth” depicts active vitritis. Right panel, “with ongoing treatment” depicts clearing of vitritis. (E) Ocular toxoplasmosis with submacular neovascular membrane. Fundus photographs shown in top row, (A D). Indocyanin green angiograph images of choroidal neovascular membrane with hemorrhage depicted in middle row, (A C). OCT images of resolution of choroiditis, depicted in middle row, with pyrimethamine, sulfadiazine, and intraocular injection of Lucentis shown in bottom row, (A D). (F) New eye lesions in children who had less than 8 to 8 weeks or more delay from diagnosis in utero to treatment. Left, Kaplan Meier plot shows the age at diagnosis of retinochoroiditis. (Solid line: delay of less than 4 weeks; dashed line: delay of 4 8 weeks; dotted line: delay of more than 8 weeks.) Right, Kaplan Meier plot estimates age at diagnosis of retinochoroiditis during the first 2 years of life among 300 infants. (G) Recurrent retinal disease and new lesions in children. Those who missed treatment in the first year of life (left panel) and those who were treated in the first year of life with pyrimethamine and sulfadiazine (right panel). Blue-shaded area represents confidence interval. OCT, Optical coherence tomography. Source: Images from (A D): Delair, E., Latkany, P., Noble, A.G., et al., 2011. Clinical manifestations of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 19 (2), 91 102, with permission; (E): Benevento, J.D., Jager, R.D., Noble, A.G., et al., 2008. Toxoplasmosisassociated neovascular lesions treated successfully with ranibizumab and antiparasitic therapy. Arch. Ophthalmol. 126, 1152 1156, with permission; (F): Kieffer, F., Wallon, M., Garcia, P., et al., 2008. Risk factors for retinochoroiditis during the first 2 years of life in infants with treated congenital toxoplasmosis. Pediatr. Infect. Dis. J., 27 32, with permission; (G): McLeod, R., Kieffer, F., Sautter, M., Hosten, T., Pelloux, H., 2009. Why prevent, diagnose and treat congenital toxoplasmosis?. Mem. Inst. Oswaldo Cruz 104, 320 344, with permission.

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FIGURE 4.5

4. Human Toxoplasma infection

(Continued).

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New England Screening Program (Guerina et al., 1994) found that up to 50% or more of those infants believed to have no signs at birth actually did have clinical findings due to their T. gondii infection when carefully examined in the perinatal period (Fig. 4.3). A recent European multicenter study reported that 19 (8%) out of 244 newborns with congenital toxoplasmosis had cerebral calcifications and that treatment within 4 weeks of prenatal diagnosis reduced the risk of neurological findings (adjusted odds ratio 0.28; CI 0.08 0.75) (Gras et al., 2005). The same study found chorioretinitis in 30 (12%) of 255 newborns with congenital toxoplasmosis; however, they reported that treatment did not reduce the risk of chorioretinitis (Gras et al., 2005). More recently, it has become clear that the more rapidly treatment is initiated, the less risk there is of eye disease due to T. gondii infection (EMSCOT, 2003; Kieffer et al., 2008). The EMSCOT SYROCOT studies included evaluations combined from centers in Europe that had variations in their standard of care diagnostic measures, treatments, and timing of implementation (Kieffer et al., 2008; SYROCOT, 2007). In the analyses of data from these differing centers that were pooled, effects on neurological signs and symptoms were not significant when durations from diagnosis to initiation of varied treatments were greater than 4 weeks. However, when data from individual centers were deconvoluted and reanalyzed separately by those centers in Paris and Lyon, beneficial effect in preventing eye disease was demonstrated (Kieffer et al., 2008). Similarly, a recent study from Lyon has demonstrated beneficial effect on prevention of infection and reduction of adverse sequelae of infection (Wallon et al., 2013). A shorter interval from diagnosis to initiation of treatment is reflected in serologic testing protocols and has resulted in the best outcomes (Kieffer et al., 2008; SYROCOT, 2007), where testing is done monthly starting at week 11 of gestation for seronegative pregnant women. Outcomes appear to improve

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with treatment in utero and postnatally (Thulliez, 2001a,b; Peyron et al., 2003, 2011; McLeod et al., 2006a, 2009, 2012; Kieffer et al., 2008; Cortina-Borja et al., 2010; Remington et al., 2011; Wallon et al., 2011). The genetic type of parasite is associated with manifestations of the infant at birth (McLeod et al., 2012), although not absolutely (Fig. 4.4). There appears to be a favorable response to treatment for both type II or non type II infections (McLeod et al., 2012; see Section 4.3). Congenital transmission from mother to fetus with chorioretinitis is more common in Brazil after primary infection in pregnancy, compared to Europe, and manifestations are more severe (Gilbert et al., 2008). In Minas Girais, 1 in B770 infants born has had congenital toxoplasmosis and half of these infants had active retinal disease at birth (Vasconcelos-Santos and Queiroz Andrade, 2011). This might reflect the different recombinant I/III genotypes of T. gondii that are prevalent in Minas Girais, Brazil, which is different from Europe and North America. About a third of the NCCCTS participants in North America has a serotype II parasitic infection. In animals in North America there are types I, II, III, IV (haplogroup X11) and other T. gondii serotypes, but type I and III parasites are not, for the most part, the same as those found in Brazil. 4.1.3.2 Congenital toxoplasmosis in different countries 4.1.3.2.1 France and Belgium

The pioneering work of Desmonts, Couvreur, Thulliez, Romand, Costa, Peyron, Wallon, Daffos, Foulon, Kieffer, McLeod, and many others (summarized above) has defined much of what we understand about congenital toxoplasmosis and how to prevent, diagnose, and treat it as well as the natural history of both treated and untreated congenital infection. This experience was derived predominantly from infections with type II T. gondii in Europe.

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The information about severity being greater and transmission less frequent in early gestation with decreasing severity later in gestation, but with higher transmission rates, is derived from data in these studies. Studies in these countries (many in collaboration with the Remington laboratory in the United States) defined the diagnostic approaches to this infection. The universal prenatal screening in France was instrumental in defining this disease and approaches to its treatment. Overall, this disease has changed from one with a poor prognosis to one in which the prognosis was for a favorable outcome (McAuley et al., 1994; Kieffer et al., 2008; Peyron et al., 2011; McLeod et al., 1992, 2000, 2006, 2009, 2012; Wallon et al., 2013). 4.1.3.2.2 Austria, Germany, The Netherlands, and Italy

Prenatal screening has also occurred in several other European countries. In Italy, there is serodiagnosis during gestation, treatment before and after birth throughout infancy and followup in a centralized manner, often in a program headed by Buffolano et al. (2013) in Naples. Koppe and Kloosterman (1982), in The Netherlands, described recurrent, new eye disease in almost all children by adolescence, even though they had no signs or symptoms of disease at birth. Aspo¨ck and Pollak (1982) described favorable outcomes for infants born to acutely infected mothers following the treatment of primary infections in pregnant women with pyrimethamine and sulfadiazine. Hotop et al. (2012) described the data of an approach used in Germany for treatment of T. gondii infection acquired during pregnancy. In this approach, spiramycin is administered until the 16th week of gestation, followed by at least 4 weeks of treatment of the pregnant woman with pyrimethamine, sulfadiazine, and leucovorin (folinic acid) (abbreviated PSL). This was independent of the timing of the primary infection during gestation. If infection of the fetus was confirmed by PCR or if fetal ultrasound

indicated severe symptoms (e.g., hydrocephalus or ventricular dilation), treatment with PSL was continued until and after delivery. In France, only spiramycin is given unless infection of the fetus is proven or if primary infection of the pregnant woman occurs after 21 weeks’ gestation. Hotop et al.’s (2012) retrospective analysis of 685 women who had a serologic profile consistent with primary infection in pregnancy and their children noted an overall transmission rate (4.8%) and rate of clinical manifestations in newborns (1.6%) that was lower than that reported in other countries. 4.1.3.2.3 United States

The NCCCTS was established in 1981 and performed a phase 1 study to determine early safety and efficacy of continued use of pyrimethamine and sulfadiazine throughout the first year of life and the influence of such treatment on later outcomes. A phase 2 randomized clinical trial followed with an additional observational study of historical untreated persons and those who were treated in the first year of life. These evaluations were performed in Chicago at prespecified time intervals (near birth, 1, 3.5, 5, 7.5, 10 years, and then continuing at 5-year intervals) for prespecified endpoints, evidence for toxicity as well as other findings. This phase 2 randomized trial uses a higher or lower dose of pyrimethamine [2 or 6 months of daily pyrimethamine, 1 mg/kg, followed by 1 mg/kg each Monday, Wednesday, Friday (MWF) for the remainder of the year of treatment] plus sulfadiazine and leucovorin begun at diagnosis near birth. Occasionally, there was also treatment of the fetus by treatment of the mother prenatally. This has allowed studies of pathogenesis, genetics and immune function in families, pharmacokinetics of medicines, outcomes as well as other studies (Sibley and Boothroyd, 1992; Silveira et al., 2002; Vallochi et al., 2005; Khan et al., 2006; Lehmann et al., 2006; Dubey et al., 2012c). This study defined parasite types in those

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participating in the NCCCTS in North America (II or non-II) (McLeod et al., 2012), the high frequency of infections with oocysts (Boyer et al., 2011), and demonstrated that outcomes with treatment are improved (McLeod et al., 2000, 2006a, 2009, 2012) with the best outcomes for those persons treated in utero. A cost benefit analysis suggested that prenatal screening and treatment would likely reduce morbidity and mortality and be cost beneficial in the United States (Stillwaggon et al., 2011). Practice guidelines regarding management of congenital toxoplasmosis and prenatal screening for the United States are being considered at present. 4.1.3.2.4 Brazil

The first case of what was later diagnosed as congenital toxoplasmosis was recognized in Brazil by Carlos Bastos Magarinos Torres in 1927 during an autopsy of a 2-day old infant. Dubey, Lagos, Jones, et al. summarized many aspects of T. gondii infections in Brazil (reviewed in Dubey et al., 2012c). Gilbert et al. (2008) described the clinical manifestations of congenital toxoplasmosis in a cohort in Brazil. In Brazil the state of Minas Gerais has very recently implemented a prenatal T. gondii screening program that will likely be using the German (Hotop et al., 2012) method for prenatal treatment. Studies surveying the seroprevalence of T. gondii in the general population of Brazil dating back to 1962 indicate that seroprevalence was, and remains, higher in Brazil than in the United States. Seroprevalence in children and pregnant women is one of the highest worldwide. Toxoplasmosis, especially congenital toxoplasmosis and eye disease, have been described as very prevalent with severe morbidity in Brazil. The variability of presentation of Brazilian T. gondii eye disease was first noted in 1992 when Glasner et al. observed a remarkably high incidence of retinal disease caused by toxoplasmosis in the Brazilian city of Erechim (Glasner et al., 1992). Congenital toxoplasmosis in the Brazilian state of Minas Gerais had a

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prevalence of 1 in 770 births with approximately 50% of infants having active eye disease at birth (Vasconcelos-Santos et al., 2009) and up to 35% having neurological findings (Dubey et al., 2012c). A similar prevalence of 1/1000 births or more has been documented in other regions of Brazil as well. There is also a remarkably high incidence of hearing loss, 40% in some series (Dubey et al., 2012c), as compared to treated congenital infection in the United States. Studies of the genotypes of T. gondii isolates has indicated that the predominant strains are different than those seen in Europe and North America (Sibley and Boothroyd, 1992; Silveira et al., 2001, 2002; Vallochi et al., 2005; Lehmann et al., 2006; Dubey et al., 2012c), and this may account for the difference in clinical presentations. The high incidence of toxoplasmosis in Brazil can partially be attributed to the high level of environmental contamination with oocysts and there have been large epidemics involving hundreds of people with substantial illness. For example, in one survey of 31 soil samples from different schoolyards, T. gondii was isolated from seven (23%) locations (dos Santos et al., 2010). Other epidemics have been documented to be due to contaminated drinking water [e.g., Santa Isabel do Ivai (Vaudaux et al., 2010)]. Another epidemic occurred among university students in Sa˜o-Jose´-dos Campos (Magaldi et al., 1969). In addition to congenital toxoplasmosis, postnatally acquired toxoplasmosis also poses a serious threat and causes severe ocular disease with recurrences of retinochoroiditis (Dubey et al., 2012c). An unusual finding, which appears to be idiosyncratic, was reported recently from Brazil (VasconcelosSantos, 2012). This group reported a chronically infected pregnant woman who transmitted T. gondii to her fetus. Whether this was due to previous exposure or reexposure to a more virulent strain of T. gondii endogenous to Brazil remains to be determined through genetic studies of both parasite and host.

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4.1.4 The special problem of ocular disease Toxoplasmic retinochoroiditis (see Chapter 5: Ocular disease due to Toxoplasma gondii) may be the result of congenitally or postnatally acquired infection (Figs. 4.5 and 4.6). Involvement of the eye occurs during the acute stage of infection and reactivation during the chronic stage of the infection may occur (Fig. 4.5; Delair et al., 2011). Appearance of eye lesions may be the same in both congenital and postnatally acquired infections. Symptoms include change in visual acuity, blurred vision, scotoma, pain, photophobia, and epiphora. There may be complete or partial loss of vision, increased intraocular pressure or glaucoma (Delair et al., 2011). There appears to be a predilection for involvement of the macula, especially the fovea, which results in impairment or a loss of visual acuity. Chorioretinitis due to T. gondii may be a relapsing disease due to reactivation of latent infection. In a study in Brazil (Holland et al., 1996), the relapses with acute, postnatally acquired infection occurred most frequently near the time of acquisition and diminished over time. This may be due to the existing genotype in the area although higher inoculation rates and infection from oocysts in the environment could be alternative explanations. Especially severe disease has been noted in the elderly. Reactivation of congenital toxoplasmosis appears to occur at school-entry age, adolescence, and other times of considerable stress (Fig. 4.5). During reactivation, bradyzoites in cysts apparently transform to tachyzoites that proliferate and cause active chorioretinal inflammation that is associated with vitritis causing the above symptoms. Impaired vision in adult patients may be due to congenital infection with T. gondii or postnatally acquired infection. One study in the United Kingdom found a lifetime risk of symptomatic T. gondii eye disease of approximately 2 per 10,000 and

a 100-fold higher risk in persons born in West Africa, living in the United Kingdom (Gilbert et al., 1999). T. gondii retinochoroiditis is more common in South America compared to North America and Europe, and differences in T. gondii genetics may be responsible (Khan et al., 2006; Vallochi et al., 2005). Eye disease due to T. gondii is highly prevalent in Brazil, and a recent study found a prevalence of retinochoroiditis in adults of 1.2% (de Amorim Garcia et al., 2004). In Erechim, Southern Brazil, 17.7% of the adult population has ocular toxoplasmosis (Glasner et al., 1992; Silveira et al., 2001). In some regions of Brazil, 80% of persons are infected, with 50% of those over the age of 50 years and 20% of the total numbers of persons having retinal disease that is recurrent and debilitating (Glasner et al., 1992; Silveira et al., 1988, 2001, 2002; Vasconcelos-Santos et al., 2009; Vasconcelos-Santos, 2012; Lavinsky et al., 2012; Ore´fice et al., 2012; Vaudaux et al., 2010; Carme et al., 2002, 2009; Gilbert et al., 2008; Lehmann et al., 2006; Demar et al., 2007; Andrade et al., 2012; Ajzenberg, 2012; reviewed in Dubey et al., 2012c; Darde et al., 1998; de Amorim Garcia et al., 2004). Ocular disease also appears to be quite severe with recurrences in persons in Colombia and Mexico (London et al., 2011). Retinal scars due to ocular toxoplasmosis were reported in 6% of adults in Quindio, Colombia (de-la-Torre et al., 2007). Separately, branch retinal artery occlusion attributed to toxoplasmosis was recently reported to have occurred in an adolescent (Chiang et al., 2012). One complication of T. gondii infection for which there is a specific treatment is choroidal neovascular membranes (CNVMs). An example of a CNVM is shown in fundus photographs and optical coherence tomography (OCT) images in Fig. 4.5E. Activity is associated with loss of visual acuity, edema, and hemorrhage. Pathogenesis appears to involve hypoxia induction factor including VegF because anti-VegF treatment results in rapid resolution of hemorrhages and other CNVM.

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FIGURE 4.6 Disease due to Toxoplasma gondii infection in immune-compromised persons can involve any organ, but frequently affects the eye and brain. (A) Fundus photograph of person with HIV/AIDS and toxoplasmosis. (B) Brain MRI of brain of person with HIV/AIDS and toxoplasmic encephalitis. Light gray area indicated by the white arrow is the only normal area of the brain in this person. (C) CT image of the brain of a seronegative person who received a heart transplant from an acutely infected donor. White arrows indicate abscesses. CT, Computed tomography; MRI, magnetic resonance image. Source: Image from (A) Roberts, F., McLeod, R., 1999. Pathogenesis of toxoplasmic retinochoroiditis. Parasitol Today 15 (2), 51 57. (B) Levin, M., McLeod, R., Young, Q., et al., 1983. Pneumocystis pneumonia: importance of gallium scan for early diagnosis and description of a new immunoperoxidase technique to demonstrate Pneumocystis carinii. Am. Rev. Respir. Dis. 128, 182 185, with permission. (C) Ryning, F.W., McLeod, R., Maddox, J.C., et al., 1979. Probable transmission of Toxoplasma gondii by organ transplantation. Ann. Intern. Med. 90, 47 49, with permission.

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4.1.5 Immune-compromised patients 4.1.5.1 HIV-infected patients The increased frequency of Toxoplasma encephalitis in patients with AIDS was reported soon after the start of the HIV epidemic (Levin et al., 1983; Roue et al., 1984; Enzensberger et al., 1985; Suzuki et al., 1988a), and Toxoplasma encephalitis was an important end-stage cause of death in HIV patients before the introduction of combination antiretroviral therapy (cART) in the United States and Europe (Luft et al., 1983, 1993; Basavaprabhu et al., 2012; Luft and Remington, 1984, 1992; Kim et al., 2012; Kovacs, 1995; Guevara-Silva et al., 2012; Leport et al., 1996; Abgrall et al., 2001; Post et al., 1983; Haverkos et al., 1987; Levy et al., 1986, 1988, 1990) (Fig. 4.6). Incidence has diminished in the era of cART. Toxoplasmosis in patients with AIDS is most often the result of reactivation of latent disease in patients with a low CD41 T-cell count. Patients with reactivated toxoplasmosis often present with signs and symptoms of encephalitis (Luft and Remington, 1992) and/or much less frequently with eye disease (Liesenfeld et al., 1999). Acute acquired infection in AIDS patients has been reported and may involve multiple organs. The disease most often presents with subacute, focal deficits including hemiparesis (39% 52%), altered mental state (30% 42%), seizures (15% 29%), cranial nerve disturbances (7% 28%), abnormalities of speech (6% 26%), cerebellar signs (9% 30%), meningismus (10% 16%), and behavioral/psychomotor manifestations (30% 42%), including psychosis, dementia, and anxiety. Pulmonary toxoplasmosis presenting as febrile illness with cough and dyspnea has also been reported (Rabaud et al., 1996). Almost all organs have been reported as being involved, although the predominance is in the brain (Luft et al., 1983, 1993; Basavaprabhu et al., 2012; Luft and Remington, 1984, 1992; Kim et al., 2012; Kovacs, 1995; Guevara-Silva et al.,

2012; Leport et al., 1996; Abgrall et al., 2001; Haverkos et al., 1987; Levy et al., 1986, 1988, 1990; Post et al., 1983; summarized in Montoya et al., 2010). 4.1.5.2 Persons with cardiac and renal transplants Toxoplasma infection has been described after heart, kidney, and liver transplantation (Aubert et al., 1996; Giordano et al., 2002; Renoult et al., 1997; Wulf et al., 2005). In most cases, infection manifests within 3 months after transplantation. Patients with organ transplants or malignancy may develop central nervous system, retina, myocardial or pulmonary involvement due to reactivation (Ferna`ndezSabe´ et al., 2012; Aubert et al., 1996; Ryning et al., 1979; Mele et al., 2002; Slavin et al., 1994; Abgrall et al., 2001; Remington, 1974; Conley et al., 1981; Lassoued et al., 2007; Wreghitt et al., 1992, 1995; Renoult et al., 1997; Singer et al., 1993; Botterel et al., 2002; Martino et al., 2000, 2005; Matsuo et al., 2007; Sing et al., 1999; Luft et al., 1983; Bretagne et al., 1995; Bautista et al., 2012; Bories et al., 2012; Busemann et al., 2012; Caselli et al., 2012; Osthoff et al., 2012; Vaughan and Wenzel, 2012; Strabelli et al., 2012; Montoya et al., 2001, 2010) (Fig. 4.6). Clinical signs of infection are similar to those in patients with AIDS and involve fever and encephalitis. The parasite may also be present in eyes, liver, heart, lungs, pancreas, adrenal, and kidney. Seronegative persons may become infected with T. gondii through transplanted organs from seropositive donors. More rarely, seropositive transplant recipients may reactivate their latent infection due to the transplantrelated immune-suppression, although typically if a seropositive person receives a transplant from a seropositive donor, there is a rise in IgG antibody titer and development of IgM antibody specific for T. gondii without illness ascribed to the parasite. Thus the frequency of transplant-related infection depends on the

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seroprevalence of infection with T. gondii in a population. Gallino et al. (1996) reported that 14 (87%) of 16 infections in T. gondii naı¨ve seronegative recipients seroconverted after receiving a cardiac transplant from a seropositive donor. The use of antiparasitic prophylaxis also impacts the rate of infection. A review of 257 cases of heart transplants between 1985 and 1993 and 33 cases of heart lung transplants found that 4.5% (13) were between a seropositive donor and a seronegative recipient. Nine of these cases were followed up and only one patient was documented as seroconverting. All these patients received trimethoprim/sulfamethoxazole prophylaxis for Pneumocystis (Orr et al., 1994). In patients receiving a cardiac transplant, 6 weeks pyrimethamine prophylaxis reduced infection from 57% (4/7) to 14% (5/37) (Wreghitt et al., 1992). When a seropositive person receives a heart transplant from a donor who is seropositive, there is an increase in T. gondii specific IgG antibody titer and a new presence of T. gondii specific IgM antibody but usually this occurs without causing illness. 4.1.5.3 Bone marrow and hematopoietic stem cell transplantation The prevalence of T. gondii in bone marrow and hematopoietic stem cell transplantation (BMT) patients also varies with the seroprevalence in the population: 0.5% in the United States to 5% in France (Ferna`ndez-Sabe´ et al., 2012; Aubert et al., 1996; Ryning et al., 1979; Mele et al., 2002; Slavin et al., 1994; Remington, 1974; Conley et al., 1981; Martino et al., 2000, 2005; Matsuo et al., 2007; Sing et al., 1999; Bretagne et al., 1995; Bautista et al., 2012; Bories et al., 2012; Busemann et al., 2012; Caselli et al., 2012; Osthoff et al., 2012; Vaughan and Wenzel, 2012; Strabelli et al., 2012; Montoya et al., 2010). Most patients are seropositive for T. gondii before transplantation and reactivate their latent infection. Symptoms of T. gondii infections in bone marrow transplant patients include fever

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(43%), seizures (14%), headache (13%), confusion (13%), and pulmonary symptoms (12%). Ninety-two percent had more than one symptom, and the average onset of symptoms was 62 days post-BMT (range 1 689 days) (Mele et al., 2002). Disseminated infection that is rapidly progressive may occur. Mortality rates are high (Chandrasekar and Momin, 1997). The European Group for Blood and Bone Marrow Transplantation reported on 106 allogenic stem cell transplants of which 55% of the recipients were Toxoplasma IgG positive. All received prophylaxis with trimethoprim and sulfamethoxazole for 6 months and 15% (16/106; 95% CI: 8% 21%) had at least one T. gondii PCR positive blood sample and 6% (6/106; 95% CI: 1% 10%) experienced clinical disease due to T. gondii infection (Martino et al., 2005). The median days to diagnosis from onset of symptoms was 42 days (range 1 178 days) and the presenting symptoms were localized encephalitis in four patients, pulmonary toxoplasmosis in one patient and one patient presenting acute disseminated disease (Martino et al., 2005). There is a recent review concerning prophylaxis to prevent and treatment of active Toxoplasma infection in hematopoietic stem cell transplant recipients (Gajurel et al., 2015).

4.2 Diagnosis of infection with Toxoplasma gondii Strategies for control and prevention of congenital toxoplasmosis vary between countries, as discussed previously, and the diagnostic challenges are different in pre- and neonatal screening programs. Systematic, prenatal screening is performed in Austria, France, Slovenia, Minas Girais, Brazil, and widespread on-demand screening takes place in Belgium, Germany, Italy, and Spain. Samples are obtained during pregnancy and analyzed for Toxoplasma-specific IgM and IgG antibodies (Table 4.1; Figs. 4.2 and 4.7). When

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TABLE 4.1 Diagnosis of toxoplasmosis.

Images from McLeod et al. (unpublished data). Equation from McLeod, R., Lee, D., Boyer, K., 2013a in press. Toxoplasmosis in the Foetus and Newborn Infant., with permission.

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FIGURE 4.7

(Continued).

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FIGURE 4.7 The French approach to treatment of congenital toxoplasmosis during gestation and infancy and outcomes. (A) A gestational screening algorithm, identifying those patients who ultimately require no treatment (blue), those who require additional testing (pink), and those for whom treatment should be strongly considered (green). This algorithm can be applied in any setting with basic laboratory capabilities, though avidity and AC/HS is more complex. In these settings, given the benign treatment profile of spriamycin, it might be appropriate to err on the side of treatment of these patients without further testing. The AC/HS test is a differential agglutination test using the ratio of two antigens present predominantly in early (AC) and late (HS) Toxoplasma gondii infection to suggest the timing of infection. It is critical to note

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seroconversion is detected, this confirms that the mother is infected, and treatment is usually started. Neonatal screening for congenital toxoplasmosis is performed in New England, was formerly performed in Denmark, and is performed in parts of Brazil by analyzing the blood samples obtained on filter paper (Guthrie cards) in the days immediately postpartum (Guerina et al.,

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1994; Lebech et al., 1999; Neto et al., 2004). Detection of Toxoplasma-specific IgM antibodies eluted from the phenylketonuria (PKU)-filter paper is followed by a request of a blood sample from both the mother and infant for confirmatory testing (Sørensen et al., 2002). Fifteen to twenty percent of these sera are found to be negative for Toxoplasma-specific IgM (Decoster et al., 1992; Lebech et al., 1999; Naot et al., 1981a,b).

that this algorithm should only be utilized for pregnant women presenting for care during the specified time period. For those presenting at different times in gestation for care, testing and treatment protocols are more nuanced. Refer to work from the Pal Alto Reference Laboratory or papers on the management of congenital toxoplasmosis for further details regarding the screening protocol. The conclusions from data using the French or Austrian approach. (B) Treatment and diagnosis regimens to follow throughout gestation. These include diagnosing and treating the pregnant women as well as the fetus. Table adapted from Remington et al. (2011). a Data from Desmonts and Couvreur (1984). b Data from Hohlfeld et al. (1994). c Data from Daffos et al. (1988). d Data from Hohlfeld et al. (1989). e From Couveur et al. (1993). f Data from Kieffer et al. (2008). g Data from SYROCOT (2007). h Data from Peyron et al. (2011). i Data from Stillwaggon et al. (2011). j Data from SYROCOT (2007). Table from Olariu et al. (2011). (C) Clinical manifestations in treated infants with congenital toxoplasmosis in European countries with screening and treatment programs and summary of effects. (D) Top: Probability of fetal infection according to gestational age at the time of maternal infection before (n 5 451) and after (n 5 1624) mid-1992—Lyon Cohort 1987 2008 Bottom: Reduction in the risk of infection and of clinical signs following changes in the retesting policy (1992) and in antenatal diagnosis and treatment procedures (1995)—Lyon Cohort 1987 2008. Source: (A) Data from Desmonts, G., Couvreur, J., 1984. Congenital toxoplasmosis. Prospective study of the outcome of pregnancy in 542 women with toxoplasmosis acquired during pregnancy. Ann. Pediatr. (Paris) 31, 805 809; Hohlfeld, P., Daffos, F., Costa, J.M., et al., 1994. Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid. N. Engl. J. Med., 331, 695 699; Daffos, F., Forestier, F., Capella-Pavlovsky, M., et al., 1988. Prenatal management of 746 pregnancies at risk for congenital toxoplasmosis. N. Engl. J. Med. 318, 271 275; Hohlfeld, P., Daffos, F., Thulliez, P., et al., 1989. Foetal toxoplasmosis: outcome of pregnancy and infant follow-up after in utero treatment. J. Pediatr. 115 (5 Pt 1), 765 769; Couveur et al. (1993); Kieffer, F., Wallon, M., Garcia, P., et al., 2008. Risk factors for retinochoroiditis during the first 2 years of life in infants with treated congenital toxoplasmosis. Pediatr. Infect. Dis. J., 27 32; SYROCOT Systemic Review on Congenital Toxoplasmosis Study Group, Thiebaut, R., Leproust, S., Chene, G., Gilbert, R., 2007. Effectiveness of prenatal treatment for congenital toxoplasmosis: a meta-analysis of individual patients’ data. Lancet 369, 115 122; Peyron, F., Garweg, J.G., Wallon, M., et al., 2011. Long-term impact of treated congenital toxoplasmosis on quality of life and visual performance. Pediatr. Infect. Dis. J. 30 (7), 597 600; Stillwaggon, E., Carrier, C.S., Sautter, M., McLeod, R., 2011. Maternal serologic screening to prevent congenital toxoplasmosis: a decision-analytic economic model. PLoS Negl. Trop. Dis., 5(9), e1333. Retrieved from https://doi.org/ 10.1371/journal.pntd.0001333. (C) Data from SYROCOT Systemic Review on Congenital Toxoplasmosis Study Group, Thiebaut, R., Leproust, S., Chene, G., Gilbert, R., 2007. Effectiveness of prenatal treatment for congenital toxoplasmosis: a meta-analysis of individual patients’ data. Lancet 369, 115 122. Table from Olariu, T., Remington, S., McLeod, R., Alam, A., Montoya, J., 2011. Severe congenital toxoplasmosis in the United State: clinical and serologic findings in untreated infants. Pediatr. Infect. Dis. J. 30 (12), 1056 1061, with permission. (D) Images from Wallon, M., Peyron, F., Cornu, C., Vinault, S., Abrahamowicz, M., Bonithon Kopp, C., et al., 2013. Congenital Toxoplasma infection: monthly prenatal screening decreases transmission rate and improves clinical outcome at 3 years. Clin. Infect. Dis. [Epub ahead of print], with permission.

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Low levels of Toxoplasma-specific IgM antibodies may be found for up to several years after acute infection, and the mere demonstration of low levels of Toxoplasma-specific-IgM antibodies is therefore not regarded as a sign by itself of acute infection with T. gondii (Liesenfeld et al., 1997, 2001a,b). One diagnostic challenge is the situation when Toxoplasma-specific IgG and IgM antibodies are found in the first sample after conception, where the time of infection is the key to estimate whether the fetus is at risk or not (Ades, 1991). The measurement of the avidity of IgG antibodies was first demonstrated for T. gondii infections in 1989 (Hedman et al., 1989, 1993; Lecolier and Pucheu, 1993) and has since then been further developed (Dannemann et al., 1990; Marcolino et al., 2000; Beghetto et al., 2003; Petersen et al., 2005) but is not reliable in the beginning of the infection when there are low levels of IgG antibodies (Press et al., 2005). Since previous studies have shown that some individuals have low avidity IgG antibodies many months after infection, Petersen et al. (2005) investigated whether treatment influences the maturation of IgG antibodies and, based on 12 untreated patients, it seems that treatment and/or pregnancy may delay IgG maturation. A study of the value of different diagnostic tests for acute infection with T. gondii, including Toxoplasma-specific IgG, IgM, IgA antibodies, and the IgG avidity index, demonstrated that the combination of a sensitive test for Toxoplasma-specific IgM antibodies and a Toxoplasma-specific IgG avidity assay has the highest predictive value of the time of infection (Robert et al., 2001).

4.2.1 Toxoplasma antigens and diagnostic assays Diagnosis of active, acute infection can be made on the basis of serologic tests or by

histopathology, isolation of T. gondii or PCR from a variety of samples (e.g., CSF, placenta, fetal tissues, or peripheral blood). T. gondii has three distinct life-stages, each with stagespecific expression of antigens (Kasper and Ware, 1989; Singh et al., 2002; Hill et al., 2011; Boyer et al., 2011). An important immunodominant antigen is the tachyzoite-specific Surface Antigen 1, SAG1 (p30, SRS29B) which comprises up to 5% of the protein of the tachyzoite (Burg et al., 1988). The antigen expressed in Escherichia coli has been shown to be recognized by natural SAG1 antibodies (Harning et al., 1996) and SAG1 is considered a prime candidate antigen in diagnostic tests because of its immunodominance and lack of known cross-reactivity to antigens from other microorganisms. Other surface antigens, SAG2 (SRS34A), SAG3 (SRS57) and SAG4 have been identified, SAG2 and SAG3 being tachyzoite specific and SAG4 being bradyzoite specific (Cesbron-Delauw, 1994, 1995; Howe and Sibley, 1994; Odberg-Ferragut et al., 1996). Two other groups of T. gondii antigens have been studied for use in diagnostic assays, the dense granule antigens, GRAs, and, in particular, GRA1, GRA6 (Lecordier et al., 2000) and GRA7 (Fischer et al., 1998) and microneme (MIC) antigens (GarciaRe`guet et al., 2000; Lourenco et al., 2001; Cerede et al., 2002). The MIC antigens have also been shown to be important in the induction of protective immunity (Beghetto et al., 2005). Bradyzoite-specific antigens like the bradyzoite antigen 1 (BAG1) (Bohne et al., 1993) and matrix antigen 1 (MAG1) (Parmley et al., 1994) should, in theory, be important in the antibody repertoire in infections past the acute stage, but they still remain to find their place in future diagnostic assays and studies have suggested their utility (Li et al., 2019b). The diagnostic value of oocyst-specific antigens has been studied in a single study of T. gondii oocyst-infected cats (Dubey et al., 1995). Hill et al. (2011,

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2012) developed a test for defining infection with sporozoites using response to an 11 kDa sporozoite protein. An additional sporozoite antigen has been identified, CCp5A, that elicits antibody in persons recently infected with oocytes (Santana et al., 2015).

4.2.2 The development of diagnostic assays The complement fixation assay was the first diagnostic test for Toxoplasma-specific antibodies (Warren and Sabin, 1942; Steen and Ka˚ss, 1951). The dye-test described by Sabin and Feldman (1948) is based on antibody-mediated killing of live T. gondii parasites in the presence of complement. Methylene blue is a vital dye. Thus if antibodies are present, parasites are lysed in the presence of complement. They then appear thin and blue as opposed to plump and robust when they are alive. The dye-test (DT) has proved a very sensitive assay, but the requirement of live, T. gondii parasites makes it more complicated and expensive to perform (Reiter-Owona et al., 1999). The DT is not included in reference panels circulated as part of external quality control programs, and multicenter studies show a considerable variability (Petithory et al., 1996; Reiter-Owona et al., 1999; Rigsby et al., 2004). Immunofluorescence assays (IFA) were introduced in the 1960s (AmbroiseThomas et al., 1966) and proved specific, but with a lower sensitivity compared to the DT. The IFA for Toxoplasma-specific IgM antibodies is still used by some centers because it is highly specific, but it has a low sensitivity (Robert et al., 2001). The enzyme immunoassay (EIA) technique became available in 1972 (Engvall and Perlmann, 1972). The first Toxoplasma-specific IgM assay was developed by Remington et al. (1968) and the first EIA based assay by Naot and Remington (1980). By the end of the 1980s

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the direct EIA measuring Toxoplasma-specific IgG antibodies and the μ-capture EIA measuring Toxoplasma-specific IgM antibodies were well established in reference centers and the first commercial test introduced (Schaefer et al., 1989). The μ-capture IgM assays were an improvement over the direct EIA IgM assays but continued to have problems with falsepositive results (Liesenfeld et al., 1997). The development of the immunosorbent agglutination assays (ISAGA) solved this by using whole cell formalin fixed T. gondii (Beghetto et al., 2003) and tests based on this technique are regarded as highly sensitive and specific for Toxoplasma-specific IgM and IgA antibodies (LeFichoux et al., 1984; Pouletty et al., 1985). Immunoblot using single antigens has also been tested as a means to improve diagnostic sensitivity (Gross et al., 1992). A method to measure the maturation of Toxoplasma-specific IgG antibodies to determine the time of infection was described by Hedman et al. (1989). The test explores the increasing avidity (sum of all affinities) by the specific IgG antibodies with the maturation of the immune response, and it was shown that the time of infection could be determined within a 3 month window after infection. The test has been adapted for automated systems (Petersen et al., 2005). Newer IgG avidity tests allow one to exclude an acute infection within the last 3 4 months in patients with high avidity antibodies. In contrast, the presence of low or intermediate avidity IgG antibodies does not necessarily allow one to diagnose an acute infection, since the maturation of IgG antibodies may show marked differences between individuals. The same principle is used in the differential agglutination test (Thulliez et al., 1989). Recently, Villard et al. (2012) compared four commercially available avidity assays and found greater than 95% sensitivity. The bioMerieux test had the greatest sensitivity and efficacy (99%). Recent studies to develop improved assays include peptides,

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recombinant proteins, and PCR (Raymond et al., 1990; Fuentes et al., 1996; Reischl et al., 2003; Flori et al., 2004; Lefevre-Pettazzoni et al., 2007; Meroni et al., 2009; Chapey et al., 2010; Dai et al., 2012a,b; Faucher et al., 2012; Fayyaz and Rafi, 2012; Holec-Gasior et al., 2012a,b; Kotresha et al., 2012; Morelle et al., 2012; Murat et al., 2012; Prusa et al., 2012; Saadatnia et al., 2012).

4.2.3 Diagnosis of Toxoplasma gondii infection in pregnant women In countries where prenatal screening programs are in place, a test of the first blood sample from pregnant women for Toxoplasmaspecific IgG and IgM antibodies in the first trimester is performed. A high avidity assay can help for date acquisition of the infection to greater than 12 weeks earlier, but a low avidity test does not prove a recent infection has not occurred. Approximately 5% of seropositive women in the first trimester have Toxoplasmaspecific IgM antibodies, but only approximately 4% of these give birth to a child with congenital Toxoplasma infection. It is, therefore, a considerable problem to diagnose whether women with specific IgM antibodies are infected before or after conception. This is particularly a problem in countries where testing of pregnant women at the beginning of pregnancy is common. This problem has been partly solved by obtaining two samples from pregnant women to see if there is any development of the specific immune response. It is generally agreed that there is a development of the Toxoplasma-specific IgG antibody response within the first 6 8 weeks after infection after which the IgG levels are maintained at a high and stable level, with or without declining IgM antibodies (Jenum and Stray-Pedersen, 1998; Jenum et al., 1997). The question of too many low-level Toxoplasma-specific IgM-positive patients and whether the diagnostic

performance could be improved by not merely repeating the same tests 2 weeks apart was investigated in a European multicenter study (Robert et al., 2001). All highly sensitive assays were found to have a low specificity, and single tests were unable to reliably distinguish between acute and latent infections. Only the sequential analysis of sera by a highly sensitive IgM assay in combination with IgG avidity testing gave excellent diagnostic performances. In contrast, IgA or IgM assays were less useful to diagnose acute infections by confirming positive IgM results. 4.2.3.1 IgG avidity index In a European multicenter study, many laboratories contributed samples from patients in whom the time of infection was known. This panel was used to determine the proportion of sera showing specific IgM and IgA antibodies to T. gondii (Beghetto et al., 2003) as well as the IgG avidity index within 1 3, 3 12, or more than 12 months after seroconversion. These data were used to propose a two-level strategy for diagnosis (Robert et al., 2001). Treatment may delay the development of high-avidity Toxoplasma-specific IgG antibodies (Meroni et al., 2009). 4.2.3.2 Combined, two-test strategies Robert et al. (2001) demonstrated that the best strategy for diagnosing acute and recent infection with T. gondii was a two-test strategy with a sensitive IgM test first followed by an IgG avidity test. Thus the study confirmed the need of the Toxoplasma-specific IgG avidity index assay in the diagnosis of acute and recent infection. The increased use of the T. gondii IgG avidity test has highlighted an inherent problem, which is that many pregnant women have long-lasting, low IgG avidity antibodies and the IgG avidity assay needs further development (Beghetto et al., 2003).

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4.2.6 Recombinant IgM and IgG assays—newborns

The problems with IgM based diagnostics in T. gondii infections have resulted in attempts to improve the tests. The accepted reference test is the ISAGA, but most analyses are performed with an EIA capture test. The assays use whole-cell, lysed T. gondii as the antigen, and attempts have been made to improve the test by using recombinant antigens (Ferrandiz et al., 2004).

4.2.5 Recombinant IgG assays—adults Conventional assays have so far used whole-cell, lysed, T. gondii antigens, which have batch variations. With increasing emphasis on the need for reproducibility, the use of recombinant antigens in diagnostics assays provides a theoretical advantage. Previous studies have shown that the GRA1, GRA7, and SAG1 molecules are immunodominant (Aubert et al., 2000; Harning et al., 1996; Jacobs et al., 1999; Johnson et al., 1992; Li et al., 2000; Suzuki et al., 1988b). Pietkiewicz et al. (2004) demonstrated that recombinant antigens, including a mixture of GRA1, GRA7, and SAG1, were not as sensitive as whole-cell, lysed, antigen if sera had an IgG titer of less than 1:1600 using an EIA test and less than 1:512 in an IgG immunofluorescence test. The test did however have a 100% sensitivity in a panel of sera from individuals who had Toxoplasma-specific IgM and/or IgA antibodies (i.e., those in whom the infection was recent) (Pietkiewicz et al., 2004). Future assays for Toxoplasma-specific IgG antibodies relying on recombinant antigens need to include a panel of antigens but this test has not yet been optimized to the same sensitivity as the whole-cell, lysed antigen assay.

Diagnostic assays based on recombinant antigens for measuring the Toxoplasmaspecific IgM antibodies were evaluated in infants with or without congenital toxoplasmosis born to mothers with toxoplasmosis acquired during pregnancy (Holec-Ga˛sior, 2013). Antigen fragments from the MIC2, MIC3, GRA3, GRA7, M2AP, and SAG1 proteins were tested in an EIA test (RecEIA) on 104 serum samples from newborns born to mothers infected with T. gondii during pregnancy (Beghetto et al., 2006). Thirty-five were congenitally infected and 34 of 35 (97%) serum samples from the congenitally infected patients reacted with at least one of the recombinant antigens (Buffolano et al., 2003). Remarkably, all sera from the 22 Toxoplasma-infected newborns who were clinically and serologically undiagnosed at birth were reactive using the IgM Rec ELISA analysis, allowing the confirmation of congenital toxoplasmosis as soon as 2 months after birth. The presence of T. gondii specific IgM antibodies against recombinant MIC2, MIC3, M2AP, and SAG1 antigens may be used for the early postnatal diagnosis of congenital toxoplasmosis. Buffolano et al. (2003 found that the newborn Toxoplasma-infected child produces primarily IgG2 and IgG3 against recombinant Toxoplasma antigens, whereas the maternally transferred antibodies primarily were IgG1. This subclass analysis of serum samples from mother and child against defined recombinant antigens may further improve diagnosis of congenital Toxoplasma infection in newborns. Peyron adapted this approach and found interferon-gamma (INF-γ) production in response to T. gondii antigens can also be used to establish diagnosis of congenital toxoplasmosis.

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4.2.7 The Toxoplasma-specific IgG avidity index The maturation of the IgG response varies considerably between individuals. A high avidity indicates infection longer than 12 14 weeks earlier but a low avidity can persist in some persons for long times. The observation that the T. gondii specific IgG-maturation is delayed in treated pregnant women compared to nontreated, nonpregnant individuals has been reported in one previous study, which found a significantly delayed IgG-maturation in treated individuals (Sensini et al., 1996). The finding that treatment may influence the IgG avidity maturation underlines the need for further studies to better clarify the avidity maturation process in pregnant women under therapy in comparison with untreated individuals. If confirmed, different cut-off values will have to be defined for treated and untreated and/or pregnant and nonpregnant individuals.

4.2.8 Molecular and other diagnostic techniques The diagnosis of acute toxoplasmosis may be established by the detection of anti T. gondii antibodies by serological tests or the detection of tachyzoites or T. gondii specific DNA in body fluids or tissue samples. In most cases of toxoplasmosis in immune-competent individuals, diagnosis is established by serological tests; however, molecular (i.e., PCR) diagnostic tests have proven useful in the diagnosis of infection in utero as well as in immunecompromised hosts. The detection of T. gondii tachyzoite DNA in body fluids and tissues by isolation and PCR amplification is effective to diagnose congenital (Grover et al., 1990), ocular (Montoya et al., 1999), and cerebral toxoplasmosis (Holliman et al., 1990). PCR is a key part of the diagnosis of in utero infection. Sensitivity in initial reports was 100%, but subsequent studies have indicated this is

dependent on gestational age of infection (Montoya, 2002; Thalib et al., 2005; Switaj et al., 2005). Sensitivity also varies with gene target (e.g., the B1 gene is present at 35 copies and AF146527 is present at 300 copies). For amniotic fluid, sensitivity appears best with PCR assay to detect the 300 copy 592 bp gene (Romand et al., 2004) (Fig. 4.2). In a French study of 2000 consecutive amniotic fluid samples, it was confirmed that a positive PCR correlates with disease and that PCR is more sensitive than any other available test (Thulliez, 2001b). Isolation of T. gondii from blood or body fluids (e.g., CSF, amniotic, or BAL fluids) also establishes diagnosis of the acute infection. Isolation can be performed by inoculation of the samples in mice or in tissue cultures. The demonstration of tachyzoites in histological sections or smears of body fluids by immunoperoxidase staining with anti T. gondii antibodies also establishes the diagnosis (Conley et al., 1981). This technique has been very useful in the diagnosis of CNS mass lesions in the setting of HIV/AIDS.

4.2.9 Diagnosis of Toxoplasma gondii infection in newborn infants Diagnosis of congenital infection with T. gondii may be difficult at birth if Toxoplasmaspecific IgM and/or IgA antibodies are not present, because present diagnostic methods can only, with difficulty, distinguish between maternal and fetal IgG. If the child has been treated continuously with sulfadiazine and pyrimethamine, the synthesis of Toxoplasmaspecific IgG antibodies often is suppressed and the serological confirmation of the infection can sometimes not be made with certainty before the second year of life (Wallon et al., 2001). This situation is found when T. gondii infection is suspected, but Toxoplasma-specific IgM and/or IgA antibodies cannot be

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demonstrated in the child and parasitological investigations, such as PCR analysis for Toxoplasma-specific nucleic acid, are negative or not appropriate. IgM and IgA antibodies do not cross the placenta, and neonatal screening programs for congenital toxoplasmosis are based on detection of Toxoplasma-specific IgM antibodies eluted from blood spots from PKUfilter papers (Guthrie cards) (Guerina et al., 1994; Lebech et al., 1999; Neto et al., 2004; Sørensen et al., 2002). Different cut-offs for maternal and newborn Toxoplasma-specific IgM antibodies have been proposed (Candolfi et al., 1993). Treatment of acute toxoplasmosis during pregnancy reduced the magnitude and duration of the Toxoplasma-specific IgM response in a number of earlier studies (Desmonts and Couvreur, 1984; Hohlfeld et al., 1989). Others did not report such an effect (Gras et al., 2004). Presumably this was due to differences in methods and cohorts. Demonstration of Toxoplasma-specific IgG antibodies with different specificities in sera from the mother and child shows that the child synthesizes her/his own IgG antibodies, confirming that the child is infected with T. gondii. Previous studies have shown that transferred maternal and neo-synthesized T. gondii specific IgG antibodies can be differentiated by immunoblot or immunocomplexing (Chumpitazi et al., 1995; Gross et al., 2000; Remington et al., 2004; Robert-Gangneux et al., 1999). Differentiation of the specificities of IgG antibodies in the mother and child can also be done by comparing T. gondii antigen precipitated with maternal or child sera before performing an electrophoresis of the antigen antibody complex (Pinon et al., 1996, 2001; Robert-Gangneux et al., 1999). The immunoblot and immunocomplexing techniques were compared in a double-blind study and found to be equally sensitive (Pinon et al., 2001). The immunoblot technique identifies newborns with congenital toxoplasmosis with a sensitivity of approximately 70%

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(Rilling et al., 2003; Tissot-Dupont et al., 2003) increasing to 85% within the first 3 months of life (Gross et al., 2000; Rilling et al., 2003; Tissot-Dupont et al., 2003). These results still leave 15% 30% of congenitally infected newborns without a confirmed diagnosis. To improve the diagnosis of congenital toxoplasmosis, a two-dimensional immunoblot (2DIB) assay was developed that is capable of distinguishing between maternal and neonate Toxoplasma-specific IgG with a better sensitivity than previous assays (Nielsen et al., 2005). The 2DIB methodology greatly increased the resolution of the antibody response by allowing identification of up to a thousand spots where the most sensitive immunoblots do not allow distinction of less than 50 bands or often considerably less (Nielsen et al., 2005). Treatment reduces the percentage of congenitally infected infants who have IgM directed against T. gondii.

4.2.10 Prompt diagnosis during gestation to facilitate treatment with unique spillover benefits There is robust evidence that screening monthly to diagnose seroconversion and rapidly initiate treatment makes toxoplasmosis a preventable and treatable disease. Those data are summarized in Figs. 4.7A D and 4.8A F. Such screening was found to be 14-fold cost saving in Austria (Prusa et al., 2017). Of course, if those making testing materials or medications to treat the disease make the cost high enough, nothing is cost-saving. Chapey et al. (2017) found that a French test had robust performance with sera from patients infected with Type II parasites. This test was then used in a study in the United States, where parasites are a mixture of Type II and Non Type II parasites, and it performed equally well under both circumstances (Begeman et al., 2017) (Fig. 4.8G N). It was then adopted to test with

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Toxoplasmosis screening in gestation.

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FIGURE 4.8

Toxoplasmosis screening in gestation.

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FIGURE 4.8 Toxoplasmosis screening in gestation. (A) Cost benefit analysis of global toxoplasmosis screening programs (Stillwaggon et al., 2011; Prusa et al., 2017; Bobi´c et al., 2019; El Bissati et al., 2018). (B) Decreasing seroprevalance in France with gestational screening protocol (El Bissati et al., 2018); data from Nogareda et al. (2014). (C) Incremental tornado analysis diagram for societal savings with screening of toxoplasmosis (Prusa et al., 2017). (D) Lifetime costs and savings from targeted and universal toxoplasmosis screening programs (Prusa et al., 2017). (E) Clinical presentation for congenital

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whole blood using black beads, rather than pink beads, where the heme in the blood obscured the ability to read the test as easily. This IgG/IgM lateral immunochromatography test (Fig. 4.8Q S) has been shown to produce results with 100% sensitivity and specificity, while only requiring 30 microliters of blood (Lykins et al., 2018a,b; Fig. 4.8Q S). The anticipated market price of these kits is approximately $5 in comparison to $650 for IgG and IgM serologies charged by some US Hospital Laboratories (Begeman et al., 2017). This will facilitate the economics of testing 8 10 times during gestation, making screening cost effective. In low/middle-income countries with high prevalence, monthly screening brings women to care, which has many other benefits (Fig. 4.8R). This test kit was also tested with a CDC set of 150 test sera for FDA approval alongside two additional test kits. These other test kits did not perform as well, particularly for IgM. When testing for Toxoplasma IgM in sera had a falsepositive result in commercial tests, the lateral chromatography test accurately indicated that

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there was no T. gondii IgM present in 50 such samples (Gomez et al., 2018; Fig. 4.8M). A new nanogold test (Fig. 4.9) used with 1 μL of saliva can detect toxoplasma-specific IgG, IgM, and IgA with an estimated cost of $6 $10 for reagents, not accounting for the cost of the machine used to perform the test (Li et al., 2019a). This requires a centralized, skilled operator and electricity. This same nanogold test performs with similar high performance of 100% sensitivity and specificity for toxoplasma IgG, and nearly as high for toxoplasma IgM. This test has been adapted for testing of cytomegalovirus (CMV), herpesvirus, and Rubella immunoglobulins, permitting multiplex testing for these pathogens of perinatal infections and the potential to utilize similar testing for HIV, Hepatitis B, Syphilis, Zika virus, and Trypanosomes at an approximate cost of $6 per pathogen. The combination of the lateral immunochromatography POC test and the multiplex nanotest is revolutionary for detection of treatable perinatal infections.

toxoplasmosis in the University of Quindio before and after implementation of evidence-based guidelines (El Bissati et al., 2018). (F) Children seen with severe neurological damage at the University of Quindio (El Bissati et al., 2018). (G) Toxoplasma ICT IgG IgM point of care test component breakdown—(1) Sample and eluent in the “sample well”; (2) Fiberglass pad—“conjugate pad” (3) A-blue latex particles coated with antirabbit goat antibodies; (4) Nitrocellulose sheet; (5) “Test line” with toxoplasma antigens; (6) “Control line” with rabbit antibodies; (7) Absorbent pad. (H) Toxoplasma ICT IgG IgM testing results using serum. A negative result is displayed on the left and a positive result on the right. C indicates the control for antibody result and T indicates the sample with antibodies reacting with Toxoplasma antigens result. The appearance of a line indicates a positive result demonstrating presence of the reactivity to the cognate antibody. Testing with sera is still not a true point of care test as there must be a centrifuge available, electricity, time for separation of sera, or the samples must be sent to an outside facility (Begeman et al. 2017). (I) Presentation of seroconversion pattern of a pregnant woman tested with LDBIO (Chapey et al., 2017). (J) Results from chronically (black symbols)/subacutely acutely (red symbols) infected patients by parasite serotype (horizontal axis) displayed from time of birth to sample acquisition(vertical access) (Begeman et al., 2017). (K) Sensitivity and specificity of point of care testing (Begeman et al., 2017). (L) Sensitivity and specificity of results with Toxoplasma ICT IgG IgM test and reference tests (Begeman et al., 2017). (M) Testing of three different POC kits against false-positive IgM results (Gomez et al., 2018). (N) Limitations in using serum in a POC test or a pink bead for whole blood. (O) Step-by-step procedure for finger prick whole blood point of care testing. The arrow points to a positive result (Lykins et al., 2018a,b). (P) Design of finger prick whole blood point of care testing study (Lykins et al., 2018a,b). (Q) Study results of whole blood point of care testing with resultant sensitivity and specificity (Lykins et al., 2018a,b). (R) Gestational screening to save mothers’ and children’s sight, cognition, lives and health care costs (Begeman et al., 2017). (S) WHO criteria for an ideal point-of-care test on the left. Characteristics of the Toxoplasma ICT IgG-IgM Finger Prick Whole Blood point-of-care test on the right which demonstrate that these ASSURED criteria are met (Lykins et al., 2018a,b). WHO, World Health Organization. Source: (G) US Patent Application, courtesy Denis Limonne, LDBio Diagnostics.

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FIGURE 4.9 Diagnosis of Toxoplasma gondii using plasmonic gold chips. (A) Plasmonic gold biochip with multiplexed T. gondii/cytomegalovirus/Rubella antigen microarray (Li et al., 2019). (B) Human IgG antibodies captured by the corresponding antigen spots on the microarray (Li et al., 2019). (C) Boxplots showing detection of IgG (above) and IgM (below) in 148 samples of serum and saliva on plasmonic gold chips and the correlation between saliva and serum detection of T. gondii antibodies (Li et al., 2019). (D) Results showing detection of IgG with 96.1% sensitivity and 93.0% specificity and detecting IgM with 100% sensitivity and 95.4% specificity (Li et al., 2019). (E) Scanning images of positive versus negative signal in serum, blood, and saliva (left) Multiplexed panel showing detection of T. gondii IgG (middle) and IgM (right) antibodies in 28 matched serum, saliva, and whole blood samples (Li et al., 2019). (F) 100% sensitivity and specificity for T. gondii IgG and 96.4% specificity for T. gondii IgM (Li et al., 2019). Source: Figure and legend adapted with permission from Springer Berlin Heidelberg publishing group.

The societal and public health impact of diagnosing and treating maternal acquisition of T. gondii during gestation without delay was summarized by Bobi´c et al. (2019) as follows: Economic analyses of health interventions, particularly "for prevention, have tended to underestimate the benefits and over-state the costs. A short-term perspective fails to recognize the lifetime costs to the individual and the community due to prenatal and

childhood injuries. The productivity losses from preventable, sometimes profound, injuries are staggering. The presumption that screening and prevention are expensive does not withstand careful examination because the lifetime costs of injury are great and the costs of screening trivial in comparison. Given the evidence of efficacy that is available, the decision to spend resources on prevention is a political choice that reflects the priorities of the society."

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4.2.11 Immune-compromised patients Because reactivation of latent Toxoplasma infection is the most common cause of toxoplasmosis in immune-compromised patients, detection of T. gondii IgG antibodies is indicated. Patients with a positive result are at risk of reactivation of the infection; patients with a negative result should be instructed on how they can prevent becoming infected, recognizing that the mode of acquisition often goes unnoticed or unrecognized. The most important factor in the management of the seropositive immune-suppressed patient is to consider T. gondii as a potential causative agent in patients presenting with nonspecific symptoms, including focal symptoms from CNS, heart, lungs, and liver. Since immunecompromised patients do not reliably produce antibodies, serology has been complemented by direct detection methods such as PCR analysis of T. gondii specific nucleic acid. The definitive diagnosis of toxoplasmosis relies on detection of T. gondii DNA by PCR or on histologic demonstration of the parasite. Whereas tachyzoites are diagnostic of the active infection, T. gondii tissue cysts may indicate latent infection. In patients with toxoplasmic encephalitis, blood, CSF, and brain tissue may be used to detect T. gondii specific DNA. Sensitivities of PCR range between 25% and 80% for blood, and 35% and 100% for CSF samples (Colombo et al., 2005; Vidal et al., 2004; Parmley et al., 1992). For brain tissue, tachyzoites and histopathology suggest active infection, but cysts will also result in a positive PCR. Pulmonary toxoplasmosis occurs in immune-compromised persons, such as those with stem cell transplantation or in HIV infected patients with low CD41 T-cell counts (Rabaud et al., 1996). A study of bronchoalveolar lavage (BAL) samples from 332 Danish HIV infected patients found 2.1% (7/332) positive samples using a new, sensitive real-time PCR method (Petersen et al., BAL). The patients

were in an advanced stage of immunesuppression with a mean CD41 T-cell count of 39 3 106/L (range 0 and 161 3 106/L; normal values greater than 650 3 106/L). Monitoring bone marrow transplant patients by PCR on peripheral blood, BAL fluid, and CSF (according to local symptoms) and treatment with pyrimethamine for positive PCR results reduced the mortality to the same levels as for T. gondii negative BMT patients. The same strategy could be applied to other immunesuppressed patients at risk of developing T. gondii infection, including those patients with HIV infection.

4.3 Treatment It is useful to separate considerations about therapy of toxoplasmosis into several categories (Table 4.2). The decision to treat is based on who has the infection or manifestations, that is, the location, activity of infection, symptomatology of the infection, severity, immune status of the patient, age, and whether or not a woman with acute acquired T. gondii infection is pregnant. There have been several studies of prophylaxis and treatment for toxoplasmosis in the setting of AIDS, congenital disease, pregnancy, and ophthalmologic disease. The recommended therapies are based on extrapolations from in vitro studies and animal models (mostly murine) and the clinical experience and practice of physicians experienced in the treatment of T. gondii infection. The standard therapeutic agents for the treatment of toxoplasmosis are the combination of pyrimethamine (administered with leucovorin) and sulfadiazine, or in the case of sulfonamide allergy, clindamycin or azithromycin or clarithromycin. Atovaquone targets cytochrome c and inhibitors of cytochrome c appear to affect cysts in animal models, at least for short times, although resistance or lack of efficacy have been described for other organisms and

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TABLE 4.2 Treatment of toxoplasmosis in different clinical settings.

a In the United States, available only on request from the USA Food and Drug Administration (telephone number 301 443-5680), and then with this approval by the physician’s request to Aventis (908-231-3365). b Adjusted for granulocytopenia; complete blood counts, including platelets, should be monitored each Monday and Thursday. c Both regimens, a higher and a lower dose, appear to be feasible and relatively safe. The duration of therapy is unknown for infants and children, especially those with AIDS. d Alternative medicines for patients with atopy or severe intolerance of sulfonamides have included pyrimethamine and leucovorin with clindamycin, or azithromycin, or atovaquone, with standard dosages as recommended according to weight. In the unusual circumstance that medicines cannot be administered orally or by intraintestinal tube feeding, trimethoprim, sulfamethoxazole, and clindamycin have been administered intravenously. e Corticosteroids should be used only in conjunction with pyrimethamine, sulfadiazine, and leucovorin treatment and should be continued until signs of inflammation (high CSF protein, $ 1 g/dL) or active chorioretinitis that threatens vision have subsided, usually B10 14 days; dosage can then be tapered and the steroids discontinued. f Image figure from Benevento et al. (2008, with permission). *However, in epidemics in Brazil, 10% of infected persons have had retinitis at 1 month and 20% at 1 year. In infections in the Maroni River and other Amazon tributaries, acute infections with hypervirulent parasites may be sever and, in that case, should be treated at 1 year, suggesting that perhaps this type of acute infection might best be treated. Adapted from Remington, J.S., McLeod, R., Thulliez, P., Desmonts, G., 2011. Toxoplasmosis. In: Remington, J., Klein, J. (Eds.), Infectious Diseases of the Foetus and Newborn Infant, seventh ed. WB Saunders, Philadelphia, PA, with permission.

4.3 Treatment

recurrences have been described in persons with AIDS during atovaquone treatment. In general, the medicines to treat toxoplasmosis are active against the rapidly replicating tachyzoite stage and have no demonstrated, or little, efficacy against established tissue cysts; therefore patients treated for toxoplasmosis will have a latent infection at the conclusion of treatment of their active disease. Pyrimethamine is a substituted phenylpyrimidine that is an inhibitor of dihydrofolate reductase. The serum half-life of pyrimethamine is 35 175 hours and serum levels on a dose of 1 mg/kg/day in infants (B25 75 mg for normal adult) range from 1000 to 4000 ng/ mL (McLeod et al., 1992). Serum levels for an individual are not completely predictable due to the wide variation in absorption and serum half-life perhaps reflecting differences in metabolism. Phenobarbital induces the enzymes that degrade pyrimethamine and thus have been associated with lower levels. Other medicines that can alter metabolism include theophylline. CSF levels of pyrimethamine are 10% 25% of the corresponding serum levels (Fig. 4.10). Dose-related bone marrow suppression primarily with neutropenia may develop. Anemia and thrombocytopenia have only been rarely reported. Leucovorin (folinic acid) is routinely given at a dose of 10 mg/day, MWF or more often, orally, to prevent these effects. Folinic acid does not inhibit the action of pyrimethamine on T. gondii, as the parasite cannot take up folinic acid at the concentrations achieved in serum. The parasite can take up folate (folic acid) that bypasses the effect of inhibitors of dihydrofolate reductase (DHFR) synthesis or other upstream enzymes. Sulfonamides inhibit dihydropteroate synthetase, which is another enzyme involved in folate synthesis. Thus sulfonamides are synergistic with pyrimethamine. Pyrimethamine and sulfadiazine, together, are eightfold more active than either compound alone. These medicines are well absorbed with good penetration

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into cerebrospinal fluid. Adverse reactions such as hypersensitivity (McLeod et al., 2006b) to sulfonamides are relatively common, particularly in AIDS patients. Bone marrow suppression is seen and this responds to folinic acid or, when severe, withholding medicines for short periods of time. Hypersensitivity reactions with rash, or less frequently, Stevens Johnson syndrome as well as renal stones have also been reported. Sulfamethazine and sulfamerazine (along with sulfadiazine) in triple sulfonamides are highly active. Other sulfonamides are less active and, when they are used in established combinations, the ratios are suboptimal for both the DHFR and dihydropteroate synthetase component, although trimethoprim sulfamethoxazole (TMP SMX) was useful for suppressing recurrent retinal disease, but is less effective in tissue culture and murine models than pyrimethamine and sulfadiazine and is not the treatment of choice for active disease (Silveira et al., 2002).

4.3.1 Asymptomatic infection or latent infection There are currently no effective treatments tested in humans that can eliminate chronic, latent infection. Thus immune-competent individuals with latent toxoplasmosis, as evidenced by positive serology, cannot be treated to completely eradicate infection. For persons with AIDS who are seropositive for T. gondii, the risk of developing encephalitis has been estimated at 10% 50%. TMP SMX, or dapsone plus pyrimethamine, is effective in preventing Toxoplasma encephalitis (Bozzette et al., 1995; Torres et al., 1993). In cardiac transplantation, prophylaxis with pyrimethamine for 6 weeks is used for T. gondii seronegative recipients receiving hearts from seropositive donors (Wreghitt et al., 1992). This was recently demonstrated to be effective in a small series from Brazil (Strabelli et al., 2012).

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(Continued).

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FIGURE 4.10

(Continued).

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FIGURE 4.10 Treatment of congenital toxoplasmosis in the United States and outcomes. (A) Design of the NCCCTS. (B) Treatment for toxoplasmosis. Top: Toxoplasmosis medications for infants. Suspended in 2% sugar solution. Suspension at usual concentration must be made up each week. Store refrigerated. Middle: Pyrimethamine serum levels (4 and 24 h after a dose) of children given 1 mg of pyrimethamine per kg daily. Bottom: Conclusions concerning administering medication to treat congenital toxoplasmosis. (C) Detectable effects of treatment for congenital toxoplasmosis in the NCCCTS Phase II RCT, 1991 2012. (D) Kaplan Meier plots showing the outcomes for each endpoint for NCCCTS patients in the pooled feasibility/observational phase and the randomized phase. Patients received either treatment 1 (solid line) or treatment 2 (dotted line). There is no visible trend for superiority or statistically significant superiority at this time. IQ, Intelligence quotient; Rx1, treatment arm 1; Rx2, treatment arm 2. (E) Endpoint outcomes and parasite serotype of persons with congenital toxoplasmosis treated in the NCCCTS cohort and untreated in literature data. (F) Manifestations at birth for newborns with in utero treatment, NCCCTS. (G) Endpoint outcomes based on treatment group, NCCCTS. (H) Resolution of hydrocephalus and calcifications is associated with shunting and treatment, NCCCTS. Part 1: CT of the brain

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4.3.2 Acute/acquired toxoplasmosis

L

In immune-competent individuals, treatment is rarely considered standard of care. However, in a rare patient whose symptoms are persistent and disabling, treatment should be as described for disseminated disease. Myocarditis, encephalitis, a sepsis-syndrome with shock and hepatitis, and pneumonia are occasionally seen. This is especially notable in South American toxoplasmosis. The standard dose for a normalsized adult would be approximately as follows:

pyrimethamine (100 mg loading dose divided BID for 2 days and, then beginning on the third day, 50 mg/day) and sulfadiazine (3 4 g/day given in divided doses, i.e., 1.5 2 g BID). For immune-competent persons, this is continued for 1 week after resolution of signs and symptoms. Dosages for children are adjusted accordingly by weight. Leucovorin (10 mg daily or administered less frequently, e.g., on MWF) should also be given while pyrimethamine is administered and in the week after it is discontinued, due to the long half-life of this drug.

at birth (A), showing development of hydrocephalus at 3 months of age with treatment (B), and at 1 year of age (C). This child had normal development. Part 2: CT of the brain at 3 months of age, showing hydrocephalus (A), at 4 months of age, after shunt placement (B), and at 8 years of age (C). This child had normal development. Part 3: CT of brain at 2 months of age before shunt placement (A), at 4 months of age after shunt placement (B), and at 14 months of age after shunt placement (C). Note the marked increase in the size of cortical mantle. This child had normal development. Part 4: CT of the brain at 1 year of age. This patient appeared normal at birth but meningoencephalitis developed and was untreated until 3 months of age. At this age, hydrocephalus and bilateral macular chorioretinitis led to the diagnosis of congenital toxoplasmosis and initiation of treatment. Note the significant residual atrophy and calcifications. This child experienced substantial motor dysfunction, developmental delays, and visual impairment. Part 5: MRI of patient at 9 months of age demonstrating changes likely due to perinatal anoxia and hypoglycemia. Such complications of toxoplasmosis and delays in shunting have been associated with the most severe sequelae. Part 6: CT of the brain at birth (A) with areas of hypolucency, mildly dilated ventricles and small calcifications, and at 1 year of age (B) with normal findings except for two small calcifications. This child had normal development. Part 7: CT of brain at birth (B) and at 1 year of age (C). Note the growth of cortex and resolution of encephalomalacia. There were no new calcifications nor an increase in the size of calcifications. Part 8: CT of the brain of a treated infant (left) and at 1 year follow-up (right). Note the diminution and/or resolution of the calcifications indicated by the arrows. Part 9: CT of the brain of a treated infant (left) and at 1 year follow-up (right). Note the diminution and/or resolution of the calcification indicated by the arrow. CT, Computed tomography; MRI, magnetic resonance image; National Collaborative Chicago-Based, Congenital Toxoplasmosis Study (NCCCTS) Source: (B) Image from (Top) McAuley, J., Boyer, K.M., Patel, D., Mets, M., Swisher, C., Roizen, N., et al., 1994. Early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis: The Chicago Collaborative Treatment Trial. Clin. Infect. Dis. 18, 38 72, with permission; (Middle) McLeod, R., Mack, D., Foss, R., et al., 1992. Levels of pyrimethamine in sera and cerebrospinal and ventricular fluids from infants treated for congenital toxoplasmosis. Antimicrob. Agents Chemother. 36, 1040 1048, with permission. (C) Data from McLeod et al., unpublished, with permission. (D) Data from McLeod, R., Khan, A.R., Noble, G.A., et al., 2006a. Severe sulfadiazine hypersensitivity in a child with reactivated congenital toxoplasmic chorioretinitis. Pediatr. Infect. Dis. J. 25 (3), 270 272, with permission. Image and caption verbatim from McLeod, R., Kieffer, F., Sautter, M., Hosten, T., Pelloux, H., 2009. Why prevent, diagnose and treat congenital toxoplasmosis?. Mem. Inst. Oswaldo Cruz 104, 320 344, with permission. (E) Image from McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Toxoplasmosis Study Group. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981 2009). Clin. Infect. Dis. 54 (11), 1595 1605, with permission. (F) Table and caption from McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Toxoplasmosis Study Group. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981 2009). Clin. Infect. Dis. 54 (11), 1595 1605, with permission. (G) Table and caption from McLeod, R., Boyer, K. M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Toxoplasmosis Study Group. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981 2009). Clin. Infect. Dis. 54 (11), 1595 1605, with permission. (H)Images of Pt 1 7 and descriptions from McAuley, J., Boyer, K.M., Patel, D., Mets, M., Swisher, C., Roizen, N., et al., 1994. Early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis: The Chicago Collaborative Treatment Trial. Clin. Infect. Dis. 18, 38 72, with permission; Images of Pt 8 9 and descriptions from Patel, D.V., Holfels, E.M., Vogel, N.P., et al., 1996. Resolution of intracranial calcifications in infants with treated congenital toxoplasmosis. Radiology 199 (2), 433 440 1996, with permission.

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Infections acquired through a laboratory accident or blood transfusion may be more severe and should also be treated as described above. As in all cases in which pyrimethamine is given, complete blood counts are monitored biweekly during treatment and in the week after it is discontinued when leucovorin is continued. Table 4.2 and Fig. 4.10 summarize the current consensus on the treatment of toxoplasmosis.

4.3.3 Acute/acquired toxoplasmosis during pregnancy There are two approaches and regimens that have been reported to be successful in reducing infection and/or manifestations of infection in newborn infants (Figs. 4.2 and 4.7 and Table 4.2). In the French approach, women are screened pre-pregnancy and/or early in pregnancy, no later than the 11th week of gestation, ideally by the 8th week of gestation. Acutely infected pregnant women are given 3 g/day of spiramycin divided three times a day once maternal infection is suspected or diagnosed to decrease transmission prior to the 18th week of gestation if there is no evidence of fetal infection (Remington et al., 2011). Spiramycin is a macrolide antibiotic that has activity against T. gondii due to its ability to inhibit apicoplast function but does not reach sufficient levels in fetal tissue to treat the fetus. Amniocentesis by 18 weeks’ gestation and fetal ultrasonography every 2 weeks should be used to assess infection in the fetus and treatment should be changed to pyrimethamine with folinic acid (leucovorin) and sulfadiazine (PSL) if there is evidence consistent with fetal infection. Ultrasonography should be performed every 2 weeks as ventricular dilation, intracerebral calcifications, necroses, and/or hepatic calcifications (echogenic areas) may develop in as little as 10 days (Remington et al., 2011). Fetal toxoplasmosis may be diagnosed by subinoculation of amniotic fluid into mice, PCR of amniotic fluid to identify presence of T. gondii genes, or

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ultrasonographic evidence of T. gondii infection including ventricular dilatation, hepatosplenomegaly, intrauterine growth retardation, or intracranial calcifications. If this occurs, then specific therapy with pyrimethamine given to the pregnant women (50 mg/day following a loading dose of 50 mg BID for 2 days), sulfadiazine (3 g/day divided BID), and folinic acid (10 mg/day) should be administered to the pregnant woman (Daffos et al., 1988; Remington et al., 2011) until delivery. Pyrimethamine is not used in the first 14 weeks of pregnancy due to concerns about teratogenicity. The majority of infants born to women treated with this regimen had subclinical disease at birth (Fig. 4.7). No group was randomized to receive a placebo because of equipoise. An alternative approach was used by Hotop et al. in Germany and Austria and is beginning to be used in Minas Gerais, Brazil. In this approach (Hotop et al., 2012), spiramycin was given until 14 16 weeks’ gestation, followed by pyrimethamine and sulfadiazine with leucovorin for all patients for 4 weeks and if PCR of amniotic fluid was positive for T. gondii DNA or ultrasound abnormal, then pyrimethamine sulfadiazine is used until birth of the infant and during the first year of life (Table 4.2). Outcomes are reported to appear to be favorable with both the French and German treatment approaches (Figs. 4.7 and 4.10). The more rapidly the treatment is initiated, the better the ocular and neurological outcomes (Kieffer et al., 2008; Cortina-Borja et al., 2010). Mandelbrot et al. (2018) describe 10% less transmission and fewer brain lesions (P , .03) when maternal infections acquired in the second trimester between 14 and 25 weeks are treated with pyrimethamine and sulfadiazine with leucovorin, compared to treatment with spiramycin (Fig. 4.11). None of 73 infants who were treated with pyrimethamine and sulfadiazine from 14 weeks onward and 6/70 (8.6%) who were treated with spiramycin after 14 weeks had cerebral lesions. Thus, continued

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FIGURE 4.11 Prenatal therapy with pyrimethamine 1 sulfadiazine versus spiramycin. (A) Transmission and severity as a function of time in gestation the infection is acquired by the mother. (B) Algorithm for gestational screening. * Ideally screening begins at ,8 weeks (El Bissati et al., 2018). (C) Transmission rate according to treatment and gestational age (Mandelbrot et al., 2018). (D) Gestational outcomes according to treatment. Source: (A) Courtesy Francois Peyron. (D) Data from Mandelbrot, L., Kieffer, F., Sitta, R., Laurichesse-Delmas, H., Winer, N., Mesnard, L., et al. (2018). Prenatal therapy with pyrimethamine 1 sulfadiazine vs spiramycin to reduce placental transmission of toxoplasmosis: a multicenter, randomized trial. Am. J. Obstet. Gynecol., 219(4), 386.e1-386.e9. https://doi.org/10.1016/j.ajog.2018.05.031

treatment with pyrimethamine and sulfadiazine after 14 weeks is now recommended in a substantial number of centers (Mandelbrot et al. (2018); Fig. 4.11D).

4.3.4 Congenital toxoplasmosis Treatment in utero, as described previously, and in infancy has benefits for congenital infection (McLeod et al., 1992, 2000, 2006a, 2009, 2012). Pathology in this infection demonstrates active infection and inflammation that is treated during fetal life and infancy (Ferguson et al., 2013, summarized in Remington et al., 2011) when the immune system is not mature and able to effectively limit this infection. This improvement is apparent when outcomes in

those treated are compared to historical controls (McLeod et al., 1990, 2000, 2006a, 2012; McAuley et al., 1994; Figs. 4.5, 4.8, and 4.10). It has been demonstrated that neonates who appear normal at birth (subclinical disease) may later demonstrate serious sequelae (primarily retinitis) if untreated (Fig. 4.3). Congenital toxoplasmosis is treated in the United States with a loading dose of pyrimethamine of (PYR) 2 mg/kg/day divided into two doses given BID for 2 days followed by 1 mg/ kg/day beginning on the third day and continued for 2 or 6 months. Dose is then decreased to 1 mg/kg every MWF for the remainder of the first year of life. In addition, sulfadiazine at 100 mg/kg/day in two divided doses and folinic acid 10 mg daily or MWF are administered

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throughout the year. Corticosteroids (1 mg/ kg/day) are added for patients who have active macular chorioretinitis or visionthreatening chorioretinitis involving the posterior pole of the eye or CSF protein greater than 1 g/dL. Table 4.2 summarizes treatment regimens. The formulations of medicines are summarized in Remington et al. (2011) and McLeod et al. (2012, 2013a in press, 2013b in press). As no pediatric solutions are made, a suspension is prepared for each medicine using the available tablets (Fig. 4.10). Medicine should be made fresh each week. Most children have an absolute neutrophil count of B1000 neutrophils/mm3 throughout the year of treatment. In the United States, CBCs are monitored biweekly using heel stick to obtain the 0.5 mL blood samples required for this test. Fansidar is not used in the United States because of concerns about serious adverse reactions with the long half-life of the sulfadoxine, and a suboptimal ratio of pyrimethamine and sulfadoxine. However, reports indicate good long-term outcomes and apparent lack of toxicity with treatment with this drug after a pre- and postnatal course of pyrimethamine and sulfadiazine for several months (Peyron et al., 2011; Wallon et al., 2013). In Paris, pyrimethamine and sulfadiazine have been utilized as in the United States (Kieffer et al., 2008; McLeod et al., 2009). This regimen is now also used in Italy. Rapid diagnosis and initiation of treatment appear to result in better ocular and central nervous system outcomes in infected children. With prenatal treatment followed by postnatal treatment in Paris, rapid diagnosis and initiation of medicines were associated with less retinal disease later (Kieffer et al., 2008). Shunting for hydrocephalus is an important adjunctive measure and it should be performed expeditiously (McLone et al., 2019; Fig. 4.12). Normalization of brain parenchyma may occur but it is not possible to predict whether this will occur based on initial appearance, as expansion of cortex can occur with less than 1 mm of cortex initially. The approach to

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aqueductal obstruction should not be a third ventriculostomy (Cinalli et al., 1999), but rather ventriculoperitoneal shunt. Third ventriculostomy often fails because of the associated inflammatory process (Renier et al., 1988).

4.3.5 Ocular toxoplasmosis Pyrimethamine (with leucovorin: folinic acid) and sulfadiazine (PSL) is the gold standard treatment that has been found to be effective in decreasing retinitis including its time course, subsequent retinal destruction, and the inflammatory response (see Chapter 5 "Ocular disease due to Toxoplasma gondii"). This was shown in a placebo trial by Perkins published in 1956 (Perkins et al., 1956). It has been confirmed by direct observation of rapid response of lesions in persons studied by the NCCCTS (e.g., Fig. 4.5). In vitro and in murine models TMP SMX is less effective than PSL (Grossman and Remington, 1979). This also appears anecdotally to be the case in treating recurrent retinal activity within the NCCCTS. For example, following 1 year of recurrences with severe vitritis and active lesions treated with repeated courses of TMP SMX, a 60-year-old patient was treated with PSL and tapering course of prednisone. In contrast to the previous year with worsening symptoms and signs while being treated with TMP SMX, within 4 weeks her vitritis resolved and her active lesions had become quiescent. Nonetheless, in Brazil, suppression with TMP SMX following treatment and resolution of active lesions was demonstrated to be effective (Silveira et al., 2002) although associated with hypersensitivity in a substantial percentage of patients. Since TMP SMX appears less effective for treatment but poses a similar risk for hypersensitivity as sulfadiazine, pyrimethamine and sulfadiazine are the preferred treatment. Other medicines such as pyrimethamine with azithromycin also appear to be effective. Suppression with azithromycin or clindamycin following treatment of active disease also anecdotally appears to be associated with lack of recurrence.

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FIGURE 4.12 Treating hydrocephalus secondary to congenital toxoplasmosis can lead to favorable outcomes. (A) Distribution of anatomical patterns of hydrocephalus (McLone et al., 2019). (B) IQ score and GMFCS level by anatomy of hydrocephalus (McLone et al., 2019). (C) Ventriculoperitoneal shunt outcomes by IQ score and GMFCS level (McLone et al., 2019). (D) Time interval from diagnosis of hydrocephalus to surgical intervention and associated outcomes by IQ score and GMFCS level (McLone et al., 2019). GMFCS, Gross Motor Function Classification Scale. Source: Figure and legend adapted with permission from JNS Publishing Group. Toxoplasma Gondii

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There appear to be fewer recurrences of retinal disease in children treated in utero and/or in the first year of life with PSL than in a cohort of persons diagnosed postnatally after the first years of life and not treated during infancy, even though these untreated children presumably had milder disease at birth that had not been recognized (Fig. 4.5G). With postnatal treatment for 1 year, B10% for those with mild disease at birth and B30% for those with moderate/severe disease at birth had reactivation of retinal disease that responded quickly to retreatment. Reactivations occur most often around the age of school entry, adolescence, and times of considerable stress (Phan et al., 2008a,b; Fig. 4.5). Corticosteroids (prednisone 1 mg/kg/day for an infant or child usually for B10 14 days) are indicated if the macula, optic nerve head, or papillomacular bundle are involved. When prednisone is given, it is tapered when vitritis or macular edema resolves. Azithromycin, clindamycin, and atovaquone (Derouin, 2001; Meneceur et al., 2008) are second-line medicines used when there is sulfonamide hypersensitivity. Azithromycin has been found to be synergistic with pyrimethamine (Derouin et al., 1992), but atovaquone has been found to be antagonistic with pyrimethamine (Romand et al., 1993). The azithromycin dose is 250 mg/day (first day’s loading dose is 500 mg/day) for adults and dose adjusted according to weight for children. Clindamycin (1200 mg/day) has been used as an alternative drug to sulfadiazine, but it was inferior to PSL (Tabbara and O’Connor, 1980). In the NCCCTS’s experience, lesions responded more slowly to second-line medicines than PSL and a recent comparative study confirmed this observation. A monthly, intraocular injection of toxoplasmic antibody to VegF (Lucentis) has been associated with prompt resolution of CNVMs secondary to T. gondii infection (Benevento et al., 2008; Fig. 4.5; Table 4.2). CNVMs due to

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T. gondii respond to Lucentis (antibody to VEGF) (Fig. 4.5; Table 4.2). Use of anti-VegF for other retinal diseases in infants has been described (Capone, 2006).

4.3.6 Toxoplasma infection in immunecompromised persons The same PSL treatment is used for T. gondii encephalitis, or other significant manifestations of disease, in the setting of immune dysfunction. The major difference is the duration of treatment. In the absence of immune reconstitution, treatment is continued to prevent relapse of infection, but if immune reconstitution occurs (i.e., in AIDS patients a CD41 T-cell count above 200) then therapy can be discontinued as it would be for an immune-competent host. For a normal-sized adult the dose of pyrimethamine is 100 mg as a loading dose (divided BID for 2 days) followed by 50 mg/day beginning on the third day with sulfadiazine 3 4 g BID (e.g., 1.5 2 g every 12 hours) and folinic acid 10 mg/ day (Liesenfeld et al., 1999). Therapy is often started empirically and response is expected within 14 days. If no resolution occurs, then brain biopsy may be required for diagnosis. In patients intolerant to sulfadiazine, clindamycin 600 1200 mg every 6 hours can be used with pyrimethamine (Remington et al., 1991). Alternative combinations with reported efficacy in case reports include pyrimethamine with one of the following: clarithromycin or azithromycin 1000 mg every day in an average size adult or dapsone. Desensitization to sulfadiazine has been reported to be successful. Corticosteroids are often used to control elevated intracranial pressure. Corticosteroids should never be used alone and should only be used if anti T. gondii medicines are given at the same time. In about 30% of patients, relapse of encephalitis occurs when treatment is stopped if the reason for immune-suppression remains, although relapse may not be evident for several weeks.

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Treatment is continued for several weeks after resolution of abnormalities. Patients may need to be maintained on pyrimethamine 25 50 mg/ day, sulfadiazine 3 4 g/day (divided BID), and folinic acid 10 mg/day after they have completed a treatment course if the cause of immunesuppression remains. With the use of antiretroviral therapy, if the CD41 is restored to over 200, secondary prophylaxes can be discontinued.

4.3.7 Future development of newer improved anti T. gondii agents Newer potential anti T. gondii compounds have been described (Harbut et al., 2012; Kavitha et al., 2012; Mahamed et al., 2012; Johnson et al., 2012; Martins-Duarte et al., 2012; Munro and Silva, 2012; Chew et al., 2012; Andrews et al., 2012; Lee et al., 2011; Barbosa et al., 2012; Lai et al., 2012; Camara et al., 2012; Doggett et al., 2012; Fomovska et al., 2012). Newer agents on the horizon may include a new echinoquinone that targets cytochrome b/c, as atovaquone (which is not synergistic with pyrimethamine) does, and eliminates cysts in an animal model (Doggett et al., 2012; McPhillie et al., 2016; McPhillie et al., in submission 2020) and a new triazine, as active alone as the synergetic combination of pyrimethamine and sulfadiazine (Mui et al., 2008). Antisense treatments with molecular transporters and small molecule inhibitors of other essential molecular targets also are being developed (Lai et al., 2012; Samuel et al., 2003; Lykins et al., 2018a,b). Rothbard, Wender, and Kumar found that molecular transporters can bring molecular cargo from the outside of the eye to the retina and across the blood brain barrier, and McLeod et al. found that they carried molecular cargoes into encysted bradyzoites (Samuel et al., 2003), but they have not yet been utilized for this infection in humans. Among the most promising findings that might lead to new antimicrobial agents include

PPMO targeting enoyl reductase, the Apetela 2 proteins, echinoquinone targeting cytochrome c, more highly active derivatives of azithromycin, calcium kinase inhibitors, new DHFR inhibitors, and a triazine (Lourido et al., 2013; Welsch et al., 2016; Stec et al., 2013; Mui et al., 2008; Vidadala et al., 2016). CRISPR mutations have revealed new molecular targets in T. gondii tachyzoites and during stage switch including a Master Regulator of Differentiation, BFD1 (Sidik et al., 2016; Waldman et al., BioRx, 2019; Fig. 4.13). An ATPase, and enzymes in the plant-like vacuole of Carruthers and Moreno are also novel targets, an ATPase sensitive to a spiroindolone being produced by a pharmaceutical company currently (Zhou et al., 2014) and many other molecular targets including essential unique organellar constituents become tractable targets both with small molecule or with antisense inhibitors (e.g., Lykins et al., 2018a,b).

4.4 Prevention Methods of avoiding this infection include cooking meat to “well done” before ingesting, washing fruits and vegetables before they are consumed (Koletzko et al., 2012), wearing gloves while gardening, avoiding contact with materials excreted by cats and avoiding raw shellfish such as mussels. There are studies concerning detection (Gallas-LIndemann et al., 2013) and disinfection of oocysts (Dumetre et al., 2008) and irradiation of meat to inactivate bradyzoites in cysts. Screening to determine whether a pregnant woman is seronegative or positive for T. gondii infection during gestation can be used to determine risk of infection as well as to diagnosis infections with a potential for congenital transmission. Prenatal screening and prompt treatment of the infected fetus and infant have improved outcomes in a number of studies. Prophylaxis of seropositive patients with immune

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dysfunction or recurrent active retinal disease can prevent reactivation of latent infection. There are currently no vaccines to prevent infection or disease in humans, but work and discovery of T. gondii peptide epitopes that bind to human HLA antigens and stimulate human CD81 and CD41 T cells and

antibodies to proteins critical for invasion or establishing infection are being optimized for delivery and protection with adjuvants such as GLA-Se. They may ultimately lead to protective preparations for humans (Fig. 4.14). Replicons and live vaccines have also conferred protection.

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4.5 Other considerations of pathogenesis in human infections In this chapter the subject thus far has been the conventional understanding of toxoplasmosis as a human disease and how

to prevent and treat it. We now address what might happen to some or all of 2 billion people who are chronically infected across their lifetimes. This next section addresses how we can gain further knowledge of the human host and parasite

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FIGURE 4.13 Novel Therapeutic Targets in and/or Treatments for Toxoplasma gondii moving toward clinical use. (A) Efficacy of pyrazolopyrimidines inhibitors of CDPK1 (Rutaganira et al., 2017). (B) Calcium Kinase Inhibitors are effective in Toxoplasma-infected mice, showing decreased parasite count and fewer cysts (Rutaganira et al., 2017). (C) Reduction in number of parasites with novel Triazine in vivo (Mui et al., 2008). (D) IC50 of novel triazine compared to Pyrimethamine against DHFR enzyme activity. Toxicity has limited further development (Mui et al., 2008). (E) Inhibition curves and binding site for DHFR inhibitors (Welsch et al., 2016). (F) Efficacy of experimental quinolones against T. gondii infection in mice compared to atovaquone Stone (Doggett et al., 2012). (G) Number of cysts per brain following treatment by atovaquone and experimental quinolones Stone (Doggett et al., 2012). (H) X-ray crystallography of MJM170 (McPhillie et al., 2016). (I) MJM170 reduces tachyzoites in tissue culture at low concentrations (McPhillie et al., 2016). (J) MJM170 eliminates cysts in EGS infected human foreskin fibroblast (McPhillie et al., 2016). (K) Intraperitoneal administration of MJM170 at 25 mg/kg daily cures active infection and decreases burden of Type II parasites (McPhillie et al., 2016). (L) Type II parasite count is reduced in infected mice following 17 days of treatment with MJM170 with 12.5 mg/kg daily (McPhillie et al., 2016). (M) VivoPMO and RNA interaction (Lykins et al., 2018a,b). (N) DHFR specific PPMO decreases immunofluorescent parasites. Red indicates antibody to DHFR staining (Lai et al., 2012). (O) Parasite burden can be decreased with DHFR PPMO in vivo (Lai et al., 2012). (P) vivoPMOs are effective knockdown agents and do not show significant toxicity at the concentrations used (Lykins et al., 2018a,b).

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interface, new information about possible interactions between host and parasite in acute, subacute and chronic infectons, and consequences of infection. This new information raises questions about pathogenesis of this human infection and its consequences (Figs. 4.15 4.17).

4.5.1 Recent studies of clinically identified associations of human brain or other diseases and presence of Toxoplasma infections (seropositivity) There are now many associations reported between seropositivity and a variety of

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FIGURE 4.14 Recent strategies and developments in creation of immunosense vaccines for humans using human cells and Mice with human transgenes (HLA Class I genes). (A) PBMC from donors who were seropositive and seronegative for Toxoplasma gondii were tested for response to predicted HLA-A03-restricted CD81 T-cell epitopes. Individual peptides were tested using IFN-γ ELISpot assay (El Bissati et al., 2016). (B) T. gondii brain cysts number was significantly reduced in HLA-A*11:01 mice immunized with pool of peptides plus Pan DR-Binding Epitope (PADRE) and GLA-SE adjuvant at 21 days after challenge with 2000 T. gondii ME49-Fluc (Type II) parasites (El Bissati et al., 2016). (C) Flow cytometry gating for CD81 memory T cells 11 weeks after immunization of HLA-A*11:01 mice with pooled adjuvanted peptides. Cells are gated on CD31CD81 T cells (El Bissati et al., 2016). (D) The orientation of the HLA-A*11:01-restricted CD81 T-cell

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behaviors and diseases (e.g., Flegr, 2007; Torrey and Yolken, 2019). These remain, for the most part, associations where cause and effect, and chronology are not determined or proven. In the large data analysis of Lykins et al. (2016) that uses CPT codes, there are odds ratios associating Toxoplasma diagnoses and treatments with malignancies, pregnancy, HIV,

epilepsy, autoimmune diseases, and psychiatric disorders including substance abuse and schizophrenia. Some of these are simply describing the circumstances in which this infection is a clinical problem, but others suggest that there may be associations when very large databases are examined that are not apparent on an individual scale (Lykins et al.,

epitopes and PADRE in the synthetic gene is shown with 2 different types of spacers, called LO and AZ, for N/KAAA and GPGPG linker, respectively (El Bissati et al., 2016). (E) SDS PAGE 4% 20% of the purified LO and AZ proteins (El Bissati et al., 2016). (F) ELISpot showing IFN-γ spot formation. PBMCs were tested using LO, AZ, and pool of peptides (El Bissati et al., 2016). (G) Two weeks after the last immunization, the transgenic mice immunized with pooled LO and AZ proteins in combination with either adjuvant GLA-SE or ALUM adjuvant or injected with phosphate buffered saline (PBS) were infected with 2000 Me49 parasites. The survival rates of the 2 groups were recorded (El Bissati et al., 2016). (H) Enumeration of cysts was performed with brains of mice challenged 21 days after final immunization. These experiments were performed at least two times, and one representative experiment of 2 is shown (El Bissati et al., 2016). (I) Splenocytes from immunized mice with LO DNA, AZ DNA, and multiepitope polypeptides alone or combined were harvested for 10 14 days after immunization and exposed to LO or AZ polypeptide for ex vivo IFN-γ expression (El Bissati et al., 2016). (J) HLA-A*11:01 transgenic mice survival curve after challenge with Type II parasites. Two weeks after the last immunization, the transgenic mice immunized with empty vector, LO DNA 1 LO polypeptide, or LO polypeptide, or were injected with PBS were infected with 2000 T. gondii ME49-Fluc (Type II) parasites (El Bissati et al., 2016). (K) CD81 memory T cells. Flow cytometry gating for CD81 memory T cells. Spleen cells are gated on CD31CD81 T cells. Memory T cells were defined as CD44hiCD45RBlo. For each group, a representative FACS plot is shown with the percent of CD81 memory T cells shown. One-way ANOVA was performed before the Student t test to determine whether there was an overall difference between the groups (El Bissati et al., 2016). (L) HLA-A*11:01 transgenic mice immunized with LO protein plus GLASE were protected compared with control mice inoculated with PBS when they were challenged with 20,000 T. gondii prugneaud strain (Fluc) luciferase expressing parasites after 4 and 6 days (El Bissati et al., 2016). (M) Assay demonstrating that GLA-SE is a TLR4 ligand that leads to production of IL-6, IL-12, and TNF-α by PBMC. Stimulation of human whole blood with GLA-SE (El Bissati et al., 2016). (N) Multiepitope proteins with GLA-SE are captured by the APCs, and the peptides contained are presented by MHC molecules on the APCs to T lymphocytes in both a class I and a class II pathway (El Bissati et al., 2016). (O) Phylogenetic tree showing 62 genetic isolates of Toxoplasma analyzed herein. These are in the multisequence alignments of proteins, and peptides derived from them, utilized to create our artificial immunogenic (smart) protein (El Bissati et al., 2017). (P) Flagellin is used as a scaffold into which epitopes are intercalated from Toxoplasma. Earlier logic for inclusion of flagellin as adjuvant and scaffold came from work with malaria (El Bissati et al., 2017). (Q) SDS PAGE of the purified protein. Lanes are as follows: Lane 1: MW (molecular weight markers). Lane 2 5: Elution fractions were from 19 to 22 (El Bissati et al., 2017). (R) Transmission electron microscopy of the nanoparticle preparation (El Bissati et al., 2017). (S) SDS PAGE 4% 20% of the purified protein. Lane 1: MW (molecular weight markers). Lane 2: CD8SAPN. Lane 3: Empty-SAPN. Samples derive from the same experiment and the gels/blots were processed in parallel (El Bissati et al., 2017). (T) CD8-SAPNs elicit restricted CD81 T and CD41 T-cell peptide-specific immune response. ELISpot showing IFN-γ spot formation (El Bissati et al., 2017). (U) Graph shows the count of spots for splenocytes of untreated, Empty-SAPN 1 GLA1 CD8-SAPN 1 GLA-SE group of mice. GLA designates GLA-SE in this figure (El Bissati et al., 2017). (V) T. gondii brain cysts luciferase expression was significantly reduced in HLA-A*1101 mice immunized with CD8SAPNplus GLA boost at 21 days after challenge with 2000 Me49 (Fluc) T. gondii expressing luciferase (El Bissati et al., 2017). (W) Xenogen imaging of brain ex vivo following the injection of luciferin into the retro-orbital plexus and then exposure of the brain to luciferin solution. This figure shows data from mice in one of the replicate experiments (n 5 4 control and 4 immunized mice) (El Bissati et al., 2017). (X) Enumeration of cyst was performed with brains of mice challenged 21 days after final immunization. SAPN reduced cyst numbers and luminescence (P , .05) (El Bissati et al., 2017). (Y) SAPN adjuvanted with GLA-SE have peptides that are presented by MHC molecules on the follicular dendritic cells to T lymphocytes. GLA-SE and flagellin are ligands of TLR-4 and TLR-5 receptors, respectively (El Bissati et al., 2017). APCs, Antigenpresenting cells; PBMC, peripheral blood mononuclear cell.

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FIGURE 4.15 Example of gene analyzed in NCCCTS that are associated with altered function: ALOX 12 and susceptibility to congenital toxoplasmosis. (A) Hap map showing single nucleotide polymorphisms. (B) Knockdown of ALOX12 alters level of arachodonic acid. (C) Effect of knockdown on Toxoplasma. (D) Effect of knockdown on Toxoplasma. (E) Effect of knockdown on Toxoplasma over time. (F) Effect of knockdown IL-1β, on IL-6, TNF-α, and caspase 1. (G) Structure showing mutations that alter function (red). NCCCTS, National Collaborative Chicago-Based, Congenital Toxoplasmosis Study. Source: From Witola, W.H., Liu, S.R., Montpetit, A., Welti, R., Hypolite, M., Roth, M., et al., 2014. ALOX12 in human toxoplasmosis. Infect. Immun., 82(7), 2670 2679. https://doi.org/10.1128/IAI.01505-13.

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FIGURE 4.16 Mutations that alter function: mother and child with toxoplasmosis with activated PI3-kinase δ syndrome type II (APDS2) and other gene mutations from the literature that result in illness. (A) Clinical and laboratory features in mother and daughter with toxoplasmosis and APDS2. (Left) Brain CT in the patient’s daughter at 3 months of age, showing marked hydrocephalus with enlarged lateral and third ventricles, profound brain atrophy and basal ganglia calcifications. (Middle) Chromatogram demonstrating heterozygosity for the c.1425 1 1g . a at the PIK3R1 locus in the patient and her daughter. (Right) Analysis of phospho-S6 in CD201 cells from a healthy control, the mother, and the daughter at resting conditions (top) and upon in vitro activation with anti-IgM (bottom) (Karanovic et al., 2019). (B) Lymph node histopathology in the mother with APDS2. (Top left) Hematoxylin and eosin stained section shows an ill-defined secondary follicle with “naked” germinal center; bottom left shows a cluster of monocytoid cells with pale cytoplasm (magnification, 10 3 ). In the inlet, cytomegalovirus-positive cells are identified by immunohistochemistry (magnification, 40 3 ). (Top middle) Double immunohistochemistry staining for CD41 (in brown) and CD81 (in red) T cells (magnification, 10 3 ). (Top right) Immunohistochemistry staining for PD-1 highlights numerous T-follicular helper cells within the germinal centers (stronger expression) as well as in the interfollicular areas (magnification, 10 3 ). (Bottom row) Immunohistochemical stains for CD201 (bottom left), IgM1 (bottom middle), and IgG1 cells (bottom right) showing a marked increase of IgM-positive plasma cells over IgG (magnification, 4 3 ) (Karanovic et al., 2019). (C) Mechanisms of macrophage-mediated response against Toxoplasma, and effects of increase PI3K signaling. (Left) In immunocompetent hosts, intracellular Toxoplasma infection with tachyzoites within parasitophorous vesicles (blue circle) elicits a macrophage response mediated by NOX4 and p22phox. Expression of the latter is controlled by the FOXO1 transcription factor. Activation of the NOX4/p22phox complex allows generation of ROS, activation of MAPK, and NF-κB signaling, and production of the pro-inflammatory MIF. (Right) In patients with APDS1/2, increased PI3K signaling induces AKT phosphorylation, which in turn mediates phosphorylation of FOXO1, impairing p22phox gene expression (in gray). This causes reduced production of ROS, defective activation of MAPK and NF-κB, and impaired production of MIF (all in gray) in response to Toxoplasma infection. Furthermore, the favorable metabolic environment supported by enhanced PI3K activity promotes intracellular replication of Toxoplasma tachyzoites (Karanovic et al., 2019). (D) Cases of toxoplasmosis in patients with primary immune deficiencies. (Karanovic et al., 2019). MAPK, MAP kinase; MIF, macrophage inhibitory factor; ROS, reactive oxygen species. Source: Figure and legend adapted with permission from Karanovic, D., Michelow, I.C., Hayward, A.R., DeRavin, S.S., Delmonte, O.M., Grigg, M.E., et al., 2019. Disseminated and congenital toxoplasmosis in a mother and child with activated PI3-kinase δ syndrome type 2 (APDS2): case report and a literature review of toxoplasma infections in primary immunodeficiencies. Front. Immunol. 10, 77. https://doi.org/10.3389/fimmu.2019.00077.

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FIGURE 4.17 Genetics, omics, and systems biology: Toxoplasma gondii in the Human Brain and its consequences and effect on human monocytic cells that may defend the brain. (A) Methodology and analyses for understanding interaction of T. gondii with the human brain (Ngoˆ et al., 2017). (B) Susceptibility genetics: expression and localization in human brain (Ngoˆ et al., 2017). (C) Transcriptomics with heat maps showing differentially expressed protein coding and miRNA genes Red and green represent genes over- or under-expressed in infected cells, respectively (Ngoˆ et al., 2017). (D) Functional enrichment analysis of transcriptomics datasets focused on KEGG pathways and GO biological processes (Ngoˆ et al., 2017). (E) Effects of EGS infection on MM6 and NSC transcriptomes (McPhillie et al., 2016). (E1a) Proteins differentially expressed during parasite infection of L-NSC (Ngoˆ et al., 2017). (E1b) Proteins differentially expressed during parasite infection of S-NSC (Ngoˆ et al., 2017). (E1c) Left panel, number of DEPs in S-NSC infected with T. gondii types I, II, and III; right panel, GO biological processes significantly overrepresented (P-value , .01) in the set of 3359 proteins differentially expressed in infected S-NSC compared with their respective uninfected controls (Ngoˆ et al., 2017). (E2a) Serum biomarkers from boys with active brain disease due to T. gondii reflect infection and neurodegeneration (Ngoˆ et al., 2017). (E2b) Changes in serum miRNA concentration between each infected child and corresponding control are expressed as the difference in RT-qPCR Ct-values for miR-124 (Ngoˆ et al., 2017). (E2c) Changes in serum miRNA concentration between each infected child and corresponding control are expressed as the difference in RT-qPCR Ct-values for miR-17, miR-19a, and miR-18b (Ngoˆ et al., 2017). (E2d) Left panel, schematic representation of the genes targeted, and pathways modulated by miRNA clusters 17 92; right panel, peptide abundances from the 10 most intense peptide ions detected by proteomics

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2016; Wing et al., 2016). Furthermore, Lafferty (2006) wrote that Toxoplasma shapes human language and cultures. In mice, aging and immune senescence influence outcomes of toxoplasma infections (Gardner and Remington, 1978; Gardner and Remington, 1978). Anecdotally, in humans, ocular disease appears to sometimes be particularly severe in older persons without other predisposing problems. Odorant receptors and the sense of smell in rodents and chimpanzees can also be influenced by Toxoplasma infection but effect on humans has not been reported (discussed in Ngoˆ et al., 2017). In the work of Kankova et al. (2015), associations with diabetes, antilipid antibodies, and other illnesses

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and behaviors have been described. Again, cause and effect were not established. Anecdotally, there are also reports of Toxoplasma occurring in conjunction with lymphoma in the eye (Sauer et al., 2010). In the Genetics, Omics, Serologic biomarkers and Systems biology analyses below, there were signature pathways of neurodegeneration, epilepsy, odorant receptors, and the largest overlap was with pathways of malignancies (Ngoˆ et al., 2017 and as discussed next). There are studies that present case reports of severe toxoplasmosis in children with calorie/protein malnutrition that are associated with severe disseminated toxoplasmosis with another parasitic infection (cryptosporidiosis).

in the three children pairs. Peptides with higher or lower abundance in ill children compared to healthy controls are depicted above or below the dashed line, respectively (Ngoˆ et al., 2017). (E2e) Bundling of upstream regulators predicted from susceptibility genes and brain biomarkers (Ngoˆ et al., 2017). (F) Reconstruction and deconvolution analyses (Ngoˆ et al., 2017). (G) “Orbital” visualization of the 25 highest statistical valued upstream regulators added to the brain infectome and graphically mapped by IPA (Ngoˆ et al., 2017). (H) Cluster deconvolution uncovers six clusters of protein protein interactions effecting brain functions and circuitry (Ngoˆ et al., 2017). (I) Upstream regulators targeting genes and proteins differentially expressed in S-NSC or L-NSC (Ngoˆ et al., 2017). (J) Gene regulatory network targeted by the 22 common upstream regulators (Ngoˆ et al., 2017). (K) Deconvolution of brain infectome by disease correlation (Ngoˆ et al., 2017). (L) Phenotypes in NSC demonstrating functions that are biologically important empirically (Ngoˆ et al., 2017). (M) Sample characteristics and the results of the questionnaires in relation to T. gondii immune status (Gajewski et al., 2014). (N) Cognitive functions in relation to T. gondii immune status (Gajewski et al., 2014). (O) Cytokine profiling of infected US cohorts (Pernas et al., 2014). (P) Cytokine profiling of infected Colombian cohorts (Pernas et al., 2014). (Q) Schematic of cytokines significantly dysregulated between US acute, US chronic, and Colombian acute cohorts in comparison to uninfected cohorts sampled within the same country (Pernas et al., 2014). (R) Transcriptome analysis identifies CCL2 as a signature response to T. gondii infection (Safronova et al., 2019). (S) S100A11 regulates monocyte recruitment in vivo (Safronova et al., 2019). (T) Role of Caspase-1 in S100A11 release. S100A11 is a CCL2-inducing molecule (Safronova et al., 2019). (U) Average frequency and absolute numbers of monocytes in T. gondii infected WT and S100a11 KO mice on day 5 post infection (Safronova et al., 2019). (V) Survival of WT (black circles) and S100a11 KO (open circles) mice infected with T. gondii (20 cysts per mouse) (Safronova et al., 2019). (X) Peripheral blood monocytes from healthy donors with serological markers of chronic toxoplasmosis (positive) or from noninfected controls (negative) differ in expression levels of CD16 and in percentages of CD62L1 or CD641 cells (Ehmen and Lu¨der, 2019). (W) Monocyte-enriched peripheral blood mononuclear cells from healthy donors with serological markers of chronic toxoplasmosis (positive) express more IL-12b mRNA in response to T. gondii infection in vitro (dark gray bars) than those from noninfected controls (negative) (Ehmen and Lu¨der, 2019). (Y) List of canonical pathways at 6 h postinfection (Syn et al., 2018). (Z) Gene network of concordant genes at 6 h postinfection (Syn et al., 2018). (AA) Example of Western blot of infected and mock-infected WERI-Rb-1 cells stained with anti-APP antibody (Syn et al., 2018). (BB) Compares expression of the 35 kDa band with control β-actin band in this representative WERI-Rb-1 cell experiment (Syn et al., 2018). (CC) Results of quantitative analysis of the Western blots using densitometry (normalized to β-actin) (Syn et al., 2018). (DD) Autophagy initiates host immunity and Toxoplasma immune evasion (Zhu et al., 2019). (EE) Resistance of T. gondii to oxidative stress (Zhu et al., 2019). (FF) Targeting of host gene expression by T. gondii. Effectors secreted by T. gondii manipulate host gene expression (Zhu et al., 2019). (GG) Balancing host cell survival and death. Inhibition of cell apoptosis preserves the intracellular parasite T. gondii (Zhu et al., 2019). DEPs, Differentially expressed proteins.

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It is clear that toxoplasma causes epilepsy in congenitally infected persons. In rodent models, alterations of GABA signaling by altering GAD67 and GLT1 are caused by experimental toxoplasmosis and lead to seizures (Brooks et al., 2015; David et al., 2016; Hermes et al., 2008). Metadata analysis of epilepsy and T. gondii seropositivity demonstrated significant associations with seropositivity (Ngoungou et al., 2015). It has been demonstrated that T. gondii damages the fetal brain and central nervous system of persons who are immune compromised. In addition, studies of cognition of humans who are seronegative versus seropositive lend credence to Toxoplasma’s effect on the brain of persons not recognized to have a predisposition to damage in the brain, or a reason for adverse consequences (Gajewsky, 2014). Inflammatory cells entering the brain may contribute to this damage based on observations in rodent models that affect neurologic function, anatomy, pathology, and synaptic plasticity (e.g., Hermes et al., 2008; Lang et al., 2018). Thus observations of peripheral blood monocytic cells and sera are also relevant to analyzing inflammatory state and damage caused by the parasite. Some association studies suggest that there are real consequences for cognition in human chronic infections (Gajewsky, 2014, Fig. 4.17M and N), but not all studies demonstrate that consistently. As in any association study, cause and effect have not been proven. Wadhawan et al. (2017) have found associations of depression and seropositivity in old order Amish men over the age of 50 years. Associations with psychological disorders have also been found in military personnel (Niebuhr et al., 2008; Duffy et al., 2015). There are multiple studies summarized by Torrey and Yolken (2019) demonstrating associations between Toxoplasma seropositivity and schizophrenia (see Chapter 24: Cerebral toxoplasmosis). Beginning with Flegr et al. (2002), there are reports that seropositivity contributes to

vehicular accidents. More recently, Coccaro et al. (2016) found that seropositivity was associated with road rage and accidents. Entrepreneurial risk taking has also been shown to be associated with Toxoplasma infection. Business school students in Boulder, Colorado, who were uninfected or infected, were tested for risk-taking behavior (Johnson et al., 2018). The infected students had greater risk-taking behavior: they were 1.4 3 more likely to major in business and 1.7 3 more likely to have an emphasis in management and entrepreneurship. Among professionals attending entrepreneurship events, T. gondii positive persons were 1.8 3 times more likely to have started their own business. Infection prevalence was a consistent predictor of entrepreneurship and intentions at the national scale. High seroprevalence countries had a lower fraction of respondents who were afraid of failure in business ventures (Johnson et al., 2018).

4.5.2 Structural and functional neuroimaging in uninfected versus infected persons without recognized clinical symptoms The NCCCTS has begun a study of structural and functional neuroimaging in high-achieving persons with and without T. gondii infection (Yang, Lu, Wu, Clouser, Zhiadong, McLeod et al., 2020 in submission). There appear to be significant differences between these groups with each of the imaging techniques utilized. In infected mice, the following behaviors, among others, have been reported: increased risk taking, increased exploratory behavior, decreased neophobia, and loss of fear of cats (Evans et al., 2014; see Chapter 24: Cerebral toxoplasmosis). This has been attributed to increased amounts of dopamine and changes in the amygdala, and inflammation or damage in the anterior cingulate cortex, and hippocampus (Ihara et al., 2016; Hermes et al., 2008; Xiao, Yolken et al., 2019 in press). Thus persons with and

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4.5 Other considerations of pathogenesis in human infections

without T. gondii infection were studied to test the hypothesis that increased dopamine levels would change function of these areas, measured by fMRI. In the fMRIs, late positive component (LPC) brain function associated with explicit recognition memory and blunted reward-seeking behavior would enhance precision of responses but decrease reward motivation. Indeed, the authors (Stock et al., 2017) results are summarized and paraphrased as follows: healthy young adults who were Toxoplasma seropositive had greatly diminished responses to monetary rewards. This was in comparison with matched uninfected persons. This selective effect reduced Toxoplasma induced speed advantages the investigators had previously observed for nonrewarded behavior; Toxoplasma seropositive persons still were found to be superior to Toxoplasma seronegative persons in their response accuracy. Event-related potential and source localization analyses demonstrated that better rewarded behavior occurred because of increased allocation of processing resources. This was reflected in increased LPC amplitudes. There were also associated activity changes in right temporoparietal junction (BA40) and left auditory cortex (BA41). In summary, seropositive persons had superior performance when circumstances required cognitive control. At the same time, persons with seropositivity had reduced sensitivity to financial motivation. The authors hypothesized that this could be explained by increased levels of dopamine (Stock et al., 2017).

4.5.3 Genetic analyses: candidate human genes in cohort and transmission disequilibrium testing studies 4.5.3.1 National Collaborative ChicagoBased, Congenital Toxoplasmosis Study (sometimes EMSCOT) gestational and congenital toxoplasmosis Human genes have profound influences on outcomes of T. gondii infections (see Roberts

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et al., 2014; Ngoˆ et al., 2017, Table 4.3): They also provide insights into pathogenesis and what is needed for protection. Over time we, in the NCCCTS, have identified susceptibility alleles in studies of families of a child with congenital toxoplasmosis. These are listed with the single nucleotide polymorphisms (SNPs) that identified these genes in Table 4.3 (the listed manuscripts provide the supporting genetic analyses and phenotypic data for these candidate genes). A number of the genetic associations were found in both the US NCCCTS (1981 to present) and EMSCOT. Each of these genes is expressed in the human brain (Fig. 4.17B). Upstream regulators of these genes were identified by Ingenuity Pathway Analysis (Ngoˆ et al., 2017). These genetic variants were used in a systems biology analysis with biomarkers that were identified by proteomics and miR analysis of sera, from ill children, relative to their healthy matched controls. These genetic analyses were considered in the context of transcriptomic and proteomic (Fig. 4.17E) analyses of human primary brain neuronal stem cells and a monocytic cell line infected with various isolates of T. gondii (see below under Toxoplasma and the human brain). Examples of phenotypic analyses defining the actual mechanisms of some of the genes identified were summarized earlier in Roberts et al. (2014) and are also shown in Fig. 4.15 and Table 4.3. The importance of the human Toxo1 region and NALP1 on early killing of Toxoplasma by a peripheral blood mononuclear cell (PBMC) line was discovered and characterized by Witola et al. (2011). The parasite ligands for rodent Nalp 1 have recently been identified by Wang et al. (2019). These ligands include GRA 35 and its associated GRA 42 and 43. Witola et al. also found that this led to a different oxidative state. The mechanisms for allelic variants of the cytoplasmic tail of P2x7r were mapped in the work of Lees et al. (2010). The role of parent of origin effect (i.e., imprinting, thus epigenetic effects) was noted in a study of the COL2a and ABC4R

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TABLE 4.3 Reason for selection of gene to test sequentially as single candidate gene in NCCCTS (1981 201).

Gene

SNP (Allele)

Reason Candidate P Value Gene

HLS Class II

DQ3

,.02

DQ1

,.0005

rs6823 (G)

,.03 (brain)

rs2276455 (A)

,.03

rs2276455 (G)*

,.0005

rs1635544 (C)

,.03

rs2070739 (T)

,.02

rs2276454 (A)

,.007

rs3803183 (T)

,.02

rs3803183 (T)*

,.003

rs952499 (C)

,.03

rs952499 (T)*

,.005

rs2297633 (G)*

,.0003

rs1761375 (G)*

,.0001

rs3112831 (C)*

,.02

rs1621388(C1772T)

,.021

rs1718119(T1068C)

,.015

A

COL2A1

ABCA4

P2RX7

HLA Class I

Replicate/Proof of Principle/Phenotype

MD

Hydrocephalous in children (DQ3), Fewer cysts in HLA transgenic mice (DQ1)

ED

EMSCOT replicates, imprinted, brain and eye disease

HC

EMSCOT replicates, imprinted, localized in human brain

OI

EMSCOT replicates for differing alleles; ATP mediated cell death, cytokine signaling, pro-inflammation

, .01

MD

Genotype association and phenotypes humans and mice. PBMC from cohort. Peptides for HLA A2, A11, B7 confer protection

rs149173(T/C)

,.0077

LfL

Genotype association and phenotypes humans and mice

Rs17481856(C/T)

.0253

rs1461567

,.023

IOID

Genotype; phenotype, cell death, inflammation

rs4251513

,.045

rs8081261

,.002

MD TRNG

rs11652907

,.02

Genotype (MD region; human); phenotype, cell death, inflammation; MD

rs9902174

,.04

rs6502997

,.0003

MD TRNG

Genotype and phenotype, cell death proinflammation

rs6502998 (C)

,.03

rs434473

,.04

B C ERAP1

IRAK4

NALP1

ALOX12

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4.5 Other considerations of pathogenesis in human infections

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TABLE 4.3 (Continued)

Gene

SNP (Allele)

Reason Candidate P Value Gene

TLR9

rs574386 (T1905C)

,.008

rs352140 (C)

,.0001

TIRAP

rs8177374(S180L)

,.006

IOID

Genotype; phenotype TLR signaling and cytokines

FOXQ1

rs920209

,.02

HC

Note NK cells mice

TREX1

rs 2242150(A/T)

.02

SPD

Related clinical and Type I IFN phenotype, LFL

NFκβ1

rs997476(C/A)

,.02

CtoPiEA

Phenotype, nuclear localization, signaling pathway

TGFβ1

rs10417924 (G overtranscribed)

.016

CtoPiEA, MD

Phenotype transcriptomics, GRA1

NOD-2 (Brazil)

rs3135499 (C/A)†

,.04

LfL Brazil

Eye Disease, IL-17, CD41

Replicate/Proof of Principle/Phenotype

TLRs

Brazil and Poland replicates; phenotype, ligand

MD, Murine model date; ED, eye disease in humans caused by mutation of this gene; HC, hydrocephalous caused in humans by gene mutation and adult macular degeneration associated with allelic variants; OI, implicated alleles for other infections; Lfl, logical to test form literature and other findings, for example, MHC Class I presentation of antigen for ERAP1; IOID, gene in other diseases in the literature; MD TRNG, Toxo 1 region not gene initially in humans based on rat Toxo 1 region; TLRs, testing TLR genes now replicated by other cohorts and proven to be important in mice; SPD, similar pattern of disease as AG brain disease due to DNA ligase mutations; CtoPIEA, central to genetic pathway identified with original analysis led to transmission disequilibrium testing analysis herein. Note: Lfl Brazil, Minas Gerais did not replicate in US cohort; no *, significant in NCCCTS not EMSCOP; * significant in EMSCOPT cohort not NCCCTS. P Values are nominal. Supporting Data [SD]: References (#) with narrative summary of findings and gene function for published work in in preparation (AM) or original data in this manuscript (OD) in Fig. 1 or online supplement.

genes (Jamieson et al., 2008). Data from studying the role of ALOX12, in the Toxoplasma 1 region, provide mechanistic insights into this susceptibility gene (Witola et al., 2014; Fig. 4.15). ALOX12 alleles are also associated with risk for schizophrenia, atherosclerosis, and cancer. The lipoxygenase, ALOX12, adds an unstable oxygen moiety to arachidonic acid, making a toxic oxidized lipid that increases inflammation in the brain and other sites, and alters monocyte destruction of T. gondii (Fig. 4.15). 4.5.3.2 Case report and literature review concerning mutations and susceptibility to severe disease when infected with Toxoplasma gondii, and antibody treatments that lead to disease a

Karanovic et al. (2019) (Fig. 4.16) reported case of disseminated and congenital

toxoplasmosis in a mother and child. This mother and child pair had activated PI3-KΔ Syndrome Type II. These authors add to this case report and provide a literature review of Toxoplasma infections occurring in persons with primary immunodeficiencies. PI3-K is integral in lymphocyte function. Mutations in the genes encoding the subunits of PI3K increase its activity and lead to immune dysregulation. PI3-Ks regulate cellular activation, development, and differentiation by affecting the generation of PIP3. Mutations in these genes lead to humoral immune deficiency, elevated IgM, diffuse lymphadenopathy, increased susceptibility to EBV and CMV, increased risk of lymphoma, short stature, increase joint flexibility, teething delay, increased upper respiratory infections, or combinations of these. The mutations identified reveal immune deficiencies and critical

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mechanisms involved in protecting humans against toxoplasma infection. These include defects in the IL-12/IFN-γ pathway involving Tcell receptors, CD-40 ligand/CD-40, and the noncanonical NF-κB signaling pathway; common variable immunodeficiency which involves humoral responses and possibly coexisting cellular deficits; Good Syndrome-Thymoma with combined cellular and humoral immunodeficiency and autoimmunity with mechanisms not well understood; other T-cell immune deficiencies and downstream molecules such as in Omenn syndrome and T-cell immunodeficiency due calcium flux defect; Activated phosphoinositide 3-kinase δ syndrome which induces activation of the PI3K/AKT signaling pathway which reduces intracellular reactive oxygen species through NOX4. This inactivates FOX01 and the resultant decrease of reactive oxygen species in macrophages causes decreased activation of AP1, MAPK, NF-κB, and MIF. AKT phosphorylation may activate mTORC1 (Karanovic et al., 2019) (see Fig. 4.16). A recent paper by Karanovic et al. (2019) identified one of our susceptibility genes, NFκB, and reviewed the literature of identified human mutations that were reported to enhance susceptibility to Toxoplasma (Fig. 4.16D). The genes identified an interrelated pathway that provides substantial insight into critical protective immunologic and other functions, and insight into how T. gondii and its host interact to create the determinants of outcomes of this infection. A schematic diagram of some of these pathways is shown in Fig. 4.16C. In addition, antibodies to TNF-α, such as Infliximab, and other critical pathways for defense against toxoplasma can lead to reactivation of cerebral toxoplasmosis (Young and McGwire, 2005). 4.5.3.3 Brazil In Brazil, TLR9 and CCR5 have been shown to have susceptibility alleles influencing ocular

toxoplasmosis. Dutra et al. (2013) noted the critical importance of NOD-2 and IL-17. Be´la et al. (2012) found an association with IRAK4 and toxoplasmosis (see Table 4.3). Killer receptors on NK cells and HLA are also critical determinants of outcomes. In Brazil, Ayo et al. (2016) investigated associations in susceptibility to ocular toxoplasmosis with the genes encoding KIR receptors and their HLA class 1 ligands. 297 patients who were seropositive for Toxoplasma were divided based on the occurrence or absence of ocular scars and/or lesions. The patients with scars and/or lesions were separated further into two groups with primary or recurrent ocular manifestation. Through genotyping by PCR-SSOP, the investigators identified that certain activating and inhibitory KIR genes may have an effect on the development of ocular toxoplasmosis. They found that the activating KIR together with their HLA ligands (KIR3DS1-Bw4 80Ile and KIR2DS11/C211 KIR3DS11/Bw4 80Ile1) was associated with increased susceptibility to ocular toxoplasmosis and its clinical manifestations. The activating KIR3DS1 gene was also identified to be associated with increased susceptibility for ocular toxoplasmosis. Ayo et al. (2016) found that KIR-HLA inhibitory pairs 2 KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3C1 2 were associated with decreased susceptibility for ocular toxoplasmosis and its clinical forms, while the KIR3DS12/KIR3DL11/ 1 Bw4 80Ile combination was associated as a protective factor against the development of ocular toxoplasmosis and specifically against recurrent manifestations (Ayo et al., 2016). More recently, a study on HLA, KIR, and MICA alleles was conducted to investigate the innate and adaptive immune responses in ocular toxoplasmosis. The MICA *002-HLA-B*35 and MICA*008BHLA-C*07 haplotypes were found to have lost their association with ocular toxoplasmosis, while HLA and KIR were found to be statistically significant in the influence of toxoplasmosis, as recently found in

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4.5 Other considerations of pathogenesis in human infections

Ayo et al. (2016) and De Mattos et al. (2019, Abstract). 4.5.3.4 Colombia The work of Naranjo-Galvis et al. (2018) demonstrates the importance of the following cytokine genes and the centrality of IL-1β to these pathways. They found IFN-γ and IL-10 alleles were present in lower frequency and IL-1β was present in higher frequency. Coexpression analysis of IL-1α, IL-1β, TNF-α, IFN-γ, and IL-10, was done using two methods including NP de novo coexpression analysis. Several other studies have shown involvement of IL-1β and a coexpression analysis identified connectivity of the IL-1α and the IL-1β, genes mediated by Ribonuclease K6. The interactions between genes are unknown; however, an expression quantitative trait locus (eQTL) study found that there was an overrepresentation of either T cell specific or monocytespecific eQTLs in susceptibility alleles for disease and coexpression was found. 4.5.3.5 Poland Polish investigators found that TLR4 and TLR9 SNPs were also associated with protection against congenital toxoplasmosis (Wujcicka et al., 2013; Wujcicka et al., 2015; Wujcicka et al., 2017). These investigators also found there is a contribution of IL6174G-C and IL1b3954C-T polymorphisms to congenital Toxoplasma infection (Wujcicka et al., 2015). Recently a genetic analysis by Wujcicka et al. (2018) demonstrated that genes encoding certain proinflammatory cytokines appeared to have polymorphisms associated with seropositivity in Polish women. This analysis began when in the literature or their own work they identified different polymorphisms in IL-1α, IL-1β, TNF-α, IL-6, IL-10, and IL-12b as being associated either with toxoplasmic retinochorioditis or other diseases associated with susceptibility alleles reflecting differences in proinflammation (Cordeiro et al., 2013; De Sa´

199

et al., 2007; Klein et al., 2001; Li et al., 2010). For the Polish investigators, IL-10 and these proinflammatory cytokine genes became candidate genes for the following analysis because each of these inflammatory mediators had been demonstrated to be important in pathogenesis of toxoplasmosis in rodent models. Specifically within a cohort of Polish pregnant women, they examined genotypes within IL-1α 2889 C . T, IL-1β 1 3954C . T, IL-6 2174G . C, IL-10 21082G . A, IL-12β 21188A . C, and TNF-α 2308G . A. This multiple-SNP analysis showed that the haplotype for certain IL-1α and IL-1β SNPs was significantly associated with a decreased risk of T. gondii infections (OR 0.41, P # .05) (Wujcicka et al., 2018). These were gIL-1α, IL-1β3954. They found that CCCAGA complex had variants that were associated with increased risk of T. gondii infection (OR8.4, P , .05). Congenital transmission of T. gondii from mother to their fetuses demonstrated that the presence of GA heterozygotic status within IL10 polymorphism increased the risk of parasitic transmission (or 5.75, P # 0.05). These proinflammatory cytokines appeared to be associated with seropositivity in Polish women.

4.5.4 Signature pathways in neuronal stem cells, peripheral blood monocytic cells, and retinal cells modified by Toxoplasma gondii infections Critical genes from genetics analyses presented previously (Table 4.3 and Fig. 4.17B), circulating serum proteins and miRNA biomarkers from congenitally infected children with active severe disease compared with matched children without illness but with congenital toxoplasma infection (Fig. 4.17E), were identified. Systems analyses of transcriptomic, proteomic, datasets from infected human primary brain neuronal stem cells and a monocytic cell line were also included in the study

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4. Human Toxoplasma infection

to better understand what T. gondii could do to the infected human brain (Fig. 4.17B from Allen Atlas). GO and KEGG pathway and David analyses were performed (Fig. 4.17C E), and interactomes and key upstream regulatory genes (Fig. 4.17G and J) were identified. Orbital, protein protein string, and disease association analyses were performed (Fig. 4.17K), and IFAs demonstrate the effect on primary human brain neuronal stem cells (Fig. 4.17L). These results demonstrate that different Toxoplasma isolates perturb their host cells in a manner that reflects the parasite modulating critical signature pathways of neurodegeneration, motor disease, epilepsy, and cancer, among others. Systems analysis demonstrated possible pathogenic mechanisms (Fig. 4.17O Q; Ngoˆ et al., 2017). Not only are there suggestions that the human brain may be altered by chronic infection but also that PBMCs are affected. PBMCs and circulating cytokines appear to differ between infected and uninfected people (Fig. 4.17O Q). These differences extend to those who have acute and chronic infections in the United States and Colombia (Pernas et al., 2014; Fig. 4.17O Q). Patterns associated with neurodegeneration in the studies with human brain stem cells were also revealed in the study of monocytic cells by Safronova et al. (2019) (Fig. 4.17R V). Transcriptomic analysis identified CCL2 as a signature response to T. gondii infection. In this response, Safronova et al. found that S100A11, a CCL2 inducing molecule, regulates monocyte recruitment in vivo and that Caspase 1 was important in S100A11 release. Knockout affected survival. Another example of peripheral blood monocytes from healthy donors having different serologic markers during chronic Toxoplasma infection compared to those from noninfected controls was demonstrated in the manuscript of Ehmen and Lu¨der (2019). These investigators searched for these differences having noted that chronically infected mice had

macrophages that increased resistance to heterologous pathogens. The authors’ results are paraphrased and summarized as follows: they found that expression levels of CD16 and of CD62L were less and CD64 was higher on PBMCs in persons who were chronically infected than in persons who were uninfected (Fig. 4.17W and X). Chronic Toxoplasma infection was not associated with a shift in classical, intermediate, and nonclassical monocytes subpopulations, however. In vitro infection of monocytes enriched PBMCs from both seropositive and seronegative blood donors with T. gondii led to an expansion of CD14 classical monocytes, and a decrease of CD14 and CD16 monocytes. Furthermore, the percentage of CCR2 monocytes sharply decreased after infection. Only monocytes from chronically infected individuals but not monocytes from naive controls upregulated MRC Class II expression following in vitro infection. IL-12 mRNA increased after infection with T. gondii, particularly in cells from those who were chronically infected, but decreased in monocytes from those who were seronegative (Fig. 4.17W and X). This demonstrates that infection of humans also leads to long term effects on their peripheral blood monocytes. Toxoplasma infection does not appear to be without consequences in chronically infected humans. Congruent with the parent of origin effects in earlier studies of COL2a and ABC4R, an additional study by Syn et al. (2018) (Fig. 4.17Y, Z, AA, BB, and CC), provides detailed mechanistic information concerning how T. gondii perturbs dopaminergic and amyloid precursor protein (APP) processing in human retinal cells. These investigators report their genome-wide analysis of host methylome and transcriptome following T. gondii infection in a retinal cell line identified genes (132, 186 and 128 genes at 2, 6, and 24 hours postinfection) concordant for methylation and expression. The authors’ results are paraphrased and summarized as follows: they

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4.6 Conclusion, unifying concepts, and toward the future

found "hypermethylated and decreased expression or hypomethylated and increased expression genes. Pathway analyses showed perturbation of two neurologically associated pathways: dopamine-DARPP32 feedback in cAMP signaling (P value 5 8.3 3 1025; adjusted P value 5 .020); and amyloid processing (P value 5 1.0 3 1023; adjusted P value 5 .043). APP decreased in level following T. gondii infection. Expression of APP early in nervous system development affects neural migration and there is a role of amyloid processing in Alzheimer’s disease. Dopamine has roles in the developing retina and in Parkinson’s disease and schizophrenia." Their results provide a possible functional link between T. gondii infection and congenital/early life and adult neurological clinical signs (Syn et al., 2018). Syn et al. (2017) also demonstrated T. gondii made mitochondria dysfunctional. This is similar to findings with human brain primary neuronal stem cells. (Ngoˆ et al., 2017). A diagram showing some of these pathways T. gondii effects is shown in Fig. 4.17DD GG.

4.6 Conclusion, unifying concepts, and toward the future T. gondii is a common infection of humans with 1/3 to 1/2 of all people infected. Manifestations of T. gondii infections vary in each clinical setting. This variation depends on genetics of the human host, genetics of the parasite, immune status of the host and probably inoculum size and parasite stage acquired, although these factors influencing pathogenesis are only partially characterized. Clinical manifestations, methods for diagnosis of primary, and chronic infections in the immunecompetent person aid in management of this infection. This active infection (e.g., in the fetus and infant, severely ill older child or adult or immune-compromised person) can be effectively treated with available medicines when

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treatment is begun expeditiously. A new pointof-care test that meets the WHO ASSURED criteria for an ideal test (Lykins et al., 2018). New inexpensive multiplex testing, including diagnosis of multiple treatable perinatal infections using saliva, present a new paradigm for prevention of congenital toxoplasmosis. Carefully performed serologic screening during gestation to diagnose primary infection in the pregnant woman, in order to facilitate treatment to eliminate disease in the fetus and infant, nontoxic medicines to eliminate encysted bradyzoites, as well as tachyzoites, and a vaccine to prevent the infection in humans are future needs. There is exciting, new work that promises to lead to elimination of human disease caused by T. gondii. These include understanding signature pathways in the brain, genetics, and immune responses, and new molecular targets defined empirically and by CRISPR analyses. New understanding of the cat intestinal cycle and understanding of epitopes, proteins, adjuvants, and novel delivery methods for vaccines provide a foundation for preventing this infection.

Acknowledgments We gratefully acknowledge patients, their families, and physicians in the NCCCTS for working with us to help us to understand the clinical manifestations and pathogenesis of this infection. We also gratefully acknowledge the participants in both EMSCOT and SYROCOT. We especially thank and also gratefully acknowledge Christine Van Tubbergan, Jose Montoya, and Eskild Petersen who contributed significantly to earlier editions of this chapter/book with us, and whose work is included in part in this version of this chapter as well. We also thank many colleagues for helpful discussions; we especially gratefully acknowledge Phillip Thulliez, Jack Remington, Jack Frenkel, Francois Kieffer, Francois Peyron, Martine Wallon, Fernand Daffos, Stephan Romand, John Costa, Herve-Pelloux, Wilma Buffolano, Kenneth Boyer, Paul Meier, Charles Swisher, Peter Heydemann, Gwen Noble, Paul Latkany, Peter Rabiah, Kristen Wroblewski, Theodore Karrison, and all the members of the NCCCTS who have generously shared their experience, skills, and insights and guided the development of the information included

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herein. We also especially acknowledge George Desmonts and Jacques Couvreur whose insights and conversations over many years helped define concepts in this chapter. This work was supported by National Institute of Allergy and Infectious Diseases (Al 16945, Al 27530 and Al 014717); March of Dimes (6 528); the US Food and Drug Administration (FD-R-000192); the Thrasher Research Fund; the United Airlines foundation; Angel Flight; United to Save Children, Gerico and Hyatt Hotel Corporation foundations; and the Michael Reese Institute Council, the Rooney Alden, Engel, Taub, Harris, Pritzker, Donelly, Cornwall, Musillami, Frieman and Mann-Cornwell families.

References Abgrall, S., Rabaud, C., Costagliola, D., 2001. Incidence and risk factors for toxoplasmic encephalitis in human immunodeficiency virus-infected patients before and during the high active anti-retroviral therapy era. Clin. Infect. Dis. 33, 1747 1755. Ades, A.E., 1991. Evaluating the sensitivity and predictive value of tests of recent infection: toxoplasmosis in pregnancy. Epidemiol. Infect. 107, 527 535. Aguirre, A.A., Longcore, T., Barbieri, M., et al., 2019. The one health approach to toxoplasmosis: epidemiology, control, and prevention strategies. Ecohealth. 16, 1 13. Ajzenberg, D., 2012. High burden of congenital toxoplasmosis in the USA: the strain hypothesis? Clin. Infect. Dis. 54 (11), 1606 1607. Alford Jr., C., Stagno, S., Reynolds, D.W., 1974. Congenital toxoplasmosis: clinical, laboratory, and therapeutic considerations, with special reference to subclinical disease. Bull. N. Y. Acad. Med. 50, 160 181. Alvarado-Esquivel, C., Sethi, S., Janitschke, K., Hahn, H., Liesenfeld, O., 2002. Comparison of two commercially available avidity tests for Toxoplasma-specific IgG antibodies. Arch. Med. Res. 33, 520 523. Alvarado-Esquivel, C., Estrada-Martinez, S., Liesenfeld, O., 2011. Toxoplasma gondii infection in workers occupationally exposed to unwashed raw fruits and vegetables: a case control seroprevalence study. Parasit. Vectors 4 (1), 235. Ambroise-Thomas, P., Garin, J.P., Rigaud, A., 1966. Improvement of the immunofluorescence technique by the use of counter dyes. Application to Toxoplasma gondii. Presse Med. 74, 2215 2216. Andrade, G.M., Vasconcelos-Santos, D.V., Carellos, E.V., Romanelli, R.M., Vitor, R.W., Carneiro, A.C., et al., 2012. Congenital toxoplasmosis from a chronically infected woman with reactivation of retinochoroiditis during pregnancy. J. Pediatr. (Rio. J.) 86 (1), 85 88.

Andrews, K.T., Haque, A., Jones, M.K., 2012. HDAC inhibitors in parasitic diseases. Immunol. Cell. Biol. 90 (1), 66 77. Aptouramani, M., Theodoridou, M., Syrogiannopoulos, G., Mentis, A., Papaevangelou, V., Gaitana, K., et al., 2012. A dedicated surveillance network for congenital toxoplasmosis in Greece, 2006 2009: assessment of the results. BMC Public Health 12 (1), 1019. Araujo, F.G., Barnett, E.V., Gentry, L.O., et al., 1971. Falsepositive anti-Toxoplasma fluorescent-antibody tests in patients with antinuclear antibodies. Appl. Microbiol. 22, 270 275. Araujo, F.G., Wong, M.M., Theis, J., Remington, J.S., 1973. Experimental Toxoplasm gondii infection in a nonhuman primate. Am. J. Trop. Med. Hyg. 22 (4), 465 472. Arslan, F., Batirel, A., Ramazan, M., Ozer, S., Mert, A., 2012. Macrophage activation syndrome triggered by primary disseminated toxoplasmosis. Scand. J. Infect. Dis. 44 (12), 1001 1004. Aspo¨ck, H., Pollak, A., 1982. Prevention of prenatal toxoplasmosis by serological screening of pregnant women in Austria. Scand. J. Infect. Dis. 84 (Suppl.), 43 45. Aubert, D., Foudrinier, F., Villena, I., Pinon, J.M., Biava, M. F., Renoult, E., 1996. PCR diagnosis and follow up of two cases of disseminated toxoplasmosis after kidney grafting. J. Clin. Microbiol. 34, 1347. Aubert, G., Maine, G.T., Villena, I., Hunt, J.C., Howard, L., Sheu, M., et al., 2000. Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J. Clin. Microbiol. 38, 1144 1150. Ayo, C.M., Frederico, F.B., Siqueira, R.C., Branda˜o de Mattos, C.C., Previato, M., Barbosa, A.P., et al., 2016. Ocular toxoplasmosis: susceptibility in respect to the genes encoding the KIR receptors and their HLA class I ligands. Sci. Rep. 6, 36632. Available from: https://doi. org/10.1038/srep36632. Bahia-Oliverira, L.M., Jones, J.L., Azevedo-Silva, J., et al., 2003. Highly endemic, waterborne transmission in north Rio de Janeiro state, Brazil. Emerg. Infect. Dis. 9, 55 62. Bahia-Oliveira, L., Gomez-Marin, J., Shapiro, K., 2017. Toxoplasma gondii. In: Rose, J.B., Jime´nez-Cisneros, B., (Eds.) Global Water Pathogen Project. ,http://www. waterpathogens.org. (Fayer, R., Jakubowski, W., (Eds.) Part 3 Protists) Michigan State University, E. Lansing, MI, UNESCO. ,http://www.waterpathogens.org/ book/toxoplasma-gondii.. Balsari, A., Poli, G., Molina, V., et al., 1980. ELISA for Toxoplasma antibody detection: a comparison with other serodiagnostic tests. J. Clin. Pathol. 33, 640 643.

Toxoplasma Gondii

References

Barbieri, M.M., Kashinsky, L., Rotstein, D.S., Colegrove, K. M., Haman, K.H., Magargal, S.L., et al., 2016. Protozoalrelated mortalities in endangered Hawaiian monk seals Neomonachus schauinslandi. Dis. Aquat. Org. 121 (2), 85 95. Retrieved from https://www.int-res.com/ abstracts/dao/v121/n2/p85-95/. Barbosa, B.F., Gomes, A.O., Ferro, E.A., Napolitano, D.R., Mineo, J.R., Silva, N.M., 2012. Enrofloxacin is able to control Toxoplasma gondii infection in both in vitro and in vivo experimental models. Vet. Parasitol. 187 (1 2), 44 52. Basavaprabhu, A., Soundarya, M., Deepak, M., Satish, R., 2012. CNS toxoplasmosis presenting with obstructive hydrocephalus in patients of retroviral disease a case series. Med. J. Malaysia 67 (2), 214 216. Bautista, G., Ramos, A., Fore´s, R., Regidor, C., Ruiz, E., de Laiglesia, A., et al., 2012. Toxoplasmosis in cord blood transplantation recipients. Transpl. Infect. Dis. 14 (5), 496 501. Bech-Nielsen, S., 2012. Toxoplasma gondii associated behavioural changes in mice, rats and humans: evidence from current research. Prev. Vet. Med. 103 (1), 78 79. Begeman, I.J., Lykins, J., Zhou, Y., Lai, B.S., Levigne, P., El Bissati, K., et al., 2017. Point-of-care testing for Toxoplasma gondii IgG/IgM using Toxoplasma ICT IgG-IgM test with sera from the United States and implications for developing countries. PLoS Negl. Trop. Dis. 11 (6), e0005670. Available from: https://doi.org/ 10.1371/journal.pntd.0005670. Retrieved from. Beghetto, E., Buffolano, W., Spadoni, A., del Pezza, M., di Cristina, M., Minenkova, O., et al., 2003. Use of an immunoglobulin G avidity assay based on recombinant antigens for diagnosis of primary Toxoplasma gondii infection during pregnancy. J. Clin. Microbiol. 41, 5414 5418. Beghetto, E., Nielsen, H.V., Del Porto, P., Buffolano, W., Guglietta, S., Felici, F., et al., 2005. A combination of antigenic regions of T. gondii microneme proteins induce protective immunity against oral infections with parasite cysts. J. Infect. Dis. 191, 637 645. Beghetto, E., Spadoni, A., Bruno, L., Buffolano, W., Gargano, N., 2006. Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant proteins. J. Clin. Microbiol. 44 (6), 2133 2140. Be´la, S.R., Dutra, M.S., Mui, E., Montpetit, A., Oliveira, F.S., Oliveira, S.C., et al., 2012. Impaired innate immunity in mice deficient in interleukin-1 receptor-associated kinase 4 leads to defective type 1 T cell responses, B cell expansion, and enhanced susceptibility to infection with Toxoplasma gondii. Infect. Immun. 80 (12), 4298 4308. Available from: https://doi.org/10.1128/ IAI.00328-12.

203

Benenson, M.W., Takafuji, E.T., Lemon, S.M., Greenup, R. L., Sulzer, A.J., 1982. Oocyst-transmitted toxoplasmosis associated with ingestion of contaminated water. N. Engl. J. Med 3007, 666 669. Benevento, J.D., Jager, R.D., Noble, A.G., et al., 2008. Toxoplasmosis-associated neovascular lesions treated successfully with ranibizumab and antiparasitic therapy. Arch. Ophthalmol. 126, 1152 1156. Benson, A., Pifer, R., Behrendt, C.L., Hooper, L.V., Yarovinsky, F., 2012. Gut commensal bacteria direct a protective immune response against Toxoplasma gondii. Cell. Host Microbe. 6 (2), 187 196. Berrebi, A., Bardou, M., Bessieres, M., et al., 2007. Outcome for children infected with congenital toxoplasmosis in the first trimester and with normal ultrasound findings: a study of 36 cases. Eur. J. Obstet. Gynaecol. Reprod. Biol. 135, 53 57. Binquet, C., Wallon, M., Quantin, C., Kodjikian, L., Garweg, J., Fleury, J., et al., 2003. Prognostic factors for the long-term development of ocular lesions in 327 children with congenital toxoplasmosis. Epidemiol. Infect. 131, 1157 1168. Bobi´c, B., Villena, I., Stillwaggon, E., 2019. Prevention and mitigation of congenital toxoplasmosis. Economic costs and benefits in diverse settings. Food Waterborne Parasitol. 16, e00058. Available from: https://doi.org/ 10.1016/J.FAWPAR.2019.E00058. Bohne, W., Heesemann, J., Gross, U., 1993. Induction of bradyzoite-specific T. gondii antigens in gamma Interferon-treated macrophages. Infect. Immun. 61, 1141 1145. Bories, P., Zink, E., Mattern, J.F., Villard, O., Berceanu, A., Bilger, K., et al., 2012. Febrile pancytopenia as uncommon presentation of disseminated toxoplasmosis after BMT. Bone Marrow Transpl. 47 (2), 301 303. Botterel, F., Ichai, P., Feray, C., et al., 2002. Disseminated toxoplasmosis, resulting from infection of allograft, after orthopedic liver transplantation: usefulness of quantitative PCR. J. Clin. Microbiol. 40, 1648 1650. Bowie, W.R., King, A.S., Werker, D.H., Isaac-Renton, J.L., Bell, A., Eng, S.B., et al., 1997. Outbreak of toxoplasmosis associated with municipal drinking water. The BC Toxoplasma Investigation Team. Lancet 350, 173 177. Boyer, K.M., Holfels, E., Roizen, N., et al., 2005. Risk factors for Toxoplasma gondii infection in mothers of infants with congenital toxoplasmosis: implications for prenatal management and screening. Am. J. Obstet. Gynecol. 192, 564 571. Boyer, K., Hill, D., Mui, E., et al., 2011. Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America. Clin. Infect. Dis. 53 (11), 1081.

Toxoplasma Gondii

204

4. Human Toxoplasma infection

Bozzette, S., Findelstein, D., Spector, S., Frame, P., Powderly, W.G., He, W., et al., 1995. A randomized trial of three antipneumocystis agents in patients with advanced human immunodeficiency virus infection. New England Journal of Medicine 332, 693 699. Bretagne, S., Costa, J.M., Keuntz, M., et al., 1995. Late toxoplasmosis evidenced by PCR in marrow transplant recipient. Bone Marrow Transpl. 15, 809 811. Brezin, A.P., Thulliez, P., Couvreur, J., et al., 2003. Ophthalmic outcome after prenatal and postnatal treatment of congenital toxoplasmosis. Am. J. Ophthalmol. 138, 779 784. Bricker-Hildalgo, H., Brenier-Pinchart, M.P., Schaal, J.P., et al., 2007. Value of Toxoplasma gondii detection in one hundred thirty-three placentas for the diagnosis of congenital toxoplasmosis. Pediatr. Infect. Dis. J. 26, 845 846. Brooks, J.M., Carrillo, G.L., Su, J., Lindsay, D.S., Fox, M.A., Blader, I.J., 2015. Toxoplasma gondii infections alter GABAergic synapses and signaling in the central nervous system. MBio 6 (6), e01428. Available from: https://doi.org/10.1128/mBio.01428-15. Brown, A.S., Schaefer, C.A., Wuesenberry Jr., C.P., et al., 2005. Maternal expsosure to toxoplasmosis and risk of schizophrenia in adult offspring. Am. J. Psychiatry 162, 767 773. Brownback, K.R., Crosser, M.S., Simpson, S.Q., 2012. A 49year-old man with chest pain and fever after returning from France. Chest 141 (6), 1618 1621. Buffolano, W., Beghetto, E., Del Pezzo, M., Spadoni, A., Di Cristina, M., Petersen, E., et al., 2003. Use of Recombinant Antigens for Early Postnatal Diagnosis of Congenital Toxoplasmosis. Burg, J.L., Perelman, L., Kasper, L.H., Ware, P.L., Boothroyd, J.C., 1988. Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. J. Immunol. 141, 3584 3591. Burnett, A.J., Shortt, S.G., Isaac-Renton, J., et al., 1998. Multiple cases of acquired toxoplasmosis retinitis presenting in an outbreak. Ophthalmology 105, 1032 1037. Burrowes, D., Boyer, K., Swisher, C.N., et al., 2012. Spinal cord lesions in congenital toxoplasmosis demonstrated with neuroimaging, including their successful treatment in an adult. J. Neuroparasitol. 3, pii: 235533. Busemann, C., Ribback, S., Zimmermann, K., Sailer, V., Kiefer, T., Schmidt, C.A., et al., 2012. Toxoplasmosis after allogeneic stem cell transplantation a single centre experience. Ann. Hematol. 91 (7), 1081 1089. Camara, D., Bisanz, C., Barette, C., Van Daele, J., Human, E., Barnard, B., et al., 2012. Inhibition of p-aminobenzoate and folate syntheses in plants and apicomplexan

parasites by natural product rubreserine. J. Biol. Chem. 287 (26), 22367 22376. Candolfi, E., Bessieres, M.H., Mart, P., Cimon, B., Gandilhon, F., Pelloux, H., et al., 1993. Determination of a new cut-off value for the diagnosis of congenital toxoplasmosis by detection of specific IgM in an enzyme immunoassay. Eur. J. Clin. Microbiol. Infect. Dis. 12, 396 398. Capone Jr., A., 2006. Treatment of neovscularization in infants with retinopathy of prematurity with antiVEGF: vitreous surgery for retinopathy of prematurity. In: Paper Presented at: Annual Meeting of the American Academy of Ophthalmology, Vegas, NV. Carme, B., Bissuel, F., Ajzenberg, D., Bouyne, R., Aznar, C., Demar, M., et al., 2002. Severe acquired toxoplasmosis in immunocompetent adult patients in French Guiana. J. Clin. Microbiol. 2002 (40), 4037 4044. Carme, B., Demar, M., Ajzenberg, D., Darde´, M.L., 2009. Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana. Emerg. Infect. Dis. 15, 656 658. Carneiro, A.C., Andrade, G.M., Costa, J.G., Pinheiro, B.V., Vasconcelos-Santos, D.V., Ferreira, A.D., et al., 2013. Genetic characterization of Toxoplasma gondii revealed highly diverse genotypes from human congenital toxoplasmosis in southeastern Brazil. J. Clin. Microbiol. 51, 901 907. Caselli, D., Andreoli, E., Paolicchi, O., Savelli, S., Guidi, S., Pecile, P., et al., 2012. Acute encephalopathy in the immune-compromised child: never forget toxoplasmosis. J. Pediatr. Hematol. Oncol. 34 (5), 383 386. Cerede, O., Dubremetz, J.F., Bout, D., Lebrun, M., 2002. The T. gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesion. EMBO J. 21, 2526 2536. Cesbron-Delauw, M.F., 1995. The SAG2 antigen of T. gondii and the 31-kDa surface antigen of sarcocystis muris share similar sequence features. Parasitol. Res. 81, 444 445. Chandrasekar, P.H., Momin, F., the Bone Marrow Transplant Team, 1997. Disseminated toxoplasmosis in marrow recipients: a report of three cases and a review of the literature. Bone Marrow Transpl. 19, 685 689. Chapey, E., Wallon, M., Debize, G., Rabilloud, M., Peyron, F., 2010. Diagnosis of congenital toxoplasmosis by using a whole-blood gamma interferon release assay. J. Clin. Microbiol. 48 (1), 41 45. Chapey, E., Wallon, M., Peyron, F., 2017. Evaluation of the LDBIO point of care test for the combined detection of toxoplasmic IgG and IgM. Clin. Chim. Acta 464, 200 201. Available from: https://doi.org/10.1016/j. cca.2016.10.023.

Toxoplasma Gondii

References

Chew, W.K., Segarra, I., Ambu, S., Mak, J.W., 2012. Significant reduction of brain cysts caused by Toxoplasma gondii after treatment with spiramycin coadministered with metronidazole in a mouse model of chronic toxoplasmosis. Antimicrob. Agents Chemother. 56 (4), 1762 1768. Chiang, E., Goldstein, D.A., Shapiro, M.J., Mets, M.B., 2012. Branch retinal artery occlusion caused by toxoplasmosis in an adolescent. Case Report Ophthalmol. 3 (3), 333 338. Chumpitazi, B.F., Boussaid, A., Pelloux, H., Racinet, C., Bost, M., Goullier-Fleuret, A., 1995. Diagnosis of congenital toxoplasmosis by immunoblotting and relationship with other methods. J. Clin. Microbiol. 33, 1479 1485. Cinalli, G., Sainte-Rose, C., Chumas, P., et al., 1999. Failure of third ventriculostomy in the treatment of aqueductal stenosis in children. Neurosurg. Focus 6 (4), e3. Coccaro, E.F., Lee, R., Groer, M.W., Can, A., CoussonsRead, M., Postolache, T.T., 2016. Toxoplasma gondii infection: relationship with aggression in psychiatric subjects. J. Clin. Psychiatry 77 (3), 334 341. Collier, S.A., Stockman, L.J., Hicks, L.A., Garrison, L.E., Zhou, F.J., Beach, M.J., 2012. Direct healthcare costs of selected diseases primarily or partially transmitted by water. Epidemiol. Infect. 140 (11), 2003 2013. Colombo, F.A., Vidal, J.E., Penalva de Oliveira, A.C., Hernandez, A.V., Bonasser-Filho, F., Nogueira, R.S., et al., 2005. Diagnosis of cerebral toxoplasmosis in AIDS patients in Brazil: importance of molecular and immunological methods using peripheral blood samples. J. Clin. Microbiol. 43, 5044 5047. Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A., et al., 2011a. Towards an immunosense vaccine to prevent toxoplasmosis: protective Toxoplasma gondii epitopes restricted by HLA-A*0201. Vaccine 29 (4), 754 762. Available from: https://doi.org/10.1016/j. vaccine.2010.11.015. Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A., et al., 2011b. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-AT1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8 1 T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii. Immunome Res. 6, 12. Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A., et al., 2012. Toxoplasma gondii HLA-B*0702restricted GRA720-28 peptide with adjuvants and a universal helper T cell epitope elicits CD8 1 T cells producing interferon-γ and reduces parasite burden in

205

HLA-B*0702 mice. Hum. Immunol. 73 (1), 1 10. Available from: https://doi.org/10.1016/j. humimm.2011.10.006. Conley, F.K., Jenkins, K.A., Remington, J.S., 1981. Toxoplasma gondii infection of the central nervous system: use of the peroxidase-antiperoxidase method to demonstrate Toxoplasma in formalin-fixed paraffin embedded tissue section. Hum. Pathol. 12, 690 698. Conrad, P.A., Miller, M.A., Kreuder, C., et al., 2005. Transmission of Toxoplasma: clues from the study of sea otters as sentinels of Toxoplasma gondii flow into the marine environment. Int. J. Parasitol. 35 (11 12), 1155 1168. Contopoulos-ioannidis, D., Wheeler, K.M., Ramirez, R., et al., 2015. Clustering of Toxoplasma gondii infections within families of congenitally infected infants. Clin. Infect. Dis. 61 (12), 1815 1824. Cook, A.J.C., Gilbert, R., Dunn, D., Buffolano, W., Zuffrey, J., Reymondin, M., et al., 2000. Sources of toxoplasmainfection in pregnant women: a European multicentre case-control study. Br. Med. J. 312, 142 147. Cordeiro, C.A., Moreira, P.R., Bessa, T.F., Costa, G.C., Dutra, W.O., Campos, W.R., et al., 2013. Interleukin-6 gene polymorphism ( 174 G/C) is associated with toxoplasmic retinochoroiditis. Acta Ophthalmol. 91 (4), e311 e314. Available from: https://doi.org/10.1111/ aos.12046. Cortina-Borja, M., Tan, H.K., Wallon, M., et al., 2010. Prenatal treatment for serious neurological sequelae of congenital toxoplasmosis: an observational prospective cohort study. PLoS Med. 7 (10), 1 11. Couvreur, J., 1955. E´tude de la toxoplasmose congenitale a` propo de 20 observation. These Paris. Couvreur, J., Desmonts, G., 1962. Congenital and maternal toxoplasmosis. A review of 300 cases. Develop. Med. Chld. Neurol. 4, 519 530. Couvreur, J., Desmonts, G., 1964. Late evolution outbreaks of congenital toxoplasmosis. Cah. Coll. Med. Hop. Paris 115, 752 758. French. Couvreur, J., Thulliez, P., 1996. Acquired toxoplasmosis with ocular or neurologic involvement. Presse Med. 25, 438 442. Couvreur, J., Desmonts, G., Girre, J.Y., 1976. Congenital toxoplasmosis in twins: a series of 14 pairs of twins: absence of infection in one twin in two pairs. J. Pediatr. 89 (2), 235 240. Couvreur, J., Desmonts, G., Aron-Rosa, D., 1984. Le prognostic oculare de la toxoplasmose congenitale: role du traitement. Ann. Pediatr. (Paris). 31, 855 858. Couvreur, J., Desmonts, G., Thulliez, P., 1988. Prophylaxis of congenital toxoplasmosis. effects of spiramycin on placental infection. J. Antimicrob. Chemother. 22, 193 200.

Toxoplasma Gondii

206

4. Human Toxoplasma infection

Couvreur, J., Thulliez, P., Daffos, F., Aufrant, C., Bompard, Y., Gesquiere, A., et al., 1993. In utero treatment of toxoplasmic fetopathy with the combination pyrimethamine-sulfadiazine. Foetal. Diagn. Ther. 8 (1), 45 50. Cunningham, T., 1982. Pancarditis in acute toxoplasmosis. Am. J. Clin. Pathol. 78, 403 405. Daffos, F., Forestier, F., Capella-Pavlovsky, M., et al., 1988. Prenatal management of 746 pregnancies at risk for congenital toxoplasmosis. N. Engl. J. Med. 318, 271 275. Dai, J., Jiang, M., Wang, Y., Qu, L., Gong, R., Si, J., 2012a. Evaluation of a recombinant multiepitope peptide for serodiagnosis of Toxoplasma gondii infection. Clin. Vaccine Immunol. 19 (3), 338 342. Dai, J.F., Jiang, M., Qu, L.L., Sun, L., Wang, Y.Y., Gong, L. L., et al., 2012b. Toxoplasma gondii: Enzyme-linked immunosorbent assay based on a recombinant multiepitope peptide for distinguishing recent from past infection in human sera. Exp. Parasitol. 133 (1), 95 100. Dannemann, B.R., Vaughan, W.C., Thulliez, P., Remington, J.S., 1990. Differential agglutination test for diagnosis of recently acquired infection with Toxoplasma gondii. J. Clin. Microbiol. 28, 1928 1933. Darde, M.L., 2008. Toxoplasma gondii, ‘new’ genotypes and virulence. Parasite 15, 366 371. Darde, M.L., Villena, I., Pinon, J.M., Beguinot, I., 1998. Severe toxoplasmosis caused by a Toxoplasma gondii strain with a new isoenzyme type acquired in French Guyana. J. Clin. Microbiol. 1, 324. David, C.N., Frias, E.S., Szu, J.I., Vieira, P.A., Hubbard, J. A., Lovelace, J., et al., 2016. GLT-1-dependent disruption of CNS glutamate homeostasis and neuronal function by the protozoan parasite Toxoplasma gondii. PLoS Pathog. 12 (6), e1005643. Available from: https://doi. org/10.1371/journal.ppat.1005643. Retrieved from. de Amorim Garcia, C.A., Orefice, F., de Oliveira Lyra, C., Gomes, A.B., Franca, M., de Amorim Garcia Filho, C.A., 2004. Socioeconomic conditions as determining factors in the prevalence of systemic and ocular toxoplasmosis in Northeastern Brazil. Ophthalmic Epidemiol. 11, 301 317. Decoster, A., Darcy, F., Caron, A., Vinatier, D., Houze de L’Aulnoit, D., Vittu, G., et al., 1992. Anti-P30 IgA antibodies as prenatal markers of congenital toxoplasma infection. Clin. Exp. Immunol. 87, 310 315. Delair, E., Monnet, D., Grabar, S., Dupouy-Camet, J., Yera, H., Brezin, A.P., 2008. Respective roles of acquired and congenital infections in presumed ocular toxoplasmosis. Am. J. Ophthalmol. 146 (6), 851 855. Delair, E., Latkany, P., Noble, A.G., et al., 2011. Clinical manifestations of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 19 (2), 91 102.

de-la-Torre, A., Gonza´lez, G., Dı´az-Ramirez, J., Go´mezMarı´n, J.E., 2007. Screening by ophthalmoscopy for toxoplasma retinochoroiditis in Colombia. Am. J. Ophthalmol. 143, 354 356. Demar, M., Ajzenberg, D., Maubon, D., Djossou, F., Panchoe, D., Punwasi, W., et al., 2007. Fatal outbreak of human toxoplasmosis along the Maroni River: epidemiological, clinical, and parasitological aspects. Clin. Infect. Dis. 45, e88 e95. Demar, M., Hommel, D., Djossou, F., Peneau, C., Boukhari, R., Louvel, D., et al., 2012. Acute toxoplasmoses in immunocompetent patients hospitalized in an intensive care unit in French Guiana. Clin. Microbiol. Infect. 18, E221 E231. De Mattos, L., Faria, G., Ayo, C., Frederico, F., Siqueira, R., Pereira-chioccola, V., 2019. Genes From Innate and Adaptive Immune Responses in Ocular Toxoplasmosis (Abstract). Denes, E., Vidal, J., Monteil, J., 2013. Spinal cord toxoplasmosis. Infection 41, 295 296. Derouin, F., 2001. Anti-toxoplasmosis drugs. Curr. Opin. Invest. Drugs 2 (10), 1368 1374. Derouin, F., Almadany, R., Chau, F., Rouveix, B., Pocidalo, J. J., 1992. Synergistic activity of azithromycin and pyrimethamine or sulfadiazine in acute experimental toxoplasmosis. Antimicrob. Agents Chemother. 36 (5), 997 1001. Derouin, F., Pelloux, H., ESCMID Study Group on Clinical Parasitology, 2008. Prevention of toxoplasmosis in transplant patients. Clin. Microbiol. Infect. 14 (12), 1089 1101. De Sa´, A.R., Moreira, P.R., Xavier, G.M., Sampaio, I., Kalapothakis, E., Dutra, W.O., et al., 2007. Association of CD14, IL1B, IL6, IL10 and TNFA functional gene polymorphisms with symptomatic dental abscesses. Int. Endod. J. 40 (7), 563 572. Available from: https://doi. org/10.1111/j.1365-2591.2007.01272.x. Desmonts, G., 1982. Acquired toxoplasmosis in pregnant women. Evaluation of the frequency of transmission of Toxoplasma and of congenital toxoplasmosis. Lyon Med. 248, 115 123. Desmonts, G., Couvreur, J., 1974a. Congenital toxoplasmosis: a prospective study of 378 pregnancies. N. Engl. J. Med. 290, 1110 1116. Desmonts, G., Couvreur, J., 1974b. Toxoplasmosis in pregnancy and its transmission to the foetus. Bull. N. Y. Acad. Med.: J. Urban Health 50, 146 159. Desmonts, G., Couvreur, J., 1979. Congenital toxoplasmosis: a prospective study of the offspring of 542 women who acquired toxoplasmosis during pregnancy. In: Thalhammer, O., Pollak, A., Baumgarten, K. (Eds.), Pathophysiology of Congenital Disease: Perinatal Medicine (6th European Congress, Vienna). Stuttgart Georg Thieme Publishers, pp. 51 60.

Toxoplasma Gondii

References

Desmonts, G., Couvreur, J., 1984. Congenital toxoplasmosis. Prospective study of the outcome of pregnancy in 542 women with toxoplasmosis acquired during pregnancy. Ann. Pediatr. (Paris) 31, 805 809. Desmonts, G., Remington, J.S., 1980. Direct agglutination test for diagnosis of Toxoplasma infection: method for increasing sensitivity. J. Clin. Microbiol. 11, 562 568. Desmonts, G., Couvreur, J., Alison, F., et al., 1965a. Etude e´pide´miologique sur la toxoplasmose: de l’Influence de la cuisson des viandes de boucherie sur la fre´quence de l’Infection Humaine. Rev. Franc. Etud. Clin. Biol 10, 952 958. Desmonts, G., Couvreur, J., Ben Rachid, M.S., 1965b. Le toxoplasme, la me`re et l’enfant. Arch. Franc¸. Pe´diatr. 22, 1183 1200. Doggett, J.S., Nilsen, A., Forquer, I., Wegmann, K.W., Jones-Brando, L., Yolken, R.H., et al., 2012. Endochinlike quinolones are highly efficacious against acute and latent experimental toxoplasmosis. Proc. Natl. Acad. Sci. U.S.A. 109 (39), 15936 15941. Available from: https://doi.org/10.1073/pnas.1208069109. Dorfman, R.F., Remington, J.S., 1973. Value of lymph-node biopsy in the diagnosis of acute toxoplasmosis. N. Engl. J. Med 289, 878 881. dos Santos, T.R., Nunes, C.M., Luvizotto, M.C., et al., 2010. Detection of Toxoplasma gondii oocysts in environmental samples from public schools. Vet. Parasitol. 171 (1-2), 53 57. Dubey, J.P., 1998. Advances in the life cycle of Toxoplasma gondii. Int. J. Parasitol. 28 (7), 1019 1024. Dubey, J.P., Beattie, C.P., 1998. Toxoplasmosis of Animals and Man. CRC Press, Boca Raton, FL. Dubey, J.P., Sharma, S.P., Juranek, D.D., Sulzer, A.J., Teutsch, S.M., 1981. Characterization of Toxoplasma gondii isolates from an outbreak of toxoplasmosis in Atlanta, Georgia. Am. J. Vet. Res. 42 (6), 1007 1010. Dubey, J.P., Lappin, M.R., Thulliez, P., 1995. Long-term antibody responses of cats fed T. gondii tissue cysts. J. Parasitol. 81, 887 893. Dubey, J.P., Ferreira, L.R., Martins, J., McLeod, R., 2012a. Oral oocyst-induced mouse model of toxoplasmosis: effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality. Parasitology 139 (1), 1 13. Available from: https://doi. org/10.1017/S0031182011001673. Dubey, J.P., Lago, E.G., Gennari, S.M., Su, C., Jones, J.L., 2012b. Toxoplasmosis in humans and animals in Brazil: high prevalence, high burden of disease, and epidemiology. Parasitology 139 (11), 1375 1424. Available from: https://doi.org/10.1017/S0031182012000765.

207

Dubey, J.P., Hill, D.E., Rozeboom, D.W., Rajendran, C., Choudhary, S., Ferreira, L.R., et al., 2012c. High prevalence and genotypes of Toxoplasma gondii isolated from organic pigs in northern USA. Vet. Parasitol. 188 (1-2), 14 18. Duffy, A.R., Beckie, T.M., Brenner, L.A., Beckstead, J.W., Seyfang, A., Postolache, T.T., et al., 2015. Relationship between Toxoplasma gondii and mood disturbance in women veterans. Mil. Med. 180 (6), 621 625. Available from: https://doi.org/10.7205/MILMED-D-14-00488. Dumetre, A., Le Bras, C., Baffet, M., Meneceur, P., Dubey, J.P., Derouin, F., et al., 2008. Effects of ozone and ultraviolet radiation treatments on the infectivity of Toxoplasma gondii oocysts. Vet. Parasitol. 153 (3 4), 209 213. Dunn, D., Newell, M.L., Gilbert, R., Mok, J., Petersen, E., Peckham, C., European Collaborative Study and Research Network on Congenital Toxoplasmosis, 1996. Low incidence of congenital toxoplasmosis in children born to women infected with human immunodeficiency virus. Eur. J. Obstet. Gyn. Reprod. Biol. 68 (1 2), 93 96. Dunn, D., Wallon, M., Peyron, F., Petersen, E., Peckham, C. S., Gilbert, R.E., 1999. Mother to child transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet 353, 1829 1833. Dutra, M.S., Be´la, S.R., Peixoto-Rangel, A.L., Fakiola, M., Cruz, A.G., Gazzinelli, A., et al., 2013. Association of a NOD2 gene polymorphism and T-helper 17 cells with presumed ocular toxoplasmosis. J. Infect. Dis. 207 (1), 152 163. Available from: https://doi.org/10.1093/ infdis/jis640. Ehmen, H.G., Lu¨der, C.G.K., 2019. Long-term impact of Toxoplasma gondii infection on human monocytes. Front. Cell. Infect. Microbiol. 9, 235. Available from: https:// doi.org/10.3389/fcimb.2019.00235. Eichenwald, H.F., 1957. Congenital toxoplasmosis: a study of 150 cases. Am. J. Dis. Chld. 94, 411 412. Eichenwald, H.F., 1960. A study of congenital toxoplasmosis, with particular emphasis on clinical manifestations, sequelae and therapy. In: Siim, J. (Ed.), Human Toxoplasmosis. Munksgaard, Copenhagen, pp. 41 49. El Bissati, K., Chentoufi, A.A., Krishack, P.A., Zhou, Y., Woods, S., Dubey, J.P., et al., 2016. Adjuvanted multiepitope vaccines protect HLA-A*11:01 transgenic mice against Toxoplasma gondii. JCI Insight 1 (15). Available from: https://doi.org/10.1172/jci.insight.85955. El Bissati, K., Zhou, Y., Paulillo, S.M., Raman, S.K., Karch, C.P., Roberts, C.W., et al., 2017. Protein nanovaccine confers robust immunity against Toxoplasma. NPJ Vaccines 2, 24. Available from: https://doi.org/ 10.1038/s41541-017-0024-6.

Toxoplasma Gondii

208

4. Human Toxoplasma infection

El Bissati, K., Levigne, P., Lykins, J., Adlaoui, E.B., Barkat, A., Berraho, A., et al., 2018. Global initiative for congenital toxoplasmosis: an observational and international comparative clinical analysis. Emerg. Microbes Infect. 7 (1), 165. Available from: https://doi.org/10.1038/ s41426-018-0164-4. Engvall, E., Perlmann, P., 1972. Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigencoated tubes. J. Immunol. 109, 129 135. Enzensberger, W., Helm, E.B., Hopp, G., Stille, W., Fischer, P.A., 1985. Toxoplasma encephalitis in patients with AIDS. Dtsch. Med. Wochenschr. 110, 83 87. European Multicentre Study on Congenital Toxoplasmosis, 2003. Effect of timing and type of treatment on the risk of mother to child transmission of Toxoplasma gondii. BJOG 110, 112 120. Evans, A.K., Strassmann, P.S., Lee, I.-P., Sapolsky, R.M., 2014. Patterns of Toxoplasma gondii cyst distribution in the forebrain associate with individual variation in predator odor avoidance and anxiety-related behavior in male Long-Evans rats. Brain Behav. Immun. 37, 122 133. Available from: https://doi.org/10.1016/j. bbi.2013.11.012. Evenga˚rd, B., Petterson, K., Engman, M.-L., Wiklund, S., Ivarsson, S.A., Tear-Fahnehjelm, K., et al., 2001. Low incidence of toxoplasma infection during pregnancy and in newborns in Sweden. Epidemiol. Infect. 127, 121 127. Eyles, D.E., Coleman, N., 1955. An evaluation of the curative effects of pyrimethamine and sulfadiazine, alone and in combination, on experimental mouse toxoplasmosis. Antibiot. Chemother. 5, 529 539. Fabiani, S., Pinto, B., Bruschi, F., 2013. Toxoplasmosis and neuropsychiatric diseases: can serological studies establish a clear relationship? Neurol. Sci. 34, 417 425. Faucher, B., Miermont, F., Ranque, S., Franck, J., Piarroux, R., 2012. Optimization of Toxoplasma gondii DNA extraction from amniotic fluid using NucliSENS easyMAG and comparison with QIAamp DNA minikit. Eur. J. Clin. Microbiol. Infect. Dis. 31 (6), 1035 1039. Fayyaz, H., Rafi, J., 2012. TORCH screening in polyhydramnios: an observational study. J. Matern. Foetal Neonatal Med. 25 (7), 1069 1072. Feldman, H.A., 1953. Congenital toxoplasmosis a study of 103 cases. Am. J. Dis. Child. 86, 487. Ferguson, D.J., Graham, D.I., Hutchison, W.M., 1991. Pathological changes in the brains of mice infected with Toxoplasma gondii: a histological, immunocytochemical and ultrastructural study. Int. J. Exp. Pathol. 72 (4), 463 474.

Ferguson, D.J., Bowker, C., Jeffery, K.J., Chamberlain, P., Squier, W., 2013. Congenital toxoplasmosis: continued parasite proliferation in the foetal brain despite maternal immunological control in other tissues. Clin. Infect. Dis. 56 (2), 204 208. Ferna`ndez-Sabe´, N., Cervera, C., Farin˜as, M.C., Bodro, M., Mun˜oz, P., Gurguı´, M., et al., 2012. Risk factors, clinical features, and outcomes of toxoplasmosis in solid-organ transplant recipients: a matched case-control study. Clin. Infect. Dis. 54 (3), 355 361. Ferrandiz, J., Mercier, C., Wallon, M., Picot, S., CesbronDelauw, M.F., Peyron, F., 2004. Limited value of assays using detection of immunoglobulin G antibodies to the two recombinant dense granule antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for distinguishing between acute and chronic infections in pregnant women. Clin. Diagn. Lab. Immunol. 11, 1016 1021. Fischer, H.G., Stachelhaus, S., Sahm, M., Meyer, H.M., Reichmann, G., 1998. GRA7, an excretory 29 kDa T. gondii dense granule antigen released by infected host cells. Mol. Biochem. Parasitol. 91, 251 262. Flament-Durand, J., Coers, C., Waelbroeck, C., van Geertruyden, J., Tousaint, C., 1967. Toxoplasmic encephalitis and myositis during treatment with immunodepressive drugs. Acta Clin. Belg. 22, 44 54. Flegr, J., 2007. Effects of toxoplasma on human behavior. Schizophr. Bull. 33 (3), 757 760. Available from: https://doi.org/10.1093/schbul/sbl074. Flegr, J., 2013. Influence of latent Toxoplasma infection on human personality, physiology and morphology: pros and cons of the Toxoplasma-human model in studying the manipulation hypothesis. J. Exp. Biol. 216 (Pt 1), 127 133. Flegr, J., Havlı´cek, J., Kodym, P., Maly´, M., Smahel, Z., 2002. Increased risk of traffic accidents in subjects with latent toxoplasmosis: a retrospective case-control study. BMC Infect. Dis. 2, 11. Available from: https://doi.org/ 10.1186/1471-2334-2-11. Flori, P., Tardy, L., Patural, H., et al., 2004. Reliability of immunoglobulin G anti-Toxoplasma avidity test and effects of treatment on avidity indexes of infants and pregnant women. Clin. Diagn. Lab. Immunol. 11 (4), 669 674. Fomovska, A., Wood, R.D., Mui, E., Dubey, J.P., Ferreira, L. R., Hickman, M.R., et al., 2012. Salicylanilide inhibitors of Toxoplasma gondii. J. Med. Chem. 55 (19), 8375 8391. Forestier, F., 1991. Foetal disease, prenatal diagnosis and practical measures. Presse Med. 1448 1454. Forsgren, M., Gille, E., Ljungstrom, I., 1991. Toxoplasma antibodies in pregnant women in Sweden in 1969, 1979 and 1987. Lancet 337, 1413 1414.

Toxoplasma Gondii

References

Foulon, W., Naessens, A., Derde, M.P., 1994. Evaluation of the possibilities for preventing congenital toxoplasmosis. Am. J. Perinatol. 11, 57 62. Foulon, W., Villena, I., Stray-Pedersen, B., et al., 1999. Treatment of toxoplasmosis during pregnancy: a multicentre study of impact on foetal transmission and children’s sequelae at age 1 year. Am. J. Obstet. Gynecol. 180, 410 415. Frenkel, J.K., Friedlander, S., 1951. Toxoplasmosis: pathology of neonatal disease, pathogenesis, diagnosis and treatment. In: USA Government Printing Office, Washington Publication n 141. US Public Health Service, p. 107. Frenkel, J.K., Hitchings, G.H., 1957. Relative reversal by vitamins (p-aminobenzoic, folic and folinic acid) of the effects of sulfadiazine and pyrimethamine on Toxoplasma, mouse and man. Antibiot. Chemother. 7, 630 638. Frenkel, J.K., Parker, B.B., 1996. An apparent role of dogs in the transmission of Toxoplasma gondii. The probable importance of xenosmophilia. Ann. N.Y. Acad. Sci. 791, 402 407. Frenkel, J.K., Dubey, J.P., Miller, N.L., 1970. Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science 167 (3919), 893 896. Fritz, H.M., Bowyer, P.W., Bogyo, M., Conrad, P.A., Boothroyd, J.C., 2012b. Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance. PLoS One 7 (1), e29955. Fuentes, I., Rodriguez, M., Domingo, C.J., et al., 1996. Urine sample used for congenital toxoplasmosis diagnosis by PCR. J. Clin. Microbiol. 34 (10), 2368 2371. Furco, A., Carmagnat, M., Chevret, S., Garin, Y.J., Pavie, J., De Castro, N., et al., 2008. Restoration of Toxoplasma gondii-specific immune responses in patients with AIDS staring HAART. AIDS 22 (16), 2087 2096. Gajewski, P.D., Falkenstein, M., Hengstler, J.G., Golka, K., 2014. Toxoplasma gondii impairs memory in infected seniors. Brain Behav. Immun. 36, 193 199. Available from: https://doi.org/10.1016/j.bbi.2013.11.019. Gajurel, K., Dhakal, R., Montoya, J.G., 2015. Toxoplasma prophylaxis in haematopoietic cell transplant recipients: a review of the literature and recommendations. Curr. Opin. Infect. Dis. 28 (4), Retrieved from ,https:// journals.lww.com/co-infectiousdiseases/Fulltext/2015/ 08000/Toxoplasma_prophylaxis_in_haematopoietic_ cell.2.aspx.. Gallas-Lindemann, C., Sotiriadou, I., Mahmoodi, M.R., Karanis, P., 2013. Detection of Toxoplasma gondii oocysts in different water resources by Loop Mediated Isothermal Amplification (LAMP). Acta Trop. 125 (2), 231 236. Available from: https://doi.org/10.1016/j. actatropica.2012.10.007.

209

Gallino, A., Maggiorini, M., Kiowski, W., Martin, X., Wunderli, W., Schneider, J., et al., 1996. Toxoplasmosis in heart transplant recipients. Eur. J. Clin. Microbiol. Infect. Dis. 15, 389 393. Garabedian, C., Le Goarant, J., Delhaes, L., Rouland, V., Vaast, P., Valat, A.S., et al., 2012. [Periconceptional toxoplasmic seroconversion: about 79 cases]. J. Gynecol. Obstet. Biol. Reprod. (Paris) 41 (6), 546 552. Garcia-Re`guet, N., Lebrun, M., Fourmaux, M.N., Mercereau-Puijalon, O., Mann, T., Beckers, J.M., et al., 2000. The microneme protein MIC3 of T. gondii is a secretory adhesion that binds to both the surface of the host cells and the surface of the parasite. Cell. Microbiol. 2, 353 364. Gard, S., Magnusson, J.H., 1951. A glandular form of toxoplasmosis in connection with pregnancy. Acta. Med. Scand. 141, 59 64. Gardner, I.D., Remington, J.S., 1978. Aging and the immune response I. Antibody formation and chronic infection in Toxoplasma gondii-infected mice. J. Immunol. 120 (3), 939 943. Retrieved from ,http://www.jimmunol.org/content/120/3/939.abstract.. Gardner, I.D., Remington, J.S., 1978. Aging and the immune response II. Lymphocyte responsiveness and macrophage activation in Toxoplasma gondii-infected mice. J. Immunol. 120 (3), 944 949. Retrieved from ,http://www.jimmunol.org/content/120/3/944. abstract.. Garin, J.P., Brossier, N., Sung, R.T.M., Moyne, T., 1985. Effect of pyrimethamine sulfadioxine (Fansidar) on an avirulent cystogenic strain of Toxoplasma gondii (Prugniaud strain) in white mice. Bull. Soc. Pathol. Exot. Filiales 78, 821 824. Garweg, J.G., Kodjikian, L., Peyron, F., Binquet, C., Fleury, J., Grange, J.D., et al., 2005. Congential ocular toxoplasmosis—ocular manifestations and prognosis after early diagnosis of infection. Klin. Monbl. Augenheilkd. 222 (9), 721 727. Gerhold, R.W., Jessup, D.A., 2012. Zoonotic Diseases Associated With Free-Roaming Cats. Zoonoses Public Health. Gilbert, R.E., Dunn, D.T., Lightman, S., Murray, P.I., Pavesio, C.E., Gormley, P.D., et al., 1999. Incidence of symptomatic toxoplasma eye disease: aetiology and public health implications. Epidemiol. Infect. 123, 283 289. Gilbert, R.E., Freeman, K., Lago, E.G., Bahia-Oliveira, L.M., Tan, H.K., Wallon, M., et al., 2008. Ocular sequelae of congenital toxoplasmosis in Brazil compared with Europe. PLoS Negl. Trop. Dis. 2, e277. Gilbert, R.E., Gras, L., Wallon, M., Peyron, F., Ades, A.E., Dunn, D.T., 2001. Effect of prenatal treatment on mother

Toxoplasma Gondii

210

4. Human Toxoplasma infection

to child transmission of Toxoplasma gondii: retrospective cohort study of 554 mother-child pairs in Lyon, France. Int. J. Epidemiol. 30 (6), 1303 1308. Giordano, L.F.C.M., Lasmar, E.P., Tavora, E.R.F., Lasmar, M.F., 2002. Toxoplasmosis transmitted via kidney allograft: case report and review. Transplant. Proc. 34, 498 499. Glasner, P.D., Silveira, C., Kruszon-Moran, D., Martins, M. C., Burnier Ju´nior, M., Silveira, S., et al., 1992. An unusually high prevalence of ocular toxoplasmosis in southern Brazil. Am. J. Ophthalmol. 114, 136 144. Godwin, R., 2012. Toxoplasma gondii and elevated suicide risk. Vet. Rec. 171 (9), 225. Gollub, E.L., Leroy, V., Gilbert, R., Cheˆne, G., Wallon, M., 2008. European Toxoprevention Study Group (EUROTOXO): effectiveness of health education on Toxoplasma-related knowledge, behaviour, and risk of seroconversion in pregnancy. Eur. J. Obstet. Gynecol. Reprod. Biol. 136 (2), 137 145. Go´mez-Marin, J.E., de-la-Torre, A., Angel-Muller, E., Rubio, J., Arenas, J., Osorio, E., et al., 2011. First Colombian multicentric newborn screening for congenital toxoplasmosis. PLoS Negl. Trop. Dis. 5 (5), e1195. Available from: https://doi.org/10.1371/journal. pntd.0001195. Retrieved from. Gomez, C.A., Budvytyte, L.N., Press, C., Zhou, L., McLeod, R., Maldonado, Y., et al., 2018. Evaluation of three point-of-care tests for detection of toxoplasma immunoglobulin IgG and IgM in the United States: proof of concept and challenges. Open Forum Infect. Dis. 5 (10), ofy215. Available from: https://doi.org/10.1093/ofid/ ofy215. Goodwin, D., Hrubec, T.C., Klein, B.G., Strobl, J.S., Werre, S.R., Han, Q., et al., 2012. Congenital infection of mice with Toxoplasma gondii induces minimal change in behaviour and no change in neurotransmitter concentrations. J. Parasitol. 98 (4), 706 712. Gras, L., Gilbert, R.E., Wallon, M., Peyron, F., CortinaBorja, M., 2004. Duration of the IgM response in women acquiring T. gondii during pregnancy: implications for clinical practices and cross sectional incidence studies. Epidemiol. Infect. 132, 541 548. Gras, L., Wallon, M., Pollak, A., Cortina-Borja, M., Evengard, B., Hayde, M., et al., 2005. Association between prenatal treatment and clinical manifestations of congenital toxoplasmosis in infancy: a cohort study in 13 European centres. Acta Paediatr. 94, 1721 1731. Greenlee, J.E., Johnson Jr., W.D., Campa, J.F., et al., 1975. Adult toxoplasmosis presenting as polymyositis and cerebellar ataxia. Ann. Intern. Med. 82, 367 371. Grigg, M.E., Ganatra, J., Boothroyd, J.C., Margolis, T.P., 2001. Unusual abundance of atypical strains associated

with human ocular toxoplasmosis. J. Infect. Dis. 184, 633 639. Gross, U., Roos, T., Heesemann, J., 1992. Improved serological diagnosis of T. gondii infection by detection of immunoglobulin A (IgA) and IgM antibodies against P30 by using the immunoblot technique. J. Clin. Microbiol. 30, 1436 1441. Gross, U., Luder, C.G., Hendgen, V., Heeg, C., Sauer, I., Weidner, A., et al., 2000. Comparative immunoglobulin G antibody profiles between mother and child (CGMC test) for early diagnosis of congenital toxoplasmosis. J. Clin. Microbiol. 38, 3619 3622. Grossman, P.L., Remington, J.S., 1979. The effect of trimethoprim and sulfamethoxazole on Toxoplasma gondii in vitro and in vivo. Am. J. Trop. Med. Hyg. 28 (3), 445 455. Grover, C.M., Thulliez, P., Remington, J.S., Boothroyd, J.D., 1990. Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reaction and amniotic fluid. J. Clin. Microbiol. 28, 2297 2301. ´ Guenter, W., Bielinski, M., Deptuła, A., Zalas-Wiecek, P., Piskunowicz, M., Szwed, K., et al., 2012. Does Toxoplasma gondii infection affect cognitive function? A case control study. Folia Parasitol. (Praha) 59 (2), 93 98. Guerina, N.G., Hsu, H.W., Meissner, H.C., Maguire, J.H., Lynfield, R., Stechenberg, B., et al., 1994. Neonatal serologic screening and early treatment for congenital T. gondii infection. The New England Regional Toxoplasma Working Group. N. Engl. J. Med. 330, 1858 1863. Guevara-Silva, E.A., Ramı´rez-Crescencio, M.A., SotoHerna´ndez, J.L., Ca´rdenas, G., 2012. Central nervous system immune reconstitution inflammatory syndrome in AIDS: experience of a Mexican neurological centre. Clin. Neurol. Neurosurg. 114 (7), 852 861. Hamdani, N., Daban-Huard, C., Lajnef, M., Richard, J.R., Delavest, M., Godin, O., et al., 2013. Relationship between Toxoplasma gondii infection and bipolar disorder in a French sample. J. Affect Disord. 148, 444 448. Harbut, M.B., Patel, B.A., Yeung, B.K., McNamara, C.W., Bright, A.T., Ballard, J., et al., 2012. Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design. Proc. Natl. Acad. Sci. U.S.A. 109 (52), 21486 21491. Harning, D., Spenter, H., Metsis, A., Vuust, J., Petersen, E., 1996. Recombinant T. gondii SAG1 (P30) expressed in Eschericia coli is recognized by human Toxoplasma-specific IgM and IgG antibodies. Clin. Diag. Lab. Immunol. 3, 355 357. Haverkos, H.W., Remington, J.S., Chan, J.C., 1987. Assessment of Toxoplasma encephalitis (TE) therapy: a cooperative study. Am. J. Med. 82, 907 914.

Toxoplasma Gondii

References

Hedman, K., Lappalainen, M., Seppala, I., Makela, O., 1989. Recent primary Toxoplasma infection indicated by a low avidity of specific IgG. J. Infect. Dis. 159, 726 739. Hedman, K., Lappalainen, M., So¨derlund, M., Hedman, L., 1993. Avidity of IgG in serodiagnosis of infectious diseases. Rev. Med. Microbiol. 4, 123 129. Henriquez, S.A., Brett, R., Alexander, J., Pratt, J., Roberts, C.W., 2009. Neuropsychiatric disease and Toxoplasma gondii infection. Neuroimmunomodulation 16 (2), 122 133. Hermes, G., Ajioka, J.W., Kelly, K.A., Mui, E., Roberts, F., Kasza, K., et al., 2008. Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection. J. Neuroinflamm. 5 (1), 48. Available from: https://doi. org/10.1186/1742-2094-5-48. Hill, D.E., Dubey, J.P., 2013. Toxoplasma gondii prevalence in farm animals in the USA. Int. J. Parasitol. 43, 107 113. Hill, D., Coss, C., Dubey, J.P., et al., 2011. Identification of a sporozoite-specific antigen from Toxoplasma gondii. J. Parasitol. 97 (2), 328 337. Hogan, M.J., Kimura, S.J., Lewis, A., Zweigart, P.A., 1957. Early and delayed ocular manifestations of congenital toxoplasmosis. Trans. Am. Ophthalmol. Soc. 55, 275 296. Hohlfeld, P., Daffos, F., Thulliez, P., et al., 1989. Foetal toxoplasmosis: outcome of pregnancy and infant follow-up after in utero treatment. J. Pediatr. 115 (5 Pt 1), 765 769. Hohlfeld, P., Daffos, F., Costa, J.M., et al., 1994. Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid. N. Engl. J. Med. 331, 695 699. Holec-Ga˛sior, L., 2013. Toxoplasma; recombinant antigens as tools for serodiagnosis of human toxoplasmosis: current status of studies. Clin. Vaccine Immunol. 20 (9), 1343 1351. Available from: https://doi.org/10.1128/ CVI.00117-13. Holec-Gasior, L., Ferra, B., Drapala, D., 2012a. MIC1MAG1-SAG1 chimeric protein, a most effective antigen for detection of human toxoplasmosis. Clin. Vaccine Immunol. 19 (12), 1977 1979. Holec-Ga˛sior, L., Ferra, B., Drapała, D., Lautenbach, D., Kur, J., 2012b. A new MIC1-MAG1 recombinant chimeric antigen can be used instead of the Toxoplasma gondii lysate antigen in serodiagnosis of human toxoplasmosis. Clin. Vaccine Immunol. 19 (1), 57 63. Holland, G.N., O’Connor, G.R., Belfort Jr., R., Remington, J.S., 1996. Toxoplasmosis. In: Pepose, J.S., Holland, G. N., Wilhelmus, K.R. (Eds.), Ocular Infection and Immunity. Mosby Year Book, St. Louis, MO, pp. 1183 1223.

211

Holland, G.N., Crespi, C.M., ten Dam-van Loon, N., et al., 2008. Analysis of recurrence patterns associated with toxoplasmic retinochoroiditis. Am. J. Ophthalmol. 145, 1007 1013. Holliman, R.E., Johnsos, J.D., Savva, D., 1990. Diagnosis of cerebral toxoplasmosis in association with AIDS using the polymerase chain reaction. Scand. J. Infect. Dis. 22, 243 244. Holmdahl, S.C., Holmdahl, K., 1955. The frequency of congenital toxoplasmosis and some viewpoints on the diagnosis. Acta Paediatr. 44, 322 329. Holub, D., Flegr, J., Dragomirecka´, E., Rodriguez, M., Preiss, M., Nova´k, T., et al., 2013. Differences in onset of disease and severity of psychopathology between toxoplasmosis-related and toxoplasmosisunrelated schizophrenia. Acta. Psychiatr. Scand. 127, 227 238. Horacek, J., Flegr, J., Tintera, J., Verebova, K., Spaniel, F., Novak, T., et al., 2012. Latent toxoplasmosis reduces gray matter density in schizophrenia but not in controls: voxel-based-morphometry (VBM) study. World J. Biol. Psychiatry 13 (7), 501 509. Hotop, A., Hlobil, H., Gross, U., 2012. Efficacy of rapid treatment initiation following primary Toxoplasma gondii infection during pregnancy. Clin. Infect. Dis. 54 (11), 1545 1552. Howe, D.K., Sibley, L.D., 1994. T. gondii analysis of different laboratory stocks of the RH strain reveals genetic heterogeneity. Exp. Parasitol. 78, 242 245. Hutchison, W.M., Dunachie, J.F., Siim, J.C., Work, K., 1970. Coccidian-like nature of Toxoplasma gondii. Br. Med. J. 1, 142 144. Hutson, S.L., Wheeler, K.M., McLone, D., Frim, D., Penn, R., Swisher, C.N., et al., 2015. Patterns of hydrocephalus caused by congenital Toxoplasma gondii infection associate with parasite genetics. Clin. Infect. Dis. 61 (12), 1831 1834. Available from: https://doi.org/10.1093/ cid/civ720. Ihara, F., Nishimura, M., Muroi, Y., Mahmoud, M.E., Yokoyama, N., Nagamune, K., et al., 2016. Toxoplasma gondii infection in mice impairs long-term fear memory consolidation through dysfunction of the cortex and amygdala. Infect. Immun. 84 (10), 2861 2870. Available from: https://doi.org/10.1128/IAI.00217-16. Isaac-Renton, J., Bowie, W.R., King, A., et al., 1998. Detection of Toxoplasma gondii oocysts in drinking water. Appl. Environ. Microbiol. 64, 2278 2280. Jacobs, D., Vercammen, M., Saman, E., 1999. Evaluation of recombinant dense granule antigen 7 (GRA7) of T. gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain. Clin. Diag. Lab. Immunol. 6, 24 29.

Toxoplasma Gondii

212

4. Human Toxoplasma infection

Jamieson, S.E., de Roubaix, L.-A., Cortina-Borja, M., Tan, H.K., Mui, E.J., Cordell, H.J., et al., 2008. Genetic and epigenetic factors at COL2A1 and ABCA4 influence clinical outcome in congenital toxoplasmosis. PLoS One 3 (6), e2285. Available from: https://doi.org/10.1371/ journal.pone.0002285. Jamieson, S.E., Cordell, H., Petersen, E., McLeod, R., Gilbert, R.E., Blackwell, J.M., 2009. Host genetic and epigenetic factors in toxoplasmosis. Memorias Do Instituto Oswaldo Cruz 104 (2), 162 169. Available from: https://doi.org/10.1590/s0074-02762009000200006. Jamieson, S.E., Peixoto-Rangel, A.L., Hargrave, A.C., Roubaix, L.-A., de, Mui, E.J., Boulter, N.R., et al., 2010. Evidence for associations between the purinergic receptor P2X(7) (P2RX7) and toxoplasmosis. Genes Immun. 11 (5), 374 383. Available from: https://doi.org/ 10.1038/gene.2010.31. Janku, J., 1923. Pathogenesa a patologicka anatomie tak nazvaneho vrozenehonalezem parasitu v sitnici. Cas. Lek. Ces. 62, 1021 1027. Jenum, P.A., Stray-Pedersen, B., 1998. Development of specific immunoglobulins G, M and A following primary T. gondii infection in pregnant women. J. Clin. Microbiol. 36, 2907 2913. Jenum, P.A., Stray-Pedersen, B., Gundersen, A.G., 1997. Improved diagnosis of primary T. gondii infection in early pregnancy by determination of anti-toxoplasma immunoglobulin G avidity. J. Clin. Microbiol. 35, 1972 1977. Johnson, A.M., Roberts, H., Tenter, A.M., 1992. Evaluation of a recombinant antigen ELISA for the diagnosis of acute toxoplasmosis and comparison with traditional ELISAs. J. Med. Microbiol. 37, 404 409. Johnson, S.M., Murphy, R.C., Geiger, J.A., DeRocher, A.E., Zhang, Z., Ojo, K.K., et al., 2012. Development of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) inhibitors with potent anti-toxoplasma activity. J. Med. Chem. 55 (5), 2416 2426. Johnson, S.K., Fitza, M.A., Lerner, D.A., Calhoun, D.M., Beldon, M.A., Chan, E.T., et al., 2018. Risky business: linking Toxoplasma gondii infection and entrepreneurship behaviours across individuals and countries. Proc. Biol. Sci. 285 (1883), 20180822. Available from: https:// doi.org/10.1098/rspb.2018.0822. Jones, J.L., Dubey, J.P., 2012. Foodborne toxoplasmosis. Clin. Infect. Dis. 55 (6), 845 851. Jones, J.L., Roberts, J.M., 2012. Toxoplasmosis hospitalizations in the USA, 2008, and trends, 1993-2008. Clin. Infect. Dis. 54 (7), e58 e61. Jones, J.L., Kruszon-Moran, D., Wilson, M., et al., 2001. Toxoplasma gondii infection in the USA: seroprevalence and risk factors. Am. J. Epidemiol. 154, 357 365.

Jones, J.L., Kruszon-Moran, D., Sanders-Lewis, K., et al., 2007. Toxoplasma gondii infection in the USA, 1999-2004, decline from the prior decade. Am. J. Trop. Med. Hyg. 77, 405 410. Jones, J.L., Dargelas, V., Roberts, J., et al., 2009. Risk factors for Toxoplasma gondii infection in the USA. Clin. Infect. Dis. 49 (6), 878 884. ˇ ´ , S., Sulc, J., Kˇrivohlava´, R., Kubˇena, A., Flegr, J., Kankova 2012. Slower postnatal motor development in infants of mothers with latent toxoplasmosis during the first 18 months of life. Early Hum. Dev. 88 (11), 879 884. Kankova, S., Flegr, J., Calda, P., 2015. An elevated blood glucose level and increased incidence of gestational diabetes mellitus in pregnant women with latent toxoplasmosis. Folia Parasitol. 62 (1), Retrieved from ,https:// folia.paru.cas.cz/artkey/fol-201501-0056.php.. Karanis, P., Aldeyarbi, H.M., Mirhashemi, M.E., Khalil, K. M., 2013. The impact of the waterborne transmission of Toxoplasma gondii and analysis efforts for water detection: an overview and update. Environ. Sci. Pollut. Res. Int. 20, 86 99. Karanovic, D., Michelow, I.C., Hayward, A.R., DeRavin, S. S., Delmonte, O.M., Grigg, M.E., et al., 2019. Disseminated and congenital toxoplasmosis in a mother and child with activated PI3-kinase δ syndrome type 2 (APDS2): case report and a literature review of toxoplasma infections in primary immunodeficiencies. Front. Immunol. 10, 77. Available from: https://doi. org/10.3389/fimmu.2019.00077. Kasper, L.H., Ware, P.L., 1989. Identification of stage specific antigens of Toxoplasma gondii. Infect. Immun. 57, 688 692. Kaushik, M., Lamberton, P.H., Webster, J.P., 2012. The role of parasites and pathogens in influencing generalised anxiety and predation-related fear in the mammalian central nervous system. Horm. Behav. 62 (3), 191 201. Kavitha, N., Noordin, R., Chan, K.L., Sasidharan, S., 2012. In vitro anti-Toxoplasma gondii activity of root extract/ fractions of Eurycoma longifolia Jack. BMC Compl. Altern. Med. 12, 91. Khan, A., Jordan, C., Muccioli, C., Vallochi, A.L., Rizzo, L. V., Belfort Jr., R., et al., 2006. Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis. Brazil Emerg. Infect. Dis. 12, 942 949. Kieffer, F., Wallon, M., Garcia, P., et al., 2008. Risk factors for retinochoroiditis during the first 2 years of life in infants with treated congenital toxoplasmosis. Pediatr. Infect. Dis. J. 27 32. Kim, J.H., Psevdos Jr., G., Gonzalez, E., Singh, S., Kilayko, M.C., Sharp, V., 2012. All-cause mortality in hospitalized HIV-infected patients at an acute tertiary care

Toxoplasma Gondii

References

hospital with a comprehensive outpatient HIV care program in New York City in the era of highly active antiretroviral therapy (HAART). Infection 41 (2), 545 551. Kimball, A., Kean, B.H., Fuchs, F., 1971. Congenital toxoplasmosis: a prospective study of 4,048 obstetric patients. Am. J. Obstet. Gynecol. 111, 211 218. Klein, W., Tromm, A., Griga, T., Fricke, H., Folwaczny, C., Hocke, M., et al., 2001. The polymorphism at position 2174 of the IL-6 gene is not associated with inflammatory bowel disease. Eur. J. Gastroenterol. Hepatol. 13 (1), Retrieved from ,https://journals.lww.com/ eurojgh/Fulltext/2001/01000/The_polymorphism_at_ position__174_of_the_IL_6_gene.8.aspx.. Kodjikian, L., Hoigne, I., Adam, O., Jacquier, P., AebiOchsner, C., Aebi, C., et al., 2004. Vertical transmission of toxoplasmosis from a chronically infected immunocompetent woman. Pediatr. Infect. Dis. 23 (3), 272 274. Kodjikian, L., Wallon, M., Fleury, M., et al., 2006. Ocular manifestations in congenital toxoplasmosis. Graefes Arch. Clin. Exp. Ophthalmol. 244, 14 21. Koletzko, B., Bauer, C.P., Bung, P., Cremer, M., Flothko¨tter, M., Hellmers, C., et al., 2012. Gesund ins Leben [Nutrition in pregnancy practice recommendations of the Network ‘Healthy Start - Young Family Network’]. Dtsch. Med. Wochenschr. 137 (25 26), 1366 1372. Kong, J.T., Grigg, M.E., Uyetake, L., Parmley, S., Boothroyd, J.C., 2003. Serotyping of Toxoplasma gondii infections in humans using synthetic peptides. J. Infect. Dis. 187 (9), 1484 1495. Koppe, J.G., Kloosterman, G.J., 1982. Congenital toxoplasmosis: long-term follow-up. Padiatr. Padol. 17, 171 179. Koppe, J.G., Loewer-Sieger, D.H., de Roever-Bonnet, H., 1986. Results of 20-year follow-up of congenital toxoplasmosis. Lancet 1 (8475), 254 256. Kortbeek, L.M., Hofhuis, A., Nijhuis, C.D., Havelaar, A.H., 2009. Congenital toxoplasmosis and DALYs in The Netherlands. Mem. Inst. Oswaldo Cruz 104 (2), 370 373. Kotresha, D., Poonam, D., Muhammad Hafiznur, Y., Saadatnia, G., Nurulhasanah, O., Sabariah, O., et al., 2012. Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis. Trop. Biomed. 29 (1), 129 137. Kovacs, J.A., 1995. Toxoplasmosis in AIDS: keeping the lid on. Ann. Intern. Med. 123 (3), 230 231. Lafferty, K.D., 2006. Can the common brain parasite, Toxoplasma gondii, influence human culture? Proc. Biol. Sci. 273 (1602), 2749 2755. Available from: https://doi. org/10.1098/rspb.2006.3641.

213

Lai, B.-S., Witola, W.H., Bissati, K., El Zhou, Y., Mui, E., et al., 2012. Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections. Proc. Natl. Acad. Sci. U.S.A. 109 (35), 14182 14187. Available from: https://doi.org/10.1073/ PNAS.1208775109. Lang, D., Schott, B.H., van Ham, M., Morton, L., Kulikovskaja, L., Herrera-Molina, R., et al., 2018. Chronic Toxoplasma infection is associated with distinct alterations in the synaptic protein composition. J. Neuroinflamm. 15 (1), 216. Available from: https://doi. org/10.1186/s12974-018-1242-1. Lass, A., Pietkiewicz, H., Szostakowska, B., Myjak, P., 2012. The first detection of Toxoplasma gondii DNA in environmental fruits and vegetables samples. Eur. J. Clin. Microbiol. Infect. Dis. 31 (6), 1101 1108. Lassoued, S., Zabraniecki, L., Marin, F., et al., 2007. Toxoplasmic chorioretinitis and antitumor necrosis factor treatment in rheumatoid arthritis. Semin. Arthritis Rheum. 36, 262 263. Lavinsky, D., Romano, A., Muccioli, C., Belfort Jr., R., 2012. Imaging in ocular toxoplasmosis. Int. Ophthalmol. Clin. 52 (4), 131 143. Leal, F.E., Cavazzana, C.L., de Andrade Jr., H.F., Galisteo Jr., A.J., de Mendonc¸a, J.S., Kallas, E.G., 2007. Toxoplasma gondii pneumonia in immunocompetent subjects: case report and review. Clin. Infect. Dis. 15, e62 e66. Lebech, M., Andersen, O., Christensen, N.C., Hertel, J., Nielsen, H.E., Peitersen, B., et al., 1999. Feasibility of neonatal screening for toxoplasma infection in the absence of prenatal treatment. Lancet 353, 1834 1837. Lecolier, B., Pucheu, B., 1993. Inte´reˆt de l’e´tude de l’avidite´ des IgG pour le diagnostic de la toxoplasmose. Pathol. Biol. Paris 41, 155 158. Lecordier, L., Fourmaux, M.P., Mercier, C., Dehecq, E., Masy, E., Cesbron-Delauw, M.F., 2000. Enzyme-linked immunosorbent assays using the recombinant dense granule antigens GRA6 and GRA1 of T. gondii for detection of immunoglobulin G antibodies. Clin. Diagn. Lab. Immunol. 7, 607 611. Lee, Y., Choi, J.Y., Fu, H., Harvey, C., Ravindran, S., Roush, W.R., et al., 2011. Chemistry and biology of macrolide antiparasitic agents. J. Med. Chem. 54 (8), 2792 2804. Lees, M.P., Fuller, S.J., McLeod, R., Boulter, N.R., Miller, C. M., Zakrzewski, A.M., et al., 2010. P2X7 receptormediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages. J. Immunol. 184 (12), 7040 7046. Available from: https:// doi.org/10.4049/jimmunol.1000012. Lefevre-Pettazzoni, M., Bissery, A., Wallon, M., et al., 2007. Impact of spiramycin treatment and gestational age on

Toxoplasma Gondii

214

4. Human Toxoplasma infection

maturation of Toxoplasma gondii immunoglobulin G avidity in pregnant women. Clin. Vaccine Immunol. 14 (3), 239 243. LeFichoux, Y., Marty, P., Chan, H., Doucet, J., 1984. De´tection des IgG anti-toxoplasmiques par I.s.ag. A. A propos de 3,786 se´rologies. Bull. Soc. Fr. Parasitol. 3, 13 18. Lehmann, T., Marcet, P.L., Graham, D.H., Dahl, E.R., Dubey, J.P., 2006. Globalization and the population structure of Toxoplasma gondii. Proc. Natl. Acad. Sci. U. S.A. 103 (30), 11423 11428. LePort, C., Chene, G., Morlat, P., et al., 1996. Pyrimethamine for primary prophylaxis of toxoplasmic encephalitis in patients with human immunodeficiency virus infection: a double-blind, randomized trial. J. Infect. Dis. 173, 91 97. Lester, D., 2012. Toxoplasma gondii and homicide. Psychol. Rep. 111 (1), 196 197. Levin, M., McLeod, R., Young, Q., et al., 1983. Pneumocystis pneumonia: importance of gallium scan for early diagnosis and description of a new immunoperoxidase technique to demonstrate Pneumocystis carinii. Am. Rev. Respir. Dis. 128, 182 185. Levy, R.M., Rosenbloom, S., Perrett, L.V., 1986. Neuroradiologic findings in AIDS: a review of 200 cases. AJNR Am. J. Neurodaiol. 147, 977 983. Levy, R.M., Bredesen, D.E., Rosenblum, M.I., 1988. Opportunistic central nervous system pathology in patients with AIDS. Ann. Neurol. 23, S7 S12. Levy, R.M., Mills, C.M., Posin, J.P., et al., 1990. The efficacy and clinical impact of brain imaging in neurologically symptomatic AIDS patients: a prospective CT/MRI study. J. Acquir. Immune. Defic. Syndr. 3, 461 471. Li, S., Galvan, G., Araujo, F.G., Suzuki, Y., Remington, J.S., Parmley, S., 2000. Serodiagnosis of recently acquired T. gondii infection. Clin. Diagn. Lab. Immunol. 7, 781 787. Li, N., He, Z., Xu, J., Liu, F., Deng, S., Zhang, H., 2010. Association of PDE4D and IL-1 gene polymorphism with ischemic stroke in a Han Chinese population. Brain Res. Bull. 81 (1), 38 42. Available from: https:// doi.org/10.1016/j.brainresbull.2009.09.009. Li, X., Pomares, C., Peyron, F., Press, C.J., Ramirez, R., Geraldine, G., et al., 2019a. Plasmonic gold chips for the diagnosis of Toxoplasma gondii, CMV, and rubella infections using saliva with serum detection precision. Eur. J. Clin. Microbiol. Infect. Dis. 38 (5), 883 890. Available from: https://doi.org/10.1007/s10096-019-03487-1. Li, Y., Severance, E.G., Viscidi, R.P., Yolken, R.H., Xiao, J., 2019b. Persistent infection of the brain induced neurodegeneration associated with activation of complement and microglia. Infect. Immun. 87 (8). Liesenfeld, O., Press, C., Montoya, J.G., Gill, R., IsaacRenton, J.L., Hedman, K., et al., 1997. False-positive

results in immunoglobulin M (IgM) toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test. J. Clin. Microbiol. 1997 (35), 174 178. Liesenfeld, O., Wong, S.Y., Remington, J.S., 1999. Toxoplasmosis in the setting of AIDS. In: Bartlett, J.G., Merigan, T.C., Bolognesi, D. (Eds.), Textbook of AIDS Medicine. Williams & Wilkins, Baltimore, MD, pp. 225 259. Liesenfeld, O., Montoya, J.G., Kinney, S., Press, C., Remington, J.S., 2001a. Effect of testing for IgG avidity in the diagnosis of T. gondii infection in pregnant women: experience in a USA reference laboratory. J. Infect. Dis. 183, 1248 1253. Liesenfeld, O., Montoya, J.G., Kinney, S., Press, C., Remington, J.S., 2001b. Confirmatory serological testing for acute toxoplasmosis and rate of induced abortions among women reported to have positive Toxoplasma immunoglobulin M antibody titres. Am. J. Obstet. Gynecol. 184, 140 145. Lindsay, D.S., Collins, M.V., Mitchell, S.M., et al., 2003. Sporulation and survival of Toxoplasmosis gondii oocysts in seawater. J. Eukaryot. Microbiol. 50, 687 688. L’Ollivier, C., Wallon, M., Faucher, B., Piarroux, R., Peyron, F., Franck, J., 2012. Comparison of mother and child antibodies that target high-molecular-mass Toxoplasma gondii antigens by immunoblotting improves neonatal diagnosis of congenital toxoplasmosis. Clin. Vaccine Immunol. 19 (8), 1326 1328. London, N.J., Hovakimyan, A., Cubillan, L.D., Siverio Jr., C.D., Cunningham Jr., E.T., 2011. Prevalence, clinical characteristics, and causes of vision loss in patients with ocular toxoplasmosis. Eur. J. Ophthalmol. 21, 811 819. Lourenco, E.V., Pereira, S.R., Faca, V.M., Coelho-Castelo, A. A.M., Mineo, J.R., Roque-Barreira, M.C., et al., 2001. T. gondii micronemal protein MIC1 is a lactose-binding lectin. Glycobiology 11, 541 547. Lourido, S., Jeschke, G.R., Turk, B.E., Sibley, L.D., 2013. Exploiting the unique ATP-binding pocket of toxoplasma calcium-dependent protein kinase 1 to identify its substrates. ACS Chem. Biol. 8 (6), 1155 1162. Available from: https://doi.org/10.1021/cb400115y. Luft, B.J., Remington, J.S., 1984. Acute Toxoplasma infection among family members of patients with acute lymphadenopathic toxoplasmosis. Arch. Intern. Med. 144, 53 56. Luft, B.J., Remington, J.S., 1992. Toxoplasmic encephalitis in AIDS (AIDS commentary). Clin. Infect. Dis. 15, 211 222. Luft, B.J., Conley, F., Remington, J.S., et al., 1983. Outbreak of central-nervous-system toxoplasmosis in western Europe and North America. Lancet 1 (8328), 781 784.

Toxoplasma Gondii

References

Luft, B.J., Hafner, R., Korzun, A.H., et al., 1993. Toxoplasmic encephalitis in patients with the acquired immunodeficiency syndrome. N. Engl. J. Med 329, 995 1000. Lykins, J., Wang, K., Wheeler, K., Clouser, F., Dixon, A., El Bissati, K., et al., 2016. Understanding toxoplasmosis in the United States through “Large Data” analyses. Clin. Infect. Dis. 63 (4), 468 475. Available from: https://doi. org/10.1093/cid/ciw356. Lykins, J.D., Filippova, E.V., Halavaty, A.S., Minasov, G., Zhou, Y., Dubrovska, I., et al., 2018a. CSGID solves structures and identifies phenotypes for five enzymes in Toxoplasma gondii. Front. Cell. Infect. Microbiol. 8, 352. Available from: https://doi.org/10.3389/fcimb.2018.00352. Lykins, J., Li, X., Levigne, P., Zhou, Y., El Bissati, K., Clouser, F., et al., 2018b. Rapid, inexpensive, fingerstick, whole-blood, sensitive, specific, point-of-care test for anti-Toxoplasma antibodies. PLoS Negl. Trop. Dis. 12 (8), e0006536. Available from: https://doi.org/10.1371/ journal.pntd.0006536. Mack, D.G., Johnson, J.J., Roberts, F., Roberts, C.W., Estes, R.G., David, C., et al., 1999. HLA-class II genes modify outcome of Toxoplasma gondii infection. Int. J. Parasitol. 29 (9), 1351 1358. Available from: https://doi.org/ 10.1016/S0020-7519(99)00152-6. Magaldi, C., Elkis, H., Pattoli, D., Coscina, A.L., 1969. Epidemic of toxoplasmosis at a university in Sa˜o-Jose´dos Campos, S.P. Brazil. 1. Clinical and serologic data. Rev. Latinoam Microbiol. Parasitol. (Mex.) 11 (1), 5 13. Mahamed, D.A., Mills, J.H., Egan, C.E., Denkers, E.Y., Bynoe, M.S., 2012. CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system. Proc. Natl. Acad. Sci. U.S.A. 109 (40), 16312 16317. Maksimov, P., Zerweck, J., Maksimov, A., Hotop, A., Gross, U., Spekker, K., et al., 2012. Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptidemicroarray. PLoS One 7 (3), e34212. Mandelbrot, L., Kieffer, F., Sitta, R., Laurichesse-Delmas, H., Winer, N., Mesnard, L., et al., 2018. Prenatal therapy with pyrimethamine 1 sulfadiazine vs spiramycin to reduce placental transmission of toxoplasmosis: a multicenter, randomized trial. Am. J. Obstet. Gynecol. 219 (4), 386.e1 386.e9. Available from: https://doi.org/ 10.1016/j.ajog.2018.05.031. Marcolino, P.T., Silva, D.A.O., Camargo, M.E., Mineo, J.R., 2000. Molecular markers in acute and chronic phases of human Toxoplasmosis: determination of immunoglobulin G avidity by Western blotting. Clin. Diag. Lab. Immunol. 7, 384 389. Martino, R., Bretagne, S., Einsele, H., et al., 2000. Toxoplasmosis after hematopoietic stem cell transplantation. Clin. Infect. Dis. 31, 1188 1195.

215

Martino, R., Bretagne, S., Einsele, H., Maertens, J., Ullmann, A.J., Parody, R., Cordonnier, C., the Infectious Disease Working Party of the European Group for Blood and Marrow Transplantation, 2005. Early detection of Toxoplasma infection by molecular monitoring of T. gondii in peripheral blood samples after allogenic stem cell transplantation. Clin. Infect. Dis. 40, 67 78. Martins-Duarte, E.S., Souza, W.D., Vommaro, R.C., 2012. Toxoplasma gondii: the effect of fluconazole combined with sulfadiazine and pyrimethamine against acute toxoplasmosis in murine model. Exp. Parasitol. 133 (3), 294 299. Masur, H., Jones, T.C., Lempert, J.A., et al., 1978. Outbreak of toxoplasmosis in a family and documentation of acquired retinochoroiditis. Am. J. Med 64, 396 402. Matsuo, Y., Takeishi, S., Miyamoto, T., et al., 2007. Toxoplasmosis encephalitis following severe graft-vs.host disease after allogeneic hematopoietic stem cell transplantation: 17 yr experience in Fukuoka BMT group. Eur. J. Haematol. 79, 317 321. McAuley, J., Boyer, K.M., Patel, D., Mets, M., Swisher, C., Roizen, N., et al., 1994. Early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis: The Chicago Collaborative Treatment Trial. Clin. Infect. Dis. 18, 38 72. McCabe, R.E., Brooks, R.G., Dorfman, R.F., Remington, J.S., 1987. Clinical spectrum in 107 cases of toxoplasmic lymphadenopathy. Rev. Infect. Dis. 9, 754 774. McLeod, R., Berry, P.F., Marshall Jr., W.H., et al., 1979. Toxoplasmosis presenting as brain abscesses. Diagnosis by computerized tomography and cytology of aspirated purulent material. Am. J. Med 67 (4), 711 714. McLeod, R., Beem, M.O., Estes, R.G., 1985. Lymphocyte anergy specific to Toxoplasma gondii antigens in a baby with congenital toxoplasmosis. J. Clin. Lab. Immunol. 17, 149 153. McLeod, R., Mack, D.G., Boyer, K., et al., 1990. Phenotypes and functions of lymphocytes in congenital toxoplasmosis. J. Lab. Clin. Med. 116 (5), 623 635. McLeod, R., Mack, D., Foss, R., et al., 1992. Levels of pyrimethamine in sera and cerebrospinal and ventricular fluids from infants treated for congenital toxoplasmosis. Antimicrob. Agents Chemother. 36, 1040 1048. McLeod, R., Boyer, K., Roizen, N., Stein, L., Swisher, C., Holfels, E., et al., 2000. The child with congenital toxoplasmosis. Curr. Clin. Top Infect. Dis. 20, 189 208. McLeod, R., Khan, A.R., Noble, G.A., et al., 2006a. Severe sulfadiazine hypersensitivity in a child with reactivated congenital toxoplasmic chorioretinitis. Pediatr. Infect. Dis. J. 25 (3), 270 272. McLeod, R., Boyer, K., Karrison, T., Kasza, K., Swisher, C., Roizen, N., et al., 2006b. Outcome of treatment for congenital toxoplasmosis, 1981-2004: the National

Toxoplasma Gondii

216

4. Human Toxoplasma infection

Collaborative Chicago-Based, Congenital Toxoplasmosis Study. Clin. Infect. Dis. 42, 1383 1394. Epub 2006 Apr 11. McLeod, R., Kieffer, F., Sautter, M., Hosten, T., Pelloux, H., 2009. Why prevent, diagnose and treat congenital toxoplasmosis? Mem. Inst. Oswaldo Cruz 104, 320 344. McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Toxoplasmosis Study Group. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981 2009). Clin. Infect. Dis. 54 (11), 1595 1605. McLeod, R., Lee, D., Boyer, K., 2013a in press. Toxoplasmosis in the Foetus and Newborn Infant. McLeod, R., Lee, D., Boyer, K., 2013b in press. Diagnosis of Congenital Toxoplasmosis: A Practical Procedural Atlas. McLeod, R., Wheeler, K.M., Boyer, K., 2015. Reply to Wallon and Peyron. Clin. Infect. Dis. 62 (6), 812 814. Available from: https://doi.org/10.1093/cid/civ1036. McLone, D., Frim, D., Penn, R., et al., 2019. Outcomes of hydrocephalus secondary to congenital toxoplasmosis. J. Neurosurg.: Pediatr. 1 8. McPhillie, M., Zhou, Y., El Bissati, K., Dubey, J., Lorenzi, H., Capper, M., et al., 2016. New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections. Sci. Rep. 6, 29179. Available from: https://doi.org/10.1038/ srep29179. Retrieved from. McPhillie, M., Zhou, Y., Hickman, M., Gordon, J., Weber, C., Li, Q., et al., 2020. Potent tetrahydroquinolone eliminates apixomplexan parasites. In Submission. Mele, A., Paterson, P.J., Prentice, H.G., Leoni, P., Kibbler, C.C., 2002. Toxoplasmosis in bone marrow transplantation: a report of two cases and systematic review of the literature. Bone Marrow Transplant 29, 691 698. Meneceur, P., Bouldouyre, M.A., Aubert, D., Villena, I., Menotti, J., Sauvage, V., et al., 2008. In vitro susceptibility of various genotypic strains of Toxoplasma gondii in pyrimethamine, sulfadiazine, and atovaquone. Antimicrob. Agents Chemother. 52 (4), 1269 1277. Mennechet, F.J., Kasper, L.H., Rachinel, N., Minns, L.A., Luangsay, S., Vandewalle, A., et al., 2004. Intestinal intraepithelial lymphocytes prevent pathogen-driven inflammation and regulate the Smad/T-bet pathway of lamina propria CD4 1 T cells. Eur. J. Immunol. 34 (4), 1059 1067. Meroni, V., Genco, F., Tinelli, C., et al., 2009. Spiramycin treatment of Toxoplasma gondii infection in pregnant women impairs the production and the avidity maturation of T. gondii-specific immunoglobulin G antibodies. Clin. Vaccine Immunol. 16 (10), 1517 1520.

Mets, M., Holfels, E., Boyer, K.M., et al., 1996. Eye manifestations of congenital toxoplasmosis. Am. J. Ophthalmol. 122, 309 324. Miller, M.A., Gardner, J.A., Kreuder, C., et al., 2002. Coastal freshwater runoff is a risk factor for Toxoplasmosis gondii infection of southern sea otters (Enhydra lutris nereis). Int. J. Parasitol. 32, 997 1006. Minot, S., Melo, M.B., Li, F., Lu, D., Niedelman, W., Levine, S.S., et al., 2012. Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity. Proc. Natl. Acad. Sci. U.S.A. 109 (33), 13458 13463. Mitchell, C.D., Erlich, S.S., Mastrucci, M.T., Hutto, S.C., Parks, W.P., Scott, G.B., 1990. Congential toxoplasmosis occurring in infants perinatally infected with human immunodeficiency virus 1. Pediatr. Infect. Dis. J. 9, 512 518. Mitra, R., Sapolsky, R.M., Vyas, A., 2012. Toxoplasma gondii infection induces dendritic retraction in basolateral amygdala accompanied by reduced corticosterone secretion. Dis. Model Mech. 6 (2), 516 520. Moncada, P.A., Montoya, J.G., 2012. Toxoplasmosis in the foetus and newborn: an update on prevalence, diagnosis and treatment. Expert Rev. Anti. Infect. Ther. 10 (7), 815 828. Montoya, J.G., 2002. Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J. Infect. Dis. 185 (Suppl. 1), S73 S82. 2002 Feb. 15. Montoya, J.G., 2018. Systematic screening and treatment of toxoplasmosis during pregnancy: is the glass half full or half empty? Am. J. Obstet. Gynecol. 219 (4), 315 319. Available from: https://doi.org/10.1016/j. ajog.2018.08.001. Montoya, J.G., Remington, J.S., 2008. Management of Toxoplasma gondii infection during pregnancy. Clin. Infect. Dis. 47 (4), 554 566. Montoya, J.G., Jordan, R., Lingamneni, S., et al., 1997. Toxoplasmic myocarditis and polymyositis in patients with acute acquired toxoplasmosis diagnosed during life. Clin. Infect. Dis. 24, 676 683. Montoya, J.G., Parmley, S., Liesenfeld, O., Jaffe, O., Remington, J.S., 1999. Use of the poluymerase chain reaction for diagnosis of ocular toxoplasmosis. Ophthalmology 106, 1554 1563. Montoya, J.G., Giraldo, L.F., Efron, B., et al., 2001. Infectious complications among 620 consecutive heart transplant patients at Stanford University Medical Centre. Clin. Infect. Dis. 33, 629 640. Montoya, J.G., Liesenfeld, O., Kinney, S., Press, C., Remington, J.S., 2002. VIDAS test for avidity of Toxoplasma-specific immunoglobulin G for confirmatory testing of pregnant women. J. Clin. Microbiol. 40, 2504 2508.

Toxoplasma Gondii

References

Montoya, J.G., Boothroyd, J.C., Kovacs, J.A., 2010. Toxoplasma gondii. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. JAMA 274 (8), 3495 3526. Morelle, C., Varlet-Marie, E., Brenier-Pinchart, M.P., Cassaing, S., Pelloux, H., Bastien, P., et al., 2012. Comparative assessment of a commercial kit and two laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis. J. Clin. Microbiol. 50 (12), 3977 3982. Morin, L., Lobry, J.R., Peyron, F., Wallon, M., 2012. Seasonal variations in acute toxoplasmosis in pregnant women in the Rhoˆne-Alpes region (France). Clin. Microbiol. Infect. 18 (10), E401 E403. Morris Jr., J.G., Greenspan, A., Howell, K., Gargano, L.M., Mitchell, J., Jones, J.L., et al., 2012. Southeastern Centre for Emerging Biologic Threats tabletop exercise: foodborne toxoplasmosis outbreak on college campuses. Biosecur. Bioterror. 10 (1), 89 97. Mosti, M., Pinto, B., Giromella, A., Fabiani, S., Cristofani, R., Panichi, M., et al., 2012. A 4-year evaluation of toxoplasmosis seroprevalence in the general population and in women of reproductive age in central Italy. Epidemiol. Infect. 11, 1 4. Mui, E.J., Schiehser, G.A., Milhous, W.K., Hsu, H., Roberts, C.W., Kirisits, M., et al., 2008. Novel triazine JPC-2067-B inhibits Toxoplasma gondii in vitro and in vivo. PLoS Negl. Trop. Dis. 2 (3), e190. Available from: https://doi. org/10.1371/journal.pntd.0000190. Retrieved from. Munro, J.B., Silva, J.C., 2012. Ribonucleotide reductase as a target to control apicomplexan diseases. Curr. Issues Mol. Biol. 14 (1), 9 26. Murat, J.B., L’Ollivier, C., Fricker Hidalgo, H., Franck, J., Pelloux, H., Piarroux, R., 2012. Evaluation of the new Elecsys Toxo IgG avidity assay for toxoplasmosis and new insights into the interpretation of avidity results. Clin. Vaccine Immunol. 19 (11), 1838 1843. Naessens, A., Jenum, P.A., Pollak, A., et al., 1999. Diagnosis of congenital toxoplasmosis in the neonatal period: a multicentre evaluation. J. Pediatr. 135, 714 719. Naot, Y., Remington, J.S., 1980. An enzyme-linked immunosorbent assay for detection of IgM antibodies to Toxoplasma gondii use for diagnosis of acute acquired toxoplasmosis. J. Infect. Dis. 142, 757 766. Naot, Y., Barnett, E.V., Remington, J.S., 1981a. Method for avoiding false-positive results occurring in immunoglobulin M enzyme-linked immunosorbent assays due to presence of both rheumatoid factor and antinuclear antibodies. J. Clin. Microbiol. 14, 73 78. Naot, Y., Desmonts, G., Remington, J.S., 1981b. IgM enzyme-linked immunosorbent assay test for the

217

diagnosis of congenital Toxoplasma infection. J. Pediatr. 98, 32 36. Naranjo-Galvis, C.A., de-la-Torre, A., Mantilla-Muriel, L.E., Beltra´n-Angarita, L., Elcoroaristizabal-Martı´n, X., McLeod, R., et al., 2018. Genetic polymorphisms in cytokine genes in Colombian patients with ocular toxoplasmosis. Infect. Immun. 86 (4), e00597 17. Available from: https://doi.org/10.1128/IAI.00597-17. Natella, V., Cozzolino, I., Sosa Fernandez, L.V., Vigliar, E., 2012. Lymph nodes fine needle cytology in the diagnosis of infectious diseases: clinical settings. Infez. Med. 20 (Suppl. 3), 12 15. Neto, E.C., Rubin, R., Schulte, J., Giugliani, R., 2004. Newborn screening for congenital infectious diseases. Emer. Infect. Dis. 10, 1069 1073. Ngoˆ, H.M., Zhou, Y., Lorenzi, H., Wang, K., Kim, T.-K., Zhou, Y., et al., 2017. Toxoplasma modulates signature pathways of human epilepsy, neurodegeneration & cancer. Sci. Rep. 7 (1), 11496. Available from: https://doi. org/10.1038/s41598-017-10675-6. Ngoungou, E.B., Bhalla, D., Nzoghe, A., Darde´, M.-L., Preux, P.-M., 2015. Toxoplasmosis and epilepsy — systematic review and meta analysis. PLoS Negl. Trop. Dis. 9 (2), e0003525. Available from: https://doi.org/ 10.1371/journal.pntd.0003525. Retrieved from. Niebuhr, D.W., Millikan, A.M., Cowan, D.N., Yolken, R., Li, Y., Weber, N.S., 2008. Selected infectious agents and risk of schizophrenia among U.S. Military Personnel. Am. J. Psychiatry 165 (1), 99 106. Available from: https://doi.org/10.1176/appi.ajp.2007.06081254. Nielsen, H.V., Schmidt, D.R., Petersen, E., 2005. Diagnosis of congenital toxoplasmosis by two-dimensional immunoblot differentiation of mother and child IgG-profiles. J. Clin. Microbiol. 2005 (43), 711 715. Noble, A.G., Latkany, P., Kusmierczyk, J., et al., 2010. Chorioretinal lesions in mothers of children with congenital toxoplasmosis in the National Collaborative Chicago-based, Congenital Toxoplasmosis Study. Sci. Med. 20 (1), 20 26. Nogareda, F., Le Strat, Y., Villena, I., De Valk, H., Goulet, V., 2014. Incidence and prevalence of Toxoplasma gondii infection in women in France, 1980 2020: model-based estimation. Epidemiol. Infect. 142 (8), 1661 1670. Available from: https://doi.org/DOI:10.1017/S0950268813002756. O’Connor, G.R., 1974. Manifestations and management of ocular toxoplasmosis. Bull. N.Y. Acad. Med. 50, 192 210. Odberg-Ferragut, C., Soete, M., Engels, A., Samyn, B., Loyens, A., van Beeumen, J., et al., 1996. Molecular cloning of the T. gondii SAG4 gene encoding an 18 kDa bradyzoite specific surface protein. Mol. Biochem. Parasitol. 82, 237 244.

Toxoplasma Gondii

218

4. Human Toxoplasma infection

Okusaga, O., Postolache, T.T., 2012. Chapter 19: Toxoplasma gondii, the immune system, and suicidal behaviour. In: Dwivdei, Y. (Ed.), The Neurological Basis of Suicide. Frontiers in Neuroscience, CRC Press, Boca Raton, FL. Olariu, T., Remington, S., McLeod, R., Alam, A., Montoya, J., 2011. Severe congenital toxoplasmosis in the United State: clinical and serologic findings in untreated infants. Pediatr. Infect. Dis. J. 30 (12), 1056 1061. Ore´fice, J.L., Costa, R.A., Scott, I.U., Calucci, D., Ore´fice, F., on behalf of the Grupo Mineiro de Pesquisa em Doenc¸as Oculares Inflamato´rias (MINAS), 2012. Spectral optical coherence tomography findings in patients with ocular toxoplasmosis and active satellite lesions (MINAS Report 1). Acta Ophthalmol 91 (1), 41 47. Orr, K.E., Gould, F.K., Short, G., Dark, J.H., Hilton, C.J., Corris, P.A., et al., 1994. Outcome of T. gondii mismatches in heart transplant recipients over a period of 8 years. J. Infect. 29, 249 253. Osthoff, M., Chew, E., Bajel, A., Kelsey, G., Panek-Hudson, Y., Mason, K., et al., 2012. Disseminated toxoplasmosis after allogeneic stem cell transplantation in a seronegative recipient. Transpl. Infect. Dis . Palma, S., Reyes, H., Guzman, L., et al., 1984. Dermatomyositis and toxoplasmosis. Rev. Med. Chil. 111, 164 167. Papoz, L., Simondon, F., Saurin, W., Sarmini, H., 1986. A simple model relevant to toxoplasmosis applied to epidemiologic results in France. Am. J. Epidemiol. 123 (1), 154 161. Parmley, S.F., Goebel, F.D., Remington, J.S., 1992. Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by polymerase chain reaction. J. Clin. Microbiol. 30, 3000 3002. Parmley, S.F., Yang, S.M., Harth, G., Sibley, L.D., Sucharczuk, A., Remington, J.S., 1994. Molecular characterization of a 65-kDa T. gondii antigen expressed abundantly in the matrix of tissue cysts. Mol. Biochem. Parasitol. 66, 283 296. Paspalaki, P.K., Mihailidou, E.P., Bitsori, M., et al., 2001. Polymyositis and myocarditis associated with acquired toxoplasmosis in an immunocompetent girl. BMC Musculoskelet Disord. 2, 8. Patel, D.V., Holfels, E.M., Vogel, N.P., et al., 1996. Resolution of intracranial calcifications in infants with treated congenital toxoplasmosis. Radiology 199 (2), 433 440. 1996. Paul, M., Petersen, E., Pawlowski, Z.S., Szczapa, J., 2000. Neonatal screening for congenital toxoplasmosis in the Poznan region of Poland by analysis of Toxoplasma gondii-specific IgM antibodies eluted from filter paper blood spots. Pediatr. Infect. Dis. J. 19, 30 36.

Pearce, B.D., Kruszon-Moran, D., Jones, J.L., 2012. The relationship between Toxoplasma gondii infection and mood disorders in the third National Health and Nutrition Survey. Biol. Psychiatry 72 (4), 290 295. Pedersen, M.G., Mortensen, P.B., Norgaard-Pedersen, B., Postolache, T.T., 2012. Toxoplasma gondii infection and self-directed violence in mothers. Arch. Gen. Psychiatry 69 (11), 1123 1130. Peixoto, L., Chen, F., Harb, O.S., Davis, P.H., Beiting, D.P., Brownback, C.S., et al., 2010. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses. Cell. Host Microbe 8 (2), 208 218. Perkins, E.S., Smith, C.H., Schofield, P.B., 1956. Treatment of uveitis with pyrimethamine (Daraprim). Br. J. Ophthalmol. 40, 577 586. Pernas, L., Ramirez, R., Holmes, T.H., Montoya, J.G., Boothroyd, J.C., 2014. Immune profiling of pregnant Toxoplasma-infected US and Colombia patients reveals surprising impacts of infection on peripheral blood cytokines. J. Infect. Dis. 210 (6), 923 931. Available from: https://doi.org/10.1093/infdis/jiu189. Petersen, E., Borobio, M.V., Guy, E., Liesenfeld, O., Meroni, V., Naessens, A., et al., 2005. European multicentre study of the LIAISONaˆ automated diagnostic system for determination of specific IgG, IgM and IgG-avidity index in toxoplasmosis. J. Clin. Microbiol. 43, 1570 1574. Petersen, E., Kijlstra, A., Stanford, M., 2012. Epidemiology of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 20 (2), 68 75. Petithory, J.C., Reiter-Owona, I., Berthelot, F., Milgram, M., De Loye, J., Petersen, E., 1996. Performance of European Laboratories testing serum samples for Toxoplasma gondii. Eur. J. Clin. Microbiol. Infect. Dis. 15, 45 49. Peyron, F., Ateba, A.B., Wallon, M., et al., 2003. Congenital toxoplasmosis in twins: a report of fourteen consecutive cases and a comparison with published data. Pediatr. Infect. Dis. J. 22, 695 701. Peyron, F., Garweg, J.G., Wallon, M., et al., 2011. Long-term impact of treated congenital toxoplasmosis on quality of life and visual performance. Pediatr. Infect. Dis. J. 30 (7), 597 600. Phan, L., Kasza, K., Jalbrzikowski, J., et al., 2008a. Longitudinal study of new eye lesions in treated congenital toxoplasmosis. Ophthalmology 115, 553 559. Phan, L., Kasza, K., Jalbrzikowski, J., et al., 2008b. Longitudinal study of new eye lesions in children with toxoplasmosis who were not treated during the first year of life. Am. J. Ophthalmol. 146, 375 384. ´ Pietkiewicz, H., Hiszczynska-Sawicka, E., Kur, J., Petersen, E., Nielsen, H.V., Stankiewicz, M., et al., 2004. Usefulness of T. gondii recombinant antigens in serodiagnosis of human toxoplasmosis. J. Clin. Microbiol. 42, 1779 1781.

Toxoplasma Gondii

References

Pinkerton, H., Weinman, D., 1940. Toxoplasma infection in man. Arch. Pathol. 30, 374 392. Pinon, J.M., Chemla, C., Villena, I., Foudrinier, F., Aubert, D., Puygauthier-Toubas, D., et al., 1996. Early neonatal diagnosis of congenital toxoplasmosis: value of comparative enzyme-linked immunofiltration assay immunological profiles and anti-T. gondii immunoglobulin M (IgM) or IgA immunocapture and implications for postnatal therapeutic strategies. J. Clin. Microbiol. 34, 579 583. Pinon, J.M., Dumon, H., Chemla, C., Franck, J., Petersen, E., Lebech, M., et al., 2001. Strategy for diagnosis of congenital toxoplasmosis: evaluation of methods comparing mothers and newborns and standard methods for postnatal detection of immunoglobulin G, M, and A antibodies. J. Clin. Microbiol. 39, 2267 2271. Pinto, B., Castagna, B., Mattei, R., Bruzzi, R., Chiumiento, L., Cristofani, R., et al., 2012. Seroprevalence for toxoplasmosis in individuals living in north west Tuscany: access to Toxo-test in central Italy. Eur. J. Clin. Microbiol. Infect. Dis. 31 (6), 1151 1156. Pollock, J.L., 1979. Toxoplasmosis appearing to be dermatomyositis. Arch. Dermatol. 115, 736 737. Pomares, C., Devillard, S., Holmes, T.H., Olariu, T.R., Press, C.J., Ramirez, R., et al., 2018. Genetic characterization of Toxoplasma gondii DNA samples isolated from humans living in North America: an unexpected high prevalence of atypical genotypes. J. Infect. Dis. 218 (11), 1783 1791. Available from: https://doi.org/10.1093/ infdis/jiy375. Post, M.J., Chan, J.C., Hensley, G.T., et al., 1983. Toxoplasma encephalitis in Haitian adults with acquired immunodeficiency syndrome: a clinical-pathologic-CT correlation. AJR Am. J. Roentgenol. 140, 861 868. Pouletty, P., Kadouche, J., Garcia-Gonzalez, M., Mihaesco, E., Desmonts, G., Thulliez, P., et al., 1985. An antihuman chain monoclonal antibody: use for detection of IgM antibodies to T. gondii by reverse immunosorbent assay. J. Immunol. Meth. 76, 289 298. Prado, S.P., Pacheco, V.C., Noemi, I.H., et al., 1978. [Toxoplasma pericarditis and myocarditis]. Rev. Chil. Pediatr. 49, 179 185. Press, C., Montoya, J.G., Remington, J.S., 2005. Use of a single serum sample for diagnosis of acute toxoplasmosis in pregnant women and other adults. J. Clin. Microbiol. 43, 3481 3483. prevention is cost-saving. PLoS Negl. Trop. Dis. 11, e0005648 (2017). Prusa, A.R., Hayde, M., Pollak, A., Herkner, K.R., Kasper, D.C., 2012. Evaluation of the liaison automated testing system for diagnosis of congenital toxoplasmosis. Clin. Vaccine Immunol. 19 (11), 1859 1863. Prusa, A.-R., Kasper, D.C., Sawers, L., Walter, E., Hayde, M., Stillwaggon, E., 2017. Congenital toxoplasmosis in Austria: prenatal screening for prevention is cost-saving.

219

PLoS Negl. Trop. Dis. 11 (7), e0005648. Available from: https://doi.org/10.1371/journal.pntd.0005648. Retrieved from. Rabaud, C., May, T., Lucet, J.C., Leport, C., AmbroiseThomas, P., Canton, P., 1996. Pulmonary toxoplasmosis in patients infected with human immunodeficiency virus: a French National Survey. Clin. Infect. Dis. 23, 1249 1254. Raymond, J., Poissonnier, M.H., Thulliez, P.H., et al., 1990. Presence of gamma interferon in human acute and congenital toxoplasmosis. J. Clin. Microbiol. 28 (6), 1434 1437. Reischl, U., Bretagne, S., Kru¨ger, D., Ernault, P., Costa, J.M., 2003. Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect. Dis. 3, 7. Reiter-Owona, I., Petersen, E., Joynson, D., Aspo¨ck, H., Darde´, M.L., Disko, R., et al., 1999. Looking back on half a century of the Sabin-Feldman dye-test: its past and present role in the serodiagnosis of toxoplasmosis. Results of an European multicentre study. Bull W. H. O. 929 935. Remington, J.S., 1974. Toxoplasmosis in the adult. Bull. N. Y. Acad. Med. 50, 211 227. Remington, J.S., Desmonts, G., 1976. Congenital toxoplasmosis. In: Remington, J.S., Klein, J.O. (Eds.), Infectious Diseases of the Foetus and Newborn Infant, first ed. WB Saunders, Philadelphia, PA, pp. 297 299. Remington, J.S., Barnett, C.G., Meikel, M., et al., 1962. Toxoplasmosis and infectious mononucleosis. Arch. Intern. Med. 110, 744 753. Remington, J.S., Miller, M.J., Brownlee, I., 1968. IgM antibodies in acute toxoplasmosis. I. Diagnostic significance in congenital cases and a method for their rapid demonstration. Pediatrics 41, 1082 1091. Remington, J.S., Araujo, F.G., Desmonts, G., 1985. Recognition of different Toxoplasma gondii antigens by IgM and IgG antibodies in mothers and their congenitally infected newborns. J. Infect. Dis. 152, 1020 1024. Remington, J.B., Wilder Antunes, F., et al., 1991. Clindamycin for toxoplasmosis encephalitis in AIDS (letter). Lancet 338, 1142 1143. Remington, J.S., McLeod, R., Thulliez, P., 2001. Toxoplasmosis. In: Remington, J.S., Klein, J. (Eds.), Infectious Diseases of the Foetus and Newborn Infant. WB Saunders, Philadelphia, PA, pp. 205 346. Remington, J.S., Thulliez, P., Montoya, J.G., 2004. Recent developments for diagnosis of toxoplasmosis. J. Clin. Microbiol. 42 (3), 941 945. Remington, J.S., McLeod, R., Thulliez, P., Desmonts, G., 2011. Toxoplasmosis. In: Remington, J., Klein, J. (Eds.), Infectious Diseases of the Foetus and Newborn Infant, seventh ed. WB Saunders, Philadelphia, PA.

Toxoplasma Gondii

220

4. Human Toxoplasma infection

Renier, D., Sainte-Rose, C., Pierre-Kahn, A., Hirsch, J.F., 1988. Prenatal hydrocephalus: outcome and prognosis. Childs Nerv. Syst. 4 (4), 213 222. Renoult, E., Georges, E., Biava, M.F., Hulin, L., Frimat, D., Hestin, D., et al., 1997. Toxoplasmosis in kidney transplant recipients: report of six cases and review. Clin. Infect. Dis. 24, 625 634. Rigsby, P., Rijpkema, S., Guy, E.C., Francis, J., Das, R.G., 2004. Evaluation of a candidate international standard preparation for human anti-Toxoplasma immunoglobulin G. J. Clin. Microbiol. 42, 5133 5138. Rilling, V., Dietz, K., Krczal, D., Knotek, F., Enders, G., 2003. Evaluation of a commercial IgG/IgM Western blot assay for early postnatal diagnosis of congenital toxoplasmosis. Eur. J. Clin. Microbiol. Infect. Dis. 22, 174 180. Robert-Gangneux, F., Commerce, V., Tourte-Schaefer, C., Dupouy-Camet, J., 1999. Performance of a Western blot assay to compare mother and newborn anti-Toxoplasma antibodies for the early neonatal diagnosis of congenital toxoplasmosis. Eur. J. Clin. Microbiol. Infect. Dis. 18, 648 654. Robert, A., Luyasu, V., Zuffrey, J., Hedman, K., Petersen, E., European Network on Congenital Toxoplasmosis, 2001. Potential of the specific markers in the early diagnosis of Toxoplasma-infection: A multicentre study using combination of isotype IgG, IgM, IgA and IgE with values of avidity assay. Eur. J. Clin. Microbiol. Infect. Dis. 20, 467 474. Roberts, C., Prasad S., Khaliq, F., et al., 2014. Chapter 25: Adaptive immunity and genetics of the host immune response. In: L. Weiss, K. Kim (Eds.). second ed., Elsevier, London, pp. 819 933. Roizen, N., Swisher, C.N., Stein, M.A., et al., 1995. Neurologic and developmental outcome in treated congenital toxoplasmosis. Pediatrics 95, 11 20. Roizen, N., Kasza, K., Karrison, T., et al., 2006. Impact of visual impairment on measures of cognitive function for children with congenital toxoplasmosis: implications for compensatory intervention strategies. Pediatrics 118, e379 e390. Rollins-Raval, M.A., Marafioti, T., Swerdlow, S.H., Roth, C. G., 2012. The number and growth pattern of plasmacytoid dendritic cells vary in different types of reactive lymph nodes: an immunohistochemical study. Hum. Pathol. 44 (6), 1003 1010. Romand, S., Pudney, M., Derouin, F., 1993. In vitro and in vivo activities of the hydroxynaphthoquinone atovaquone alone or combined with pyrimethamine, sulfadiazine, clarithromycin, or minocycline against Toxoplasma gondii. Antimicrob. Agents Chemother. 37 (11), 2371 2378.

Romand, S., Chosson, M., Franck, J., et al., 2004. Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of foetal infection with Toxoplasma gondii. Am. J. Obstet. Gynecol. 190, 797 802. Roue, R., Debord, T., Denamur, E., Ferry, M., Dormont, D., Barre-Sinoussi, F., et al., 1984. Diagnosis of Toxoplasma encephalitis in absence of neeurological signs by early computerised tomography scanning in patients with AIDS. Lancet ii, 1472. Roux, C., Desmont, G., Mulliez, N., et al., 1976. [Toxoplasmosis and pregnancy. Evaluation of 2 years of prevention of congenital toxoplasmosis in the maternity ward of Hoˆpital Saint-Antoine (1973 1974)]. J. Gynecol. Obstet. Biol. Reprod. (Paris) 5 (2), 249 264. Ruskin, J., Remington, J.S., 1976. Toxoplasmosis in the compromised host. Ann. Intern. Med. 84, 193 199. Rutaganira, F.U., Barks, J., Dhason, M.S., Wang, Q., Lopez, M.S., Long, S., et al., 2017. Inhibition of Calcium Dependent Protein Kinase 1 (CDPK1) by pyrazolopyrimidine analogs decreases establishment and reoccurrence of central nervous system disease by Toxoplasma gondii. J. Med. Chem. 60 (24), 9976 9989. Available from: https://doi.org/10.1021/acs.jmedchem.7b01192. Ryning, F.W., McLeod, R., Maddox, J.C., et al., 1979. Probable transmission of Toxoplasma gondii by organ transplantation. Ann. Intern. Med. 90, 47 49. Saadatnia, G., Mohamed, Z., Ghaffarifar, F., Osman, E., Moghadam, Z.K., Noordin, R., 2012. Toxoplasma gondii excretory secretory antigenic proteins of diagnostic potential. APMIS 120 (1), 47 55. Sabin, A.B., 1941. Toxoplasmic encephalitis in children. J. Am. Med. Assoc. 116, 801 807. Sabin, A.B., Feldman, H.A., 1948. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoan parasite (Toxoplasma). Science 108, 660 663. Safronova, A., Araujo, A., Camanzo, E.T., Moon, T.J., Elliott, M.R., Beiting, D.P., et al., 2019. Alarmin S100A11 initiates a chemokine response to the human pathogen Toxoplasma gondii. Nat. Immunol. 20 (1), 64 72. Available from: https://doi.org/10.1038/s41590-0180250-8. Samuel, B.U., Hearn, B., Mack, D., Wender, P., Rothbard, J., Kirisits, M.J., et al., 2003. Delivery of antimicrobials into parasites. Proc. Natl. Acd. Sci. U.S.A. 100 (24), 14281 14286. Santana, S.S., Gebrim, L.C., Carvalho, F.R., Barros, H.S., Barros, P.C., Pajuaba, A.C.A.M., et al., 2015. CCp5A Protein from Toxoplasma gondii as a serological marker of oocyst-driven infections in humans and domestic animals. Front. Microbiol. 6, 1305Retrieved from. Available from: https://www.frontiersin.org/article/10.3389/ fmicb.2015.01305.

Toxoplasma Gondii

References

Sauer, T.C., Meyers, S.M., Shen, D., Vegh, S., Vygantas, C., Chan, C.-C., 2010. Primary intraocular (retinal) lymphoma after ocular toxoplasmosis. Retinal Cases Brief Rep. 4 (2), 160 163. Available from: https://doi.org/ 10.1097/ICB.0b013e3181ad3916. Saxon, S.A., Knight, W., Reynolds, D.W., Stagno, S., Alford, C.A., 1973. Intellectual deficits in children born with subclinical congenital toxoplasmosis: a preliminary report. J. Pediatr. 85 (5), 792 797. Schaefer, L.E., Dyke, J.W., Meglio, F.D., Murray, P.R., Crafts, W., Niles, A.C., 1989. Evaluation of microparticle enzyme immunoassays for immunoglobulins G and M to rubella virus and Toxoplasma gondii on the Abbott IMx automated analyzer. J. Clin. Microbiol. 27, 2410 2413. Schreiner, M., Liesenfeld, O., 2009. Small intestinal inflammation following oral infection with Toxoplasma gondii does not occur exclusively in C57BL/6 mice: review of 70 reports from the literature. Mem. Inst. Oswaldo Cruz 104 (2), 221 233. Sensini, A., Pascoli, S., Marchetti, D., Castronari, A., Marangi, M., Sbaraglia, G., et al., 1996. IgG avidity in the serodiagnosis of acute T. gondii infection: a multicentre study. Clin. Microbiol. Infect. 2, 25 29. Severance, E.G., Kannan, G., Gressitt, K.L., Xiao, J., Alaedini, A., Pletnikov, M.V., et al., 2012. Anti-gluten immune response following Toxoplasma gondii infection in mice. PLoS One 7 (11), E50991. Shapiro, K., Conrad, P.A., Mazet, J.A., et al., 2010. Effect of estuarine wetland degradation on transport of Toxoplasma gondii surrogates from land to sea. Appl. Environ. Microbiol. 76 (20), 6821 6828. Shapiro, K., Miller, M.A., Packham, A.E., et al., 2016. Dual congenital transmission of Toxoplasma gondii and Sarcocystis neurona in a late-term aborted pup from a chronically infected southern sea otter (Enhydra lutris nereis). Parasitology 143 (3), 276 288. Sharma, R., Loseto, L.L., Ostertag, S.K., Tomaselli, M., Bredtmann, C.M., Crill, C., et al., 2018. Qualitative risk assessment of impact of Toxoplasma gondii on health of beluga whales, Delphinapterus leucas, from the Eastern Beaufort Sea, Northwest Territories. Arctic Sci. 4 (3), 321 337. Available from: https://doi.org/10.1139/as2017-0037. Sibley, L.D., Boothroyd, J.C., 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 359, 82 85. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.-H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166 (6), 1423 1435.e12. Available from: https://doi.org/10.1016/j.cell.2016.08.019.

221

Siim, J.C., 1951. Acquired toxoplasmosis: report of seven cases with strongly positive serologic reactions. J. Am. Med. Assoc. 147, 1641 1645. Silveira, C., Belfort Jr., R., Burnier Jr., M., et al., 1988. Acquired toxoplasmic infection as the cause of toxoplasmic retinochoroiditis in families. Am. J. Ophthalmol. 106, 362 364. Silveira, C., Belfort Jr., R., Muccioli, C., Abreu, M.T., Martins, M.C., et al., 2001. A follow-up study of Toxoplasma gondii infection in southern Brazil. Am. J. Ophthalmol. 131, 351 354. Silveira, C., Belfort Jr., R., Muccioli, C., Holland, G.N., Victora, C.G., et al., 2002. The effect of long-term intermittent trimethoprim/sulfamethoxazole treatment on recurrences of toxoplasmic retinochoroiditis. Am. J. Ophthalmol. 134 (1), 41 46. Sing, A., Leitritz, L., Roggenkamp, A., et al., 1999. Pulmonary toxoplasmosis in bone marrow transplant recipients: report of two cases and review. Clin. Infect. Dis. 29, 429 433. Singer, M.A., Hagler, W.S., Grossniklaus, H.E., 1993. Toxoplasma gondii retinochoroiditis after liver transplantation. Retina 13, 40 45. Singh, U., Brewer, J.L., Boothroyd, J.C., 2002. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in T. gondii reveals a hierarchy of gene induction. Mol. Microbiol. 44, 721 733. Slavin, M.A., Meyers, J.D., Remington, J.S., et al., 1994. Toxoplasma gondii infection in marrow transplant recipients: a 20 year experience. Bone Marrow Transplant 13, 549 557. Sørensen, T., Spenter, J., Jaliashvili, I., Christiansen, M., Nørgarrd-Pedersen, B., Petersen, E., 2002. An automated time-resolved immunofluometric assay for detection of T. gondii-specific IgM and IgA antibodies in filterpaper samples from newborns. Clin. Chem. 48, 1981 1986. Stagno, S., Reynolds, D.W., Amos, C.S., et al., 1977. Auditory and visual defects resulting from symptomatic and subclinical congenital cytomegaloviral and Toxoplasma infections. Pediatrics 669 678. Stec, J., Fomovska, A., Afanador, G.A., Muench, S.P., Zhou, Y., Lai, B.-S., et al., 2013. Modification of triclosan scaffold in search of improved inhibitors for enoyl-acyl carrier protein (ACP) reductase in Toxoplasma gondii. ChemMedChem 8 (7), 1138 1160. Available from: https://doi.org/10.1002/cmdc.201300050. Steen, E., Ka˚ss, E., 1951. A new Toxoplasma antigen for complement fixation test. Acta Path. Microbiol. Scand. 28, 36 39. Stepick-Biek, P., Thulliez, P., Araujo, F.G., Remington, J.S., 1990. IgA antibodies for diagnosis of acute congenital and acquired toxoplasmosis. J. Infect. Dis. 162 (1), 270 273.

Toxoplasma Gondii

222

4. Human Toxoplasma infection

Stillwaggon, E., Carrier, C.S., Sautter, M., McLeod, R., 2011. Maternal serologic screening to prevent congenital toxoplasmosis: a decision-analytic economic model. PLoS Negl. Trop. Dis. 5 (9), e1333. Available from: https:// doi.org/10.1371/journal.pntd.0001333. Retrieved from. Stock, A.-K., Dajkic, D., Ko¨hling, H.L., von Heinegg, E.H., Fiedler, M., Beste, C., 2017. Humans with latent toxoplasmosis display altered reward modulation of cognitive control. Sci. Rep. 7 (1), 10170. Available from: https://doi.org/10.1038/s41598-017-10926-6. Stommel, E.W., Seguin, R., Thadani, V.M., Schwartzman, J. D., Gilbert, K., Ryan, K.A., et al., 2001. Cryptogenic epilepsy: an infectious etiology? Epilepsia 42 (3), 436 438. Strabelli, T.M., Siciliano, R.F., Vidal Campos, S., Bianchi Castelli, J., Bacal, F., Bocchi, E.A., et al., 2012. Toxoplasma gondii myocarditis after adult heart transplantation: successful prophylaxis with pyrimethamine. J. Trop. Med. 2012, 853562. Suzuki, Y., Israelski, D.M., Dannemann, B.R., Stepick-Biek, P., Thulliez, P., Remington, J.S., 1988a. Diagnosis of toxoplasmic encephalitis in patients with acquired immunodeficiency syndrome by using a new serological method. J. Clin. Microbiol. 26, 2541 2543. Suzuki, Y., Thulliez, P., Desmonts, G., Remington, J.S., 1988b. Antigen(s) responsible for immunoglobulin G responses specific for the acute stage of Toxoplasma infection in humans. J. Clin. Microbiol. 26 (5), 901 905. Swisher, C.N., Boyer, K., McLeod, R., The Toxoplasmosis Study Group, 1994. Congenital toxoplasmosis. Semin. Pediatr. Neurol. 14 25. Syn, G., Anderson, D., Blackwell, J.M., Jamieson, S.E., 2017. Toxoplasma gondii infection is associated with mitochondrial dysfunction in-vitro. Front. Cell. Infect. Microbiol. 7, 512. Syn, G., Anderson, D., Blackwell, J.M., Jamieson, S.E., 2018. Epigenetic dysregulation of host gene expression in Toxoplasma infection with specific reference to dopamine and amyloid pathways. Infect. Genet. Evol. 65, 159 162. Available from: https://doi.org/10.1016/j. meegid.2018.07.034. SYROCOT Systemic Review on Congenital Toxoplasmosis Study Group, Thiebaut, R., Leproust, S., Chene, G., Gilbert, R., 2007. Effectiveness of prenatal treatment for congenital toxoplasmosis: a meta-analysis of individual patients’ data. Lancet 369, 115 122. Tabbara, K.F., O’Connor, G.R., 1980. Treatment of ocular toxoplasmosis with clindamycin and sulfadiazine. Opthalmology 87, 129 134. Tan, T.G., Mui, E., Cong, H., Witola, W.H., Montpetit, A., Muench, S.P., et al., 2010. Identification of T. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans. Vaccine 28 (23),

3977 3989. Available from: https://doi.org/10.1016/j. vaccine.2010.03.028. Teutsch, S.M., Juranek, D.D., Sulzer, A., Dubey, J.P., Sikes, R.K., 1979. Epidemic toxoplasmosis associated with infected cats. N. Engl. J. Med 300, 695 699. Thalhammer, O., 1975. Die Toxoplasmose-Untersuchung von Schwangeren und neugeborenen. Wien Klein. Wochenschr. 87, 676 681. Thulliez, P., 1992. Screening programme for congenital toxoplasmosis in France. Scand. J. Infect. Dis. 84 (Suppl.), 43 45. Thulliez, P., 2001a. Commentary: Efficacy of prenatal treatment for toxoplasmosis: a possibility that cannot be ruled out. Int. J. Epidemiol. 30, 1315 1316. Thulliez, P., 2001b. Maternal and foetal infection. In: Joynson, D.H.M., Wreghitt, T.G. (Eds.), Toxoplasmosis: A Comprehensive Clinical Guide. Cambridge University Press, pp. 193 213. Thulliez, P., Remington, J.S., Santoro, F., et al., 1986. A new agglutination test for the diagnosis of acute and chronic Toxoplasma infection. Pathol. Biol. (Paris) 34, 173 177. Thulliez, P., Remington, J.S., Santoro, F., Ovlaque, G., Sharma, S., Desmonts, G., 1989. A new agglutination reaction for the diagnosis of the development stage of acquired toxoplasmosis. Pathol. Biol. Paris 34, 173 177. Tissot-Dupont, D., Fricker-Hidalgo, H., Brenier-Pinchart, M.P., Bost-Bru, C., Ambroise-Thomas, P., Pelloux, H., 2003. Usefulness of Western blot in serological follow-up of newborns suspected of congenital toxoplasmosis. Eur. J. Clin. Microbiol. Infect. Dis. 22, 122 125. Toporovski, J., Romano, S., Hartmann, S., Benini, W., Chieffi, P.P., 2012. Nephrotic syndrome associated with toxoplasmosis: report of seven cases. Rev. Inst. Med. Trop. Sao Paulo 54 (2), 61 64. Torres, R., Barr, M., Thorn, M., Gregory, G., Kiely, S., Chanin, E., et al., 1993. Randomized trial of dapsone and aerosolized pentamidine for the prophylaxis of Pneumocystis carinii pneumonia and toxoplasmic encephalitis. Am. J. Med. 95, 573 583. Torrey, E.F., Yolken, R.H., 2019. Schizophrenia as a pseudogenetic disease: a call for more gene-environmental studies. Psychiatry Res. 278, 146 150. Available from: https://doi.org/10.1016/j.psychres.2019.06.006. Torrey, E.F., Bartko, J.J., Yolken, R.H., 2012. Toxoplasma gondii and other risk factors for schizophrenia: an update. Schizophr. Bull. 38 (3), 642 647. Townsend, J.J., Wolinsky, J.S., Baringer, J.R., Johnson, P.C., 1975. Acquired toxoplasmosis. A neglected cause of treatable nervous system disease. Arch. Neurol. 32 (5), 335 343.

Toxoplasma Gondii

References

Trivin˜o-Valencia, J., Lora, F., Zuluaga, J.D., Gomez-Marin, J.E., 2016. Detection by PCR of pathogenic protozoa in raw and drinkable water samples in Colombia. Parasitol. Res. 115 (5), 1789 1797. Available from: https://doi.org/10.1007/s00436-016-4917-5. Vallochi, A.L., Muccioli, C., Martins, M.C., Silveira, C., Belfort Jr., R., et al., 2005. The genotype of Toxoplasma gondii strains causing ocular toxoplasmosis in humans in Brazil. Am. J. Ophthalmol. 139, 350 351. Vasconcelos-Santos, D.V., 2012. Ocular manifestations of systemic disease: toxoplasmosis. Curr. Opin. Ophthalmol. 23 (6), 543 550. Vasconcelos-Santos, D.V., Queiroz Andrade, G.M., 2011. Geographic differences in outcomes of congenital toxoplasmosis. Pediatr. Infect. Dis. J. 30 (9), 816 817. Vasconcelos-Santos, D.V., Machado Azevedo, D.O., Campos, W.R., Ore´fice, F., Queiroz-Andrade, G.M., et al., 2009. Congenital toxoplasmosis in southeastern Brazil: results of early ophthalmologic examination of a large cohort of neonates. Ophthalmology 116, 2199 2205. Vaudaux, J.D., Muccioli, C., James, E.R., et al., 2010. Identification of an atypical strain of Toxoplasma gondii as the cause of a waterborne outbreak of toxoplasmosis in Santa Isabel do Ivai. Brazil. J. Infect. Dis. 202, 1226 1233. Vaughan, L.B., Wenzel, R.P., 2012. Disseminated toxoplasmosis presenting as septic shock five weeks after renal transplantation. Transpl. Infect. Dis. 15 (1), E20 24. Vidadala, R.S.R., Rivas, K.L., Ojo, K.K., Hulverson, M.A., Zambriski, J.A., Bruzual, I., et al., 2016. Development of an orally available and central nervous system (CNS) penetrant Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) inhibitor with minimal human ether-a-go-go-related gene (hERG) activity for the treatment of toxoplasmosis. J. Med. Chem. 59 (13), 6531 6546. Available from: https://doi.org/10.1021/ acs.jmedchem.6b00760. Vidal, J.E., Colombo, F.A., de Oliveira, A.C., Focaccia, R., Pereira-Chioccola, V.L., 2004. PCR assay using cerebrospinal fluid for diagnosis of cerebral toxoplasmosis in Brazilian AIDS patients. J. Clin. Microbiol. 42, 4765 4768. Vietzke, W.M., Geldermann, A.H., Grimley, P.M., et al., 1968. Toxoplasmosis complicating malignancy: experience at the National Cancer Institute. Cancer 21, 816 827. Villard, O., Breit, L., Cimon, B., Franck, J., Fricker-Hidalgo, H., Godineau, N., et al., 2012. Comparison of four commercially available avidity tests for Toxoplasma-specific IgG antibodies. Clin. Vaccine Immunol. 20 (2), 197 204.

223

Villena, I., Ancelle, T., Delmas, C., et al., 2010. Congenital toxoplasmosis in France in 2007: first results from a national surveillance system. Euro. Surveill. 15, 19600. Vogel, N., Kirisits, M., Michael, E., Bach, H., Hostetter, M., Boyer, K., et al., 1996. Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception. Clin. Infect. Dis. 23, 1055 1060. Vyas, A., Sapolsky, R., 2010. Manipulation of host behaviour by Toxoplasma gondii: what is the minimum a proposed proximate mechanism should explain? Folia Parasitol. (Praha) 57 (2), 88 94. Wadhawan, A., Dagdag, A., Duffy, A., Daue, M.L., Ryan, K.A., Brenner, L.A., et al., 2017. Positive association between Toxoplasma gondii IgG serointensity and current dysphoria/hopelessness scores in the Old Order Amish: a preliminary study. Pteridines 28 (3 4), 185 194. Available from: https://doi.org/10.1515/pterid-2017-0019. Wainwright, K.E., Miller, M.A., Kreuder, C., et al., 2007. Chemical inactivation of Toxoplasma gondii oocysts in water. J. Parasitol. 93, 925 931. Wallon, M., Peyron, F., 2015. Effect of antenatal treatment on the severity of congenital toxoplasmosis. Clin. Infect. Dis. 62 (6), 811 812. Available from: https://doi.org/ 10.1093/cid/civ1035. Wallon, M., Cozon, G., Ecochard, R., Lewin, P., Peyron, F., 2001. Serological rebound in congenital toxoplasmosis: long-term follow-up of 133 children. Eur. J. Pediatr. 160, 534 540. Wallon, M., Kodjikian, L., Binquet, C., et al., 2004. Longterm ocular prognosis in 327 children with congenital toxoplasmosis. Pediatr. 113, 1567 1572. Wallon, M., Franck, J., Thulliez, P., et al., 2010. Accuracy of real-time polymerase chain reaction for Toxoplasma gondii in amniotic fluid. Obstet. Gynecol. 115, 727 733. Wallon, M., Kieffer, F., Binquet, C., Thulliez, P., GarciaMeric, P., Dureau, P., et al., 2011. Congenital toxoplasmosis: randomized comparison of strategies for retinochoroiditis prevention. Therapie 66 (6), 473 480. Wallon, M., Peyron, F., Cornu, C., Vinault, S., Abrahamowicz, M., Bonithon Kopp, C., et al., 2013. Congenital Toxoplasma infection: monthly prenatal screening decreases transmission rate and improves clinical outcome at 3 years. Clin. Infect. Dis. 56 (9), 1223 1231. Walls, K.W., Bullock, S.L., English, D.K., 1977. Use of the enzyme-linked immunosorbent assay (ELISA) and its microadaptation for the serodiagnosis of toxoplasmosis. J. Clin. Microbiol. 5, 273 277.

Toxoplasma Gondii

224

4. Human Toxoplasma infection

Wang, Y., Cirelli, K.M., Barros, P.D.C., Sangare´, L.O., Butty, V., Hassan, M.A., et al., 2019. Three Toxoplasma gondii dense granule proteins are required for induction of Lewis rat macrophage pyroptosis. MBio 10 (1), e02388 18. Available from: https://doi.org/10.1128/mBio.02388-18. Warren, J., Sabin, A.B., 1942. The complement fixation reaction in Toxoplasma infection. Proc. Soc. Exp. Biol. Med. 51, 11 14. Webster, J.P., Lamberton, P.H., Donnelly, C.A., Torrey, E. F., 2006. Parasites as causative agents of human affective disorders? The impact of anti-psychotic, moodstabilizer and anti-parasite medication on Toxoplasma gondii’s ability to alter host behaviour. Proc. Biol. Sci. 273 (1589), 1023 1030. Webster, J.P., Kaushik, M., Bristow, G.C., McConkey, G. A., 2013. Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour? J. Exp. Biol. 216 (Pt 1), 99 112. Weiss, L.M., Dubey, J.P., 2009. Toxoplasmosis: a history of clinical observations. Int. J. Parasitol. 39 (8), 895 901. Weiss, L.M., Harris, C., Berger, M., Tanowitz, H.B., Wittner, M., 1988. Pyrimethamine concentration in serum and cerebrospinal fluid during treatment of Toxoplasma encephalitis in patients with AIDS. J. Infect. Dis. 157, 580 583. Welsch, M.E., Zhou, J., Gao, Y., Yan, Y., Porter, G., Agnihotri, G., et al., 2016. Discovery of potent and selective leads against Toxoplasma gondii dihydrofolate reductase via structure-based design. ACS Med. Chem. Lett. 7 (12), 1124 1129. Available from: https://doi. org/10.1021/acsmedchemlett.6b00328. Wilson, C., Remington, J.S., Stagno, S., Reynolds, D.W., 1980a. Development of adverse sequelae in children born with subclinical congenital Toxoplasma infection. Pediatrics 66, 767 774. Wilson, C.B., Desmonts, G., Couvreur, J., Remington, J.S., 1980b. Lymphocyte transformation in the diagnosis of congenital Toxoplasma infection. N. Engl. J. Med 302 (14), 785 788. Wing, M.E.J., 2016. Editorial Commentary: Toxoplasmosis: Cats Have It, Humans Get It, but How Much Disease Does It Cause? Clin. Infect. Dis. 63 (4), 476 477. Available from: https://doi.org/10.1093/cid/ciw358. Witola, W.H., Mui, E., Hargrave, A., Liu, S., Hypolite, M., Montpetit, A., et al., 2011. NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells. Infect. Immun. 79 (2), 756 766. Available from: https://doi.org/10.1128/ IAI.00898-10. Witola, W.H., Liu, S.R., Montpetit, A., Welti, R., Hypolite, M., Roth, M., et al., 2014. ALOX12 in human

toxoplasmosis. Infect. Immun. 82 (7), 2670 2679. Available from: https://doi.org/10.1128/IAI.01505-13. Wolf, A., Cowen, D., Paige, B.H., 1939. Toxoplasmic encephalomyelitis III. A new case of ganulomatous encephalitis due to a protozoon. Am. J. Pathol. 15, 657 694. Wong, S.Y., Hajdu, M.P., Ramirez, R., et al., 1993. Role of specific immunoglobulin E in diagnosis of acute Toxoplasma infection and toxoplasmosis. J. Clin. Microbiol. 31 (11), 2952 2959. Wong, W.K., Upton, A., Thomas, M.G., 2012. Neuropsychiatric symptoms are common in immunocompetent adult patients with Toxoplasma gondii acute lymphadenitis. Scand. J. Infect. Dis. . Wreghitt, T.G., Gray, J.J., Pavel, P., Balfour, A., Fabbri, A., Sharples, L.D., et al., 1992. Efficacy of pyrimethamine for the prevention of donor-acquired T. gondii infection in heart and heart-lung transplant patients. Transplant. Int. 5, 197 200. Wreghitt, T.G., McNeil, K., Roth, C., et al., 1995. Antibiotic prophylaxis for the prevention of donor-acquired Toxoplasma gondii infection in transplant patients. [letter, comment]. J. Infect. 31, 253 254. ´ Wujcicka, W., Gaj, Z., Wilczynski, J., Nowakowska, D., 2015. Contribution of IL6 2174 G . C and IL1B 13954 C . T polymorphisms to congenital infection with Toxoplasma gondii. Eur. J. Clin. Microbiol. Infect. Dis. 34 (11), 2287 2294. Available from: https://doi.org/ 10.1007/s10096-015-2481-z. ´ Wujcicka, W., Wilczynski, J., Nowakowska, D., 2013. SNPs in toll-like receptor (TLR) genes as new genetic alterations associated with congenital toxoplasmosis? Eur. J. Clin. Microbiol. Infect. Dis. 32 (4), 503 511. ´ Wujcicka, W., Wilczynski, J., Nowakowska, D., 2017. Genetic alterations within TLR genes in development of Toxoplasma gondii infection among Polish pregnant women. Adv. Med. Sci. 62 (2), 216 222. ´ ´ Wujcicka, W., Wilczynski, J., Spiewak, E., Nowakowska, D., 2018. Genetic modifications of cytokine genes and Toxoplasma gondii infections in pregnant women. Microb. Pathog. 121, 283 292. Available from: https:// doi.org/10.1016/J.MICPATH.2018.05.048. Wulf, M.W.H., van Crevel, R., Portier, R., Meulen, C.G., Melchers, W.J.G., van der Ven, A., et al., 2005. Toxoplasmosis after renal transplantation: Implications of a missed diagnosis. J. Clin. Microbiol. 43, 3544 3547. Xiao, J., Kannan, G., Jones-Brando, L., Brannock, C., Krasnova, I.N., Cadet, J.L., et al., 2012a. Sex-specific changes in gene expression and behaviour induced by chronic Toxoplasma infection in mice. Neuroscience 206, 39 48.

Toxoplasma Gondii

Further reading

Xiao, J., Viscidi, R.P., Kannan, G., Pletnikov, M.V., Li, Y., Severance, E.G., et al., 2012b. The Toxoplasma MAG1 peptides induce sex-based humoral immune response in mice and distinguish active from chronic human infection. Microbes. Infect. 15 (1), 74 83. Yang, Lu, Clouser, WuZhiaodong, McLeod, 2020. DTI MRS, fMRI and structural MRI in persons with toxoplasma infection sand matched controls. In Submission. Young, J.D., McGwire, B.S., 2005. Infliximab and reactivation of cerebral toxoplasmosis. N. Engl. J. Med. 353 (14), 1530 1531. Available from: https://doi.org/10.1056/ NEJMc051556. Zhou, Y., Fomovska, A., Muench, S., Lai, B.S., Mui, E., Mcleod, R., 2014. Spiroindolone that inhibits PfATPase4 is a potent, cidal inhibitor of Toxoplasma gondii tachyzoites in vitro and in vivo. Antimicrob. Agents Chemother. 58 (3), 1789 1792. Zhu, W., Li, J., Pappoe, F., Shen, J., Yu, L., 2019. Strategies developed by Toxoplasma gondii to survive in the host. Front. Microbiol. 10, 899. Available from: https://doi. org/10.3389/fmicb.2019.00899.

Further reading Brown, C.R., McLeod, R., 1990. Class I MHC genes and CD8 1 T cells determine cyst number in Toxoplasma gondii infection. J. Immunol. 145 (10), 3438 3441Retrieved from. Available from: http://www.jimmunol.org/content/ 145/10/3438.abstract. Cavaille`s, P., Sergent, V., Bisanz, C., Papapietro, O., Colacios, C., Mas, M., et al., 2006. The rat Toxo1 locus directs toxoplasmosis outcome and controls parasite proliferation and spreading by macrophage-dependent mechanisms. Proc. Natl. Acad. Sci. U.S.A. 103 (3), 744 749. Available from: https://doi.org/10.1073/ pnas.0506643103. Cavailles, P., Flori, P., Papapietro, O., Bisanz, C., Lagrange, D., Pilloux, L., et al., 2014. A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage. PLoS Pathog. 10 (4), e1004005. Available from: https://doi.org/10.1371/journal. ppat.1004005. Retrieved from. Cesbron-Delauw, M.F., Tomavo, S., Beauchamps, P., Fourmaux, M.P., Camus, D., Capron, A., et al., 1994. Similarities between the primary structure of two distinct major surface proteins of Toxoplasma gondii. J. Biol. Chem. 269, 16217 16222. Cirelli, K.M., Gorfu, G., Hassan, M.A., Printz, M., Crown, D., Leppla, S.H., et al., 2014. Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii. PLoS Pathog. 10 (3), e1003927. Available from: https://doi.org/10.1371/journal. ppat.1003927. Retrieved from.

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Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A., et al., 2010. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8 1 T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii. Immun. Res. 6, 12. Available from: https://doi.org/ 10.1186/1745-7580-6-12. D’Angelillo, A., De Luna, E., Romano, S., Bisogni, R., Buffolano, W., Gargano, N., et al., 2011. Toxoplasma gondii dense granule antigen 1 stimulates apoptosis of monocytes through autocrine TGF-β signaling. Apoptosis 16 (6), 551 562. Available from: https://doi. org/10.1007/s10495-011-0586-0. Demirel, E., Kolo¨ren, Z., Karaman, U., Ayaz, E., 2014. Investigation on Toxoplasma gondii by polymerase chain reaction and loop-mediated isothermal amplification in water samples from Giresun, Turkey. Mikrobiyol. Bul. 48 (4), 661 668. Retrieved from https://www.ncbi.nlm. nih.gov/pubmed/25492661. Dickerson, F., Stallings, C., Origoni, A., et al., 2014. Antibodies to Toxoplasma gondii and cognitive functioning in schizophrenia, bipolar disorder, and nonpsychiatric controls. J. Nerv. Ment. Dis. 202 (8), 589 593. El Bissati, K., Zhou, Y., Dasgupta, D., Cobb, D., Dubey, J.P., Burkhard, P., et al., 2014. Effectiveness of a novel immunogenic nanoparticle platform for Toxoplasma peptide vaccine in HLA transgenic mice. Vaccine 32 (26), 3243 3248. Available from: https://doi.org/10.1016/j. vaccine.2014.03.092. Ewald, S.E., Chavarria-Smith, J., Boothroyd, J.C., 2014. NLRP1 is an inflammasome sensor for Toxoplasma gondii. Infect. Immun. 82 (1), 460 468. Available from: https://doi.org/10.1128/IAI.01170-13. Fritz, H., Barr, B., Packham, A., Melli, A., Conrad, P.A., 2012a. Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts. J. Microbiol. Methods 88 (1), 47 52. Garcia-lopez, L., Cardona, N., Hernandez, A., OsorioMendez, J., Jorge, G.-M., 2019. Expression of inhibitory receptors on CD8 1 T cells from ocular toxoplasmosis individuals. Int. Toxoplasma Congress 15 23 (2), 36. Goldszmid, R.S., Caspar, P., Rivollier, A., White, S., Dzutsev, A., Hieny, S., et al., 2012. NK cell-derived interferon-γ orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection. Immunity 36 (6), 1047 1059. Available from: https://doi.org/10.1016/j.immuni.2012.03.026. Gorfu, G., Cirelli, K.M., Melo, M.B., Mayer-Barber, K., Crown, D., Koller, B.H., et al., 2014. Dual role for inflammasome sensors NLRP1 and NLRP3 in murine resistance to Toxoplasma gondii. MBio 5 (1), e01117 13. Available from: https://doi.org/10.1128/mBio.01117-13.

Toxoplasma Gondii

226

4. Human Toxoplasma infection

Gov, L., Karimzadeh, A., Ueno, N., Lodoen, M.B., 2013. Human innate immunity to Toxoplasma gondii is mediated by host caspase-1 and ASC and parasite GRA15. MBio 4 (4), . Available from: https://doi.org/10.1128/ mBio.00255-13e00255-13. Hernandez-Cortazar, I.B., Acosta-Viana, K.Y., GuzmanMarin, E., Ortega-Pacheco, A., Segura-Correa, J.C., Jimenez-Coello, M., 2017. Presence of Toxoplasma gondii in drinking water from an endemic region in Southern Mexico. Foodborne Pathog. Dis. 14 (5), 288 292. Available from: https://doi.org/10.1089/fpd.2016.2224. Holliman, R.E., Raymond, R., Renton, N., Johnson, J.D., 1994. The diagnosis of toxoplasmosis using IgG avidity. Epidemiol. Infect. 112, 399 408. Khan, I., Khan, A.M., Ayaz, S., Khan, S., Anees, M., Khan, S.A., et al., 2013. Molecular detection of Toxoplasma gondii in water sources of District Nowshehra, Khyber Pakhtunkhwa, Pakistan. J. Toxicol. Environ. Health, A 76 (14), 837 841. Available from: https://doi.org/ 10.1080/15287394.2013.821962. Korhonen, M.H., Brunstein, J., Haario, H., Katnikov, A., Rescaldani, R., Hedman, K., 1999. A new method with general diagnostic utility for the calculation of immuglobulin G avidity. Clin. Diag. Lab. Immunol. 6, 725 728. Kourenti, C., Karanis, P., 2006. Evaluation and applicability of a purification method coupled with nested PCR for the detection of Toxoplasma oocysts in water. Lett. Appl. Microbiol. 43 (5), 475 481. Available from: https://doi. org/10.1111/j.1472-765X.2006.02008.x. Lafferty, K.D., 2006. Can the common brain parasite, Toxoplasma gondii, influence human culture? Proc. Biol. Sci. 273 (1602), 2749 2755. ¨ mma¨la¨, P., Lappalainen, M., Koskela, P., Koskiniemi, M., A Hiilesmaa, V., Teramo, K., et al., 1993. Toxoplasmosis acquired during pregnancy: improved serodiagnosis based on avidity of IgG. J. Infect. Dis. 167, 691 697. Li, X., Pomares, C., Gonfrier, G., Koh, B., Zhu, S., Gong, M., et al., 2016. Multiplexed anti-Toxoplasma IgG, IgM, and IgA assay on plasmonic gold chips: towards making mass screening possible with dye test precision. J. Clin. Microbiol. 54 (7), 1726 1733. Available from: https:// doi.org/10.1128/JCM.03371-15. McLeod, R., Skamene, E., Brown, C.R., Eisenhauer, P.B., Mack, D.G., 1989. Genetic regulation of early survival and cyst number after peroral Toxoplasma gondii infection of A x B/B x A recombinant inbred and B10 congenic mice. J. Immunol. 143 (9), 3031 3034. Retrieved from http://www.jimmunol.org/content/143/9/3031. abstract. Melo, M.B., Nguyen, Q.P., Cordeiro, C., Hassan, M.A., Yang, N., McKell, R., et al., 2013. Transcriptional analysis of murine macrophages infected with different

Toxoplasma strains identifies novel regulation of host signaling pathways. PLoS Pathog. 9 (12), e1003779. Available from: https://doi.org/10.1371/journal. ppat.1003779. Retrieved from. Neal, L.M., Knoll, L.J., 2014. Toxoplasma gondii profilin promotes recruitment of Ly6Chi CCR2 1 inflammatory monocytes that can confer resistance to bacterial infection. PLoS Pathog. 10 (6), e1004203. Available from: https://doi.org/10.1371/journal.ppat.1004203. Retrieved from. Nimgaonkar, V.L., Yolken, R.H., Wang, T., et al., 2016. Temporal cognitive decline associated with exposure to infectious agents in a population-based, aging cohort. Alzheimer Dis. Assoc. Disord. 30 (3), 216 222. Norrby, R., Eilard, T., Svedhem, A., Lycke, E., 1975. Treatment of toxoplasmosis with trimethoprimsulphamethoxazole. Scand. J. Infect. Dis. 7, 72 75. Peixoto-Rangel, A.L., Miller, E.N., Castellucci, L., Jamieson, S.E., Peixe, R.G., Elias, L., et al., 2009. Candidate gene analysis of ocular toxoplasmosis in Brazil: evidence for a role for toll-like receptor 9 (TLR9). Memo´rias Do Instituto Oswaldo Cruz 104, 1187 1190. scielo. Pelloux, H., Brun, E., Vernet, G., Marcillat, S., Jolivet, M., Guergour, D., et al., 1998. Determination of anti-T. gondii immunoglobulin G avidity: adaption to the Vidas system (bioMe´rieux). Diag. Microbiol. Infect. Dis. 32, 69 73. Petersen, E., Dubey, J.P., 2001. Biology of Toxoplasmosis. In: Joynson, D.H.M., Wreghitt, T.G. (Eds.), Clinical Toxoplasmosis: Prevention and Management. Cambridge University Press, Cambridge, pp. 1 42. Peyron, F., L’ollivier, C., Mandelbrot, L., Wallon, M., Piarroux, R., Kieffer, F., et al., 2019. Maternal and congenital toxoplasmosis: diagnosis and treatment recommendations of a French Multidisciplinary Working Group. Pathogens 8. Available from: https://doi.org/ 10.3390/pathogens8010024. Prince, H.E., Wilson, M., 2001. Simplified assay for measuring T. gondii immunoglobulin G avidity. Clin. Diag. Lab. Immunol. 8, 904 908. Remington, J.S., Miller, M.J., 1966. 19S and 7S antiToxoplasma antibodies in diagnosis of acute congenital and acquired toxoplasmosis. Proc. Soc. Exp. Biol. Med. 121, 357 363. Rosowski, E.E., Lu, D., Julien, L., Rodda, L., Gaiser, R.A., Jensen, K.D.C., et al., 2011. Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208 (1), 195 212. Available from: https://doi.org/10.1084/jem.20100717. Rossi, C.L., 1998. A simple, rapid enzyme-linked immunosorbent assay for evaluating immunoglobulin G antibody avidity in Toxoplasmosis. Diag. Microbiol. Infect. Dis. 30, 25 30.

Toxoplasma Gondii

Further reading

Ruskin, J., Remington, J.S., 1968. Immunity and intracellular infection: resistance to bacteria in mice infected with a protozoan. Science 160 (3823), 72 74. Available from: https://doi.org/10.1126/science.160.3823.72. Sa´nchez, C., Lo´pez, M.C., Galeano, L.A., Qvarnstrom, Y., Houghton, K., Ramı´rez, J.D., 2018. Molecular detection and genotyping of pathogenic protozoan parasites in raw and treated water samples from southwest Colombia. Paras. Vec. 11 (1), 563. Available from: https://doi.org/10.1186/s13071-018-3147-3. Siegel, S.E., Lunde, M.N., Gelderman, A.H., et al., 1971. Transmission of toxoplasmosis by leukocyte transfusion. Blood 37, 388 394. Sotiriadou, I., Karanis, P., 2008. Evaluation of loopmediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunofluorescence test (IFT). Diagn. Microbiol. Infect. Dis. 62 (4), 357 365. Available from: https://doi.org/10.1016/j. diagmicrobio.2008.07.009. Stetson, D.B., Ko, J.S., Heidmann, T., Medzhitov, R., 2008. Trex1 prevents cell-intrinsic initiation of autoimmunity. Cell 134 (4), 587 598. Available from: https://doi.org/ 10.1016/j.cell.2008.06.032.

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Switaj, K., Master, A., Skrzypczak, M., Zaborowski, P., 2005. Recent trends in molecular diagnostics for Toxoplasma gondii infections. Clin. Microbiol. Infect. 11 (3), 170 176. Thalib, L., Gras, L., Romand, S., Prusa, A., Bessieres, M.H., Petersen, E., et al., 2005. Prediction of congenital toxoplasmosis by polymerase chain reaction analysis of amniotic fluid. BJOG 112 (5), 567 574. Villavedra, M., Battistoni, J., Nieto, A., 1999. IgG recognizing 21 24 kDa and 30 33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis. Rev. Inst. Med. Trop. Sao Paulo 41, 297 303. Villena, I., Aubert, D., Gomis, P., Ferte´, H., Inglard, J.-C., Denis-Bisiaux, H., et al., 2004. Evaluation of a strategy for Toxoplasma gondii oocyst detection in water. Appl. Environ. Microbiol. 70 (7), 4035 4039. Available from: https://doi.org/10.1128/AEM.70.7. 4035-4039.2004. Waldman, B.S., Schwarz, D., Wadsworth, M.H., Saeij, J.P., Shalek, A.K., Lourido, S., 2019. Identification of a master regulator of differentiation in Toxoplasma. BioRxiv 660753. Available from: https://doi.org/10.1101/ 660753.

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C H A P T E R

5 Ocular disease due to Toxoplasma gondii Jorge Enrique Gomez-Marin1 and Alejandra de-la-Torre2 1

GEPAMOL Group, Centro de Investigaciones Biome´dicas, Universidad del Quindı´o, Armenia, Colombia 2NeURos Group, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogota´, Colombia

5.1 Introduction

5.2 Historical landmarks in ocular toxoplasmosis

The eye is an important target of Toxoplasma gondii infection. Given the high prevalence of this parasite in the human population, it is not unexpected that ocular toxoplasmosis is the most common etiology of posterior uveitis in the world and an important cause of visual impairment and blindness (Mahittikorn et al., 2015; Scherrer et al., 2007). Research into this disease has significantly increased our understanding of the basis for retinal damage, genetic susceptibility to infection, the immune response to this pathogen, differences in disease manifestations due to different strains of this organism, and how treatment paradigms have been tailored depending on the geographic origin of the infecting strain. Based on current nomenclature for the pathogenesis of ocular disease, we utilize the term retinochoroiditis rather than chorioretinitis for the ocular manifestation of T. gondii infection.

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00005-0

Ocular involvement in congenital toxoplasmosis was first described in 1923 by the Czech pathologist Janku, who described an 11-monthold boy who died of hydrocephalus and whose ocular biopsy demonstrated organisms that he called sporocysts (Jankuˆ, 1923). This case was subsequently reviewed by Levadit (1928) who identified the parasite as T. gondii. However, it was not until 1939 that ocular disease became a widely appreciated manifestation of congenital toxoplasmosis (Wolf et al., 1939). The prevalence of these lesions in congenital infection led others to look for this disease in adults. Jakob Frenkel, a pathologist, developed the Toxoplasmin skin hypersensitivity test (Frenkel, 1948) and used this test in 28 patients with retinochoroiditis and 90 control patients. This demonstrated T. gondii infection in 70% of the retinochoroiditis patients and 10% of the controls (Frenkel, 1949). Hogan

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(1950) provided the first detailed clinical description of ocular toxoplasmosis. Rieger (1951) then originated the concept of postnatally acquired T. gondii as well as the theory that recurrence may be related to an immunecompromised state in these patients. These ideas were revolutionary in 1951 and remain topics of inquiry today. Hogan et al. (1952) studied 125 patients with retinochoroiditis and 201 controls using the T. gondii Dye test. Their careful clinical and pathological analyses of cases were fundamental in establishing the basis of what we know as the characteristics of this disease. It is interesting to read the words of Hogan’s lecture from the Memorial Jackson Conference in 1958 (Hogan, 1958) that captures the research issues at that time: “Human toxoplasmosis is one of the most interesting diseases of modern medicine. Its manifestations are protean and, in the acquired form, resemble those of a number of well-known disease entities. At a first glance, it is surprising that diagnostic studies have failed to show the frequency of this disease. However, one of the important diagnostic tests, the methylene blue dye test, is technically difficult and involves the risk of handling live organisms. These factors probably have restricted widespread testing and have prevented more rapid progress in the diagnosis of the disease.” While the Toxoplasmin skin test was critical for the initial clinical descriptions of ocular toxoplasmosis and linked clinical observations to positive reactions, direct evidence of the parasite in pathologic specimens was scant. In 1952 Helenor Campbell Wilder Foerster, a technician in the registry of Ophthalmic Pathology at the Armed Forces Institute of Pathology (AFIP, Washington DC, United States), in a landmark case series clearly demonstrated the presence of T. gondii on pathological specimens of retinochoroiditis (Wilder, 1952a,b). Wilder made her findings after putting enormous effort into the identification of microbes in “tuberculous” eyes submitted to the AFIP, but never identified bacteria or spirochetes by special staining until she

identified T. gondii in the retinas of these eyes (Wilder, 1952a,b). She examined 53 eyes that had been enucleated due to pain and blindness, establishing a strong relationship between T. gondii and these ocular manifestations. Each eye in Wilder’s cohort had lesions that were granulomatous with central necrosis, and T. gondii was consistently found in the necrotic areas. Serologic testing on these patients revealed all of them to test positive for T. gondii antibodies. Through careful laboratory techniques and persistent investigation, she not only provided some answers to the enigma of Toxoplasma retinochoroiditis but put forth central questions that remain unanswered. As a result of Wilder’s work, ocular toxoplasmosis resulting from congenital infection became accepted as the leading cause of posterior uveitis in otherwise healthy adults. This work solidified the hypothesis that toxoplasmosis, not tuberculosis, causes ocular disease characterized by choriorretinal lesions. Prior to Wilder’s report, tuberculosis was routinely erroneously ascribed as the source of what Wilder ultimately demonstrated was ocular toxoplasmosis. Postnatally acquired infection with ocular involvement, as well as ocular manifestations of congenital disease, were fully characterized by Hogan in 1958 (Hogan, 1958). During the 1960s Hogan and associates made the incorrect assumption that ocular symptoms of toxoplasmosis occur largely in the presence of systemic symptoms and rarely alone (Hogan et al., 1964). Based on this assumption and the fact that most postnatally acquired T. gondii infections are asymptomatic, they believed that ocular involvement in patients with acquired infection was uncommon. This classic teaching that most, if not all, Toxoplasma retinochoroiditis is congenital was given further support in 1973 when Perkins concluded that nearly all cases of Toxoplasma ocular involvement in the United Kingdom resulted from congenital infection (Perkins, 1973). Based on the belief that ocular involvement only occurs immediately

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5.3 Epidemiology

after infection, episodes of recurrent retinochoroiditis in children and adults were attributed to congenital infection that went undetected at birth (Hogan, 1961). These assumptions are now known to be incorrect and have been refuted by subsequent studies that investigated the nature and course of Toxoplasma retinochoroiditis. For example, it is now well established that the majority of cases of ocular toxoplasmosis are due to postnatally acquired infections (Arantes et al., 2015; Go´mez-Marı´n et al., 2000, 2018; Montoya and Remington, 1996; Ongkosuwito et al., 1999) and that retinal lesions can develop long after the initial infection (Arantes et al., 2015; Go´mez-Marı´n et al., 2018; Silveira et al., 2015a). Warren and Sabin (1942) were the first to test a large number of antiprotozoal drugs and chemotherapeutic agents for their in vitro and in vivo effectiveness against T. gondii and found that sulfonamide compounds were the most active compounds. Following this publication, several other groups found that sulfonamides were active, but that they had variable efficacy. Although pyrimethamine was shown in experimental infections to be superior to that of sulfonamides, the effective dose of pyrimethamine alone as a therapeutic agent had a narrow therapeutic window. Eyles and Coleman (1953) searched for a combination of drugs that would be effective, and they discovered that sulfadiazine acts synergistically with pyrimethamine, allowing the use of a much smaller dose of each to control T. gondii infection in a mouse model. The combination of pyrimethamine and sulfadiazine has subsequently been demonstrated to be effective in T. gondii infection in humans for many different manifestations of this disease.

5.3 Epidemiology The epidemiology of the disease has been reviewed recently (Kijlstra and Petersen, 2014;

231

Petersen et al., 2012). Surveys on the general population employing funduscopic screening and serological analysis have found that people with chorioretinal scars are often unaware of the presence of this condition. In the United States, in 842 residents from Maryland, 5 (0.6%) had chorioretinal scars consistent with Toxoplasma lesions (Smith and Ganley, 1972). A study in the south of Brazil found a prevalence of 17.7% (Glasner et al., 1992). In Colombia in the general population a prevalence of 6% was found (de-la-Torre et al., 2007). In military personnel in Colombia, toxoplasmic choriorretinal lesions were found in four soldiers that operated in the jungle (1%; CI 95%: 0.2 2.5 of the IgG anti Toxoplasma positive soldiers) and in one urban soldier (0.4%; CI 95%: 0.2 2.4 of the IgG anti Toxoplasma positive soldiers) (Go´mez-Marı´n et al., 2012). A problem with surveys using only funduscopic eye screening is that some lesions that are seen as typical of toxoplasmosis are due to other illness; therefore funduscopic screening can overestimate the prevalence of lesions due to toxoplasmosis. The number of people with chorioretinal scars due to acquired toxoplasmosis is larger than the number of people with scars due to congenital infection or the proportion of newborns with congenital infection. For example, in Colombia, it is estimated that 5.5% of the population have chorioretinal scars after noncongenital infection and that 20% of these persons have reduced visual capacity, while 0.5% of the population have scars coming from congenital infection (de-la-Torre et al., 2009a,b). Longitudinal studies examining patients with seroconversion (indicating acquired infection) indicate that toxoplasmosis is by far the most important cause of chorioretinal hyperpigmented scars (Arantes et al., 2015; Glasner et al., 1992). Outbreaks of acquired ocular toxoplasmosis have been reported in Canada (Bowie et al., 1997), Brazil (De Moura et al., 2006; Vaudaux et al., 2010), and India (Palanisamy et al., 2006). Serologic findings

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also support that many patients displaying only ocular symptoms of toxoplasmosis have recently acquired infection, as opposed to recurring congenital infection (Go´mez-Marı´n et al., 2000, 2018; Jones et al., 2015; Montoya and Remington, 1996). An important epidemiologic parameter is the frequency of ocular toxoplasmosis in a clinical series of uveitis. In these series, it appears that ocular toxoplasmosis is more common in South America, Central America, the Caribbean, and parts of tropical Africa, as compared to Europe and Northern America and that it is quite rare in China (Arevalo et al., 2010; Jabs, 2008). Ocular toxoplasmosis is responsible for 30% 50% of posterior uveitis cases in immune-competent individuals. In some countries, such as Colombia, it is one of the most important causes of visual impairment (de-la-Torre et al., 2009a, b). The impact of this disease on the quality of life was demonstrated by a study that found worse vision-related quality of life in patients with ocular toxoplasmosis, especially if they had bilateral lesions and more recurrences, than in people without this condition (de-la-Torre et al., 2011a,b). Ocular disease in South America is more severe than in other continents due to the presence of extremely virulent genotypes of the parasite (de-la-Torre et al., 2013; Go´mez-Marı´n et al., 2018; Pfaff et al., 2014). There are significant differences between countries and regions in the incidence of new cases of ocular toxoplasmosis, for instance in Colombia (Quindio region) the incidence of ocular toxoplasmosis has been estimated to be 3 new cases per 100,000 inhabitants per year (de-la-Torre et al., 2009a,b), while in British-born patients, it was estimated to be 0.4 cases per 100,000 inhabitants per year (Gilbert et al., 1999). Toxoplasma retinochoroiditis is the most common clinical consequence of congenital infection, with significant impact on the quality of life (de-la-Torre et al., 2011a,b; Peyron et al., 2011; Wallon et al., 2004, 2014). The number of children annually with this consequence of

congenital infection in the world was estimated by metaanalysis to be 190,100 (95% credible interval, CI: 179,300 206,300) with high burdens in South America and in some MiddleEastern and low-income countries (Torgerson and Mastroiacovo, 2013). Congenital and acquired ocular toxoplasmosis differ significantly in South America and Europe necessitating different clinical and public health decisions for management in these regions (Go´mez-Marı´n et al., 2018; Pfaff et al., 2014; Sauer et al., 2011). Congenital toxoplasmosis in South America is more symptomatic than in Europe as demonstrated by comparing cohorts of congenitally infected children. The Systematic Review on Congenital Toxoplasmosis (SYROCOT) (2007) was an international collaborative study that analyzed data from 25 cohorts of infected mothers in Europe, North America, and South America. The risk of ocular lesions was much higher among Colombian and Brazilian children (47%) than among European children (14%). In another comparative prospective cohort study of congenitally infected children in Brazil and Europe, it was found that Brazilian children had eye lesions that were larger, more numerous, and more likely to affect the part of the retina responsible for central vision, compared with their counterparts in Europe (Gilbert et al., 2008). More children developed retinochoroiditis sooner in Brazil than in Europe, and choriorential lesions recurred at an earlier age in Brazil than in Europe. By 4 years of age, the probability of a second lesion among children with a first lesion was 43% in Brazil compared with 29% in Europe, and the risk of multiple recurrences was also greater in Brazil (Gilbert et al., 2008). A separate report of 178 congenitally infected children in the Southeastern region of Brazil, found a high rate of early retinochoroidal involvement (80%), and 47% of them had active lesions during the first 3 months of life (Vasconcelos-Santos et al., 2009).

Toxoplasma Gondii

5.3 Epidemiology

There are several modes of transmission of Toxoplasma to humans, the contribution of each mean of transmission varies depending on region as reviewed recently by the World Health Organization (Hald et al., 2016). Most transmission is estimated to occur by ingestion, whether it is from contaminated water or undercooked meat (Guo et al., 2016; Hald et al., 2016; Opsteegh et al., 2011). Vegetarians and nonvegetarians had similar prevalence rates providing evidence that foods other than meats serve as a source of infection (Hald et al., 2016). The accumulated evidence and reports on parasite transmission by ingestion of water contaminated with T. gondii oocysts have led to the recognition that toxoplasmosis is also a waterborne disease (Baldursson and Karanis, 2011; Karanis et al., 2013; Trivin˜oValencia et al., 2016). An important factor that increases the transmission of the parasite is rainfall (Afonso et al., 2010; Go´mez-Marin et al., 2011; Tizard et al., 1976; VanWormer et al., 2016). In Argentina, high rainfall periods were linked to an increase in the number of recurrences in ocular toxoplasmosis (Rudzinski et al., 2013). This can be explained by the findings of a study that reported that 43.7% (95% CI: 35.6, 53.5) of oocysts survived under damp conditions after 100 days, whereas survival was only 7.4% (95%CI: 5.1, 10.8) under dry conditions (Le´lu et al., 2012). During the Colombian national newborn screening, which was performed in seven different cities, it was found that geographical factors such as altitude or average temperature were not correlated with the incidence of congenital infection, but that mean annual rainfall was strongly associated (Go´mez-Marin et al., 2011). Cities with a low mean annual rainfall (48 806 mm3/year) had a low frequency of congenital toxoplasmosis (0.5% 0.7% of newborns) and cities with high mean annual rainfall (over 2500 mm/ year) had a high frequency of congenital toxoplasmosis (3% 6% of newborns) (Go´mezMarin et al., 2011). This epidemiological

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association finding was confirmed by a metagenomics analysis of the diversity of soil protists, where Toxoplasma was highly abundant in more humid soils (Bates et al., 2013). A large outbreak of ocular toxoplasmosis affecting patients residing in Tamil Nadu in Southern India in 2004 also pointed to contaminated municipal drinking water as the most plausible source (Balasundaram, 2010; Palanisamy et al., 2006). 198 out of 248 patients with active retinochoroiditis were seropositive for IgM antiToxoplasma, suggesting recently acquired infections (Balasundaram, 2010). Interestingly, only 35 patients (14.1%) had prodrome fever prior to onset of retinitis (Balasundaram, 2010). Conventional serological commercial tests recognize antigens from tachyzoites but cannot determine the predominant route (oocysts or tissue cysts) in transmission. A technique for understanding the origin of infection is to use a serological test specifically for antibodies against oocyst proteins (Boyer et al., 2011; Mun˜oz-Zanzi et al., 2010). Evaluating the relative epidemiologic importance of oocyst versus tissue cysts transmission in populations has recently become feasible by means of an ELISA using a recombinant protein discriminating between antibodies against tissue cyst specific proteins versus those from oocyst-specific proteins (Boyer et al., 2011). These ELISA assays use recombinant sporozoite-specific embryogenesis-related protein (Hill et al., 2011) or recombinant CCp5A protein (Santana et al., 2015). Discriminating the route of infection represents an essential tool for implementing effective prevention measures for reducing exposure to the parasite in more vulnerable groups, such as pregnant women and immunocompromised patients. In the United States the ingestion of infective oocysts is responsible for most human infections (Guo et al., 2016; Jones et al., 2014a,b). Unrecognized ingestion of T. gondii oocysts leading to congenital toxoplasmosis and causing ocular disease outbreaks has been reported (Botto´s et al., 2009; Boyer et al., 2011; De Moura et al., 2006;

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Palanisamy et al., 2006). Oocysts were the predominant source of T. gondii infection in four North American outbreaks, and in mothers of children in the National Collaborative Chicago based Congenital Toxoplasmosis Study (Boyer et al., 2011). This latter study demonstrated that 59 (78%) of 76 mothers of congenitally infected infants had a primary infection due to oocysts, and more importantly, that of the 76 infected mothers only 49% (37 mothers) identified significant risk factors for sporozoite acquisition (Boyer et al., 2011). In an endemic setting in Brazil, where exposure to water contaminated with T. gondii oocysts has been reported to be a significant risk factor, it was observed that more than 50% of the population (older than 20 years) infected with T. gondii (evaluated by conventional serology) had antibodies against sporozoites (Garcia BahiaOliveira et al., 2003; Santos et al., 2010) and that among individuals younger than 20 years over 65% had IgA antibodies to sporozoite antigens detectable in their saliva (Mangiavacchi et al., 2016), indicating exposure to infective oocysts. In women who acquired T. gondii during pregnancy in Chile, 43% had oocyst-specific antibodies (Mun˜oz-Zanzi et al., 2010). Important questions about Toxoplasma retinochoroiditis are “What it is the percentage of people that develop retinochoroiditis after postnatal toxoplasmosis and what it is the period between infection and apparition of ocular symptoms (or retinal scars)?”, which can be addressed by examining reports on outbreaks or clusters of ocular toxoplasmosis. In an outbreak in Vancouver (Canada) the number of symptomatic patients with ocular toxoplasmosis was 19 of 100 acute outbreak-related cases (19%) with a median time between systemic symptoms and ocular manifestations of 6 weeks (Bowie et al., 1997). A similar time of 7 weeks was found between the presence of lymphadenopathy and the development of retinochoroiditis in a study of Colombian cases (Go´mez-Marı´n et al., 2018). In an important

representative study performed in Brazil the risk of new chorioretinal lesions after diagnosis was 10 per 100 person/year (i.e., 10%) (Arantes et al., 2015). A recent retrospective review of cases of ocular toxoplasmosis seen in a referral center in the United States reported that 11.7% of patients with ocular disease had recently acquired T. gondii infection (Jones et al., 2014a, b). In Colombian patients a similar risk is reported and we estimated that 12% of Toxoplasma seropositive people in Colombia develop chorioretinal scars during the course of their infection (de-la-Torre et al., 2007). The range of time to develop retinochoroiditis following symptomatic acute infection is between 1 and 60 weeks (Table 5.1) (Balasundaram, 2010; Burnett et al., 1998; Couvreur and Thulliez, 1996; Marx-Chemla et al., 1998; Masur et al., 1978).

5.4 Pathophysiology: lessons from animal models and clinical studies The eye is an unusual immunologic environment that is designed to reduce inflammation in that in ocular cells transforming growth factor beta is constitutively expressed, a high concentration of Fas ligand is present, and Class I MHC molecules are downregulated (Streilein, 2003, 1993). Thus this unusual immunologic microenvironment may decrease clearing of T. gondii infection. Studies performed both in murine model and in humans have given insights on the pathogenesis of ocular toxoplasmosis. After the inoculation of tachyzoites, mice developed minor uveitis and retinal vasculitis (Norose et al., 2011). This uveitis is characterized by an infiltration of CD4 1 T lymphocytes and macrophages into the retina and the expression of IFN-γ and TNF-α mRNA in retinal lymphocytes (Gazzinelli et al., 1994; Norose et al., 2011). In most cases the inflammation becomes destructive, with chorioretinal scarring and modification of the pigmentary

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5.4 Pathophysiology: lessons from animal models and clinical studies

TABLE 5.1 Literature reports of the interval between systemic symptoms and the development of ocular retinochoroiditis. % Ocular involvement

Time from systemic symptoms to ocular disease (weeks)

Family outbreak, New York (United States)

1/7 (14.2%)

16

Akstein et al. (1982)

Outbreak Atlanta (United States)

1/37 (2.7%)

208 (?)a

Couvreur and Thulliez (1996)

Hospital (France)

6/45 (13%)

Not reported

Burnett et al. (1998)

Water-related outbreak (Canada)

19/100 (19%)

Median: 6; range: 2 12

Marx-Chemla et al. (1998)

Case report, France

Reference

Setting

Masur et al. (1978)

Balasundaram (2010)

60

Outbreak Coimbatore (India)—water related?

248/ (?)

b

1 22

a

(?) Retinochoroiditis was recorded after ophthalmoscopic examination 4 years later, patient did not have periodical follow-up. (?) Denominator was not reported.

b

epithelium. Parasites are rarely been detected in situ in these infected mice (Gazzinelli et al., 1994). Importantly, in humans the lymphocytes of patients with ocular toxoplasmosis react not only with T. gondii antigens but also with retinal antigens, providing the basis for hypersensitivity reactions which participate in tissue damage along with the direct cytolytic effects of this parasite (Nussenblatt et al., 1989). Depletion of both cytokines and lymphocytes has been tested in a murine model of T. gondii choriorentitis. Treating mice with anti-CD4 1 or anti-CD8 1 antibodies led to an increase in the number of ocular cysts, whereas treatment with anti-IFN-γ or anti-TNF-α antibodies produced lesions containing tachyzoites (Gazzinelli et al., 1994). The histopathological characteristics of mice treated with antibodies to produce immune depression resemble those observed in the lesions of immunocompromised patients. The latter develop multiple lesions characterized by retinal necrosis and marked inflammation of the retina, the vitreous humor and the subjacent choroid (Kikumura et al., 2012). The

model supports the conclusion that the retinochoroiditis that develops in immune-competent subjects must be considered independently of cases arising in immunocompromised patients (Kikumura et al., 2012). Altogether, the results on murine and human ocular toxoplasmosis indicate that hypersensitivity and inflammation exacerbate the destructive process that takes place in otherwise immune-competent hosts (Garweg and Candolfi, 2009). In one study of ocular cytokine concentrations in 27 French patients with ocular toxoplasmosis, no correlation was found with age, sex, and region of origin of the patients, time from symptom onset to the sampling, the degree of uveal inflammation, or the etiology of the infection (primary acquired or congenital), but a characteristic local cytokine profile in human ocular toxoplasmosis was observed, compared to other causes of uveitis (Lahmar et al., 2009). High levels of IFN-γ, IL6, and macrophage inflammatory protein 1β were frequently detected in samples from patients with ocular toxoplasmosis and in

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samples from those with viral uveitis, whereas IL-17 was frequently detected in samples from patients with ocular toxoplasmosis and in samples from those with intermediate uveitis (Lahmar et al., 2009). This profile notably included IL-17A overexpression (Sauer et al., 2012). In another prospective study in French patients, it was found that Th1 (IL-2, IFN-γ) and Th2 (IL-13) cytokines as well as inflammatory (IL-6, IL-17) and downregulating (IL-10) cytokines were upregulated in aqueous humor of patients with confirmed ocular toxoplasmosis (Sauer et al., 2015). In contrast, TNF-α was not upregulated. These studies were done in patients infected by the relatively benign type II strain, which predominates in both Europe and North America. In contrast, South American patients usually suffer from more virulent strains and develop more severe clinical symptoms (de-la-Torre et al., 2013). Intraocular IFN-γ and IL-17 expression were lower, while higher levels of IL-13 and IL-6 were detected in aqueous humor of Colombian patients (de-la-Torre et al., 2014). A study on the peripheral lymphocyte response in 19 Colombian patients with ocular toxoplasmosis demonstrated a preferential Th2 response (Torres-Morales et al., 2014). In a human retinal pigment epithelial (HRPE) cell culture assay system IFN-γ was shown to cause L-tryptophan starvation through induction of indoleamine 2,3-dioxygenase, an enzyme that converts tryptophan to N-formylkynurenine, suggesting that this is the mechanism of INF-γ protection in the eye (Nagineni et al., 1996). Infliximab, a chimeric antibody against TNF-α, is used to treat some forms of uveitis as well as rheumatoid arthritis and Crohn’s disease, and it has been reported to be a cause of biopsyconfirmed CNS toxoplasmosis (Pulivarthi et al., 2015; Young, 2005). But, despite the large prevalence of T. gondii, by 2005 there were only eight known cases of T. gondii associated reactivation with over 600,000 patients having received the drug (Lassoued et al., 2007).

5.5 Host factors Since T. gondii infection is quite common, but only a fraction of the infected population develops ocular diseases, it is likely that host genetic susceptibility plays a significant role in disease penetrance. Genetic linkage studies to identify host susceptibility markers are difficult to conduct, due to the low number of cases in Europe and North America. Chances are much better in regions with a high prevalence of ocular toxoplasmosis, and nearly all genetic studies have been undertaken in these regions. Obviously, genes coding for known immune mediators or their promoter regions were checked for association with clinically apparent ocular toxoplasmosis. Polymorphisms in genes encoding various cytokines have been shown to be connected with susceptibility to ocular toxoplasmosis. In Brazil, patients homozygous for the A allele (1874T/A) of the IFN-γ gene had a higher risk of ocular toxoplasmosis if they possessed the A/A genotype, compared to a negative control group (Peixe et al., 2014). IL-10 gene polymorphism (IL-10 21082A allele, AA 1 AG genotypes) was associated with the occurrence of ocular toxoplasmosis (Cynthia A Cordeiro et al., 2008a,b,c). In Brazil, an IL-6 polymorphism (2174 G/C) was associated with ocular toxoplasmosis, but not the recurrence of ocular toxoplasmosis (Wujcicka et al., 2015). These authors also showed that the recurrence of toxoplasmic retinochoroiditis was associated with the IL-1α (2889C/T) polymorphism, related to an increase in IL-1α expression (Wujcicka et al., 2015). Analysis of TNF-α gene polymorphism (2308G/A) in Brazilian patients demonstrated that the occurrence or recurrence of Toxoplasma retinochoroiditis was not associated with this polymorphism (Cordeiro et al., 2008a,b,c). In Colombian patients with ocular toxoplasmosis, one polymorphism in the IL-10 gene-promoter (21082G/A) was significantly more prevalent in ocular toxoplasmosis patients than in

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5.6 Parasite factors

controls; in contrast, the haplotype “AG” of the IL-10 gene promoter polymorphisms (rs1800896, rs1800871) was significantly less frequent (Naranjo-Galvis et al., 2018). In the same Colombian population the polymorphism (1874A/T) of IFN-γ and the haplotype “GAG” of the IL-1β gene promoter polymorphisms (rs1143634, rs1143627, and rs16944) were significantly associated with ocular toxoplasmosis (Naranjo-Galvis et al., 2018). Other studies have addressed the question of genetic polymorphisms of innate immune response proteins. The TLR-9 gene, located on chromosome 3p21.3, is a receptor for hypomethylated cytosine guanine (CpG) DNA motifs and is expressed by B cells, acting as a sensor for infection, that leads to the innate immunity activation (Akira and Takeda, 2004). Polymorphism of TLR9 was associated with Toxoplasma retinochoroiditis in patients from the state of Rio de Janeiro, Brazil (PeixotoRangel et al., 2009). Another study found an association with the intracellular pattern recognition receptor NOD2 in patients from the same region, as well as from the Belo Horizonte region, Brazil (Dutra et al., 2013). T. gondii infected TLR-9 deficient mice were more susceptible to the infection and revealed a 50% decrease in IFN-γ production (Minns et al., 2006). A study performed in Brazilian children with ocular toxoplasmosis, showed that the TLR-9 2848G . A variation was associated with Toxoplasma retinochoroiditis (Peixoto-rangel et al., 2009). Taking into account these studies, it is plausible that the TLR-9 2848G . A polymorphism is involved in the development of T. gondii infection, simultaneously with other molecular changes, as this polymorphism results in synonymous amino acid changes with no alterations at the regulatory site (Wujcicka et al., 2017). The P2X7 receptor is highly expressed by cells of the hematopoietic lineage and can mediate cell death, killing of infectious organisms, and regulation of the inflammatory

response (Elena et al., 2009). P2X7 KO mice have showed increased susceptibility to toxoplasmosis characterized by an impaired production of proinflammatory cytokines (IL-1b, IL-12, TNF-α, and IFN-γ) and increased tissue damage and parasitic load (Huang et al., 2017). Studies of patients with congenital toxoplasmosis in the United States provide congruent data on proinflammatory and down modulatory cytokines involved in immune response to Toxoplasma in humans. These genes include P2RX7 (Jamieson et al., 2010; Lees et al., 2010), NALP1 (Gorfu et al., 2014; Witola et al., 2011), ALOX12 (Witola et al., 2014), and HLA classes I and II (Cong et al., 2010; Tan et al., 2010). Altogether, these studies suggest that genetic control of immune response is relevant for the pathogenesis of T. gondii retinochoroiditis. A summary of linkage studies between genetic polymorphisms of genes of immune response and human ocular toxoplasmosis is depicted in Table 5.2. It should be noted that given the complexity of the immune response to parasites with complex biological traits it is unlikely that genetic variation at a single locus, as shown by an SNP analysis, would provide an adequate explanation for the interindividual differences in the host immune response that results in diverse clinical manifestations. For a better description of the disease susceptibility traits and unambiguous identification of factors responsible for both causality and predisposition to a disease, functional appraisal of disease-associated polymorphisms remains essential, but the studies performed until now remain limited at this regard.

5.6 Parasite factors The variability in the clinical presentations of ocular and nonocular toxoplasmosis is not fully explained by host-genetic variations in immune response, and it is likely that the diversity of the parasite population plays a

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5. Ocular disease due to Toxoplasma gondii

TABLE 5.2 Studies of the association between human genetic polymorphisms and susceptibility to ocular toxoplasmosis. Genotypes (OR) associated with OT

Reference

Gene

SNP

Peixe et al. (2014)

IFN-γ

rs2069718 rs3181035

rs2069718 AA (0,36), AG (0,28), A (5,49), GG (3,3) rs3181035 CT (2,79), TT (4,8), C (0,3), CC (0,3)

Jamieson et al. (2010)

P2RX7

rs1718119

1068T . C (0,27)

Cordeiro et al. (2008a,b,c)

TNF

2 308G/A

No differences

Cordeiro et al. (2013)

IL6

2 174 G/C

CG

Neves et al. (2012)

IFN-γ

1 874T/A

No differences

Cordeiro et al. (2008a,b,c)

IL10

2 1082G/A

AA 1 AG (2,5)

Cordeiro et al. (2008a,b,c)

IL1

IL1a-889; IL1A 1 3954

No for frequency of OT but recurrences with 2889C/T

Peixoto-Rangel et al. (2009)

TRL (2, 4,5 and 9)

Rs352140, s3804099, rs1053954

rs352140 C (7)

Ayo et al. (2015)

KIR receptor

KIR3DS1 2 Bw4 80Ile KIR2DS1 1 / C211 KIR3DS1 1 /Bw4 80Ile 1 )

KIR3DS1

significant role in the manifestations of infection. The first molecular analysis of strain diversity of Toxoplasma was performed based on restriction fragment length polymorphism (RFLP) and classified T. gondii into three genetic types (I, II, III) associated with differences in mouse virulence: type I isolates were 100% lethal to mice, irrespective of the dose, while types II and III in general were not virulent (Sibley and Boothroyd, 1992). These clonal types were then associated to different clinical presentations of human disease (Howe and Sibley, 1995). However, this classification was biased, because it was based on strains isolated mainly in Europe and North America and was not representative of T. gondii diversity. We now have better information about the diversity of parasite populations and their biological characteristics. A large multilocus and haplotype analysis of 950 strains collected from around the world, found 15 haplogroups that collectively defined six major clades using microsatellite and multilocus

RFLP assays (Su et al., 2012). This work demonstrated that T. gondii populations existed with a biphasic geographical pattern distribution that correspond to a few highly clonal genotypes that predominate in the northern hemisphere, whereas in South America the parasite populations are characterized by a diverse assemblage that shows greater evidence of recombination and a greater genetic diversity (Su et al., 2012). The Southern Hemisphere strains lack signs of the recent genetic bottleneck and clonal structure that are present in strains from the Northern Hemisphere (Sibley and Ajioka, 2008). Thereafter, a phylogeographic analysis indicated that T. gondii originated in South America, by using haplotype richness as a measure of genetic diversity in a large number of hypothetical origins across the world (Bertranpetit et al., 2017). This approach identified South America, and more specifically Colombia, as the most likely origin of the modern strains and that continent separation during hundreds of millions of years

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5.6 Parasite factors

allowed the genetic evolution of the various T. gondii strains that are seen today (Bertranpetit et al., 2017). Specific virulent factors have been identified that correlate well with pathogenicity in mice and humans (Alvarez et al., 2015a,b; de-laTorre et al., 2013; Niedelman et al., 2012; Saeij et al., 2005; Sa´nchez et al., 2014). These virulent factors are predominantly kinase proteins that interact directly with host transcription factors, modulating the immune response (Saeij et al., 2005). Toxoplasma genotypes analyzed in South-American patients with severe clinical forms, correlated with virulent genotypes of ROP proteins and modification of the cytokine immune response (Alvarez et al., 2015a,b; dela-Torre et al., 2013, 2014; Torres-Morales et al., 2014). In 12 samples of patients with ocular toxoplasmosis a higher inflammatory reaction on eye was associated to the existence of ROP18 mouse-virulence related allele (Sa´nchez et al., 2014). The intraocular cytokine Th2 response was related to more severe clinical characteristics in patients infected by type I/III strains, as determined by serotype analysis (de-la-Torre et al., 2014). A comparative analysis of the ocular cytokinome between French and Colombian patients showed that Colombian patients had an intraocular polarized Th2 cytokine response (de-la-Torre et al., 2013). Furthermore, Colombian patients with ocular toxoplasmosis showed a similar peripheral Th2 skewed response (Torres-Morales et al., 2014). Moreover, 83.3% of the ROP16 sequences from patients with ocular toxoplasmosis clustered with those of the mousevirulent ROP16 protein (Alvarez et al., 2015a, b). Altogether, these results are consistent with the hypothesis that South-American strains cause more severe ocular toxoplasmosis due to an inhibition of the protective effect of IFN-γ, giving an explanation as to why Colombian patients have more severe clinical characteristics than European patients, including greater inflammation, higher number and size of

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lesions (de-la-Torre et al., 2013). This severe form of ocular parasite infection is linked to the Th2 cytokine profile (de-la-Torre et al., 2014) and to infection by type I/III strains, including higher levels of IL-1β (de-la-Torre et al., 2013, 2014). Pure type I strains are rare in South America and are usually called “atypical” strains as they possess mixed alleles, including those responsible for the virulent phenotype of clonal lineages (Su et al., 2012). South American atypical strains often have the virulence chromosome Ia (Khan et al., 2014, 2006a). The higher virulence of atypical strains has been suggested to explain the higher burden of ocular disease in congenitally infected children and in postnatal infected adults in Brazil when compared to those infected in Europe by type II strains (Gilbert et al., 2008; Khan et al., 2006b). In congenitally infected infants in the United States the serotyping assay showed that severe disease and eye severity at birth were more common in infants with non type II serotypes than in those with type II serotypes (McLeod et al., 2012). Highly virulent strains exist in Amazonian rainforest in French Guiana where wild strains are responsible for life-threatening disseminated toxoplasmosis in otherwise healthy people (Carme et al., 2002; Demar et al., 2007). The difficulty for clinical application of strain characterization is not the method, such as microsatellites, polymerase chain reaction (PCR) RFLP or intron sequencing, but is the amount of parasite in the clinical sample (Ajzenberg et al., 2010; Rajendran et al., 2012). Tachyzoites are present only during the very short period of acute infection and T. gondii strains can be isolated from human cases of toxoplasmosis almost only during the acute phase of infection. Genotyping the DNA extracted directly from clinical samples is an alternative to genotyping the DNA extracted from strains isolated after bioassay in mice, but direct genotyping is not possible for the

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majority of cases because the amount of parasitic DNA is often too low in clinical samples, below the limit of detection of PCR assays with the single-copy markers used in genotyping analyses (Alvarez et al., 2015a,b; Sa´nchez et al., 2014). The serotype assay based on the detection of antibodies against strain-specific peptides was developed to circumvent the difficulty to isolate Toxoplasma strains in clinical samples (Grigg et al., 2001). However, the most used ELISA assay for serotypes cannot completely distinguish between serotype I and III (Sousa et al., 2008). As such, there has not been any clinical utility for this test (Peyron et al., 2006), and it has only proven useful for examining geographical differences of infecting strains (Morisset et al., 2008) by examining antibodies against the polymorphic GRA6 protein. Some attempts have been made also to detect antibodies against virulent factors such as ROP18 or ROP16 of Toxoplasma, but the low antigenicity of these virulent factors has hampered the sensitivity of these tests (Alvarez et al., 2015a,b; Sa´nchez et al., 2014).

5.7 Animal models Several intraocular models of toxoplasmosis have been developed. The primate (Culbertson et al., 1982), mouse, and rabbit (Nozik and O’Connor, 1968) models suffer from the lack of similarity to the human toxoplasmosis where ocular seeding occurs from systemic endogenous infection. There are no ideal small animal models of human ocular toxoplasmosis. The macula is the central anatomical segment of retina within the visual axis that gives humans their most fine acuity and only primates have macular, and macula disease is an important feature of congenital toxoplasmosis. The proportional volume of lens is much greater and the proportional vitreous volume much less in rodents than in the human eye. Despite these

shortcomings, the murine model of ocular toxoplasmosis has been used for many studies, usually with an injection of tachyzoites into the anterior chamber of the mouse (Hu et al., 1999). Studies using the intraocular inoculation murine model have demonstrated that genetic factors of the host mouse as well as the parasite strain are significant in determining susceptibility to experimental ocular toxoplasmosis (Lu et al., 2005). Surprisingly, there appears to be no detectable difference in measured intraocular infection by either the topical or intravitreal route of infection in mice. Both routes had detectable parasites within intraocular vessels, glial reaction of the inner plexiform layer by day 7, and disruption of the retinal pigmented epithelium (RPE). Intravitreal injection of PBS alone also resulted in glial changes within the inner plexiform layer (Tedesco et al., 2005). A congenital model of ocular toxoplasmosis has been reported in which infected dams are inoculated during gestation (Hay et al., 1984). Unfortunately, a wide range of clinical diseases occurred in this model. In vivo imaging of the mouse fundus is possible and is greatly facilitated by the appropriate imaging set-up systemic infection using in vivo imaging employing the IVIS system (Xenogen, Alameda, California). While this paper demonstrates some bioluminescence due to luciferase transgenic parasites with pixels correlating with ocular involvement no ex vivo imaging of the eyes to confirm this localization was reported (Saeij et al., 2005). Frenkel (1955) was first to report hamsters as an ocular model for toxoplasmosis. Although that the RH strain he used required therapy to prevent mortality, the CJ strain did not and consistently produce ocular lesions. As with the mouse model, the ocular disease does not exactly mimic human disease. This model, however, is attractive as hamsters reliably develop ocular lesions with little systemic disease and resolve spontaneously without

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treatment. Unlike humans, ocular toxoplasmosis in the hamsters does not result in pigmentation, the overlying retina alone appears atrophic, and the disease is bilateral. Hogan (1951) was the first to create a published animal model of ocular toxoplasmosis by injecting tachyzoites into the carotid arteries. However, the RH strain used in this model frequently resulted in meningoencephalitis and rapid mortality. A feline model of intracarotid inoculation of 5000 tachyzoites was successful in producing a reliable model of lesions of ocular toxoplasmosis (Davidson et al., 1993). Because the feline model has primarily choroidal involvement, it differs from human ocular infection. As expected, nonhuman primates have the most similar ocular environment mimicking the human eye. The nonhuman primate models support the theory that recurrent ocular disease is from the direct presence of T. gondii and not from indirect antigenic immunogenicity (Holland et al., 1988).

5.8 Clinical characteristics The most common clinical presentation of ocular infection due to T. gondii is a unilateral retinochoroiditis associated with a preexisting chorioretinal scar and an overlying vitritis. In addition, the clinical ophthalmic diagnosis of retinal vasculitis, of both arterioles and veins, is commonly made in active disease secondary to interaction between antibodies and antigens (O’Connor, 1974). T. gondii accounts for greater than one-quarter of all cases of posterior uveitis. Lesions can occur in any part of the fundus, but in patients with congenital infection, severe macular lesions appear more common than in acquired infection. One study (Mets et al., 1997) found that macular lesions were present in 58% of a cohort of 94 children with congenital toxoplasmosis, 76 of whom had 1

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year of treatment with pyrimethamine and sulfadiazine. Peripheral scars were present in 64%. This could be due to the early vascularization of the posterior pole during fetal development, or to the unique vascularization of the fetal macula, which contains end arterioles or to the higher concentration of cells. Evidence of bilateral infection, without simultaneous active disease but with the presence of bilateral scars, was found in 46% of eyes from one study (Hogan et al., 1964). A patient with typical ocular toxoplasmosis consults an ophthalmologist because of floaters and blurred vision. The ophthalmological examination (Fig. 5.1) usually reveals a focal necrotizing retinochoroiditis accompanied by vitreous inflammatory reaction, frequently associated with adjacent old scars indicative of recurrent attacks in satellite positions in 70% 80% of cases seen at first consultation (Delair et al., 2011). The clinical picture is typically characterized by periods of 8 16 weeks of recurrent intraocular inflammation. Ocular toxoplasmosis can be clinically classified as primary, if there exists an active creamy-white focal retinal lesion without associated pigmented retinochoroidal scars in either eye (Fig. 5.2A), or

FIGURE 5.1 Toxoplasma gondii retinochoroidal scar. Ocular funduscopic photograph of a typical Toxoplasmic retinochoroidal scar (black arrow). Source: Courtesy Jorge E. Gomez-Marin.

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FIGURE 5.2 Typical lesions observed in ocular toxoplasmosis. Ocular funduscopic photographs (A) Primary lesion: white cream lesion without hyper pigmented scar (arrow). (B) Recurrent lesion: creamy lesion with accompanying hyper-pigmented scar (arrow). Source: Courtesy Jorge E GomezMarin.

recurrent, if an active retinochoroidal lesion occurs in the presence of old pigmented retinochoroidal scars in either eye (Fig. 5.2B). Lesions should be described according to their size in optic disk diameters. Central lesions are defined as being located within the large vascular arcades. The most common symptoms during the active phase are blurred vision, floaters, ocular pain, and photophobia. During the ophthalmological examination, vitritis, anterior uveitis, vasculitis, or papillitis can be found. The most frequent complications after the resolution of inflammation are posterior synechiae, ocular hypertension, cataracts; less frequently, cystoid macular edema and, in congenital cases, strabismus. There are no differences in gender distribution of the disease and in the age of presentation. Definitive unilateral blindness can be present in 24% 37% of cases. Complications such as granulomatous iritis, high intraocular pressure, retinal vasculitis and vascular occlusions, rhegmatogenous and serous retinal detachments, and diverse forms of secondary pigmentary retinopathies might disguise the original T. gondii lesion and make the correct diagnosis difficult. The causes of visual loss include the location of T. gondii lesion in the macular area and retinal detachment. In a cross-sectional analysis of a cohort of 178 newborns with confirmed congenital toxoplasmosis from 146,307 screened babies

(95% of live births) from Minas Gerais state, southeastern Brazil, a high prevalence of congenital toxoplasmosis was encountered (1/770) with high rates of early choroiretinal involvement (approximately 80%) and many active lesions (in approximately 50%), indicating a possibly more severe ocular involvement by CT in Brazil than in other parts of the world (Vasconcelos-Santos et al., 2009). Ocular toxoplasmosis is characterized by recurrences that cause further visual loss and thus seriously affect the quality of life. The risk of a recurrence is the highest soon after an episode and then declines as the patient continues to remain recurrence free, this is called a clustering of clinical episodes. A previous use of systemic steroids without antibiotics or the application of subconjunctival injections of steroids (Reich et al., 2015a), as well as the use of systemic corticosteroid monotherapy, is related to a higher index of recurrences (Reich et al., 2016). Other factors related to the recurrence of episodes have still to be identified. The classic symptoms of ocular toxoplasmosis are like the classic symptoms of uveitis in general. They depend on the location, the degree, and the extension of the ocular inflammation. When ocular toxoplasmosis is active and posterior segment compromise (retinochoroiditis with or without vitritis) is accompanied by anterior segment inflammation,

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usually there is pain, redness, photophobia, and decreased vision. When there is only posterior segment compromise without anterior uveitis, the main symptoms are floaters, blurred vision and decreased visual acuity. The pain and photophobia are minimal unless there is severe iridocyclitis. It can be difficult to detect the early onset of any of these symptoms in children as they cannot articulate their symptoms appropriately. As a congenital toxoplasmosis study cohort demonstrated, if children are instructed to promptly report any change in their vision to their caregivers, this can increase the detection of active disease (Mets et al., 1997). Ocular toxoplasmosis can present with unusual manifestations of retinal pathology. Instead of the classic severely involved focal retinochoroiditis, it has also been reported to resemble unilateral acute idiopathic maculopathy (Lieb et al., 2004) or might present as intraocular inflammation only (anterior chamber cells and flare, vitreous inflammatory reactions, and retinal whitening), without clinically apparent necrotizing retinochoroiditis, at initial or baseline ocular examination (Arantes et al., 2015). After resolution of an active lesion, patients will have decreased vision within the area of retinochoroiditis. If the lesion is small and, in the periphery, then the patient will probably be asymptomatic. If, however, the lesion is small but within the macula, then the patient will probably be symptomatic. It is useful to have patients check their vision one eye at a time on a daily basis. Toxoplasma lesions within one disk diameter of the optic disk result in very significant “downstream” visual field defects. This means that an entire region of retina away from the actual lesion, but whose communicating nerve fibers pass over the lesion, can have a loss of input as measured by visual field testing (Stanford et al., 2005). Because of overlapping visual fields and the fact that ocular toxoplasmosis is usually active unilaterally not bilaterally, a change in vision

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may not be initially detected unless unilateral daily screening occurs. An Amsler grid, a graph paper like grid of boxes, mounted to a flat surface may help compliance with self-vision screening. In addition to decreased vision in the area of reactivation, patients will likely have floaters or other media opacity related complaints that will vary depending on the degree of inflammation when the lesion was active and may persist after resolution of the underlying reactivation secondary to inflammatory debris being trapped within the vitreous. Vision loss can be caused by many of the complications associated with ocular toxoplasmosis. Involvement of the macula or optic nerve can directly decrease central vision. Complications secondary to inflammation can indirectly affect vision. These include macular edema, vitreous opacity, epiretinal membrane, and retinal detachment (Bosch-Driessen et al., 2000; Friedmann and Knox, 1969; Mets et al., 1997). Under normal circumstances, peripheral scars can affect the visual field but do not impair central vision. However, in rare cases, peripheral scarring may lead to central vision loss. An example of a rare manifestation of ocular toxoplasmosis is one case wherein central vision loss occurred due to a giant macular hole (Blaise et al., 2005). This macular hole was the result of vitreous traction caused by peripheral ocular toxoplasmosis. Subretinal neovascularization in ocular toxoplasmosis has been reported as an unusual cause of vision loss (Cotliar and Friedman, 1982). Ocular involvement has been shown to occur long after the time of infection, either acquired or congenital infection. New lesions are likely to occur near the borders of existing lesions, and a larger lesion surrounded by smaller satellite lesions has been considered the hallmark of a recurrence of both congenital or postnatally acquired disease. Little is known about what influences the rate of recurrence of ocular toxoplasmosis. Though Bosch-Driessen

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et al. (2002) report a cumulative increase in the prevalence of chorioretinal recurrence, it cannot be assumed that the risk of recurrence in an individual patient increases over time. Holland’s (2003) impression was that the pattern of recurrence decreased over time. One possible explanation for this pattern is that tissue cysts in human hosts have a limited lifespan and lose their ability to reactivate. There is no evidence that short-term therapy at the time of infection has any effect on the pattern of recurrence (Bosch-Driessen et al., 2002).

5.8.1 Recurrence The classic description of recurrent active Toxoplasma retinochoroiditis is a focus of retinitis or retinochoroiditis appearing at the border of a retinochoroidal scar; however, there are several reports illustrating the variance in the clinical features of this disease. Active chorioretinitis might resolve without treatment and usually leaves a hyperpigmented scar; and recurrences develop as “satellite” lesions. A recurrence is usually symptomatic with floaters and blurry vision when there is a presence of retinochoroiditis; and with additional redness, pain, and light sensitivity when there is anterior segment inflammation or any generalized panuveitis. Recurrence of retinochoroiditis can lead to vision loss (Friedmann and Knox, 1969) and blindness. The risk of a recurrence is higher soon after an episode and then declines as the patient continues to remain recurrence free (clustering). The frequency of recurrence in ocular toxoplasmosis in Colombia was two episodes every 11 years, with recurrences clustering soon after an active attack (de la Torre et al., 2009a,b). There is a limited understanding between the many factors that exist between infection and recurrence of disease. There is a strong pattern of recurrence during the teenage and adult years (Bosch-Driessen and Rothova, 1999; Bosch-Driessen et al., 2002; Garweg et al.,

2008). Although it was thought that women with ocular toxoplasmosis were at a higher risk of recurrence during pregnancy (O’Connor, 1983), recurrence rates of ocular toxoplasmosis are probably not higher during pregnancy, in contrast to traditional beliefs (Braakenburg et al., 2014; Reich et al., 2015b). With regards to the fetus, except for rare reports (Andrade et al., 2010; Silveira et al., 2003), it appears to be at risk only during a mother’s initial infection. Initially, it was unclear what recurrence actually was, and it certainly remains unclear why recurrence occurs. Frenkel’s (1974) theory that recurrence represents a hypersensitivity reaction appears unlikely to be the central cause of recurrent ocular toxoplasmosis. Release of T. gondii antigen into the retina is not associated with a hypersensitivity reaction. Prior to the AIDS epidemic there was controversy as to whether recurrence represented an autoimmune process without the presence of actively replicating parasites, but it is now accepted that when recurrence occurs actively replicating parasites are present in the eye. Histopathology of ocular toxoplasmosis in the setting of HIV infection has demonstrated parasites in areas of involved retina. Eyes that received corticosteroid treatment alone had very poor outcomes associated with parasites demonstrated on tissue biopsy (Sabates et al., 1981). The use of systemic steroids without antibiotics and subconjunctival injection of steroids were identified as the main factors related to recurrences in a group of patients with ocular toxoplasmosis (de-la-Torre et al., 2009a,b). Moreover, patients had a higher risk of recurrence when receiving systemic corticosteroid monotherapy (despite being younger when receiving this therapy) compared with either receiving T. gondii specific antibiotic treatment or no therapy (Reich et al., 2015a). Intermittent use of trimethoprim/sulfamethoxazole, every 2 3 days, after an active lesion of ocular toxoplasmosis significantly reduces the

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risk of recurrence, at least for 1 year. Therefore it is appropriate to give prophylaxis, for at least the first year, and possibly the first 2 years, after suffering a recurrence (Kopec et al., 2003; Silveira et al., 2002, 2015b). Reactivation is thought to represents a shift of T. gondii from the dormant phase known as bradyzoites to the more active phase as a tachyzoite. There is not a clear understanding of how this shift from bradyzoite to tachyzoite occurs within the eye or what factors influence or cause this shift. Evidence of prior recurrence is the presence of inactive satellite lesions, which are local areas of retinochoroidal scars. Recurrent lesions usually occur close in proximity to prior areas of retinochoroiditis as is evident in the usual clusters of scars that exist (Fig. 5.3). Recurrent disease occurs when new areas of retina are involved in an infectious inflammatory process that results in permanent destruction of involved tissue. Resolution of the inflammatory process will usually occur spontaneously after several weeks. Although the general eye inflammation will resolve, the area of retina with focal retinochoroiditis is irreversibly impaired. If the lesion or recurrence is in the peripheral retina the impact of the recurrence on the infected individual’s vision can be minimal, or even asymptomatic, because the

FIGURE 5.3 Recurrent ocular toxoplasmosis. Ocular funduscopic photograph of a recurrent lesion (black arrow) at the borders of an old chorioretinal scar (black arrow). Source: Courtesy Jorge E Gomez-Marin.

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impaired vision exists in a small area of the peripheral field. Usually, the new lesion is a focal retinochoroiditis; however, a more generalized vitritis and anterior uveitis usually develop secondarily as a generalized intraocular inflammatory process. This more generalized intraocular inflammatory process is responsible for the complaint of decreased vision in patients with ocular toxoplasmosis. The secondary inflammatory process can lead to secondary serous or rhegmatogenous retinal detachments (Kianersi et al., 2012) or other ocular morbidities such as epiretinal membranes, fibrous bands, optic neuritis and neuropathy, cataracts, increased intraocular pressure during active infection, and choroidal neovascular membranes (Delair et al., 2011).

5.8.2 Congenital ocular toxoplasmosis Congenital toxoplasmosis appears to be the highest risk of a systemic infection for the development of ocular lesions. The risk of retinochoroiditis from intrauterine infection is 20% in the early childhood years and can rise to as high as 80% in adolescence. Congenital infection of ocular toxoplasmosis has been estimated to affect 3000 infants born in the United States each year with a resultant annual cost of between 400 million and 8.8 billion dollars each year (Roberts et al., 1994; Roberts and Frenkel, 1990; Wilson and Remington, 1980). Toxoplasma retinochoroiditis is present in 70% 90% of patients with congenital Toxoplasma infection, and it is the most common manifestation of disease (Alford et al., 1974). Though 85% of congenitally infected infants appear normal at birth, studies indicate that if these patients are not treated, approximately 85% of them will go on to develop retinochoroidal lesions, some resulting in vision loss, by adulthood (Koppe et al., 1974; Wilson et al., 1980). Congenital infection is more common later in gestation, but disease manifestations are

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worse if acquired earlier in gestation (Dunn et al., 1999). Classically, congenital disease is associated with bilateral macular scarring, but acquired infection can also result in macular disease and rarely bilateral scarring as well (Glasner et al., 1992). Other manifestations include optic neuritis, iritis, neuroretinitis, retinal vasculitis, acute retinal necrosis (ARN), recurrent iridocyclitis, and persistent vitritis. Long-term follow-up of congenitally infected children results in the identification of further ocular sequelae not present at birth, for example, in one study four of six untreated congenitally infected children developed scars subsequent to birth during the next 20 years (Koppe et al., 1986). It is estimated that 85% of infants untreated and without ocular lesions at birth will subsequently develop ocular toxoplasmosis (Koppe et al., 1974; Wilson et al., 1980). Microphthalmos and microcornea can occur as a consequence of severe congenital eye disease (Suhardjo and Agni, 2003). Nystagmus and strabismus and amblyopia secondary to congenital toxoplasmosis are more complex than even most expert ophthalmologists’ realize (O’Neill, 1998). There is a tendency for clinicians not to struggle with the complex care involved in trying to achieve optimal visual outcome in congenital infection. Exposure to Toxoplasma six months prior to conception is thought to eliminate the possibility of congenital transmission secondary to lifelong immunity in immunocompetent individuals. Rarely, reactivation of toxoplasmosis in previously infected immunodeficient women can result in congenital transmission of toxoplasmosis (Mitchell et al., 1990). There is one case report of treated acquired ocular toxoplasmosis during pregnancy occurring in the mom without any subsequent fetal disease (Ramchandani et al., 2002). The exact mechanism of transmission is not yet understood but is thought to be secondary to transplacental transmission of the parasite. The severity of ocular manifestations

parallels the severity of CNS disease in congenital infection (Roberts et al., 2001). Mets et al. (1997) highlighted the ophthalmic findings of congenital T. gondii infection in treated and untreated individuals. 79% of children had retinochoroidal scars. 28% of individuals had significant unilateral vision loss. 29% of children had bilateral vision loss. The presence of inactive chorioretinal lesions in congenitally infected newborns indicates that the complete cycle of infection, activation, and resolution of retinochoroidal lesions may occur in utero (Guerina, 1994; Mets et al., 1997). The New England Regional Toxoplasma Working Group detected 100 of 635,000 infants who were seropositive for IgG and IgM against Toxoplasma. Four of 39 treated children observed for as long as 6 years had new postnatally developed retinal scars, and a separate 9 of 48 patients had retinal lesions at birth (Guerina, 1994). In another study in England, after 20 years of follow-up, 9 of 11 patients with congenital toxoplasmosis had evidence of retinochoroiditis and 4 had severe impairment (Koppe et al., 1986). The largest report of congenital toxoplasmosis in twins highlights that multiple factors beyond the time of exposure during gestation influence the ocular outcome in congenital infection (Peyron et al., 2003). Although there are possible confounding issues of shared placentas and mortality, as is true of any infectious congenital disease involving twins, if concordance of the disease is more common among monozygotic twin than among dizygotic ones, then genetic susceptibility is likely more important than environmental influence in disease outcome. While there is no rigorous protocol that has been published focusing on a cohort of ocular outcome in twins (Rieger, 1959), it appears there is a lack of identical outcome between eyes and between twins. It is, therefore, not time or inoculum alone that leads to the presence or absence of ocular disease, size of lesions, or location of lesions

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(Couvreur et al., 1976). There are differences in specific ocular outcomes in both dizygotic and monozygotic twins. The general disease impact with respect to symptomatic involvement and eventual ocular involvement appears more concordant in dizygotic than in monozygotic twins. To definitively answer patterns of ocular toxoplasmosis in twins a long rigorous followup report remains to be published. In settings where infants receive ante- and postnatal treatment, congenital toxoplasmosis is rarely severe, but patients are at risk of new or relapsing ocular lesions during their life. In a cohort of 485 cases of congenital toxoplasmosis, first lesions appeared at a median age of 3.1 (range: 0.0 20.7) years. In 33.8% of cases, recurrence or first ocular lesion appeared up to 12 years (Wallon et al., 2014; Wallon and Peyron, 2018). Visual performance using the VF14 questionnaire was evaluated on 126 adults presenting with congenital toxoplasmosis and monitored since birth. Among them, 58.8% presented with at least one ocular lesion with a foveal localization in 15.7% of cases. Visual function was slightly impaired with a VF14 global score of 97.3 (out of 100) (Peyron et al., 2011; Wallon and Peyron, 2018). In an early ophthalmologic examination of a large cohort of neonates in Brazil, of 146,307 neonates screened, 190 had congenital toxoplasmosis, yielding a prevalence of 1 in 770 live births, of whom 178 (93.7%) underwent standardized ophthalmologic examination at an average age of 55.6 6 16.6 days. Of these 178 infants, 142 (79.8%) had retinochoroidal lesions consistent with congenital toxoplasmosis in at least 1 eye. Bilateral involvement was noted in 113 patients (63.5%). Macular involvement was seen in 165 eyes (46.3%) of 111 patients (62.4%). Active lesions were observed in 142 eyes (39.9%) of 85 patients (47.8%). These lesions were located in the macula of 75 eyes (21.1%) and were associated with retinal vascular sheathing in 44 eyes (12.4%). A high prevalence of congenital toxoplasmosis was

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encountered (1/770) with high rates of early retinochoroidal involvement (approximately 80%) and many active lesions (in approximately 50%), indicating a possibly more severe ocular involvement by congenital toxoplasmosis in Brazil than in other parts of the world, due probably to a higher parasite virulence and increased individual susceptibility (Vasconcelos-Santos et al., 2009). The treatment regimens for congenital toxoplasmosis (see Chapter 4: Human Toxoplasma infection) include spiramycin to prevent congenital transmission from an infected mother to the fetus, pyrimethamine (with folinic acid to ameliorate bone marrow suppression) combined with sulfadoxine to treat an infected fetus in utero or a child with congenital infection; CSF shunting for the treatment of hydrocephalus; or combinations of pyrimethamine, azithromycin, and corticosteroids for treating ocular toxoplasmosis after birth (Khan and Khan, 2018). It is unclear why macular lesions commonly occur in congenital infection. Other frequently involved areas in the brain are the periaqueductal, periventricular, and basal ganglia regions. One theory is that secondary to a high-affinity transport protein for putrescein, T. gondii thrives in the putrescein-rich fetal retina (Henrique Seabra et al., 2004). Another theory is that the macula is the first part of the retina that is vascularized as the vasculogenesis spreads peripherally from central posterior retina to the far periphery. The macula is, therefore, affected because it is the region that is vascularized longest and more likely to be infected than the peripheral retina.

5.8.3 Ocular presentation in the elderly The role of patient age on a number of features of ocular toxoplasmosis has been a matter of study for several years. Interactions between age and other factors are complex and will

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require continued study to understand observed patterns (Holland, 2009). Although the symptoms of ocular toxoplasmosis may occur at any stage of life (Ferreira et al., 2014), the deterioration of the immune system in the elderly likely increases its occurrence and severity (Holland, 2009; Maenz et al., 2014). Thus the main factor in the elderly causing ocular disease is the decreased surveillance and control of infection from parasites that emerge from tissue cysts due to immune senescence. A number of studies suggest that ocular toxoplasmosis is a more severe disease at the extremes of age (Holland, 2009). Studies indicate that most patients with ocular toxoplasmosis who have serologic evidence of recent infection are older (Bosch-Driessen et al., 2002; Ongkosuwito et al., 1999; Ronday et al., 1995). Patients whose ocular toxoplasmosis was first observed—when they had serologic proof of remotely acquired infection had a mean age of 29.9 years, while patients whose ocular toxoplasmosis was first observed while they had serologic proof of recent infection—had a mean age of 50.6 years (Bosch-Driessen et al., 2002). In addition, patients with primary retinal lesions were older than those with recurrent lesions) (Bosch-Driessen et al., 2002; Ongkosuwito et al., 1999). In an epidemic infection of T. gondii in Victoria, British Columbia, Canada, the mean age of infected patients without retinal lesions was approximately 28 years, whereas the mean age of infected individuals with eye disease was 54 years (Burnett et al., 1998). Additional studies from Brazil support an increased risk of ocular involvement among older patients following T. gondii infection. Patients without retinal lesions at the time of first positive IgM test who were older than 40 years were at higher risk of developing retinal lesions during follow-up than were younger age groups (Arantes et al., 2015). Several studies strongly suggest that older patients have a higher risk of developing

ocular lesions following recently acquired T. gondii infection (Holland, 2009). The severity of retinochoroiditis (in terms of lesion size, location, and associated inflammation) is affected by patient age at the time of initial infection or recurrence. In an international, multicenter study of 210 patients with active T. gondii retinochoroiditis there was a relationship between age and lesion size (Dodds et al., 2008) with patients $ 60 years of age having a significantly higher percentage of lesions .1 DA in size (77% vs 42% for patients , 60 years of age; P 5 .020). A relationship between older age and the area of “scarring” might be explained by a history of progressive enlargement in older patients having long-standing disease and multiple reactivations, but such factors would not explain a relationship between age and the area of disease activity during a recurrence. It is more likely that the relationship reflects immune senescence in elderly patients, with a decreased ability to limit parasite proliferation (Holland, 2009). There also was a relationship between patient age and signs of intraocular inflammation. Eyes with more severe vitreous humor cells and vitreous humor haze were from older patients than those with less severe scores. Patients older than 40 years of age at the time of an active episode were at higher risk of recurrence after an active episode than younger patients. Increased risk among older patients may be related to decreased host immunity, as discussed above (Dodds et al., 2008). Relapses are seen less commonly among older patients with T. gondii retinochoroidal scars than among young patients with such scars. A study found that significantly more recurrences were seen among patients less than 30 years of age than among older individuals (Garweg et al., 2008). Univariate analysis of this cohort demonstrated that older age at an active episode was associated with a reduced risk of subsequent recurrence. This apparent contradiction could be explained by the complex interaction between age at first episode and age at any subsequent,

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active episode. These ages are apparently linked by the interval between the first episode and recurrence. It has been hypothesized that younger individuals are at increased risk for recurrences following initial infection because of agespecific activities that lead to ingestion of more parasites and thus, a greater parasite burden in the retina, or because of age-specific activities that result in exposure to sources contaminated by more virulent parasite strains. These factors have to be clarified, but they may still be age related in some way. Variations in innate immunity might also influence both the age of infection and parasite burden after ingestion. The interaction of age-related factors contributes to the variability of recurrence patterns observed by clinicians and was interpreted previously as randomness (Holland, 2009).

5.8.4 Atypical presentations of ocular toxoplasmosis 5.8.4.1 Immunocompromised patients Ocular toxoplasmosis typically presents with characteristic findings of unilateral and focal retinochoroiditis with an adjacent healed retinochoroidal scar and vitreous inflammation. In rare cases, particularly in immunocompromised patients (e.g., patients with HIV/ AIDS, immune modulatory therapy, severe underlying illness, or advanced age), OT can present as aggressive retinochoroiditis that could be bilateral, multifocal and/or extensive (Smith and Cunningham, 2002). Other unusual manifestations in immunocompromised patients are punctate outer retinal toxoplasmosis (Moraes, 1999; Smith and Cunningham, 2002) neuroretinitis and other forms of optic neuropathy (Grossniklaus et al., 1990). 5.8.4.2 Acute retinal necrosis Toxoplasmosis can mimic ARN, a devastating blinding retinochoroiditis usually caused by herpes viruses (Moshfeghi et al., 2004). Viruses

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of the herpes family, especially Herpes simplex and Herpes zoster, may lead to extensive retinal involvement, in the form of ARN, characterized by peripheral and rapidly progressive retinitis along with severe intraocular inflammation and occlusive retinal vasculopathy, according to the American Uveitis Society (AUS) criteria (Holland, 1994). ARN may be difficult to differentiate from severe T. gondii retinochoroiditis (Fig. 5.4), unless patients are followed to demonstrate rapid progression, which is not advisable due to the high risk of retinal damage and detachment. Similar cases not fulfilling the AUS criteria for ARN may be classified as necrotizing herpetic retinopathy (NHR) (Holland, 1994) and should also be differentiated from severe T. gondii retinochoroiditis. In these cases, lack of visible chorioretinal scars and extensive areas of retinal involvement may lead to a diagnosis of ARN or NHR and further treatment with antivirals (Moshfeghi et al., 2004). Because of the large size of the lesions and the overlying vitritis, the lesions caused by T. gondii may be difficult to distinguish from ARN caused by Herpes viruses. OT retinochoroiditis lesions often do not follow the vascular spread seen in CMV or

FIGURE 5.4 Active retinochoroiditis simulating acute retinal necrosis. Ocular funduscopic photograph of extensive retinochoroidal lesions (black arrow), retinal hemorrhages (white arrows) and vitriitis in a patient with ocular toxoplasmosis simulating ARN. ARN, Acute retinal necrosis. Source: Courtesy Dr. Alejandra de-la-Torre.

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the axonal spread seen in VZV and HSV. Atypical toxoplasmosis needs to be considered in cases of necrotizing retinitis not responding appropriately to antiviral treatment along with syphilis, lymphoma, and CMV retinitis in non HIV patients. Since diagnosis of ARN is usually made by clinical appearance alone, therapy is usually directed first against the herpes virus, and subsequently, a broader differential is entertained if instituted therapy fails to stop progression of disease. An anti Toxoplasma regimen may be instituted in severe posterior uveitis with ARN as empiric therapy (Burke et al., 2016). In addition to a therapeutic trial, alternative methods to establish a definite diagnosis in these challenging cases include analysis of intraocular antibody synthesis, PCR of ocular fluids, and even a retinal biopsy (Fardeau et al., 2002; Harper et al., 2009; Moshfeghi et al., 2004; Palkovacs et al., 2008). 5.8.4.3 Punctate outer retinal toxoplasmosis Some reports (Doft and Gass, 1985) have described a distinctly different category of T. gondii lesions known as “punctuate outer retinal toxoplasmosis” (Fig. 5.5). This condition

FIGURE 5.5 Active retinochoroiditis with punctate lesions. Ocular fundus photography of active whitening retinochoroidal lesion adjacent to the optic disk (black arrow) accompanied by punctate outer retinal toxoplasmosis (white arrow). Source: Courtesy Dr. Alejandra de-la-Torre.

consists of multifocal lesions that are gray to white in color and less than 1000 μm in size. These lesions appear at the deep level of the retina and retinal pigment epithelium. There is little inflammatory reaction in the vitreous, and involvement is sometimes bilateral. Though these lesions appear as a cluster, there is generally only one focus of active disease at any point in time. 5.8.4.4 Other atypical clinical presentations Other unusual forms of posterior eye involvement include neuroretinitis (Moreno et al., 1992; Smith and Cunningham, 2002), occlusive retinal vasculitis (Braunstein and Gass, 1980) retinal and subretinal neovascularization (Gaynon et al., 1984; Lafaut et al., 1999), rhegmatogenous and serous retinal detachment (Fig. 5.6) (BoschDriessen et al., 2000; Frezzotti et al., 1974; Kraushar et al., 1979), pigmentary retinopathy that may mimic retinitis pigmentosa (Silveira et al., 1989), and various optic nerve pathologies (Confavreux et al., 1985; Mansour et al., 1993; Rehder et al., 1988; Roach et al., 1985; Song et al., 2002). In addition, T. gondii may proliferate in other parts of the eye, producing anterior uveitis, intermediate uveitis (Holland et al., 1999), and endophthalmitis (Moorthy et al., 1993).

FIGURE 5.6

Retinochoroidal lesion with retinal detachment. Ocular fundus photography of active retinochoroidal lesion (white arrow) and secondary retinal detachment in a recurrent ocular toxoplasmosis (black arrows). Source: Courtesy Dr. Alejandra de-la-Torre.

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There is a documented, although poorly understood, association between Fuchs heterochromic iridocyclitis and ocular toxoplasmosis (Schwab, 1991). A dramatic and unusual periarteritis, eponymously named Kyrieleis-type periarteritis, with deposition of focal plaque like deposits can occur along the major arcades and may persist after resolution of active disease (Schwartz, 1977). Rarely, a very opaque form called frosted branch angiitis (Ysasaga and Davis, 1999) may occur without retinochoroidal lesions (Holland et al., 1999). A neuroretinitis can also occur with its classic stellate exudative like appearance within the macula as a consequence of T. gondii infection (Ku¨c¸u¨kerdo¨nmez et al., 2002; Perrotta et al., 2003). Other atypical presentations include retinal vascular occlusions, rhegmatogenous with serous retinal detachments, unilateral pigmentary retinopathy mimicking retinitis pigmentosa, neuroretinitis, and additional forms of optic neuropathy and scleritis (Bonfioli and Orefice, 2005; Crosson et al., 2015; Smith and Cunningham, 2002). Very infrequently, scleritis has been reported to be due to T. gondii infection (Schuman et al., 1988; Smith and Cunningham, 2002).

5.8.5 Classification systems for uveitis and retinochoroiditis The Standardization of Uveitis Nomenclature (SUN) Project was started in 2004 by a working group of uveitis specialists who intent on developing an international consensus for the use of terms to report on uveitis at academic meetings and in the literature. Subsequently, the “Standardization of Uveitis Nomenclature for Reporting Clinical Data: Results of the First International Workshop” was published in 2005 (Jabs et al., 2005; Okada and Jabs, 2013). Although still a work in development and hence subject to periodic revision, investigators are adopting these guidelines. This allows for a measure of comparability

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between articles and/or clinical trials. The terms defined included many that we take for granted, such as acute, recurrent, and chronic. These SUN guidelines also affirmed the classification of uveitis by location within the eye and standardized the use of clinical grading as a tool for assessing degree of inflammation, for the most part by adopting previously published single institution “expert panel” driven definitions (Bloch-Michel and Nussenblatt, 1987; Hogan et al., 1959; Nussenblatt et al., 1985). Consensus was also achieved on how to document and report intraocular pressure abnormalities, outcomes of treatment, and visual acuity (Okada and Jabs, 2013). The main subdivisions of uveitis are brokendown by anatomic location: anterior, posterior, intermediate uveitis or pan-uveitis. T. gondii may cause inflammation in any subdivision (either primarily or secondarily). Anterior uveitis refers to inflammation in the front of the eye anterior to the vitreous (iritis or iridocyclitis). Intermediate uveitis refers to inflammation in the vitreous and peripheral retina or in the pars plana (tissue located just anterior to the retina). Posterior uveitis refers to an inflammation within the retina or choroid (retinitis, choroiditis, retinochoroiditis, or retinochoroiditis) and pan-uveitis refers to inflammation of all layers of the uvea, which includes the iris, ciliary body, and choroid. There was a consensus that an anatomic classification of uveitis should be used and should serve as a framework for subsequent work on diagnostic criteria for specific uveitic diagnoses. The International Uveitis Study group (IUSG) anatomic classification (Table 5.3) was endorsed, and it was agreed that the classification of the anatomic location of the uveitis should be on the basis of the site(s) of inflammation and not on the presence of structural complications (Jabs et al., 2005). The terms “acute” and “chronic” have been used inconsistently in the literature and have been used variably to refer to the onset of the uveitis, the duration of an attack of uveitis, or

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TABLE 5.3 The SUN Working Group anatomic classification of uveitis*. Type

Primary site of inflammation

Includes

Anterior uveitis

Anterior chamber

Iritis Iridocyclitis Anterior cyclitis

Intermediate uveitis

Vitreous

Pars planitis Posterior cyclitis Hyalitis

Posterior uveitis

Retina or choroid

Focal, multifocal, or diffuse choroiditis

Panuveitis

Anterior chamber, vitreous and retina or choroid

*Group, Standardization of Uveitis Nomenclature (SUN) Working (2005) (Jabs et al., 2005).

TABLE 5.4 The SUN Working Group descriptors of uveitis*. Category

Descriptor

Onset

Sudden

Comment

Insidious Duration

Course

Limited

# 3 months duration

Persistent

.3 months duration

Acute

Episode characterized by sudden onset and limited duration

Recurrent

Repeated episodes separated by periods of inactivity without treatment $ 3 months duration

Chronic

Persisten uveitis with relapse , 3 months after discontinuing treatment

*Group, Standardization of Uveitis Nomenclature (SUN) Working (2005) (Jabs et al., 2005).

to the course of uveitis. Consensus was obtained that the use of these terms should be reserved for the description of the clinical course of the uveitis, and that other terms should be used to describe the onset of the uveitis and the duration of an attack of uveitis (Table 5.4) (Jabs et al., 2005). Standard methods for grading anterior chamber cells (Table 5.5) and anterior chamber flare (Table 5.6) should be considered. Although the goal of treatment of uveitis is to suppress the inflammation completely (“inactive” disease), for the short-term evaluation of

new therapies, it may be appropriate to determine whether the inflammation has improved or worsened (Table 5.7). Ocular toxoplasmosis can cause several kinds of uveitis, according to the SUN classification as discussed in the following subsections. 5.8.5.1 Anterior uveitis The severity of anterior uveitis can range from a quiet anterior chamber to intense anterior uveitis masking inflammation of the posterior segment and can be either granulomatous

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TABLE 5.5 The SUN Working Group grading scheme for anterior chamber cells*. Grade

Cells in fielda

0

,1

0.5 1

1 5

11

6 15

21

16 25

31

16 50

41

.50

a

Field size is a 1 mm by 1 mm sit beam. *Group, Standardization of Uveitis Nomenclature (SUN) Working (2005) (Jabs et al., 2005).

TABLE 5.6 The SUN Working Group grading scheme for anterior chamber flare*,a. Grade

Description

0

None

11

Faint

21

Moderate (iris and lens details clear)

31

Marked (iris and lens details hazy)

41

Intense (fibrin or plastic aqueous)

a

Field size is a 1 mm by 1 mm sit beam. *Group, Standardization of Uveitis Nomenclature (SUN) Working (2005) (Jabs et al., 2005).

TABLE 5.7 The SUN Working Group activity of uveitis terminology*. Term

Definition

Inactive

Grade 0 cells

Worsening activity

Two-step increase in level of inflammation (e.g., anterior chamber cells, vitreous haze) or increase from grade 3 1 to 4 1

Improved activity

Two-step decrease in level of inflammation (e.g., anterior chamber cells, vitreous haze) or decrease to grade 0

Remission

Inactive disease for $ 3 months after discontinuing all treatments for eye disease

*Group, Standardization of Uveitis Nomenclature (SUN) Working (2005) (Jabs et al., 2005).

or nongranulomatous inflammation. If the diagnosis is delayed, prolonged inflammation of the anterior chamber may lead to irreversible iris synechiae. In a study of 210 patients

with active T. gondii retinochoroiditis, intraocular inflammation was more intense in older patients and increased with the size of areas of retinochoroiditis and in peripheral lesions

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FIGURE 5.7 Photograph of the anterior segment of the eye. Ocular fundus photography of keratic precipitates in a patient with anterior uveitis related with ocular toxoplasmosis. Source: Courtesy Dr. Alejandra de-la-Torre.

(Dodds et al., 2008). Secondary ocular hypertension was associated with inflammation of the anterior segment and occurred in 30% of cases. Overall, more intense inflammation may be observed in cases of ocular toxoplasmosis in patients from certain areas of Central and South America. Also, intense anterior inflammation may occur secondary to the retinochoroiditis near the ora serrata, which may be missed on initial examination (Delair et al., 2011) (Fig. 5.7). 5.8.5.2 Vitritis Inflammation of the vitreous is usually more intense near the active retinochoroiditis. However, there may be no vitritis if the retinal inflammation does not extend to the inner limiting membrane. In cases of intense vitritis, epiretinal membranes may develop and vitreoretinal traction adjacent to the area of retinochoroiditis may occur. The intensity of the vitritis is associated to the vitreous haze, and it also appears to reflect duration of the process prior to diagnosis and treatment, with more intense inflammatory response associated with longer intervals before starting the treatment. “Headlight in the fog” was a phrase for

FIGURE 5.8 Vitritis. Ocular fundus photography of vitritis associate with ocular toxoplasmosis. Grade 3 according to Nussenblatt et al. (1985) and Grade 7 according to Davis et al. (2010). Source: Courtesy Dr. Alejandra dela-Torre.

severe vitritis coined by Richard O’Connor to describe a bright white reflex seen when one shines the light of the indirect ophthalmoscope into the back of the eye (Delair et al., 2011) (Fig. 5.8). Vitreous haze is the obscuration of fundus details by vitreous cells and protein exudation. It impacts vision more profoundly than anterior inflammation and therefore used commonly as an outcome measure for clinical trials involving intermediate, posterior, or panuveitis (Davis et al., 2010). The usefulness of photographic grading of vitreous haze can delineate vitreal inflammatory activity and have been found to be most helpful in standardizing our clinical findings (Nussenblatt et al., 1985). Gradations of vitreal haze from no haze (0 1 ) to severe haze (4 1 ) were proposed by Nussenblatt et al (1985) by selecting fundus photographs from uveitis patients that depicted varying degrees of clarity of the optic nerve and retina. Davis et al. (2010) proposed vitreous haze scale grades from 0 through 8. In 2011 validation of a photographic vitreous haze grading technique for clinical trials in uveitis was proposed by Madow et al. (2011), suggesting that this photographic grading with a nine-step logarithmic scale is a highly reproducible methodology that may be adaptable to

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use in future clinical trials. Some examples of this gradation are seen in Fig. 5.9, a photography that shows grade 0 vitreal haze according to all the proposed scales, and Fig. 5.8, a photography that shows grade 7 vitreal haze according to Davis et al. (2010) and grade 4 vitreal haze, according to the Nussenblatt’s proposal (Nussenblatt et al., 1985). 5.8.5.3 Retinochoroiditis Active retinochoroidal lesions are typically seen as whitish foci without well-limited borders, usually adjacent to a pigmented and/or atrophic scar (Fig. 5.10). When retinal vessels are close to the lesion, signs of vasculitis contiguous to the lesion may be seen (Fig. 5.11). Periphlebitis is more frequent than arteritis

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and retinal hemorrhages can also be seen. In some cases, with very intense inflammation, vasculitis may be generalized to retinal areas distant from the primary site of retinochoroiditis. An active retinochoroidal lesion usually results in an atrophic retinochoroidal scar, which resolves from the periphery to the center of the lesion, usually leading to pigmentation (Fig. 5.12). The average time for the scarring of an active toxoplasmic area of retinochoroiditis often appears to be related to the function of the lesion size and resolution often occurs in approximately 3 4 weeks. With prompt diagnosis and treatment, lesions may resolve more rapidly and even heal without creating a scar (Delair et al., 2011; McLeod et al., 2009). It is convenient to describe the number of lesions, the location (fovea, macula, posterior pole, and periphery), and the size of the retinochoroidal lesions. The size of the lesion can be measured in “disk diameters” (comparing the size of the lesion with the size of the optic disk) or in millimeters on fundus photography.

5.8.6 Optic nerve involvement in ocular toxoplasmosis FIGURE 5.9 Macular scar due to toxoplasmosis. Ocular fundus photography of macular scar with hyperpigmented and atrophic areas (white oval) and no vitritis. Source: Courtesy Dr. Alejandra de-la-Torre.

The optic nerve is populated by approximately 1.3 million nerves that originate in the retina ganglion cell layer and connect the eye to the brain. In congenital toxoplasmosis, because eyes available for autopsy are from individuals FIGURE 5.10 Fundus photography and fluorescein angiography of active retinochoroiditis. Active retinochoroidal lesion seen as whitish foci without well-limited borders (black arrow) adjacent to a pigmented scar (white arrow). Source: Courtesy Dr. Alejandra de-laTorre.

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FIGURE 5.11 Fluorescein angiography demonstrating vasculitis. Hyperfluorescent lesion (black arrow) with surrounding leakage (white arrow) and staining of retinal vessels indicating retinal vasculitis secondary to ocular toxoplasmosis (gray arrows). Source: Courtesy Dr. Alejandra de-la-Torre.

FIGURE 5.12 Epiretinal membrane. Ocular fundus photography of multiple macular scars (white arrows) and a secondary epiretinal membrane due to ocular toxoplasmosis (white triangles). Source: Courtesy Dr. Alejandra de-laTorre.

with severe CNS involvement, it is not possible to definitively determine whether optic nerve changes are secondary to direct infection or secondarily to active CNS processes such as toxoplasmic encephalitis. Optic nerve atrophy was present in 20% of individuals in one congenital toxoplasmosis cohort (Mets et al., 1997). The diagnosis of ocular toxoplasmosis is difficult in the presence of papillitis without other characteristic signs of retinochoroidal inflammation (Delair et al., 2011). Whitish inflammatory lesions located on the disk with associated vitritis suggest the diagnosis (Song et al., 2002). Such lesions located on the border of the disk are responsible for visual defects occasionally referred to as Jensen scotoma (Delair et al., 2011). T. gondii may cause a lesion in the optic

disk because of contiguousness, (Banta et al., 2002; Uchida et al., 1978) by direct involvement (Berengo and Frezzotti, 1962; Fish et al., 1993; Folk and Lobes, 1984; Moreno et al., 1992; Song et al., 2002; Williams and Miller, 1996), or become involved when a retinochoroiditis lesion is located distant from the optic nerve (Gonc¸alves et al., 1995; Lavinsky, 2002). The contiguous form was described by Jensen long time ago (Abraham, 1929) as a specific entity in four cases of juxtapapillary choroiditis, later considered as probably due to tuberculosis. In 1952 Wilder recognized T. gondii histopathologically in necrotic retinochoroidal lesion in adult patients (Holland et al., 2002; Wilder, 1952a,b). Lesions had a granulomatous appearance and manifested necrosis in many occasions leading to a pathologic diagnosis of tuberculosis at that time. Currently, however, it is accepted that most cases of Jensen’s choroiditis are due to T. gondii infection. This type of lesion consists of a typical area of retinochoroiditis in contact with a swollen optic disk resulting in a typical sectorial deficit in the visual field (Eckert et al., 2007). The direct involvement of the optic nerve by T. gondii was demonstrated histopathologically by the presence of different forms of the parasite inside it (Manschot and Daamen, 1965). This involvement may be subclassified into pure anterior neuritis or papillitis and neuroretinitis (Banta

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et al., 2002). The optic disk involvement has also been reported associated to an active distant retinal lesion (Eckert et al., 2007). Pure anterior neuritis occurs when the parasitic infection affects the optic disk directly, generating a swollen papilla with sheathing of the peripapillary veins, with or without simultaneous active retinochoroiditis lesion (Eckert et al., 2007; Gonc¸alves et al., 1995). Neuroretinitis is frequently characterized by a swollen optic nerve accompanied of papillomacular or serous macular detachment of the retina associated to star-shape hard exudates in the macula (Fish et al., 1993; Ku¨c¸u¨kerdo¨nmez et al., 2002; Maitland and Miller, 1984; Moreno et al., 1992). Papillitis associated to an active distant retinal lesion is an additional form of involvement of the optic disk that has been reported associated to an active distant retinal lesion, with an active focal necrotizing retinochoroiditis lesion situated at different distances from the optic disk (Eckert et al., 2007).

5.8.7 Toxoplasma and glaucoma Glaucoma, defined by the presence of ocular hypertension associated with visual field defects or by the presence of a reduced thickness of the retinal nerve fiber layer (RNFL), is measured by optical coherence tomography. Unlike herpetic uveitis which is frequently associated with glaucoma, a retrospective study of ocular toxoplasmosis found the highest incidence, 38%, of elevated intraocular pressure, in patients with active ocular toxoplasmosis (Westfall et al., 2005). There was, however, no associated glaucomatous nerve damage. The elevated intraocular pressure appeared to resolve with resolution of the retinochoroiditis. Glaucoma has been reported to be associated with ocular toxoplasmosis in 1% 5.4% of patients (Kovaˇcevi´c-Pavi´cevi´c et al., 2012; Schlaen et al., 2018; Westfall et al., 2005). In children with congenital ocular

toxoplasmosis, glaucoma was found in 15% of the patients as a complication associated with retinochoroiditis (Vutova et al., 2002). A prospective study will be required to further examine any possible association in this population. Nonglaucomatous retinal lesions may simulate glaucomatous RNFL atrophy and visual field loss, particularly when focal damage to the retinal ganglion cells occurs. Careful inspection of the retina and optic disk, with attention to the integrity of the neural rim, and analysis of the pattern of RNFL loss in proximity to a retinal lesion may enable the clinician to differentiate glaucomatous and nonglaucomatous pathogenic mechanisms (Sheets et al., 2009).

5.9 Diagnostic tests 5.9.1 Histopathology Destructive retinochoroiditis secondary to T. gondii infection has been characterized by pathology in several reports (Hogan, 1951; Rao and Font, 1977; Wilder, 1952a,b; Zimmerman, 1961). Organisms in immune-competent individuals have been identified in the retina and optic nerve, but not in the choroid. T. gondii cysts have been demonstrated in the RPE (Nicholson and Wolchok, 1976). Granulomatous choroidal inflammation and scleral thickening can occur adjacent to the retinal lesions. After resolution of active disease the involved retina shows severe destruction with retinal atrophy and chorioretinal adhesions. In a murine congenital model of ocular toxoplasmosis, surprisingly the photoreceptors (not the T. gondii cysts) appeared to be the focal activity of the mononuclear intraocular infiltrate (Dutton et al., 1986). Wilder’s case series highlighted the value of thicker sections to identify tachyzoites. She used celloidin which permits 18 μm sections versus the 8 μm performed with paraffin thin sections (Holland et al., 2002); however, celloidin requires 6 weeks before sectioning and paraffin

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thin sections have subsequently been shown (Holland et al., 1988) to be sensitive enough to detect parasites. Wilder (1952a,b) noted in her report of severe disease that active disease can extend into the sclera. The original identification of T. gondii as an etiology of ocular disease came through histopathologic examination of ocular tissues in congenital infection which revealed organisms in the retina and adjacent choroid (Wolf et al., 1939). Parasites were identified in 10 of 18 eyes of infants and fetuses in one report (Roberts et al., 2001). In this report, 8 of 15 eyes had focal retinal lesions some with retinal necrosis. A lesion 7 mm large was identified in a 22-weekold fetus. Only one of four cases had bilateral disease. In addition, 10 of 15 eyes had clinical lesions in the peripheral retina only. Vitritis was identified in 7 of the 18 eyes. In five of eight eyes where there was optic nerve present in sections, a leptomeningitis was identified, and three of the eyes had disruption of the optic nerve architecture. Diffuse choroiditis was identified in all cases. Eyes from a 7- and 5-day-old infant showed similar findings to the fetuses with more evidence of organization and a retinal detachment. A 2-year-old child demonstrated areas of an end stage continuum compared to the fetal eyes with evidence of retinal atrophy, retinal pigmentary epithelial changes, and overlying gliosis. The inflammatory cells present in the eyes consisted of lymphocytes, plasma cells, and macrophages. Immunohistochemical staining showed both T and B cells with the B cells less in number and confined primarily to the choroid. The T-cell population contained both CD4 1 and CD8 1 cells. No tissue cysts were identified in the group of eyes. However, immunohistochemical staining confirmed the presence of parasites in ten of fifteen eyes. Parasites were not identified in the choroid or substance of the optic nerve of any eye. A key point in understanding the complexity of ocular toxoplasmosis is that normal-appearing retina can harbor T. gondii (McMenamin et al., 1986).

The typical clinical presentation in acquired toxoplasmosis in retina is a “head-light-in-fog” appearance, and pathological feature in this disease entity is a well-demarcated area of coagulative necrosis with adjacent choroiditis, vasculitis, hemorrhage, and vitritis (Bonfioli and Orefice, 2005; Das et al., 2016). Viable tachyzoites and tissue cysts may be found in superficial layers of the infected retina along with an intense mononuclear inflammatory cell reaction seen in the involved retina as well as adjacent choroid and vitreous (Rao and Font, 1977). T. gondii can be seen in tissue as isolated single organisms, or as multiple organisms in an intracellular vacuole or as multiple organisms in a tissue cyst (Yanoff and Sassani, 2009). Tissues cysts usually lack any inflammatory reaction (Borkowski, 2001). Subretinal aspirates obtained during pars-plana vitrectomy (PPV) as well as vitreous cytology smears can be useful in detecting the organism and are an excellent tool to aid in diagnosis of toxoplasmosis. T. gondii cysts were detected in eyes with necrotizing retinitis that developed secondary to imprudent use of corticosteroids (Nijhawan et al., 2013). In recent studies, bradyzoites which have been traditionally considered to be dormant or nonreplicating entities were found to be surprisingly active (Das et al., 2016; Watts et al., 2015).

5.9.2 Ocular biopsies Since most cases of ocular toxoplasmosis can be diagnosed based on clinical features in combination with serologic tests, the need of intraocular biopsies to make the diagnosis is unusual. Analysis of the intraocular fluids samples is a useful tool in the diagnostic of ocular toxoplasmosis in cases of atypical clinical presentation. There are two intraocular compartments that can be sampled, the anterior chamber and the vitreous. The anterior chamber is in the front of the eye and it contains the aqueous humor, a transparent, watery fluid

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similar to plasma, but containing a low protein concentration. The aqueous humor is produced by the ciliary-body epithelium; entering the posterior chamber, it passes through the pupil into the anterior chamber and then peripherally toward the anterior chamber angle. It fills both the anterior and the posterior chambers of the eye. The vitreous humor is a clear, avascular, gelatinous body that comprises two-thirds of the volume and weight of the eye. It fills the space bounded by the lens, retina, and optic disk, also known as the posterior cavity or vitreous chamber (Cunningham et al., 2017). Although the risk of complications with intraocular procedures is low, reported complications are retinal detachment, cataracts (Van der Lelij and Rothova, 1997), corneal infections (Azuara-Blanco and Katz, 1997), endophthalmitis (McLeod et al., 1995), phthisis bulbil, and secondary irreversible blindness (Johnston et al., 2004). An anterior chamber paracentesis or aqueous humor tap is usually performed under local anesthesia, obtaining a volume of 100 200 μL aqueous humor from the anterior chamber with a 27-ga 1.2-in. needle on a tuberculin syringe. Blood samples can be also taken if a Goldmann Witmer coefficient (GWC) is to be calculated from the paired samples of blood and aqueous humor. The procedure takes less than 10 minutes and no additional treatment is required. It is preferred to puncture the anterior chamber, when the pupil is not dilated in order to avoid touching the anterior capsule of the lens with the needle and the formation of subsequent cataract. Aqueous sampling is recommended only by an experienced ophthalmologist, preferably intraocular surgeons or specialists in uveitis, who regularly perform this diagnostic procedure (Van der Lelij and Rothova, 1997). While there is a risk of complications from sampling this fluid, there is less risk than a vitreous biopsy. A vitreous biopsy is performed by sampling the posterior chamber or vitreous cavity. Even though the vitreous is 99% water by weight, it

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is a collagen gel matrix, and sampling the vitreous runs the risk of other complications such as retinal detachment, this is in contrast to an anterior chamber paracentesis which would not be likely to cause a retinal detachment. Based on widely used medicines, for macular degeneration, which requires intraocular injections, a consensus on using topical povidone and a lid speculum are important steps in any intraocular procedure to prevent bacterial endophthalmitis (Aiello et al., 2004). A vitreal biopsy can be performed as an office-based procedure, depending on the local office setting, operating room access, and clinical scenario as to where the vitreal biopsy should be performed. The vitreal biopsy has more risks than an anterior chamber paracentesis because it can cause traction on the retina. Analysis of T cells recovered after vitrectomy in 10 patients with active recurrent T. gondii retinochoroiditis demonstrated the presence of T. gondii specific T-cell clones (Feron et al., 2001) and an absence of T-cell clones against retinal antigens. Three of eight patients were positive for T. gondii by PCR. Using ocular fluids, DNA extraction for genotyping analysis, immunoblotting, and cytokine-chemokine profile measurement was performed to examine strain-specific parasite virulence and intraocular immune responses in French and Columbian patients with ocular toxoplasmosis in order to compare clinical, parasitological, and immunological responses (de-la-Torre et al., 2013).

5.9.3 Serology Fig. 5.13 shows an algorithm for T. gondii serology in patients with suspected ocular toxoplasmosis. Typical clinical findings and serological confirmation are the commonest way to make the diagnosis of ocular toxoplasmosis. Especially in cases of atypical clinical presentations, the diagnosis can be confirmed by detection of either Toxoplasma DNA using

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Patients with suspected uveitis of infectious origin

ELISA assay in serum for anti-Toxoplasma IgG and IgM antibodies

Positive

Negative

AH sample for qPCR negative

AH sample tested for:

for pathogens

VZV, HVS2, CMV, EBV

tested or

or Mycobacterium

insufficient AH

Toxoplasma and other virus and Mycobacterium tuberculosis

tuberculosis

samples

Insufficient AH or no sample of AH because only one eye

Undetermined ocular

Positive for other uveitis

Undertemined other uveitis

PCR or GWC negative for Toxoplasma but

Toxoplasma

positive for other

and/or

virus (VZV, HSV2, EBV

GWC

PCR or GWC negative PCR positive

for toxoplasma and

for

negative for other

Toxoplasma

pathogens (VZV, HSV2,

or CMV) or

PCR positive

Mycobacterium

and other

EBV, or CMV) or

in AH

pathogens or

Mycobacterium

positive qPCR

tuberculosis

tuberculosis

for other pathogens

Confirmed other uveitis Confirmed

Undetermined

case of ocular

ocular

toxoplasmosis

toxoplasmosis

Confirmed other uveitis Coinfection

Other uveitis

FIGURE 5.13 Algorithm for Toxoplasma gondii serology in patients with suspected ocular toxoplasmosis. AH, Aqueous humor; GWC, Goldmann Witmer coefficient test; PCR, polymerase chain reaction; qPCR, quantitative real-time PCR.

PCR or the demonstration of local production of IgG and/or IgA antibodies. Diagnosis enables targeted treatment against T. gondii to be initiated to limit the cytolysis of retinal tissues and development of larger scars, thereby improving visual acuity outcome (Villard et al.,

2016). Serologic testing in these settings is unique since the assays need to detect intraocular T. gondii antibody production in aqueous humor. Serum should always be sampled simultaneously. If serum tests are negative for T. gondii antibodies, all investigations should

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be stopped in immune-competent patients. When Toxoplasma antibodies are detected, a PCR assay and detection of IgG antibodies can be conducted on ocular fluid (Villard et al., 2016). A positive PCR confirms the etiology of the lesion as being due to T. gondii (Bastien, 2002; Bourdin et al., 2014). Genotyping the strain involved in the disease provides important information on the impact of various T. gondii genotypes on retinochoroiditis (Ajzenberg et al., 2009; Fekkar et al., 2011). In the case of a negative PCR, classical criteria should be applied to the serum to determine if an infection is acute or recurrent (Gilbert and Stanford, 2000; Witola et al., 2011). Two methods can be used to determine local Toxoplasma antibody production versus systemic production confirming ocular toxoplasmosis. The first method, the GWC (Desmonts, 1966), is calculated based on the determination of the ratio of T. gondii specific versus total IgG levels in the serum and aqueous humor. Its sensitivity is around 50% (Garweg et al., 2000; RobertGangneux et al., 2004; Talabani et al., 2009). The second method is a similar procedure using ELISA to compare the levels of Toxoplasma-specific antibodies versus mumps virus specific antibodies, instead of measuring total IgG (Turunen et al., 1983; Villard et al., 2003). If the blood retinal barrier is ruptured the GWC (Desmonts, 1966) is unable to distinguish between systemic and local T. gondii -specific antibodies in the aqueous humor. Therefore using immunoblot (Western blot) to determine a T. gondii specific antibody recognition profile is of value in this context. In addition to having been developed for the diagnosis of congenital toxoplasmosis, an immunoblot analysis of serum and paired aqueous humor sampled on the same day is able to determine the recognition profile (Villard et al., 2016), and an observed difference, namely, one or more different bands, signifies the presence of T. gondii specific ocular antibodies, confirming local production and

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ocular toxoplasmosis (Talabani et al., 2009; Villard et al., 2003). Although the sensitivity of immunoblot is similar to GWC, the former achieves higher specificity ( . 95%) and is less influenced by rupture of the blood-retinal barrier (Robert-Gangneux and Darde´, 2012). A combination of GWC, PCR, and immunoblot has been shown to improve the sensitivity of biological diagnosis, which can reach up to 83% (Fekkar et al., 2011; Villard et al., 2003). If both the serology and PCR are negative, other causative infectious and noninfectious diseases should be investigated (Kijlstra et al., 1989; Villard et al., 2016). There is clinically an important, but limited role, for serologic testing in recurrent ocular toxoplasmosis. It is important obtaining a confirmatory IgG ELISA in suspected recurrent disease; however, there is no test available that can confirm that ocular inflammation is in fact from ocular toxoplasmosis. High avidity ( . 40%) antibodies are associated with infections that are over 6 months old (Liesenfeld et al., 2001; Mun˜oz-Zanzi et al., 2010; Paul, 1999). Documented seroconversion is a scenario where serologic testing is useful in suspected cases of acquired ocular disease. Patients concomitantly infected with HIV and T. gondii can have a negative serology (Moshfeghi et al., 2004).

5.9.4 Immunoblotting No universal pattern exists based on immunoblotting to aid the diagnosis of ocular toxoplasmosis; one report highlighted bands below 16 kDa and above 116 kDa (De Marco et al., 2003), a different report highlighted a 28-kDa band (Klaren et al., 1998), and two further papers did not detail findings (Riss et al., 1995; Villard et al., 2003). IgA in the aqueous humor has been demonstrated in from 26% to 63% of patients and IgM has been reported from ,1% to 11% in the aqueous humor (Garweg et al., 2000; Ronday et al., 1999). IgE has been

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reported from 0% to 14% in the vitreous fluid (Go´mez-Marı´n et al., 2000; Liotet et al., 1992). In a study comparing the intraocular immune response between French and Colombian patients, immunoblotting was performed in aqueous humor samples in order to detect intraocular synthesis of Toxoplasma-specific antibodies (LDBIO Diagnosis, Lyon, France). The authors noticed that intraocular antibody responses demonstrated major differences in T. gondii antigen recognition in different populations (de-la-Torre et al., 2013). In a retrospective study in which all aqueous humor samples were analyzed by both GWC and immunoblot, the GWC was significant in 47.6% of 42 patients with suspected ocular toxoplasmosis, and intraocular production of specific antibody anti T. gondii IgG and IgA was revealed by immunoblot in 71.4% of samples. The combination of these two methods increased the sensitivity to 76.2%. Based on the interval between symptom onset and paracentesis, immunoblot had a greater sensitivity than GWC when sample of aqueous humor was taken in the first 3 weeks (64.7% vs 23.5%, P 5 .039), while the difference between the sensitivity of immunoblot and GWC was less significant in cases with an interval .3 weeks (76% vs 64% P 5 .625). Immunoblot seems to be more useful than the GWC if only one of these methods can be performed, especially during the first 3 weeks after symptom onset (Mathis et al., 2018).

5.9.5 Polymerase chain reaction Since PCR testing requires small sample volume, it is well suited for the small samples available from most invasive ocular biopsies (Bre´zin et al., 1991; Burg et al., 1989). Unfortunately, the vitreous being 99% by weight water does not have the dense cellular substrate ideally suited for the presence of T. gondii (Bre´zin et al., 1991). However, large

atypical lesions have more cells and in one series five of seven patients with severe toxoplasmosis, had positive vitreous PCR results (Montoya et al., 1999). Should a sensitive specific test requiring intraocular sampling be developed for the eye, sampling aqueous humor would be safest; however, PCR testing of the aqueous has a low yield in ocular toxoplasmosis (Aouizerate et al., 1993; Bou et al., 1999; Figueroa et al., 2000; Montoya et al., 1999). The yield of positive PCR testing has been reported to range from 17% in patients with retinochoroiditis (Bre´zin et al., 1991) to 100% of patients with large lesions (Fardeau et al., 2002). In elderly patients, larger lesions also had a higher aqueous PCR positive rate compared to smaller lesions (60% vs 25%) (Labalette et al., 2002). In a study comparing the intraocular immune response between Colombian and French patients, aqueous humor samples from Colombian patients revealed the presence of T. gondii DNA in 11 out of 23 samples (47.8%). In French patients, T. gondii DNA could be detected in aqueous humor samples of 7 out of 19 patients (36.8%). This difference was not statistically significant. In contrast, parasite loads in aqueous humor were significantly higher in Colombian patients, 4.53 parasites 6 2 per 100 mL versus 0.35 6 0.13 parasites per 100 mL (P 5 .0006). In PCR positive patients, it is possible to calculate the number of parasites per mL in aqueous humor by quantitative PCR using T. gondii specific primers (de-la-Torre et al., 2013). When a fulminant vitritis transforms the normally transparent vitreous to an opaque structure, it can be impossible to examine the retina. Without the availability of detecting clinical features on fundus examination, more dependence on ancillary tests is necessary. A multiplex-PCR has been established to help differentiate between the more common causes of retinitis (Dabil et al., 2001). The T. gondii based primer is based on the repetitive B1 gene, which has been found to have a

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sensitivity of 60% 70% (Montoya et al., 1999). Fifteen minutes of boiling of the vitreous is required to remove PCR reaction inhibitors. Modifications to this PCR including more recent primers have increased its sensitivity to about one tachyzoite (Jones et al., 2000). The most common use of PCR in ocular disease is to differentiate T. gondii from herpes family viruses (Van Gelder, 2003). Short tandem multiple pathogen PCR analysis has been validated as a possible approach given the inherently limited sample from ocular tissues (Dabil et al., 2001). While PCR can be less sensitive for ocular toxoplasmosis, it appears to be very sensitive for herpetic ocular disease with reported sensitivities as high as 97% (Abe et al., 1998; Ganatra et al., 2000).

5.9.6 Clinical tissue culture systems Tissue culture systems developed for viral culture are widely available in most eye centers and have been used to culture T. gondii (Miller et al., 2000). Because it can take several weeks before a result becomes positive, therapy should likely be initiated empirically while awaiting results.

5.9.7 Ocular imaging Imaging in ocular toxoplasmosis is valuable mainly for the follow-up and to contribute to the accurate description of the different features of the lesions secondary to the complications of the infection and inflammation. Imaging can also be useful in the discussion and recommendations concerning improved diagnosis and therapy of the patients with ocular toxoplasmosis (Lavinsky et al., 2012). 5.9.7.1 Fundus color photographs Fundus color photographs can be useful in the detection of retinochoroiditis lesions when findings such as the presence of hyperpigmented old scars are missed at clinical examination.

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FIGURE 5.14 Retinochoroiditis with vasculitis. Ocular fundus photography of an active retinochoroidal lesion (white arrow) with secondary peripheral vasculitis (black arrows) in a patient with ocular toxoplasmosis. Source: Courtesy Dr. Alberto Castro, Retinologist from Cali, Colombia.

They can be useful to document the location, number of lesions, extent, evolution of infection, and secondary findings (Fig. 5.14) (Lavinsky et al., 2012). 5.9.7.2 Fluorescein angiography and indocyanine green angiography Fluorescein angiography (FA) reveals leakage of retinal vessels such as in vasculitis and any disturbance of the RPE layer (window defect) and neovascularization (Hassenstein and Meyer, 2009). Indocyanine green angiography (ICGA) provides images of stunning crisp clarity of the choroidal circulation, including dynamic images (movie) from the uptake to the late-stage phase. One of the strongest applications of ICGA is to visualize choroidal neovascularization (CNV). ICGA can often reveal greater details of a CNV, which may remain invisible on FA. The specific properties of ICGA with longer wavelength fluorescence and a limited diffusion within the choriocapillaris enhance the visualization of structures beneath blood, exudates or RPE detachments in greater detail (Hassenstein and Meyer, 2009).

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FA and ICG can be used to monitor the evolution of the lesions, although in most of the cases the clinical follow-up is sufficient. FA can be valuable to confirm complications, for instance: papillitis, vasculitis (Fig. 5.15), shunts, vascular occlusions, macular edema, and neovascular membranes. In some cases, they can also be useful in the diagnosis of associated macular edema and papillitis (Foster and Vitale, 2013). Early fluorescence is blocked by the edema, and, after that, the lesion will stain progressively, initiating from the borders. In some occasions, retinitis can be accompanied by a serous retinal detachment of the overlying area; in such cases, the whole area of detached retina stains in late sequences. After the acute stage of toxoplasmosis the edema gradually reverts. Pigmentary changes become evident and are initially best seen with FA. After the inflammation resolves, a retinochoroidal scar is present (Lavinsky et al., 2012). ICG may be useful in evaluating the extent of choroidal

involvement and the evolution of lesions as an adjunctive follow-up parameter in some special situations. 5.9.7.3 Confocal scanning laser ophthalmoscopy Confocal scanning laser ophthalmoscopy (CSLO) can capture images of the retina with a high degree of spatial sensitivity. This imaging system uses an optically pumped solid-state laser to generate excitation at an appropriate wavelength, such as 488 nm for blue reflectancy for fundus autofluorescence (FAF) and FA, 787 nm for ICG angiography, and 830 nm for infrared reflectancy (IR). The confocal principle uses these light sources focused on the target and suppresses different layers using pinhole optical system. The higher emission of light within the safety thresholds of retina enables better acquirement of images with media opacities compared with conventional techniques (Lavinsky et al., 2012). Blue

FIGURE 5.15 Fundus photography and fluorescein angiography of retinochoroiditis with vasculitis. Ocular fundus photography (A) and fluorescein angiography (B) demonstrating an active retinochoroidal lesion (black arrows). Fluorescein angiography (C and D) shows secondary vasculitis (white arrow) and papilitis (gray arrow). Source: Courtesy Dr. Alejandra de-laTorre.

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5.9 Diagnostic tests

reflectancy (red-free mode) records light reflected directly from the retina with no barrier filters. It is useful for determining retina surface pathologies such as retinal folds, cysts, internal limiting membrane irregularities, and epiretinal membranes, which are often related to macular toxoplasmosis lesions (Lavinsky et al., 2012). IR uses invisible wavelength, which increases patient cooperation during examination, as well as enables imaging of retina features that cannot be detected using conventional light sources, specifically lens and vitreous opacities, which frequently occur during the early stages of ocular toxoplasmosis (Lavinsky et al., 2012). IR has been associated with melanin contents of RPE and choroid, which is the primary pigment that produces increased reflectivity with IR mode; therefore it is valuable for studying these structures (Weinberger et al., 2006). In the acute phases, IR frequently shows an augmented reflectivity in the center of the lesion, with decreased signal over the adjacent tissues. Vascular abnormalities such as tortuosity and increased reflectivity over the walls of the vessels are also visible noninvasively by IR reflectivity (Lavinsky et al., 2012).

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5.9.7.4 Fundus autofluorescence FAF can be useful for detecting the RPE defects (Schmitz-Valckenberg et al., 2008). In ocular toxoplasmosis, RPE abnormalities can be noticed as an extensive granulomatous inflammatory infiltration of the RPE and choroid leading to substantial subretinal fibrosis. The FAF signal may be different depending on the phase and level of activity of the infection. In acute stages, it could be hard to recognize the lesion due to severe vitreous opacities, but a patch of reduced FAF signal with the presence of increased perilesional autofluorescence may be observed in the early stages of the disease. Later, retinal scarring and atrophy lead to a mostly low FAF signal combined with retinal fibrosis in advanced stages. Satellite lesions could be identified by FAF as a smaller increased or decreased FAF patchy lesion depending on the time point of onset (Fig. 5.16) (Lavinsky et al., 2012). 5.9.7.5 Optical coherent tomography Optical coherent tomography (OCT) is a noninvasive imaging technique that allows investigation of retinal morphology and pathologies.

FIGURE 5.16 Fundus autofluorescence and ocular toxoplasmosis. (A) Fundus autofluorescence demonstrates macular hyperautofluorescent lesions (white arrows) with increased intralesional autofluorescence, as well as areas of decreased fundus autofluorescence, which may correspond to a previous scar (black arrow). (B) Fundus autofluorescence shows a hypoautofluorescent lesion (white arrow) in the macula corresponding to retinal scarring and atrophy secondary to ocular toxoplasmosis. Source: Courtesy Fundacio´n Oftalmolo´gica Nacional, Bogota´-Colombia.

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FIGURE 5.17 Infrared imaging and optical coherent tomography. (A) Infrared image of an ocular toxoplasmosis lesion inferior temporal to the optic disk (white arrow). (B) OCT of acute ocular toxoplasmosis demonstrates: (1) vitritis (vitreal cells) (white circle), (2) posterior vitreous detachment, (3) Disruption of the retinal layers, and (4) subretinal fluid and serous retinal detachment (black arrows). OCT, Optical coherent tomography. Source: Courtesy Dr. Alejandra de-la-Torre.

OCT has become one of the most valuable imaging techniques, both in uveitis and in OT. This technique is helpful in the study of the vitreoretinal interface, retinal, and choroid (Fig. 5.17). It is also crucial to diagnose and differentiate between epiretinal membranes, vitreoretinal tractions, and new vessels membranes. In addition, it permits to measure lesions such as the retinal thickness at the macula and monitor changes over time along with the response to therapies. Moreover, OCT can be of help in the differentiation between small old scars and new foci of inflammation (Diniz et al., 2011). The OCT technique has advanced in the last years, but it is still far from achieving the required resolution to identify retinal T. gondii cysts. OCT has the advantage of not being invasive and can be repeated often without side effects (Lavinsky et al., 2012). Spectral domain optical coherence tomography (SD-OCT) has higher axial resolution and faster acquisition speed compared with time domain (TD-OCT), which allows SD-OCT to provide not only detailed views of intraretinal microstructure, but also three-dimensional images of the macula and retinochoroiditis lesions. These enhanced views should offer physicians a better view than previous TD-OCT devices (Diniz et al., 2011).

5.9.7.6 Ultrasonography Ultrasonography (US) allows the diagnosis of inflammatory activity and complications in some situations, such as in patients with opaque media. US is an important diagnostic tool when lens or vitreous opacities do not allow proper visualization of the ocular fundus. It is very helpful to diagnose vitreous inflammation/hemorrhages, posterior vitreous detachment, vitreoretinal tractions, retinal holes, and detachments with or without tractions in addition to choroidal thickening, choroidal detachments, macular edema, changes at the optic disk (edema and glaucoma), and it can also suggest areas of retinal thickness (Fig. 5.18). In addition, this technique is useful in the differential diagnosis of ocular toxoplasmosis to identify masquerade syndromes such as retinoblastoma, melanoma, and other intraocular syndromes presenting as inflammatory diseases. US biomicroscopy can be useful to identify complications in the anterior segment of the eye, such as ciliary body detachment, intraocular lens placement, or angle-closure glaucoma. B-scan US is used for patients where the fundus examination is difficult or impossible due to the presence of posterior synechiae, cataract, or corneal opacification (Hercos et al., 2004).

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FIGURE 5.18

Ocular ultrasonography: (A) choroidal thickening (white arrow), (B) vitreous inflammation (white arrow), (C) posterior vitreous detachment (white arrow), and (D) retinal holes detachment without traction (white arrow). Source: Courtesy Dr. Alejandra de-la-Torre.

5.10 Differential diagnosis The diagnosis of ocular toxoplasmosis is based mostly on clinical and serological findings and the exclusion of other diseases in the differential diagnoses. Since seropositivity for T. gondii is very frequent worldwide (Holland, 2003), the presence of specific IgG antibodies is useful only to confirm previous exposure to the parasite; therefore it only supports the diagnosis but does not establish the diagnosis by itself (VasconcelosSantos et al., 2011). The variety of clinical presentations of ocular toxoplasmosis and the potential overlap with features of other infectious, noninfectious, and neoplastic entities have to be considered for an appropriate differential diagnosis (Vasconcelos-Santos et al., 2011). Congenital toxoplasmosis must be differentiated from other possible causes with similar signs and symptoms, which are represented in the classic clinical acronym “TORCH”. The acronym includes Toxoplasma, rubella, cytomegalovirus, syphilis, and herpes simplex

virus. However, emerging pathogens such as West Nile Virus must also now be considered as part of any differential in known congenital infection (Alpert et al., 2003). Recurrent toxoplasmosis with its unilateral active lesion associated with multiple adjacent chorioretinal scars with the appropriate clinical history is virtually pathognomonic. However, clinical syndromes such as serpiginous retinochoroiditis and other infectious etiologies such as cytomegalovirus may occasionally be considered. For the many other possible and unusual manifestations of ocular toxoplasmosis such as pars planitis the differential diagnosis is even broader and includes autoimmune disorders such as multiple sclerosis and infections such as Lyme disease. Importantly, there are likely many cases of unusual manifestations of ocular toxoplasmosis that remain undiagnosed because of the limits of our noninvasive assays. Analyses of intraocular fluids, including PCR and evaluation of intraocular antibody synthesis through the GWC, may be helpful in ambiguous cases

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in order to establish the definite diagnosis (Garweg et al., 2000; Harper et al., 2009). A close follow-up is essential, particularly of individuals on systemic corticosteroids, when the possibility of another infectious etiology cannot be ruled out (Vasconcelos-Santos et al., 2011). New imaging modalities, such as OCT, can also be helpful to more accurately demarcate the vitreoretinal changes (Ore´fice et al., 2006). When required, empiric treatment should be started with caution, and patients under systemic steroids should be closely observed, especially if other infectious etiologies are of concern. Table 5.8 summarizes the most significant differential diagnoses of T. gondii retinochoroiditis. Particularly in immunosuppressed individuals, especially those with AIDS, T. gondii is an important opportunistic agent leading to posterior uveitis. T. gondii retinochoroiditis in these patients may present with atypical features, such as large, multiple, and even bilateral lesions, which may be produced by several other microorganisms, including viruses, fungi, bacteria, and parasites (Vasconcelos-Santos et al., 2011).

5.11 The treatment and management of ocular toxoplasmosis Most experts consider the combination of pyrimethamine and sulfadiazine to be the gold standard for the treatment of ocular toxoplasmosis. However, a survey of the members of the AUS highlights the lack of uniformity of ophthalmologists regarding therapy. The most common regimen used in the 1991 published survey was pyrimethamine, sulfadiazine, prednisone, and folinic acid in 32% of respondents and an additional 27% added clindamycin to the most common regimen (Engstrom et al., 1991). Other agents used include quinolones, and macrolides. Adjunctive therapies such as laser treatment or cryotherapy (Jacklin, 1975)

within and adjacent to chorioretinal scars are rarely used. A very controversial review of the literature in 2003 highlighted that only three designed prospective randomized placebocontrolled studies existed at the time of the review (Stanford et al., 2003) and much of the literature was deemed inappropriate for their analysis because of a lack of placebo. The conclusion of this metaanalysis went against what most would consider standard of care. The recent data of difference between strains pathogenicity between strains form South America and strains from other parts of the world illuminates why these European and North American trial were equivocal about the benefit of therapy (Go´mez-Marı´n et al., 2018). Nowadays, most clinical scientists would not have sufficient ethical equipoise to design a placebo trial for the management of ocular toxoplasmosis, but some clinicians do not administer specific drug therapy when a peripheral Toxoplasma retinochoroiditis recurrence occurs in an immunocompetent person; however, we believe that, at least in patients from South America, antiparasitic treatment should always be administered to these patients (Go´mez-Marı´n et al., 2018). A recent survey highlights the uncertainty around the treatment and understanding of toxoplasmosis (Lum et al., 2005). This survey of 1000 ophthalmologists in the United States, completed in 2000, has a 48% response rate. During 1999 and 2000 there were an estimated 253,000 visits to ophthalmologists in the United States for ocular toxoplasmosis, 24,000 of which were for active disease. There was surprising lack of understanding among surveyed respondents regarding the importance of acquired disease (50%), the elderly as a highrisk group (16%), and the unlikelihood of transmission to fetus from recurrence of ocular toxoplasmosis during pregnancy (30%). Only 19% of respondents compared to 15% of uveitis subspecialists treated all patients with ocular toxoplasmosis (Holland and Lewis, 2002).

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TABLE 5.8 Differential diagnosis of Toxoplasma gondii retinochoroiditis*. Early presentation (congenital toxoplasmosis)

Late presentation (congenital/postnatally acquired toxoplasmosis)

Infectious

Infectious

Rubella

Bacterial

CMV

Syphilis

Herpes

Tuberculosis

Syphilis

Bartonellosis (neuroretinitis, foci of retinitis, and angiomatous lesions)

West Nile virus fever

Lyme disease

Acute lymphocytic choriomeningitis

Endogenous endophthalmitis Others Viral Acute retinal necrosis/necrotizing herpetic retinopathy CMV retinitis Progressive outer retinal necrosis Others Fungal Candidiasis (especially endogenous endophthalmitis) Aspergillosis coccidioidomycosis Histoplasmosis Sporotrichosis Paracoccidioidomycosis Others Parasitic DUSN Toxocariasis Cysticercosis Schistosomiasis Onchocerciasis Others

Noninfectious

Noninfectious

Retinochoroidal colobomata

Behc¸et disease

Persistent hyperplastic vitreous

Sarcoidosis (Continued)

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TABLE 5.8 (Continued) Early presentation (congenital toxoplasmosis)

Late presentation (congenital/postnatally acquired toxoplasmosis) Serpiginous choroiditis, ampiginous choroiditis, and others Multifocal choroiditis and panuveitis Punctate inner choroidopathy Multiple evanescent white dots syndrome Unilateral acute idiopathic maculopathy Others

Neoplastic Retinoblastoma/retinocytoma

Neoplastic Primary vitreoretinal lymphoma Others

*Modified from Vasconcelos-Santos et al. (2011).

Surprisingly, in a zone of recurrence called the papillomacular bundle which is a vital area for vision, only 51% of respondents indicated they would offer treatment. In our opinion, this area of recurrence should always warrant treatment, due to the high risk of visual loss. There are many different regimens that are used in the treatment of ocular toxoplasmosis. A 2001 survey of uveitis subspecialists reported that 9 different commercially available drugs were used in 24 different possible combinations as the treatment of choice for the treatment of typical ocular toxoplasmosis by different uveitis subspecialists (Holland and Lewis, 2002). For the 80 responding specialists, this included (in descending order of frequency): clindamycin [74 (94%)], pyrimethamine [71 (90%)], sulfadiazine [64 (81%)], trimethoprim/sulfamethoxazole [64 (81%)], sulfadiazine/sulfamerazine/sulfamethazine [“triple sulfa,” 37 (47%)], doxycycline [27 (34%)], atovaquone [26 (33%)], tetracycline [25 (32%)], minocycline [20 (25%)], azithromycin [15 (19%)], sulfasoxazole [14 (18%)], pyrimethamine/sulfadoxine, clarithromycin [6 (8%)], spiramycin [6 (8%)], trimethoprim [6 (8%)], dapsone [5 (6%)], and trimetrexate [1 (1%)]. Comparing results between the 1991 and 2001

survey of uveitis specialists indicates a trend toward more aggressive treatment of uveitis among respondents. There was decreased use of clindamycin between the two surveys. The initial enthusiasm for clindamycin was the finding that clindamycin appeared both to achieve good intraocular concentrations and enter into cysts well (Tabbara and O’connor, 1975); however, the decrease in use of clindamycin is presumed to be the lack of evidence of improved outcome and the fear of side effects with its use such as pseudomembranous enterocolitis. The most commonly used treatment regimen was a combination of sulfadiazine, pyrimethamine, corticosteroids and folinic acid (Holland and Lewis, 2002). This regimen has demonstrated in vitro synergy for its activity against T. gondii. The plasma half-life of pyrimethamine in adults is 100 hours and in children is about 60 hours (McLeod et al., 1992). In a recent study from France where serologic testing for T. gondii is a routine part of prenatal care, 18 of 24 consecutive congenitally infected patients were examined for ocular outcome with treatment (Bre´zin et al., 2003). An oral regimen was used to treat mothers prenatally. Pyrimethamine (50 mg/day) was

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alternated throughout gestation with 4 weeks of concomitant sulfadiazine (3 g/day) and folinic acid followed by a 2-week cycle of spiramycin (9 million IU/day). Postnatal treatment was continued for 1 year with a regimen of 1 mg/ kg/day of pyrimethamine, 50 mg/kg/day of sulfadiazine, and 50 mg/week of folinic acid. The ocular outcome was 61% had no lesions, peripheral lesions were seen in nine eyes of five children (four eyes also had posterior pole lesions), posterior pole lesions were detected in six eyes of five children (all of which had good visual acuity). Only one patient had a severe visual impairment which was associated with sensory deprivation nystagmus. In a different study where 15 of 39 cases of congenital Toxoplasma infection did not result in termination of gestation the treatment regimen was 3 g of spiramycin per day when infection was suspected and pyrimethamine plus sulfonamides were added when diagnosis in the fetus was confirmed with a shorter median follow-up of 12 months only two patients had eye lesions (Daffos et al., 1988). For infants, the pyrimethamine dose is usually 1 mg/kg/day and for sulfadiazine 100 mg/kg/day in two equal doses. Folinic acid is given 10 mg day with apple juice. This infant regimen is derived from the Chicago Collaborative Treatment Trial from which there is a helpful dispensing aide based on weekly weight assessment (McAuley et al., 1994) In addition, treatment is not dictated by presence or absence of eye involvement alone in congenital toxoplasmosis, as extended treatment appears to be indicated to provide optimal outcome for the multiple systemic complications (Mets et al., 1997; Remington et al., 2004). The regimen can result in prompt resolution of active ocular toxoplasmosis in newborns (Mets et al., 1997). The most common side effect from the use of pyrimethamine is bone marrow toxicity. Folinic acid is commonly used to help ward off the toxicity associated with pyrimethamine

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therapy (Holland and Lewis, 2002). Folinic acid can be used at doses of 15 mg (2 7 times per week). Folinic acid does not cross the cellular membrane of T. gondii and therefore has no impact on pyrimethamine efficacy (Allegra et al., 1987). Sulfadiazine can cause a crystalluria which usually promptly responds to alkalinazation of the urine, and there is one report in the ophthalmic literature of acute ureteric obstruction soon after initiation of therapy for ocular toxoplasmosis (Smith et al., 2001). Sulfonamides can cause Stevens Johnson severe skin and mucosal necrolytic reactions, which appear 1 or 2 weeks after the beginning of therapy (Peters et al., 2007). An ideal objective in patients with ocular toxoplasmosis is to eradicate the cysts in retina that are responsible of recurrences; however, there is no current therapy that eradicates tissue cysts (Foster and Vitale, 2013). Most studies indicate that standard durations of current therapies do not reduce the incidence and recurrence of ocular toxoplasmosis. Thus there is still a need of a drug capable to eliminate the cysts of parasite (Foster and Vitale, 2013; Pradhan et al., 2016; Stanford and Gilbert, 2009). There are some reports that longer durations of therapy may decrease the rate of recurrence of patients after their acute episodes of ocular toxoplasmosis (Rothova et al., 1993), possibly due to a decrease in tachyzoite infection of new retinal cell (de-la-Torre et al., 2011a,b).

5.11.1 Drug treatment of ocular toxoplasmosis Treatment should be started and close monitoring continued for an active lesion that affects or is located in the vicinity of optic nerve between two diameters of disk, for an active lesion in the temporary arcades, for an active lesion affecting large retinal vessels or which has resulted in bleeding, for an active

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lesion with severe inflammation and an associated severe vitreous haze, for extensive or multiple active lesions with visual acuity impairment related to loss of at least two lines of vision, for intraocular inflammation, for congenital toxoplasmosis in the first year of life and for any injury in immunocompromised patient (Foster et al., 2012; Foster and Vitale, 2013). Similarly, in the case of the presence of a lesion or several lesions with the presence of persistent inflammation for more than 1 month, it is recommended to treat until resolution of the inflammatory process, due to the possible association of decreased visual acuity, the potential for macular edema and macular traction, and the possibility that free tachyzoites coming from the active lesions will infect other parts of the retina causing further damage (Ore´fice, 2005; Soheilian et al., 2005). Therapy should take into account the origin of the patient to be treated, since virulent T. gondii strains from South America have been associated with increased risk of developing of ocular lesions in children with congenital toxoplasmosis up to five times above the European population (Gilbert et al., 2008). Inactive lesions should not be treated. In active retinochoroiditis treatment is indicated to reduce the damage of retina and optic nerve (Holland and Lewis, 2002). Regular management is started with inhibitors of dihydrofolate, sulfa drugs and steroids (de-la-Torre et al., 2011a,b), regularly for a period of between 4 and 8 weeks depending on the severity of the infection (de-la-Torre et al., 2011a,b; Foster and Vitale, 2013). The most common therapeutic scheme is pyrimethamine/sulfadiazine, at an initial dose of pyrimethamine of 75 100 mg per day for 2 days, followed by 25 50 mg a day, with sulfadiazine 1 g every 6 hours, and 5 10 mg of folinic acid daily for a total of 4 8 weeks. Oral prednisolone is often given from the third day of treatment at a dose of 1 mg/kg/day with a duration of 2 6 weeks. Improvement of disease

can be seen within 4 6 weeks (de-la-Torre et al., 2011a,b). The combination of pyrimethamine and sulfadiazine can have hematologic toxicity (leucopenia and thrombocytopenia) and the folinic acid helps mitigate this effect. Allergic reactions can occur to sulfadiazine that can range from mild to severe and may even compromise the life of the patient as in the case of Steven Johnson Syndrome (de-la-Torre et al., 2011a,b). An alternative therapy that is often used is trimethoprim/sulfamethoxazole at a dose of 80 mg/400 mg every 12 hours, associated with 1 mg/kg day of prednisolone started 3 days after the onset of the antibiotic. This treatment has had similar efficacy to pyrimethamine/sulfadiazine in some randomized trials (de-laTorre et al., 2011a,b; Soheilian et al., 2011). An additional therapy that is used is clindamycin 300 mg every 6 hours along with pyrimethamine/sulfadiazine. Other therapies with reported efficacy alone and in combinations include clindamycin, atovaquone, azithromycin, and clarithromycin (de-la-Torre et al., 2011a,b). However, these medications tend to be difficult to obtain in some countries and most of them are not available as pediatric formulations. Cryotherapy and laser therapy have also been reported as an adjunct treatments (Holland and Lewis, 2002). A special condition to be considered is the treatment of a pregnant woman with high levels of antibodies against T. gondii and active chorioretinal lesions, due to the effect of medications on the fetus. It is unusual for recurrent active lesions in the mother to cause congenital infection. If there is a risk of visual impairment in the mother as a result of active ocular toxoplasmosis (Go´mez, 2007; Soheilian et al., 2011) in the first 18 weeks of pregnancy, spiramycin should be used, instead of pyrimethamine/sulfadiazine (Go´mez, 2007; Zuluaga et al., 2017). Spiramycin has been shown to prevent the development of chorioretinal lesions in the fetus, as revealed in a study of 23 women with

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acute toxoplasmosis in which 1/15 children born to mothers treated with spiramycin and 8/8 children to untreated mothers developed ocular toxoplasmosis (Zuluaga et al., 2017). An alternative regimen proposed for active retinochoroiditis in pregnancy is a combination of intravitreal clindamycin with dexamethasone (Soheilian et al., 2011).

5.11.2 Corticosteroids Intravitreal, topical, oral, and periocular corticosteroids are often used as part of the regimen to treat ocular toxoplasmosis. In surveys of experienced ophthalmologists, topical corticosteroids were used by 80% of respondents presumably to prevent presumed complications of anterior segment inflammation such as posterior synechiae (scarring of the iris to the underlying lens). Only 17% of respondents used corticosteroids in all patients regardless of severity of inflammation. 71% of uveitis specialist respondents would use therapy in severe vitreous inflammation. Highlighting how ocular toxoplasmosis is secondary to actively replicating parasites are several reports about poor outcome with patients treated with corticosteroids alone without a concomitant antiparasitic regimen (Nozik, 1977; O’Connor and Frenkel, 1976; Sabates et al., 1981). In early studies, steroids alone were used to treat ocular toxoplasmosis, as this disease was thought to be a hypersensitivity reaction (Gordon, 1970). It later became clear that steroids alone, without concomitant antimicrobials, can have negative effects on vision, including development of endophthalmitis (Bosch-Driessen and Rothova, 1998; Nozik, 1977). Monotherapy with systemic steroids is also associated with a higher risk of recurrence (de la Torre et al., 2009a,b; Reich et al., 2015a). Careful use of steroids together with antimicrobial therapy for severe inflammation or when the lesions are near the fovea or optic disk may

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suppress the inflammation and can be beneficial, but excessive doses can yield a suboptimal response. While the optimal dose is not established, most experts use 0.5 1 mg/kg/day. Steroids are generally started a few days after initiation of antimicrobial therapy and continued for about 1 month, with gradual tapering (Holland and Lewis, 2002; Winterhalter et al., 2010). It should be acknowledged that the evidence for adjuvant steroids is not based on high-quality data, (Jasper et al., 2017) and steroids must not be used without concomitant antimicrobial therapy, as this can lead to a complete loss of vision (Dunay et al., 2018).

5.11.3 Laser treatment In the past, laser treatment was given to the retina surrounding T. gondii scars (Spalter, 1966). The theory was that laser photocoagulation can destroy cysts and tachyzoites and thus inhibit the spread of infection; thus the destruction of retina by local laser treatment would dramatically decrease if not eliminate the risk of recurrence. Since T. gondii bradyzoites have been demonstrated in normal-appearing retina, the practice of laser treatment of retina as a means of prophylaxis against recurrence is rarely if ever employed today. This procedure has proven to have limited effectiveness in OT. Its use is considered especially in recurrences in pregnant women, intolerance to drugs and the formation of neovascular membranes in the retina. Among the complications of this procedure we can find vitreous hemorrhage, epiretinal membrane development and choroidal neovascular membrane (Foster and Vitale, 2013; Ghartey and Brockhurst, 1980).

5.11.4 Subconjunctival therapy Clindamycin has been administered subconjuctivally with an every other day injection of 50 mg for 15 injections over 30 days

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(Ferguson, 1981). The benefit of subconjunctival injection as a route of local medicine administration is that if a medicine is able to penetrate the sclera and get sufficient intraocular concentrations, the complications associated with subconjunctival injection are much less than those associated with intravitreal injection. However, large amounts of subconjunctival clindamycin (150 mg) have resulted in corneal edema (Tabbara and O’connor, 1975). This therapy is not widely used at present, due to the lack of evidence of its effectiveness and its possible secondary side effects.

5.11.5 Surgical therapy PPV is useful for removing vitreous opacities, as well as to decrease the risk of vitreoretinal traction and retinal detachment. This technique can be useful for the elimination of proteins and inflammatory cells (Ada´n et al., 2009). In addition to antiparasitic therapy the complications of retinochoroiditis may require different types of clinical management and therapy. Some of these complications may require surgical interventions, such as retinal detachment, epiretinal membranes, and neovascularization (Delair et al., 2011).

5.11.6 Intravitreal therapy Each intravitreal injection has the risk of irreversible blindness as well as other potential complications. However, intravitreal therapy is the mode of administration of medicines for a variety of eye diseases. The benefit of intravitreal therapy is that it has excellent bioavailability and has almost no risk of systemic side effects. Clindamycin and dexamethasone applied directly within the eyeball have been demonstrated to have high cellular penetration. Intravitreal clindamycin/dexamethasone, clindamycin/triamcinolone acetonide with

systemic anti Toxoplasma therapy (Aggio et al., 2006), and liposomal-encapsulated clindamycin (Peyman et al., 1988) have all been used in ocular toxoplasmosis. Intravitreal administration of clindamycin was used as an adjunct therapy in a retrospective case series of six patients, who were either intolerant or unresponsive to systemic therapy (Sobrin et al., 2007). All six patients, with or without concomitant PPV, demonstrated resolution of active toxoplasmic retinochoroiditis. Another small case series recruited 12 patients with vision-threatening disease, with active retinochoroidal lesions located within 3000 μm from fovea, or 1500 μm from the optic disk (damage within the central retina often leads to permanent visual impairment or distortion). Three of the patients had a contraindication to systemic anti Toxoplasma therapy secondary to pregnancy, and the remaining patients either showed lack of response after at least 30 days of systemic therapy or intolerance to the standard therapy (Lasave et al., 2010). These patients received intravitreal injections of a combination of 1.5 mg of clindamycin and 400 μg of dexamethasone. Five patients also continued with concurrent systemic therapy to minimize damage to the fovea or optic nerve. All 12 patients showed resolution of active retinochoroiditis with a mean of 3.6 intravitreal injections, with a range of two to five injections. Visual acuity either improved or stabilized in almost all of the patients except one with lesion at the fovea. In a more recent randomized single-masked clinical trial that included 68 patients with active ocular toxoplasmosis, the efficacy of intravitreal clindamycin/dexamethasone was studied against a regimen of the more classic systemic regimen consisting of pyrimethamine, sulfadiazine, and prednisolone (Soheilian et al., 2011). Patients that were randomized to receive intravitreal 1 mg of clindamycin and 400 μg of dexamethasone injections had comparable reduction of active retinal lesions, visual acuity

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improvement, and resolution of vitreous inflammation. The mean number of injections required for resolution of active retinal lesions was 1.6 with the range of one to three injections. During the 2-year follow-up period, both intravitreal and systemic groups had the same disease recurrence rate (5.9%). The limited number of studies thus far suggests that intravitreal injection of clindamycin/dexamethasone may be as effective as systemic therapy for recurrent ocular toxoplasmosis, with much less adverse side-effects, and less demand for patient compliance. An international survey of leading uveitic experts in 2011 reports that only 9 out of 32 respondents had experience with intravitreal clindamycin (Wakefield et al., 2011). A larger trial with a longer follow-up period will need to be conducted to better know how intravitreal therapy fits into standard therapy. In conclusion, local intravitreal therapy is an alternative treatment for ocular toxoplasmosis that is effective as second-line therapy and has the advantage of lower toxicity and adverse effects. In patients with recent T. gondii with elevated IgM, systemic instead of intravitreal therapy is recommended (de-laTorre et al., 2011a,b; Soheilian et al., 2011). Intravitreal treatments are also useful for the treatment of some complications of ocular toxoplasmosis such as choroidal neovascular membrane (CNVM). CNVM is a rare but potentially vision threatening complication of ocular toxoplasmosis. Off-label use of intravitreal ranibizumab [an anti VEGF (vascular endothelial growth factor) agent] along with antiparasitic therapy demonstrated satisfactory results in treating patients with CNVM secondary to ocular toxoplasmosis (Benevento et al., 2008; Yahia et al., 2008). Treating toxoplasmosis associated CNVM borrows from the success of treating other retinal diseases with anti VEGF medication (Rosenfeld et al., 2006). One common factor that has been found to play a major role in the pathophysiology in the diseases that are associated with retinochoroidal ischemia is

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elevated intraocular VEGF (Adamis and Shima, 2005). Elevated intraocular VEGF can lead to increased vascular permeability and growth of abnormal blood vessels, which disrupt regular retinal anatomy and decrease vision. In an on-going large multicenter randomized trial, intravitreal ranibizumab or bevacizumab injections preserved vision in patients with neovascular age-related macular degeneration (CATT Group 2012). In 1185 patients randomized to receive either ranibizumab or bevacizumab intravitreal injections regularly or as needed based on clinical exam every 4 weeks most patients showed improvement of choroidal neovascularization and decrease in subretinal fluids. 60% of the patients achieved 20/40 or better visual acuity at 2 years after initiation of treatments. If left untreated under 10% of these patients with macular degeneration would have been able to maintain the same level of visual acuity.

5.11.7 Prophylactic therapy Prophylactic therapy is administered in order to reduce or prevent relapses in patients with ocular toxoplasmosis. Several clinical trials support the benefit of long-term secondary prophylaxis to prevent ocular toxoplasmosis recurrences. An important study described the follow-up of 95 patients in Campinas (Brazil) randomized to trimethoprim/sulfamethoxazole tablet every 2 days or identical placebo tablet every 2 days. The incidence of recurrent toxoplasmosis retinochoroiditis within 12 months was 0 of 46 (0%) in the trimethoprim-sulfamethoxazole and 6 of 47 (12.8%) in the placebo groups, no treatment limiting toxicity was observed (Felix et al., 2014). In another study, the outcome of secondary prophylaxis was described for 124 patients with a history of recurrent T. gondii retinochoroiditis who were randomized to treatment with one tablet of trimethoprim (160 mg)/

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sulfamethoxazole (800 mg) every 3 days (61 patients) or to observation without treatment (63 patients) and followed monthly for up to 20 consecutive months for clinical signs of disease recurrence. Recurrences were observed in 4 (6.6%) treated patients and in 15 (23.8%) controls (Silveira et al., 2002). In a survey, 15 out of 32 uveitis specialists indicate that they would initiate prophylactic treatment in patients with high number of recurrences (Wakefield et al., 2011). Other indications suggested by survey respondents include immunocompromised patients (8 out of 32), vision threatening eye lesions (11 out of 32), monocular patients (1 out of 32), and prior to cataract or vitrectomy surgery (3 out of 32). Hematological, gastrointestinal, and dermatological side effects need to be monitored when using trimethoprim/sulfamethoxazole. The time during which it is most appropriate to give prophylaxis appears to be the first year and possibly the first 2 years after suffering a recurrence (Kopec et al., 2003; Silveira et al., 2002, 2015b). The administration of prophylactic antibiotic therapy should be discussed with the patient in the following cases: (1) during the first year after active ocular disease, especially in the case of a primary lesion; (2) in elderly patients, especially if they have a primary infection (Reich et al., 2015a); and (3) in the setting of extensive macular damage in children with congenital toxoplasmosis to preserve its remnants of visual function.

5.12 Conclusion The devastation of ocular toxoplasmosis even though widespread in our societies remains without appropriate attention. Perhaps this is because ocular toxoplasmosis is a disease that crosses several disciplines: epidemiology, infectious disease, ophthalmology, pediatrics, internal medicine, pathology, and parasitology. Also, ocular toxoplasmosis is a

disease whose active component will resolve; however, visual morbidity remains and is often permanent after inflammation resolves. With the identification of unique intracellular targets (Roberts et al., 1998) to effectively kill T. gondii with little likelihood of any impact on our own cellular machinery, the future of care of patients with ocular toxoplasmosis will likely change dramatically. Indeed, it is possible in the future that regimens may not only treat active disease but could also by effectively killing all stages of the parasite eliminate the frustrating possibility of recurrence as well.

Acknowledgments We would like to thank Juliana Mun˜oz Ortiz for her manuscript and references editing work and Dr. Louis Weiss, MD for his support, enthusiasm, and editing work.

References Abe, T., Sato, M., Tamai, M., 1998. Correlation of varicellazoster virus copies and final visual acuities of acute retinal necrosis syndrome. Graefes Arch. Clin. Exp. Ophthalmol. 236, 747 752. Abraham, S.V., 1929. Retinochoroiditis Juxtapapillaris— Jensen. Arch. Ophthalmol. 2, 452 455. Adamis, A.P., Shima, D.T., 2005. The role of vascular endothelial growth factor in ocular health and disease. Retina 25, 111 118. Ada´n, A., Giralt, J., Alvarez, G., Alforja, S., Bure´s-Jesltrup, A., Casaroli-Marano, R.P., et al., 2009. Pars plana vitrectomy for vitreoretinal complications of ocular toxoplasmosis. Eur. J. Ophthalmol. 19, 1039 1043. Afonso, E., Thulliez, P., Gilot-Fromont, E., 2010. Local meteorological conditions, dynamics of seroconversion to Toxoplasma gondii in cats (Felis catus) and oocyst burden in a rural environment. Epidemiol. Infect. 138, 1105 1113. Available from: https://doi.org/10.1017/ S0950268809991270. Aggio, F.B., Muccioli, C., Belfort Jr, R., 2006. Intravitreal triamcinolone acetonide as an adjunct in the treatment of severe ocular toxoplasmosis. Eye 20, 1080. Aiello, L.P., Brucker, A.J., Chang, S., Cunningham Jr., E.T., D’amico, D.J., Flynn Jr., H.W., et al., 2004. Evolving guidelines for intravitreous injections. Retina 24, S3 S19. Ajzenberg, D., Yera, H., Marty, P., Paris, L., Dalle, F., Menotti, J., et al., 2009. Genotype of 88 Toxoplasma gondii

Toxoplasma Gondii

References

isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings. J. Infect. Dis. 199, 1155 1167. Ajzenberg, D., Collinet, F., Mercier, A., Vignoles, P., Darde´, M.-L., 2010. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. J. Clin. Microbiol. 48, 4641 4645. Available from: https://doi.org/10.1128/JCM.01152-10. Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499 511. Available from: https://doi.org/10.1038/nri1391. Akstein, R.B., Wilson, L.A., Teutsch, S.M., 1982. Acquired toxoplasmosis. Ophthalmology 89, 1299 1302. Alford Jr, C.A., Stagno, S., Reynolds, D.W., 1974. Congenital toxoplasmosis: clinical, laboratory, and therapeutic considerations, with special reference to subclinical disease. Bull. N.Y. Acad. Med. 50, 160. Allegra, C.J., Kovacs, J.A., Drake, J.C., Swan, J.C., Chabner, B.A., Masur, H., 1987. Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate. J. Clin. Invest. 79, 478 482. Alpert, S.G., Fergerson, J., Noe¨l, L.-P., 2003. Intrauterine West Nile virus: ocular and systemic findings. Am. J. Ophthalmol. 136, 733 735. Alvarez, C., de-la-Torre, a, Vargas, M., Herrera, C., UribeHuertas, L.D., Lora, F., et al., 2015a. Striking divergence in Toxoplasma ROP16 nucleotide sequences from human and meat samples. J. Infect. Dis. 211, 2006 2013. Available from: https://doi.org/10.1093/infdis/jiu833. Alvarez, C., de-la-Torre, a, Vargas, M., Herrera, C., UribeHuertas, L.D., Lora, F., et al., 2015b. Striking divergence in Toxoplasma ROP16 nucleotide sequences from human and meat samples. J. Infect. Dis. 211, 1 8. Available from: https://doi.org/10.1093/infdis/jiu833. Andrade, G.M.Q., Vasconcelos-Santos, D.V., Carellos, E.V. M., Romanelli, R., Vitor, R.W.A., Carneiro, A.C.A.V., et al., 2010. Congenital toxoplasmosis from a chronically infected woman with reactivation of retinochoroiditis during pregnancy an underestimated event? J. Pediatr. (Rio. J). 86, 85 88. Aouizerate, F., Cazenave, J., Poirier, L., Verin, P.H., Cheyrou, A., Begueret, J., et al., 1993. Detection of Toxoplasma gondii in aqueous humour by the polymerase chain reaction. Br. J. Ophthalmol. 77, 107 109. Arantes, T.E.F., Silveira, C., Holland, G.N., Muccioli, C., Yu, F., Jones, J.L., et al., 2015. Ocular involvement following postnatally acquired Toxoplasma gondii infection in southern Brazil: a 28-year experience. Am. J. Ophthalmol. 159, 1002 1012. Arevalo, J.F., Belfort, R., Muccioli, C., Espinoza, J.V., 2010. Ocular toxoplasmosis in the developing world. Int. Ophthalmol. Clin. 50, 57 69. Available from: https:// doi.org/10.1097/IIO.0b013e3181d26bf4.

277

Ayo, C.M., Camargo, A.V., da, S., Frederico, F.B., Siqueira, R.C., Previato, M., et al., 2015. MHC class I chainrelated gene a polymorphisms and linkage disequilibrium with HLA-B and HLA-C alleles in ocular toxoplasmosis. PLoS One 10, e0144534. Available from: https:// doi.org/10.1371/journal.pone.0144534. Azuara-Blanco, A., Katz, L.J., 1997. Infectious keratitis in a paracentesis tract. Ophthalmic Surgery, Lasers Imaging Retin. 28, 332 333. Balasundaram, M.B., 2010. Outbreak of acquired ocular toxoplasmosis involving 248 patients. Arch. Ophthalmol. 128, 28. Available from: https://doi.org/ 10.1001/archophthalmol.2009.354. Baldursson, S., Karanis, P., 2011. Waterborne transmission of protozoan parasites: review of worldwide outbreaks—an update 2004 2010. Water Res. 45, 6603 6614. Available from: https://doi.org/10.1016/j.watres.2011.10.013. Banta, J.T., Davis, J.L., Lam, B.L., 2002. Presumed toxoplasmosic anterior optic neuropathy. Ocul. Immunol. Inflamm. 10, 201 211. Bastien, P., 2002. Molecular diagnosis of toxoplasmosis. Trans. R. Soc. Trop. Med. Hyg. 96, S205 S215. Bates, S.T., Clemente, J.C., Flores, G.E., Walters, W. a, Parfrey, L.W., Knight, R., et al., 2013. Global biogeography of highly diverse protistan communities in soil. ISME J. 7, 652 659. Available from: https://doi.org/ 10.1038/Ismej.2012.147. Benevento, J.D., Jager, R.D., Gwendolyn Noble, A., Latkany, P., Mieler, W.F., Sautter, M., et al., 2008. Toxoplasmosis associated neovascular lesions treated successfully with ranibizumab and anti-parasitic therapy. Arch. Ophthalmol. 126, 1152 1156. Available from: https://doi.org/10.1001/archopht.126.8.1152. Berengo, A., Frezzotti, R., 1962. Active neuro-ophthalmic toxoplasmosis. A clinical study on nineteen patients. Bibl. Ophthalmol. 59, 265. Bertranpetit, E., Jombart, T., Paradis, E., Pena, H., Dubey, J., Su, C., et al., 2017. Phylogeography of Toxoplasma gondii points to a South American origin. Infect. Genet. Evol. 48, 150 155. Available from: https://doi.org/ 10.1016/j.meegid.2016.12.020. Blaise, P., Comhaire, Y., Rakic, J.-M., 2005. Giant macular hole as an atypical consequence of a toxoplasmic chorioretinitis. Arch. Ophthalmol. 123, 863 864. Bloch-Michel, E., Nussenblatt, R.B., 1987. International Uveitis Study Group recommendations for the evaluation of intraocular inflammatory disease. Am. J. Ophthalmol. 103, 234 235. Bonfioli, A.A., Orefice, F., 2005. Toxoplasmosis. Semin. Ophthalmol. 20, 129 141. Available from: https://doi. org/10.1080/08820530500231961. Borkowski, P.K., 2001. New trends in ocular toxoplasmosis—the review. Przegl. Epidemiol. 55, 483 493.

Toxoplasma Gondii

278

5. Ocular disease due to Toxoplasma gondii

Bosch-Driessen, E.H., Rothova, A., 1998. Sense and nonsense of corticosteroid administration in the treatment of ocular toxoplasmosis. Br. J. Ophthalmol. 82, 858 860. Bosch-Driessen, E.H., Rothova, A., 1999. Recurrent ocular disease in postnatally acquired toxoplasmosis. Am. J. Ophthalmol. 128, 421 425. Available from: https://doi. org/10.1016/S0002-9394(99)00271-8. Bosch-Driessen, L.H., Karimi, S., Stilma, J.S., Rothova, A., 2000. Retinal detachment in ocular toxoplasmosis. Ophthalmology 107, 36 40. Bosch-Driessen, L.E.H., Berendschot, T.T.J.M., Ongkosuwito, J.V., Rothova, A., 2002. Ocular toxoplasmosis: clinical features and prognosis of 154 patients. Ophthalmology 109, 869 878. Botto´s, J., Miller, R.H., Belfort, R.N., Macedo, A.C., Belfort, R., Grigg, M.E., et al., 2009. Bilateral retinochoroiditis caused by an atypical strain of Toxoplasma gondii. Br. J. Ophthalmol. 93, 1546 1550. Available from: https:// doi.org/10.1136/bjo.2009.162412. Bou, G., Figueroa, M.S., Martı´-Belda, P., Navas, E., Guerrero, A., 1999. Value of PCR for detection of Toxoplasma gondii in aqueous humor and blood samples from immunocompetent patients with ocular toxoplasmosis. J. Clin. Microbiol. 37, 3465 3468. Bourdin, C., Busse, A., Kouamou, E., Touafek, F., Bodaghi, B., Le Hoang, P., et al., 2014. PCR-based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis. J. Clin. Microbiol. 52, 3987 3991. Bowie, W.R., King, A.S., Werker, D.H., Isaac-Renton, J.L., Bell, A., Eng, S.B., et al., 1997. Outbreak of toxoplasmosis associated with municipal drinking water. The BC Toxoplasma Investigation Team. Lancet 350, 173 177. Boyer, K., Hill, D., Mui, E., Wroblewski, K., Karrison, T., Dubey, J.P., et al., 2011. Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America. Clin. Infect. Dis. 53, 1081 1089. Available from: https://doi.org/ 10.1093/cid/cir667. Braakenburg, A.M.D., Crespi, C.M., Holland, G.N., Wu, S., Yu, F., Rothova, A., 2014. Recurrence rates of ocular toxoplasmosis during pregnancy. Am. J. Ophthalmol. 157, 767 773. Braunstein, R.A., Gass, J.D., 1980. Branch artery obstruction caused by acute toxoplasmosis. Arch. Ophthalmol. 98, 512 513. Bre´zin, A.P., Eqwuagu, C.E., Silveira, C., Thulliez, P., Martins, M.C., Mahdi, R.M., et al., 1991. Analysis of aqueous humor in ocular toxoplasmosis. N. Engl. J. Med. 324, 699. Bre´zin, A.P., Thulliez, P., Couvreur, J., Nobre´, R., Mcleod, R., Mets, M.B., 2003. Ophthalmic outcomes after

prenatal and postnatal treatment of congenital toxoplasmosis. Am. J. Ophthalmol. 135, 779 784. Burg, J.L., Grover, C.M., Pouletty, P., Boothroyd, J.C., 1989. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J. Clin. Microbiol. 27, 1787 1792. Burke, S.P., Fine, H.F., Albini, T.A., 2016. Toxoplasmosis retinitis masquerading as acute retinal necrosis. Ophthalmic Surg. Lasers Imaging Retina 47, 895 899. Burnett, A.J., Shortt, S.G., Isaac-Renton, J., King, A., Werker, D., Bowie, W.R., 1998. Multiple cases of acquired toxoplasmosis retinitis presenting in an outbreak. Ophthalmology 105, 1032 1037. Carme, B., Bissuel, F., Ajzenberg, D., Bouyne, R., Aznar, C., Demar, M., et al., 2002. Severe acquired toxoplasmosis in immunocompetent adult patients in French Guiana. J. Clin. Microbiol. 40, 4037 4044. Available from: https://doi.org/10.1128/JCM.40.11.4037.4044.2002. Confavreux, C., Girard-Madoux, P., Moulin, T., Boisson, D., Vighetto, A., Aimard, G., et al., 1985. Acute acquired toxoplasmosis causing neuroptico-meningoencephalitis in an immunocompetent boy. J. Neurol. Neurosurg. Psychiatry 48, 715. Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A., et al., 2010. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted C. Immunome Res. 6. Available from: https://doi.org/10.1186/1745-7580-6-12. Cordeiro, C.A., Moreira, P.R., Andrade, M.S., Dutra, W.O., Campos, W.R., Ore´fice, F., et al., 2008a. Interleukin-10 gene polymorphism (21082G/A) is associated with toxoplasmic retinochoroiditis. Invest. Ophthalmol. Vis. Sci. 49, 1979 1982. Available from: https://doi.org/ 10.1167/iovs.07.1393. Cordeiro, C.A., Moreira, P.R., Costa, G.C., Dutra, W.O., Campos, W.R., Ore´fice, F., et al., 2008b. TNF-alpha gene polymorphism (2308G/A) and toxoplasmic retinochoroiditis. Br. J. Ophthalmol. 92, 986 988. Available from: https://doi.org/10.1136/bjo.2008.140590. Cordeiro, C.A., Moreira, P.R., Costa, G.C., Dutra, W.O., Campos, W.R., Ore´fice, F., et al., 2008c. Interleukin-1 gene polymorphisms and toxoplasmic retinochoroiditis. Mol. Vis. 14, 1845 1849. Cordeiro, C.A., Moreira, P.R., Bessa, T.F., Costa, G.C., Dutra, W.O., Campos, W.R., et al., 2013. Interleukin-6 gene polymorphism (2174 G/C) is associated with toxoplasmic retinochoroiditis. Acta Ophthalmol. 311 314. Available from: https://doi.org/10.1111/ aos.12046.

Toxoplasma Gondii

References

Cotliar, A.M., Friedman, A.H., 1982. Subretinal neovascularisation in ocular toxoplasmosis. Br. J. Ophthalmol. 66, 524. Couvreur, J., Thulliez, P., 1996. [Acquired toxoplasmosis of ocular or neurologic site: 49 cases]. Presse Med. 25, 438 442. Couvreur, J., Desmonts, G., Girre, J.Y., 1976. Congenital toxoplasmosis in twins: a series of 14 pairs of twins: absence of infection in one twin in two pairs. J. Pediatr. 89, 235 240. Crosson, J.N., Laird, P.W., Grossniklaus, H.E., Hendrick, A. M., 2015. Toxoplasma chorioretinitis diagnosed by histopathology in a patient with AIDS. Retin. Cases Br. Rep. 9, 162 163. Culbertson, W.W., Tabbara, K.F., O’connor, G.R., 1982. Experimental ocular toxoplasmosis in primates. Arch. Ophthalmol. 100, 321 323. Cunningham, E.T., Augsburger, J.J., Correˆa, Z.M., Pavesio, C., 2017. Uveal Tract & Sclera. In: Riordan-Eva P, Augsburger JJ, editors. Vaughan & Asbury’s General Ophthalmology, 19e [Internet]. New York, NY: McGraw-Hill Education; 2017. Available from: http://accessmedicine.mhmedical. com/content.aspx?aid=1144468073. Dabil, H., Boley, M.L., Schmitz, T.M., Van Gelder, R.N., 2001. Validation of a diagnostic multiplex polymerase chain reaction assay for infectious posterior uveitis. Arch. Ophthalmol. 119, 1315 1322. Daffos, F., Forestier, F., Capella-Pavlovsky, M., Thulliez, P., Aufrant, C., Valenti, D., et al., 1988. Prenatal management of 746 pregnancies at risk for congenital toxoplasmosis. N. Engl. J. Med. 318, 271 275. Das, D., Ramachandra, V., Islam, S., Bhattacharjee, H., Biswas, J., Koul, A., et al., 2016. Update on pathology of ocular parasitic disease. Indian J. Ophthalmol. 64, 794. Davidson, M.G., Lappin, M.R., English, R.V., Tompkins, M. B., 1993. A feline model of ocular toxoplasmosis. Invest. Ophthalmol. Vis. Sci. 34, 3653 3660. Davis, J.L., Madow, B., Cornett, J., Stratton, R., Hess, D., Porciatti, V., et al., 2010. Scale for photographic grading of vitreous haze in uveitis. Am. J. Ophthalmol. 150, 637 641. Delair, E., Latkany, P., Noble, A.G., Rabiah, P., McLeod, R., Bre´zin, A., 2011. Clinical manifestations of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 19, 91 102. Available from: https://doi.org/10.3109/09273948.2011.564068. de-la-Torre, A., Gonza´lez, G., Dı´az-Ramirez, J., Go´mezMarı´n, J.E., 2007. Screening by ophthalmoscopy for Toxoplasma retinochoroiditis in Colombia. Am. J. Ophthalmol. 143, 354 356. Available from: https://doi. org/10.1016/j.ajo.2006.09.048. de-la-Torre, A., Lo´pez-Castillo, C.A., Go´mez-Marı´n, J.E., 2009a. Incidence and clinical characteristics in a

279

Colombian cohort of ocular toxoplasmosis. Eye 23, 1090 1093. Available from: https://doi.org/10.1038/ eye.2008.219. de-la-Torre, A., Rios-Cadavid, A.C., Cardozo-Garcı´a, C.M., Gomez-Marı´n, J.E., 2009b. Frequency and factors associated with recurrences of ocular toxoplasmosis in a referral centre in Colombia. Br. J. Ophthalmol. 93, 1001 1004. Available from: https://doi.org/10.1136/ bjo.2008.155861. de-la-Torre, A., Gonza´lez-Lo´pez, G., Montoya-Gutie´rrez, J. M., Marı´n-Arango, V., Go´mez-Marı´n, J.E., 2011a. Quality of life assessment in ocular toxoplasmosis in a Colombian population. Ocul. Immunol. Inflamm. 19, 262 266. Available from: https://doi.org/10.3109/ 09273948.2011.582220. de-la-Torre, A., Stanford, M., Curi, A., Jaffe, G.J., GomezMarin, J.E., 2011b. Therapy for ocular toxoplasmosis. Ocul. Immunol. Inflamm. 19, 314 320. Available from: https://doi.org/10.3109/09273948.2011.608915. de-la-Torre, A., Sauer, A., Pfaff, A.W., Bourcier, T., Brunet, J., Speeg-Schatz, C., et al., 2013. Severe South American ocular toxoplasmosis is associated with decreased IFNγ/IL-17a and increased IL-6/IL-13 intraocular levels. PLoS Negl. Trop. Dis. 7, e2541. Available from: https:// doi.org/10.1371/journal.pntd.0002541. de-la-Torre, A., Pfaff, A.W., Grigg, M.E., Villard, O., Candolfi, E., Gomez-Marin, J.E., 2014. Ocular cytokinome is linked to clinical characteristics in ocular toxoplasmosis. Cytokine 68, 23 31. Available from: https:// doi.org/10.1016/j.cyto.2014.03.005. Demar, M., Ajzenberg, D., Maubon, D., Djossou, F., Panchoe, D., Punwasi, W., et al., 2007. Fatal outbreak of human toxoplasmosis along the Maroni River: epidemiological, clinical, and parasitological aspects. Clin. Infect. Dis. 45, e88 e95. Available from: https://doi. org/10.1086/521246. De Marco, R., Ceccarelli, R., Frulio, R., Palmero, C., Vittone, P., 2003. Retinochoroiditis associated with congenital toxoplasmosis in children: IgG antibody profiles demonstrating the synthesis of local antibodies. Eur. J. Ophthalmol. 13, 74 79. De Moura, L., Garcia Bahia-Oliveira, L.M., Wada, M.Y., Jones, J.L., Tuboi, S.H., Carmo, E.H., et al., 2006. Waterborne toxoplasmosis, Brazil, from field to gene. Emerg. Infect. Dis. 12, 326 329. Available from: https://doi.org/10.3201/eid1202.041115. Desmonts, G., 1966. Definitive serological diagnosis of ocular toxoplasmosis. Arch. Ophthalmol. 76, 839 851. Diniz, B., Regatieri, C., Andrade, R., Maia, A., 2011. Evaluation of spectral domain and time domain optical coherence tomography findings in toxoplasmic retinochoroiditis. Clin. Ophthalmol. (Auckland, NZ) 5, 645.

Toxoplasma Gondii

280

5. Ocular disease due to Toxoplasma gondii

Dodds, E.M., Holland, G.N., Stanford, M.R., Yu, F., Siu, W. O., Shah, K.H., et al., 2008. Intraocular inflammation associated with ocular toxoplasmosis: relationships at initial examination. Am. J. Ophthalmol. 146, 856 865. Doft, B.H., Gass, J.D.M., 1985. Punctate outer retinal toxoplasmosis. Arch. Ophthalmol. 103, 1332 1336. Dunay, I.R., Gajurel, K., Dhakal, R., Liesenfeld, O., Montoya, J.G., 2018. Treatment of toxoplasmosis: historical perspective, animal models, and current clinical practice. Clin. Microbiol. Rev. 31, e00057-17. Dunn, D., Wallon, M., Peyron, F., Petersen, E., Peckham, C., Gilbert, R., 1999. Mother-to-child transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet 353, 1829 1833. Dutra, M.S., Bela, S.R., Peixoto-Rangel, A.L., Fakiola, M., Cruz, A.G., Gazzinelli, A., et al., 2013. Association of a NOD2 gene polymorphism and T-helper 17 cells with presumed ocular toxoplasmosis. J. Infect. Dis. 207, 152 163. Available from: https://doi.org/10.1093/ infdis/jis640. Dutton, G.N., Hay, J., Hair, D.M., Ralston, J., 1986. Clinicopathological features of a congenital murine model of ocular toxoplasmosis. Graefes Arch. Clin. Exp. Ophthalmol. 224, 256 264. Eckert, G.U., Melamed, J., Menegaz, B., 2007. Optic nerve changes in ocular toxoplasmosis. Eye 21, 746. Elena, B., Vadim, B., Zane, O., Friedrich, K.-N., Friedrich, H., Silvia, B.-P., 2009. ATP induces P2X7 receptorindependent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells. J. Leukoc. Biol. 85, 692 702. Available from: https:// doi.org/10.1189/jlb.0808470. Engstrom, R.E., Holland, G.N., Nussenblatt, R.B., Jabs, D. A., 1991. Current practices in the management of ocular toxoplasmosis. Am. J. Ophthalmol. 111, 601 610. Eyles, D.E., Coleman, N., 1953. Synergistic effect of sulfadiazine and daraprim against experimental toxoplasmosis in the mouse. Antibiot. Chemother. (Northfield, IL) 3, 483 490. Fardeau, C., Romand, S., Rao, N.A., Cassoux, N., Bettembourg, O., Thulliez, P., et al., 2002. Diagnosis of toxoplasmic retinochoroiditis with atypical clinical features. Am. J. Ophthalmol. 134, 196 203. Fekkar, A., Ajzenberg, D., Bodaghi, B., Touafek, F., Le Hoang, P., Delmas, J., et al., 2011. Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France. J. Clin. Microbiol. 49, 1513 1517. Felix, J.P.F., Lira, R.P.C., Zacchia, R.S., Toribio, J.M., Nascimento, M.A., Arieta, C.E.L., 2014. Trimethoprimsulfamethoxazole versus placebo to reduce the risk of recurrences of Toxoplasma gondii retinochoroiditis: randomized controlled clinical trial. Am. J. Ophthalmol.

157, 762 766. Available from: https://doi.org/10.1016/ j.ajo.2013.12.022. Ferguson, J.J.G., 1981. Clindamycin therapy for toxoplasmosis. Ann. Ophthalmol. 13, 95 100. Feron, E.J., Klaren, V.N., Wierenga, E.A., Verjans, G.M., Kijlstra, A., 2001. Characterization of Toxoplasma gondiispecific T cells recovered from vitreous fluid of patients with ocular toxoplasmosis. Invest. Ophthalmol. Vis. Sci. 42, 3228 3232. Available from: https://doi.org/ 10.1007/s00384-016-2573-y. Ferreira, A.I.C., De Mattos, C.C.B., Frederico, F.B., Meira, C.S., Almeida, G.C., Nakashima, F., et al., 2014. Risk factors for ocular toxoplasmosis in Brazil. Epidemiol. Infect. 142, 142 148. Figueroa, M.S., Bou, G., Marti-Belda, P., Lopez-Velez, R., Guerrero, A., 2000. Diagnostic value of polymerase chain reaction in blood and aqueous humor in immunocompetent patients with ocular toxoplasmosis. Retina 20, 614 619. Fish, R.H., Hoskins, J.C., Kline, L.B., 1993. Toxoplasmosis neuroretinitis. Ophthalmology 100, 1177 1182. Folk, J.C., Lobes, L.A., 1984. Presumed toxoplasmic papillitis. Ophthalmology 91, 64 67. Foster, C.S., Vitale, A.T., 2013. Diagnosis & Treatment of Uveitis. JP Medical Ltd. Frenkel, J.K., 1948. Dermal hypersensitivity to toxoplasma antigens (Toxoplasmins). Exp. Biol. Med. 68, 634 639. Available from: https://doi.org/10.3181/00379727-6816579. Frenkel, J.K., 1949. Uveitis and toxoplasmin sensitivity. Am. J. Ophthalmol. 32 (Pt. 2), 127 135. Available from: https://doi.org/10.1016/S0002-9394(14)78365-5. Frenkel, J.K., 1955. Ocular lesions in hamsters; with chronic toxoplasma and besnoitia infection. Am. J. Ophthalmol. 39, 203 225. Frenkel, J.K., 1974. Pathology and pathogenesis of congenital toxoplasmosis. Bull. N.Y. Acad. Med. 50, 182. Frezzotti, R., Guerra, R., Terragna, A., Tosi, P., 1974. A case of congenital toxoplasmosis with active chorioretinitis. Ophthalmologica 169, 321 325. Friedmann, C.T., Knox, D.L., 1969. Variations in recurrent active toxoplasmic retinochoroiditis. Arch. Ophthalmol. 81, 481 493. Ganatra, J.B., Chandler, D., Santos, C., Kuppermann, B., Margolis, T.P., 2000. Viral causes of the acute retinal necrosis syndrome. Am. J. Ophthalmol. 129, 166 172. Garcia Bahia-Oliveira, L.M., Jones, J.L., Azevedo-Silva, J., Alves, C.C.F., Ore´fice, F., Addiss, D.G., 2003. Highly endemic, waterborne toxoplasmosis in North Rio de Janeiro State, Brazil. Emerg. Infect. Dis. 9, 55 62. Available from: https://doi.org/10.3201/eid0901.020160. Garweg, J.G., Candolfi, E., 2009. Immunopathology in ocular toxoplasmosis: facts and clues. Mem. Inst. Oswaldo

Toxoplasma Gondii

References

Cruz 104, 211 220. Available from: https://doi.org/ 10.1590/S0074-02762009000200014. Garweg, J.G., Jacquier, P., Boehnke, M., 2000. Early aqueous humor analysis in patients with human ocular toxoplasmosis. J. Clin. Microbiol. 38, 996 1001. Garweg, J.G., Scherrer, J.N., Halberstadt, M., 2008. Recurrence characteristics in European patients with ocular toxoplasmosis. Br. J. Ophthalmol. 92, 1253 1256. Available from: https://doi.org/10.1136/bjo.2007.123661. Gaynon, M.W., Boldrey, E.E., Strahlman, E.R., Fine, S.L., 1984. Retinal neovascularization and ocular toxoplasmosis. Am. J. Ophthalmol. 98, 585 589. Gazzinelli, R.T., Brezin, A., Li, Q., Nussenblatt, R.B., Chan, C.C., 1994. Toxoplasma gondii: acquired ocular toxoplasmosis in the murine model, protective role of TNF-α and IFN-γ. Exp. Parasitol. 78, 217 229. Available from: https://doi.org/10.1006/expr.1994.1022. Ghartey, K.N., Brockhurst, R.J., 1980. Photocoagulation of active toxoplasmic retinochoroiditis. Am. J. Ophthalmol. 89, 858 864. Gilbert, R.E., Stanford, M.R., 2000. Is ocular toxoplasmosis caused by prenatal or postnatal infection? Br. J. Ophthalmol. 84, 224 226. Gilbert, R.E., Dunn, D.T., Lightman, S., Murray, P.I., Pavesio, C.E., Gormley, P.D., et al., 1999. Incidence of symptomatic toxoplasma eye disease: aetiology and public health implications. Epidemiol. Infect. 123, 283 289. Available from: https://doi.org/10.1017/ S0950268899002800. Gilbert, R.E., Freeman, K., Lago, E.G., Bahia-Oliveira, L.M. G., Tan, H.K., Wallon, M., et al., 2008. Ocular sequelae of congenital toxoplasmosis in Brazil compared with Europe. PLoS Negl. Trop. Dis. 2, e277. Glasner, P.D., Silveira, C., Kruszon-Moran, D., Martins, M.C., Burnier, M., Silveira, S., et al., 1992. An unusually high prevalence of ocular toxoplasmosis in southern Brazil. Am. J. Ophthalmol. 114, 136 144. Available from: https://doi.org/10.1016/S0002-9394 (14)73976-5. Go´mez-Marin, J.E., 2007. Guı´a de pra´ctica clı´nica para toxoplasmosis durante el embarazo y toxoplasmosis conge´nita en Colombia: Clinical practice guidelines for toxoplasmosis during pregnancy and congenital toxoplasmosis in Colombia. Infectio 11, 129 141. Go´mez-Marı´n, J.E., Montoya-de-London˜o, M.T., Castan˜oOsorio, J.C., Heine, F.A., Duque, A.M., Chemla, C., et al., 2000. Frequency of specific anti-Toxoplasma gondii IgM, IgA and IgE in Colombian patients with acute and chronic ocular toxoplasmosis. Mem. Inst. Oswaldo Cruz 95, 89 94. Available from: https://doi.org/10.1590/ S0074-02762000000100014. Go´mez-Marin, J.E., de-la-Torre, A., Angel-Muller, E., Rubio, J., Arenas, J., Osorio, E., et al., 2011. First

281

Colombian multicentric newborn screening for congenital toxoplasmosis. PLoS Negl. Trop. Dis. 5, e1195. Available from: https://doi.org/10.1371/journal.pntd.0001195. Go´mez-Marı´n, J.E., de-la-Torre, A., Barrios, P., Cardona, ´ lvarez, C., Herrera, C., 2012. Toxoplasmosis in milN., A itary personnel involved in jungle operations. Acta Trop. 122, 46 51. Available from: https://doi.org/ 10.1016/j.actatropica.2011.11.019. Go´mez-Marı´n, J.E., Zuluaga, J.D., Pechene´ Campo, E.J., Trivin˜o, J., de-la-Torre, A., 2018. Polymerase chain reaction (PCR) in ocular and ganglionar toxoplasmosis and the effect of therapeutics for prevention of ocular involvement in South American setting. Acta Trop. 184, 83 87. Available from: https://doi.org/10.1016/j. actatropica.2018.01.013. Gonc¸alves, E.C., Ore´fice, F., Mendes, A.G., Pedroso, E.P., 1995. Toxoplasmose ocular adquirida tardia: Relato de treˆs casos simultaˆneos em membros da mesma famı´lia. Rev. Bras. Oftalmol. 54, 57 60. Gordon, D.M., 1970. The treatment of toxoplasmic uveitis. Int. Ophthalmol. Clin. 10, 639 646. Gorfu, G., Cirelli, K.M., Melo, M.B., Mayer-Barber, K., Crown, D., Koller, B.H., et al., 2014. Dual role for inflammasome sensors NLRP1 and NLRP3 in murine resistance to Toxoplasma gondii. mBio 5, e01117-13. Available from: https://doi.org/10.1128/mBio.01117-13. Grigg, M.E., Ganatra, J., Boothroyd, J.C., Margolis, T.P., 2001. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J. Infect. Dis. 184, 633 639. Available from: https://doi.org/10.1086/322800. Grossniklaus, H.E., Specht, C.S., Allaire, G., Leavitt, J.A., 1990. Toxoplasma gondii retinochoroiditis and optic neuritis in acquired immune deficiency syndrome: report of a case. Ophthalmology 97, 1342 1346. Guerina, N.G., 1994. Congenital infection with Toxoplasma gondii. Pediatr. Ann. 23, 138 151. Guo, M., Mishra, A., Buchanan, R.L., Dubey, J.P., Hill, D.E., Gamble, H.R., et al., 2016. A systematic meta-analysis of Toxoplasma gondii prevalence in food animals in the United States. Foodborne Pathog. Dis. 13, 109 118. Available from: https://doi.org/10.1089/fpd.2015.2070. Hald, T., Aspinall, W., Devleesschauwer, B., Cooke, R., Corrigan, T., Havelaar, A.H., et al., 2016. World Health Organization estimates of the relative contributions of food to the burden of disease due to selected foodborne hazards: a structured expert elicitation. PLoS One 11, . Available from: https://doi.org/10.1371/journal. pone.0145839e0145839. Harper, T.W., Miller, D., Schiffman, J.C., Davis, J.L., 2009. Polymerase chain reaction analysis of aqueous and vitreous specimens in the diagnosis of posterior segment infectious uveitis. Am. J. Ophthalmol. 147, 140 147.

Toxoplasma Gondii

282

5. Ocular disease due to Toxoplasma gondii

Hassenstein, A., Meyer, C.H., 2009. Clinical use and research applications of Heidelberg retinal angiography and spectral-domain optical coherence tomography—a review. Clin. Exp. Ophthalmol 37, 130 143. Hay, J., Aitken, P.P., Hair, D.M., Hutchison, W.M., Graham, D.I., 1984. The effect of congenital Toxoplasma infection on mouse activity and relative preference for exposed areas over a series of trials. Ann. Trop. Med. Parasitol. 78, 611 618. Henrique Seabra, S., DaMatta, R.A., de Mello, F.G., de Souza, W., 2004. Endogenous polyamine levels in macrophages is sufficient to support growth of Toxoplasma gondii. J. Parasitol. 90, 455 460. Hercos, B.V., Muinos, S.J., Casaroli-Marano, R.P., 2004. Utility of ultrasonography in toxoplasmic uveitis. Arch. Soc. Esp. Oftalmol. 79, 59 65. Hill, D., Coss, C., Dubey, J.P., Wroblewski, K., Sautter, M., Hosten, T., et al., 2011. Identification of a sporozoitespecific antigen from Toxoplasma gondii. J. Parasitol. 97, 328 337. Available from: https://doi.org/10.1645/GE2782.1. Hogan, M., 1951. Ocular Toxoplasmosis. Columbia Univ. Press. Hogan, M.J., 1950. Toxoplasmosis; ocular manifestations. Trans. Am. Acad. Ophthalmol. Otolaryngol. 54, 183 189. Hogan, M.J., 1958. Ocular toxoplasmosis. Am. J. Ophthalmol. 46, 467 494. Available from: https://doi. org/10.1016/0002-9394(58)91127-9. Hogan, M.J., 1961. Ocular toxoplasmosis in adult patients. Surv. Ophthalmol. 6, 835 851. Hogan, M.J., Thygeson, P., Kimura, S., 1952. Ocular toxoplasmosis. Trans. Am. Acad. Ophthalmol. Otolaryngol. 56, 863 874. Hogan, M.J., Kimura, S.J., Thygeson, P., 1959. Signs and symptoms of uveitis: I. Anterior uveitis. Am. J. Ophthalmol 47, 155 170. Hogan, M.J., Kimura, S.J., O’Connor, G.R., 1964. Ocular toxoplasmosis. Arch. Ophthalmol. 72, 592 600. Holland, G.N., 1994. Standard diagnostic criteria for the acute retinal necrosis syndrome. Am. J. Ophthalmol. 117, 663 666. Holland, G.N., 2003. Ocular toxoplasmosis: a global reassessment. Part I: Epidemiology and course of disease. Am. J. Ophthalmol. 136, 973 988. Holland, G.N., 2009. Ocular toxoplasmosis: the influence of patient age. Mem. Inst. Oswaldo Cruz 104, 351 357. Holland, G.N., Lewis, K.G., 2002. An update on current practices in the management of ocular toxoplasmosis. Curr. Pract. Manag. Ocul. 134, 102 114. Holland, G.N., O’Connor, G.R., Diaz, R.F., Minasi, P., Wara, W.M., 1988. Ocular toxoplasmosis in immunosuppressed nonhuman primates. Invest. Ophthalmol. Vis. Sci. 29, 835 842.

Holland, G.N., Muccioli, C., Silveira, C., Weisz, J.M., Belfort, R.J., O’Connor, G.R., 1999. Intraocular inflammatory reactions without focal necrotizing retinochoroiditis in patients with acquired systemic toxoplasmosis. Am. J. Ophthalmol. 128, 413 420. Holland, G.N., Lewis, K.G., O’connor, G.R., 2002. Ocular toxoplasmosis: a 50th anniversary tribute to the contributions of Helenor Campbell Wilder Foerster. Arch. Ophthalmol. 120, 1081 1084. Howe, D.K., Sibley, L.D., 1995. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J. Infect. Dis. 172, 1561 1566. Hu, M.S., Schwartzman, J.D., Lepage, A.C., Khan, I.A., Kasper, L.H., 1999. Experimental ocular toxoplasmosis induced in naive and preinfected mice by intracameral inoculation. Ocul. Immunol. Inflamm. 7, 17 26. Huang, S.-W., Walker, C., Pennock, J., Else, K., Muller, W., Daniels, M.J., et al., 2017. P2X7 receptor-dependent tuning of gut epithelial responses to infection. Immunol. Cell Biol. 95, 178 188. Available from: https://doi.org/ 10.1038/icb.2016.75. Jabs, D.A., 2008. Epidemiology of uveitis. Ophthalmic Epidemiol. 15, 283 284. Available from: https://doi. org/10.1080/09286580802478724. Jabs, D.A., Nussenblatt, R.B., Rosenbaum, J.T., Standardization of Uveitis Nomenclature (SUN) Working Group, 2005. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop. Am. J. Ophthalmol. 140, 509 516. Jacklin, H.N., 1975. Cryotreatment of toxoplasmosis retinochoroiditis. Ann. Ophthalmol. 7, 853 855. Jamieson, S.E., Peixoto-Rangel, A.L., Hargrave, A.C., Roubaix, L.-A., de, Mui, E.J., Boulter, N.R., et al., 2010. Evidence for associations between the purinergic receptor P2X(7) (P2RX7) and toxoplasmosis. Genes Immun. 11, 374 383. Available from: https://doi.org/10.1038/ gene.2010.31. Jankuˆ, J., 1923. Pathogenesa a Pathologicka´ Anatomie T. ´ ˇ Zv. Vrozeneho Kolobomu Zlute Skurny Oku Norma´l nˇe Velike´m a Mikrophtalmickˇem s Nǎlezem Parasitu v ˇ 62, 1021 1027. Sitnici. Cǎs Le´k Ces Jasper, S., Vedula, S.S., John, S.S., Horo, S., Sepah, Y.J., Nguyen, Q.D., 2017. Corticosteroids as adjuvant therapy for ocular toxoplasmosis. Cochrane Database Syst, Rev. 1, CD007417. Johnston, R.L., Tufail, A., Lightman, S., Luthert, P.J., Pavesio, C.E., Cooling, R.J., et al., 2004. Retinal and choroidal biopsies are helpful in unclear uveitis of suspected infectious or malignant origin. Ophthalmology 111, 522 528. Jones, C.D., Okhravi, N., Adamson, P., Tasker, S., Lightman, S., 2000. Comparison of PCR detection methods for B1,

Toxoplasma Gondii

References

P30, and 18S rDNA genes of T. gondii in aqueous humor. Invest. Ophthalmol. Vis. Sci. 41, 634 644. Jones, J.L., Bonetti, V., Holland, G.N., Press, C., Sanislo, S. R., Khurana, R.N., et al., 2014a. Ocular toxoplasmosis in the United States: recent and remote infections. Clin. Infect. Dis. 60, 271 273. Available from: https://doi. org/10.1093/cid/ciu793. Jones, J.L., Parise, M.E., Fiore, A.E., 2014b. Neglected parasitic infections in the United States: toxoplasmosis. Am. J. Trop. Med. Hyg. 90, 794 799. Jones, J.L., Bonetti, V., Holland, G.N., Press, C., Sanislo, S. R., Khurana, R.N., et al., 2015. Ocular toxoplasmosis in the United States: recent and remote infections. Clin. Infect. Dis. 60, 271 273. Available from: https://doi. org/10.1093/cid/ciu793. Karanis, P., Aldeyarbi, H.M., Mirhashemi, M.E., Khalil, K. M., 2013. The impact of the waterborne transmission of Toxoplasma gondii and analysis efforts for water detection: an overview and update. Environ. Sci. Pollut. Res. Int. 20, 86 99. Available from: https://doi.org/ 10.1007/s11356-012-1177-5. Khan, W., Khan, K., 2018. Congenital toxoplasmosis: an overview of the neurological and ocular manifestations. Parasitol. Int. 67, 715 721. Khan, A., Bo¨hme, U., Kelly, K.A., Adlem, E., Brooks, K., Simmonds, M., et al., 2006a. Common inheritance of chromosome Ia associated with clonal expansion of Toxoplasma gondii. Genome Res. 16, 1119 1125. Available from: https://doi.org/10.1101/gr.5318106. Khan, A., Jordan, C., Muccioli, C., Vallochi, A.L., Rizzo, L. V., Belfort, R., et al., 2006b. Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil. Emerg. Infect. Dis. 12, 942 949. Khan, A., Ajzenberg, D., Mercier, A., Demar, M., Simon, S., Darde´, M.L., et al., 2014. Geographic separation of domestic and wild strains of Toxoplasma gondii in French Guiana correlates with a monomorphic version of chromosome 1a. PLoS Negl. Trop. Dis. 8, e3182. Available from: https://doi.org/10.1371/journal. pntd.0003182. Kianersi, F., Naderi Beni, A., Ghanbari, H., Fazel, F., 2012. Ocular toxoplasmosis and retinal detachment: five case reports. Eur. Rev. Med. Pharmacol. Sci. 16, 84 89. Kijlstra, A., Petersen, E., 2014. Epidemiology, pathophysiology, and the future of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 22, 138 147. Available from: https://doi.org/10.3109/09273948.2013.823214. Kijlstra, A., Luyendijk, L., Baarsma, G.S., Rothova, A., Schweitzer, C.M.C., Timmerman, Z., et al., 1989. Aqueous humor analysis as a diagnostic tool in toxoplasma uveitis. Int. Ophthalmol. 13, 383 386. Kikumura, A., Ishikawa, T., Norose, K., 2012. Kinetic analysis of cytokines, chemokines, chemokine receptors and

283

adhesion molecules in murine ocular toxoplasmosis. Br. J. Ophthalmol. 96, 1259 1267. Available from: https://doi.org/10.1136/bjophthalmol-2012-301490. Klaren, V.N.A., van Doornik, C.E.M., Ongkosuwito, J.V., Feron, E.J., Kijlstra, A., 1998. Differences between intraocular and serum antibody responses in patients with ocular toxoplasmosis. Am. J. Ophthalmol. 126, 698 706. Kopec, R., Caro, G., De, Chapnick, E., Ghitan, M., Saffra, N., 2003. Prophylaxis for ocular toxoplasmosis. Clin. Infect. Dis. 37, e147 e148. Koppe, J.G., Kloosterman, G.J., de Roever-Bonnet, H., Eckert-Stroink, J.A., Loewer-Sieger, D.H., De Bruijne, J. I., 1974. Toxoplasmosis and pregnancy, with a longterm follow-up of the children. Eur. J. Obstet. Gynecol. 4, 101 109. Koppe, J.G., Loewer-Sieger, D.H., de Roever-Bonnet, H., 1986. Results of 20-year follow-up of congenital toxoplasmosis. Lancet 327, 254 256. Kovaˇcevi´c-Pavi´cevi´c, D., Radosavljevi´c, A., Ili´c, A., Kovaˇcevi´c, I., Djurkovi´c-Djakovi´c, O., 2012. Clinical pattern of ocular toxoplasmosis treated in a referral centre in Serbia. Eye 26, 723. Kraushar, M.F., Gluck, S.B., Pass, S., 1979. Toxoplasmic retinochoroiditis presenting s serous detachment of the macula. Ann. Ophthalmol. 11, 1513 1514. Ku¨c¸u¨kerdo¨nmez, C., Akova, Y.A., Yilmaz, G., 2002. Ocular toxoplasmosis presenting as neuroretinitis: report of two cases. Ocul. Immunol. Inflamm. 10, 229 234. Labalette, P., Delhaes, L., Margaron, F., Fortier, B., Rouland, J.-F., 2002. Ocular toxoplasmosis after the fifth decade. Am. J. Ophthalmol. 133, 506 515. Lafaut, B.A., Meire, F.M., Leys, A.M., Dralands, G., De Laey, J.-J., 1999. Vasoproliferative retinal tumors associated with peripheral chorioretinal scars in presumed congenital toxoplasmosis. Graefes Arch. Clin. Exp. Ophthalmol. 237, 1033 1038. Lahmar, I., Abou-Bacar, A., Abdelrahman, T., Guinard, M., Babba, H., Ben Yahia, S., et al., 2009. Cytokine profiles in toxoplasmic and viral uveitis. J. Infect. Dis. 199, 1239 1249. Available from: https://doi.org/10.1086/ 597478. Lasave, A.F., Dı´az-Llopis, M., Muccioli, C., Belfort Jr, R., Arevalo, J.F., 2010. Intravitreal clindamycin and dexamethasone for zone 1 toxoplasmic retinochoroiditis at twenty-four months. Ophthalmology 117, 1831 1838. Lassoued, S., Zabraniecki, L., Marin, F., Billey, T., 2007. Toxoplasmic chorioretinitis and antitumor necrosis factor treatment in rheumatoid arthritis. Semin. Arthritis Rheum. 36, 262 263. Available from: https://doi.org/ 10.1016/j.semarthrit.2006.08.004. Lavinsky, D., Romano, A., Muccioli, C., Belfort Jr, R., 2012. Imaging in ocular toxoplasmosis. Int. Ophthalmol. Clin. 52, 131 143.

Toxoplasma Gondii

284

5. Ocular disease due to Toxoplasma gondii

Lavinsky J., 2002. Doenc¸as prevalentes da retina e vı´treo Editora Cultura Medica (Rio Janeiro) 1st Edition. p. 455 465 [in Portuguese]. Lees, M.P., Fuller, S.J., McLeod, R., Boulter, N.R., Miller, C. M., Zakrzewski, A.M., et al., 2010. P2X7 receptormediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages. J. Immunol. 184, 7040 7046. Available from: https://doi. org/10.4049/jimmunol.1000012. Le´lu, M., Villena, I., Darde´, M.L., Aubert, D., Geers, R., Dupuis, E., et al., 2012. Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil. Appl. Environ. Microbiol. 78, 5127 5132. Available from: https://doi.org/10.1128/AEM.00246-12. Levadit, I.C., 1928. Au sujet de certaines protozooses Hereditaires humaines a localization oculaires et nerveuses. C. R. Soc. Biol. 98, 297 299. Lieb, D.F., Scott, I.U., Flynn Jr, H.W., Davis, J.L., Demming, S.M., 2004. Acute acquired toxoplasma retinitis may present similarly to unilateral acute idiopathic maculopathy. Am. J. Ophthalmol. 137, 940 942. Liesenfeld, O., Montoya, J.G., Tathineni, N.J., Davis, M., Brown Jr, B.W., et al., 2001. Confirmatory serologic testing for acute toxoplasmosis and rate of induced abortions among women reported to have positive Toxoplasma immunoglobulin M antibody titers. Am. J. Obstet. Gynecol. 184, 140 145. Liotet, S., Bloch-Michel, E., Petithory, J.C., Batellier, L., Chaumeil, C., 1992. Biological modifications of the vitreous in intraocular parasitosis: preliminary study. Int. Ophthalmol. 16, 75 80. Lu, F., Huang, S., Hu, M.S., Kasper, L.H., 2005. Experimental ocular toxoplasmosis in genetically susceptible and resistant mice. Infect. Immun. 73, 5160 5165. Lum, F., Jones, J.L., Holland, G.N., Liesegang, T.J., 2005. Survey of ophthalmologists about ocular toxoplasmosis. Am. J. Ophthalmol. 140, 724-e1. Madow, B., Galor, A., Feuer, W.J., Altaweel, M.M., Davis, J. L., 2011. Validation of a photographic vitreous haze grading technique for clinical trials in uveitis. Am. J. Ophthalmol. 152, 170 176. Maenz, M., Schlu¨ter, D., Liesenfeld, O., Schares, G., Gross, U., Pleyer, U., 2014. Ocular toxoplasmosis past, present and new aspects of an old disease. Prog. Retin. Eye Res. 39, 77 106. Mahittikorn, A., Mori, H., Popruk, S., Roobthaisong, A., Sutthikornchai, C., Koompapong, K., et al., 2015. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loopmediated isothermal amplification (LAMP). PLoS One 10, e0120997. Available from: https://doi.org/10.1371/ journal.pone.0120997.

Maitland, C.G., Miller, N.R., 1984. Neuroretinitis. Arch. Ophthalmol. 102, 1146 1150. Mangiavacchi, B.M., Vieira, F.P., Bahia-Oliveira, L.M.G., Hill, D., 2016. Salivary IgA against sporozoite-specific embryogenesis-related protein (TgERP) in the study of horizontally transmitted toxoplasmosis via T. gondii oocysts in endemic settings. Epidemiol. Infect. 144, 2568 2577. Available from: https://doi.org/10.1017/ S0950268816000960. Manschot, W.A., Daamen, C.B.F., 1965. Connatal ocular toxoplasmosis. Arch. Ophthalmol. 74, 48 54. Mansour, A.M., Ansari, N.H., Egwuagu, C.E., Kaspar, H. G., Harmon, M.K., 1993. Bacterial optic disk mass and toxoplasmic-like encephalitis in an HIV-seropositive subject. Graefes Arch. Clin. Exp. Ophthalmol. 231, 668 671. Marx-Chemla, C., Villena, I., Foudrinier, F., Pinon, J.M., Gotzamanis, A., Hamon, F., et al., 1998. Overt chorioretinitis after patient acquired toxoplasmosis in an immunocompetent subject. Br. J. Ophthalmol. 82, 1446 1447. Masur, H., Jones, T.C., Lempert, J.A., Cherubini, T.D., 1978. Outbreak of toxoplasmosis in a family and documentation of acquired retinochoroiditis. Am. J. Med. 64, 396 402. Available from: https://doi.org/10.1016/ 0002-9343(78)90218-8. Mathis, T., Beccat, S., Se`ve, P., Peyron, F., Wallon, M., Kodjikian, L., 2018. Comparison of immunoblotting (IgA and IgG) and the Goldmann-Witmer coefficient for diagnosis of ocular toxoplasmosis in immunocompetent patients. Br. J. Ophthalmol. 102, 1454 1458. Available from: https://doi.org/10.1136/bjophthalmol2017-311528. McAuley, J., Boyer, K.M., Patel, D., Mets, M., Swisher, C., Roizen, N., et al., 1994. Early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis: the Chicago Collaborative Treatment Trial. Clin. Infect. Dis. 18, 38 72. McLeod, R., Mack, D., Foss, R., Boyer, K., Withers, S., Levin, S., et al., 1992. Levels of pyrimethamine in sera and cerebrospinal and ventricular fluids from infants treated for congenital toxoplasmosis. Toxoplasmosis Study Group. Antimicrob. Agents Chemother. 36, 1040 1048. McLeod, S.D., Flowers, C.W., Lopez, P.F., Marx, J., McDonnell, P.J., 1995. Endophthalmitis and orbital cellulitis after radial keratotomy. Ophthalmology 102, 1902 1907. McLeod, R., Kieffer, F., Sautter, M., Hosten, T., Pelloux, H., 2009. Why prevent, diagnose and treat congenital toxoplasmosis? Mem. Inst. Oswaldo Cruz 104, 320 344. Available from: https://doi.org/10.1590/S007402762009000200029.

Toxoplasma Gondii

References

McLeod, R., Boyer, K.M., Lee, D., Mui, E., Wroblewski, K., Karrison, T., et al., 2012. Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981-2009). Clin. Infect. Dis. 54, 1595 1605. Available from: https://doi.org/10.1093/cid/cis258. McMenamin, P.G., Dutton, G.N., Hay, J., Cameron, S., 1986. The ultrastructural pathology of congenital murine toxoplasmic retinochoroiditis. Part I: The localization and morphology of Toxoplasma cysts in the retina. Exp. Eye Res. 43, 529 543. Mets, M.B., Holfels, E., Boyer, K.M., Swisher, C.N., Roizen, N., Stein, L., et al., 1997. Eye manifestations of congenital toxoplasmosis. Am. J. Ophthalmol. 123, 1 16. Miller, D., Davis, J., Rosa, R., Diaz, M., Perez, E., 2000. Utility of tissue culture for detection of Toxoplasma gondii in vitreous humor of patients diagnosed with toxoplasmic retinochoroiditis. J. Clin. Microbiol. 38, 3840 3842. Minns, L.A., Menard, L.C., Foureau, D.M., Darche, S., Ronet, C., Mielcarz, D.W., et al., 2006. TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii. J. Immunol. 176, 7589 7597. Mitchell, C.D., Erlich, S.S., Mastrucci, M.T., Hutto, S.C., Parks, W.P., Scott, G.B., 1990. Congenital toxoplasmosis occurring in infants perinatally infected with human immunodeficiency virus 1. Pediatr. Infect. Dis. J. 9, 512 518. Montoya, J.G., Remington, J.S., 1996. Toxoplasmic chorioretinitis in the setting of acute acquired toxoplasmosis. Clin. Infect. Dis. 23, 277 282. Available from: https:// doi.org/10.1093/clinids/23.2.277. Montoya, J.G., Parmley, S., Liesenfeld, O., Jaffe, G.J., Remington, J.S., 1999. Use of the polymerase chain reaction for diagnosis of ocular toxoplasmosis. Ophthalmology 106, 1554 1563. Moorthy, R.S., Smith, R.E., Rao, N.A., 1993. Progressive ocular toxoplasmosis in patients with acquired immunodeficiency syndrome. Am. J. Ophthalmol. 115, 742 747. Moraes Jr, H.V., 1999. Punctate outer retinal toxoplasmosis in an HIV-positive child. Ocul. Immunol. Inflamm. 7, 93 95. Moreno, R.J., Weisman, J., Waller, S., 1992. Neuroretinitis: an unusual presentation of ocular toxoplasmosis. Ann. Ophthalmol. 24, 68 70. Morisset, S., Peyron, F., Lobry, J.R., Garweg, J., Ferrandiz, J., Musset, K., et al., 2008. Serotyping of Toxoplasma gondii: striking homogeneous pattern between symptomatic and asymptomatic infections within Europe and South America. Microbes Infect. 10, 742 747. Available from: https://doi.org/10.1016/j.micinf.2008.04.001. Moshfeghi, D.M., Dodds, E.M., Couto, C.A., Santos, C.I., Nicholson, D.H., Lowder, C.Y., et al., 2004. Diagnostic

285

approaches to severe, atypical toxoplasmosis mimicking acute retinal necrosis. Ophthalmology 111, 716 725. Mun˜oz-Zanzi, C.A., Fry, P., Lesina, B., Hill, D., 2010. Toxoplasma gondii oocyst-specific antibodies and source of infection. Emerg. Infect. Dis. 16, 1591 1593. Available from: https://doi.org/10.3201/eid1610. 091674. Nagineni, C.N., Pardhasaradhi, K., Martins, M.C., Detrick, B., Hooks, J.J., 1996. Mechanisms of interferon-induced inhibition of Toxoplasma gondii replication in human retinal pigment epithelial cells. Infect. Immun. 64, 4188 4196. Available from: https://doi.org/10.1089/ cpb.2005.8.15. Naranjo-Galvis, C.A., de-la-Torre, A., Mantilla-Muriel, L.E., Beltra´n-Angarita, L., Elcoroaristizabal-Martı´n, X., McLeod, R., et al., 2018. Genetic polymorphisms in cytokine genes in colombian patients with ocular toxoplasmosis. Infect. Immun. 86, e00597 17. Available from: https://doi.org/10.1128/IAI.00597-17. Neves, E., de, S., Curi, A.L.L., Albuquerque, M.C., de, Palhano-Silva, C.S., Silva, L.B., et al., 2012. Genetic polymorphism for IFNγ 1874T/A in patients with acute toxoplasmosis. Rev. Soc. Bras. Med. Trop. 45, 757 760. Nicholson, D.H., Wolchok, E.B., 1976. Ocular toxoplasmosis in an adult receiving long-term corticosteroid therapy. Arch. Ophthalmol. 94, 248 254. Nijhawan, R., Bansal, R., Gupta, N., Beke, N., Kulkarni, P., Gupta, A., 2013. Intraocular cysts of Toxoplasma gondii in patients with necrotizing retinitis following periocular/intraocular triamcinolone injection. Ocul. Immunol. Inflamm. 21, 396 399. Norose, K., Kikumura, A., Luster, A.D., Hunter, C.A., Harris, T.H., 2011. CXCL10 is required to maintain Tcell populations and to control parasite replication during chronic ocular toxoplasmosis. Investig. Opthalmol. Vis. Sci. 52, 389. Available from: https://doi.org/ 10.1167/iovs.10-5819. Nozik, R.A., 1977. Results of treatment of ocular toxoplasmosis with injectable corticosteroids. Trans. Sect. Ophthalmol. Am. Acad. Ophthalmol. Otolaryngol. 83, 811 818. Nozik, R.A., O’Connor, G.R., 1968. Experimental toxoplasmic retinochoroiditis. Arch. Ophthalmol. 79, 485 489. Nussenblatt, R.B., Palestine, A.G., Chan, C.-C., Roberge, F., 1985. Standardization of vitreal inflammatory activity in intermediate and posterior uveitis. Ophthalmology 92, 467 471. Nussenblatt, R.B., Mittal, K.K., Fuhrman, S., Sharma, S.D., Palestine, A.G., 1989. Lymphocyte proliferative responses of patients with ocular toxoplasmosis to parasite and retinal antigens. Am. J. Ophthalmol. 107, 632 641. Available from: https://doi.org/10.1016/ 0002-9394(89)90260-2.

Toxoplasma Gondii

286

5. Ocular disease due to Toxoplasma gondii

O’Connor, G.R., 1974. Manifestations and management of ocular toxoplasmosis. Bull. N.Y. Acad. Med 50, 192. O’Connor, G.R., 1983. Ocular toxoplasmosis. Trans. New Orleans Acad. Ophthalmol. 31, 108. O’Connor, G.R., Frenkel, J.K., 1976. Dangers of steroid treatment in toxoplasmosis: periocular injections and systemic therapy. Arch. Ophthalmol. 94, 213. Okada, A.A., Jabs, D.A., 2013. The standardization of uveitis nomenclature project: the future is here. JAMA Ophthalmol. 131, 787 789. O’Neill, J.F., 1998. The ocular manifestations of congenital infection: a study of the early effect and long-term outcome of maternally transmitted rubella and toxoplasmosis. Trans. Am. Ophthalmol. Soc. 96, 813. Ongkosuwito, J.V., Bosch-Driessen, E.H., Kijlstra, A., Rothova, A., 1999. Serologic evaluation of patients with primary and recurrent ocular toxoplasmosis for evidence of recent infection. Am. J. Ophthalmol. 128, 407 412. Opsteegh, M., Prickaerts, S., Frankena, K., Evers, E.G., 2011. A quantitative microbial risk assessment for meatborne Toxoplasma gondii infection in The Netherlands. Int. J. Food Microbiol. 150, 103 114. Available from: https://doi.org/10.1016/j.ijfoodmicro.2011.07.022. Ore´fice, F., 2005. Uveı´te clı´nica e ciru´rgica: texto e atlas. 2 ed. Rio de Janeiro: Cultura Me´dica; vol 2, 709 712;1111 1130. Ore´fice, J.L., Costa, R.A., Campos, W., Calucci, D., Scott, I. U., Ore´fice, F., 2006. Third-generation optical coherence tomography findings in punctate retinal toxoplasmosis. Am. J. Ophthalmol. 142, 503 505. Palanisamy, M., Madhavan, B., Balasundaram, M., Andavar, R., Venkatapathy, N., 2006. Outbreak of ocular toxoplasmosis in Coimbatore, India. Indian J. Ophthalmol. 54, 129 131. Available from: https://doi. org/10.4103/0301-4738.25839. Palkovacs, E.M., Correa, Z., Augsburger, J.J., Eagle, R.C., 2008. Acquired toxoplasmic retinitis in an immunosuppressed patient: diagnosis by transvitreal fine-needle aspiration biopsy. Graefes Arch. Clin. Exp. Ophthalmol 246, 1495 1497. Paul, M., 1999. Immunoglobulin G avidity in diagnosis of toxoplasmic lymphadenopathy and ocular toxoplasmosis. Clin. Diagn. Lab. Immunol. 6, 514 518. Peixe, R.G., Boechat, M.S.B., Rangel, A.L.P., Rosa, R.F.G., Petzl-Erler, M.L., Bahia-Oliveira, L.M.G., 2014. Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection. Mem. Inst. Oswaldo Cruz 109, 99 107. Available from: https://doi.org/10.1590/0074-0276140539. Peixoto-Rangel, A.L., Miller, E.N., Castellucci, L., Jamieson, S.E., Peixe, R.G., Elias, L., et al., 2009. Candidate gene analysis of ocular toxoplasmosis in Brazil: evidence for a role for toll-like receptor 9 (TLR9). Mem. Inst. Oswaldo Cruz 104, 1187 1190.

Perkins, E.S., 1973. Ocular toxoplasmosis. Br. J. Ophthalmol. 57, 1. Perrotta, S., Nobili, B., Grassia, C., Sebastiani, A., Parmeggiani, F., Costagliola, C., 2003. Bilateral neuroretinitis in a 6-year-old boy with acquired toxoplasmosis. Arch. Ophthalmol. 121, 1493 1496. Peters, P.J., Thigpen, M.C., Parise, M.E., Newman, R.D., 2007. Safety and toxicity of sulfadoxine/pyrimethamine. Drug Saf. 30, 481 501. Available from: https:// doi.org/10.2165/00002018-200730060-00003. Petersen, E., Kijlstra, A., Stanford, M., 2012. Epidemiology of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 20, 68 75. Available from: https://doi.org/10.3109/ 09273948.2012.661115. Peyman, G.A., Charles, H.C., Liu, K.-R., Khoobehi, B., Niesman, M., 1988. Intravitreal liposome-encapsulated drugs: a preliminary human report. Int. Ophthalmol. 12, 175 182. Peyron, F., Ateba, A.B., Wallon, M., Kodjikian, L., Binquet, C., Fleury, J., et al., 2003. Congenital toxoplasmosis in twins: a report of fourteen consecutive cases and a comparison with published data. Pediatr. Infect. Dis. J. 22, 695 701. Peyron, F., Lobry, J.R., Musset, K., Ferrandiz, J., GomezMarin, J.E., Petersen, E., et al., 2006. Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America). Microbes Infect. 8, 2333 2340. Available from: https://doi.org/10.1016/j. micinf.2006.03.023. Peyron, F., Garweg, J.G., Wallon, M., Descloux, E., Rolland, M., Barth, J., 2011. Long-term impact of treated congenital toxoplasmosis on quality of life and visual performance. Pediatr. Infect. Dis. J. 30, 597 600. Pfaff, A.W., de-la-Torre, A., Rochet, E., Brunet, J., Sabou, M., Sauer, A., et al., 2014. New clinical and experimental insights into Old World and neotropical ocular toxoplasmosis. Int. J. Parasitol. 44, 99 107. Available from: https://doi.org/10.1016/j.ijpara.2013.09.007. Pradhan, E., Bhandari, S., Re, G., Stanford, M., 2016. Antibiotics versus no treatment for toxoplasma retinochoroiditis (Review) 20, CD002218. https://doi. org/10.1002/14651858.CD002218.pub2.www.cochrane library.com. Pulivarthi, S., Reshi, R.A., McGary, C.T., Krishna Gurram, M., 2015. Cerebral toxoplasmosis in a patient on methotrexate and infliximab for rheumatoid arthritis. Intern. Med. 54, 1433 1436. Available from: https://doi.org/ 10.2169/internalmedicine.54.3977. Rajendran, C., Su, C., Dubey, J.P., 2012. Molecular genotyping of Toxoplasma gondii from Central and South America revealed high diversity within and between populations. Infect. Genet. Evol. 12, 359 368. Available from: https://doi.org/10.1016/j. meegid.2011.12.010.

Toxoplasma Gondii

References

Ramchandani, M., Weaver, J.B., Joynson, D.H.M., Murray, P.I., 2002. Acquired ocular toxoplasmosis in pregnancy. Br. J. Ophthalmol. 86, 938 939. Rao, N.A., Font, R.L., 1977. Toxoplasmic retinochoroiditis: electron-microscopic and immunofluorescence studies of formalin-fixed tissue. Arch. Ophthalmol. 95, 273 277. Rehder, J.R., Burnier, M.B.J., Pavesio, C.E., Kim, M.K., Rigueiro, M., Petrilli, A.M., et al., 1988. Acute unilateral toxoplasmic iridocyclitis in an AIDS patient. Am. J. Ophthalmol. 106, 740 741. Reich, M., Becker, M.D., Mackensen, F., 2015a. Influence of drug therapy on the risk of recurrence of ocular toxoplasmosis. Br. J. Ophthalmol. 100, 195 199. Available from: https://doi.org/10.1136/bjophthalmol2015-306650. Reich, M., Ruppenstein, M., Becker, M.D., Mackensen, F., 2015b. Risk of recurrence of preexisting ocular toxoplasmosis during pregnancy. Ocul. Immunol. Inflamm. 23, 240 245. Reich, M., Dodds, E.M., Stanford, M.R., Yu, F., Rothova, A., Muccioli, C., et al., 2016. Intraocular inflammation associated with ocular toxoplasmosis: a longitudinal evaluation. Invest. Ophthalmol. Vis. Sci. 57. Remington, J.S., Thulliez, P., Montoya, J.G., 2004. Recent developments for diagnosis of toxoplasmosis. J. Clin. Microbiol. 42, 941 945. Rieger, H., 1951. Zur Klinik der im Erwachsenenalter erworbenen Toxoplasmose. Klin. Monatsbl. Augenh. 119, 459 476. Rieger, H., 1959. Toxoplasmosis congenita und Zwillingsschwangerschaft. Klin. Monatsbl. Augenh. 134, 862 871. Riss, J.M., Carboni, M.E., Franck, J.Y., Mary, C.J., Dumon, H., Ridings, B., 1995. Ocular toxoplasmosis: value of immunoblotting for the determination of an intra-ocular synthesis of antibodies. Pathol. Biol. (Paris) 43, 772 778. Roach, E.S., Zimmerman, C.F., Troost, B.T., Weaver, R.G., 1985. Optic neuritis due to acquired toxoplasmosis. Pediatr. Neurol. 1, 114 116. Robert-Gangneux, F., Darde´, M.-L., 2012. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin. Microbiol. Rev. 25, 264 296. Robert-Gangneux, F., Binisti, P., Antonetti, D., Brezin, A., Yera, H., Dupouy-Camet, J., 2004. Usefulness of immunoblotting and Goldmann-Witmer coefficient for biological diagnosis of toxoplasmic retinochoroiditis. Eur. J. Clin. Microbiol. Infect. Dis. 23, 34 38. Roberts, T., Frenkel, J.K., 1990. Estimating income losses and other preventable costs caused by congenital toxoplasmosis in people in the United States. J. Am. Vet. Med. Assoc. 196, 249 256. Roberts, C.W., Brewer, J.M., Alexander, J., 1994. Congenital toxoplasmosis in the Balb/c mouse: prevention of

287

vertical disease transmission and fetal death by vaccination. Vaccine 12, 1389 1394. Roberts, F., Roberts, C.W., Johnson, J.J., Kyle, D.E., Krell, T., Coggins, J.R., et al., 1998. Evidence for the shikimate pathway in apicomplexan parasites. Nature 393, 801. Roberts, F., Mets, M.B., Ferguson, D.J.P., O’grady, R., O’grady, C., Thulliez, P., et al., 2001. Histopathological features of ocular toxoplasmosis in the fetus and infant. Arch. Ophthalmol. 119, 51 58. Ronday, M.J.H., Luyendijk, L., Baarsma, G.S., Bollemeijer, J.-G., Van der Lelij, A., Rothova, A., 1995. Presumed acquired ocular toxoplasmosis. Arch. Ophthalmol. 113, 1524 1529. Ronday, M.J.H., Ongkosuwito, J.V., Rothova, A., Kijlstra, A., 1999. Intraocular anti Toxoplasma gondii IgA antibody production in patients with ocular toxoplasmosis. Am. J. Ophthalmol. 127, 294 300. Rosenfeld, P.J., Brown, D.M., Heier, J.S., Boyer, D.S., Kaiser, P.K., Chung, C.Y., et al., 2006. Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355, 1419 1431. Rothova, A., Meenken, C., Buitenhuis, H.J., Brinkman, C.J., Baarsma, G.S., Boen-Tan, T.N., et al., 1993. Therapy for ocular toxoplasmosis. Am. J. Ophthalmol. 115, 517 523. Rudzinski, M., Meyer, A., Khoury, M., Couto, C., 2013. Is reactivation of toxoplasmic retinochoroiditis associated to increased annual rainfall? Parasite 20, 44. Available from: https://doi.org/10.1051/parasite/2013044. Sabates, R., Pruett, R.C., Brockhurst, R.J., 1981. Fulminant ocular toxoplasmosis. Am. J. Ophthalmol. 92, 497 503. Saeij, J.P.J., Boyle, J.P., Grigg, M.E., Arrizabalaga, G., Boothroyd, J.C., 2005. Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains. Infect. Immun. 73, 695 702. Sa´nchez, V., de-la-Torre, A., Go´mez-Marı´n, J.E., 2014. Characterization of ROP18 alleles in human toxoplasmosis. Parasitol. Int. 63, 463 469. Available from: https://doi.org/10.1016/j.parint.2013.10.012. Santana, S.S., Gebrim, L.C., Carvalho, F.R., Barros, H.S., Barros, P.C., Pajuaba, A.C.A.M., et al., 2015. CCp5A protein from Toxoplasma gondii as a serological marker of oocyst-driven infections in humans and domestic animals. Front. Microbiol. 6, 1305. Available from: https:// doi.org/10.3389/fmicb.2015.01305. Santos, T.R., dos, Nunes, C.M., Luvizotto, M.C.R., Moura, A.B., de, Lopes, W.D.Z., Costa, A.J., et al., 2010. Detection of Toxoplasma gondii oocysts in environmental samples from public schools. Vet. Parasitol. 171, 53 57. Available from: https://doi.org/10.1016/j. vetpar.2010.02.045. Sauer, A., De La Torre, A., Gomez-Marin, J., Bourcier, T., Speeg-Schatz, Garweg, J., et al., 2011. Prevention of

Toxoplasma Gondii

288

5. Ocular disease due to Toxoplasma gondii

retinochoroiditis in congenital toxoplasmosis: Europe Versus South America. Pediatr. Infect. Dis. J. 30, 601 603. Available from: https://doi.org/10.1097/ INF.0b013e3182129e70. Sauer, A., Pfaff, A.W., Villard, O., Creuzot-Garcher, C., Dalle, F., Chiquet, C., et al., 2012. Interleukin 17A as an effective target for anti-inflammatory and antiparasitic treatment of toxoplasmic uveitis. J. Infect. Dis. 206, 1319 1329. Available from: https://doi.org/10.1093/ infdis/jis486. Sauer, A., Villard, O., Creuzot-Garcher, C., Chiquet, C., Berrod, J.P., Speeg-Schatz, C., et al., 2015. Intraocular levels of interleukin 17A (IL-17A) and IL-10 as respective determinant markers of toxoplasmosis and viral uveitis. Clin. Vaccine Immunol. 22, 72 78. Available from: https://doi.org/10.1128/CVI.00423.14. Scherrer, J., Iliev, M.E., Halberstadt, M., Kodjikian, L., Garweg, J.G., 2007. Visual function in human ocular toxoplasmosis. Br. J. Ophthalmol. 91, 233 236. Available from: https://doi.org/10.1136/bjo.2006. 100925. Schlaen, A., Colombero, D., Ormaechea, S., Ladeveze, E., Rudzinski, C., Ingolotti, M., et al., 2018. Regional differences in the clinical manifestation of ocular toxoplasmosis between the center and northeast of Argentina. Ocul. Immunol. Inflamm. 1 9. Schmitz-Valckenberg, S., Holz, F.G., Bird, A.C., Spaide, R. F., 2008. Fundus autofluorescence imaging: review and perspectives. Retina 28, 385 409. Schuman, J.S., Weinberg, R.S., Ferry, A.P., Guerry, R.K., 1988. Toxoplasmic scleritis. Ophthalmology 95, 1399 1403. Schwab, I.R., 1991. The epidemiologic association of Fuchs’ heterochromic iridocyclitis and ocular toxoplasmosis. Am. J. Ophthalmol. 111, 356 362. Schwartz, P.L., 1977. Segmental retinal periarteritis as a complication of toxoplasmosis. Ann. Ophthalmol. 9, 157 162. Sheets, C., Grewal, D., Greenfield, D.S., 2009. Ocular toxoplasmosis presenting with focal retinal nerve fiber atrophy simulating glaucoma. J. Glaucoma 18, 129. Sibley, L.D., Boothroyd, J.C., 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 359, 82 85. Available from: https://doi.org/ 10.1038/359082a0. Silveira, C., Belfort, R., Nussenblatt, R., Farah, M., Takahashi, W., Imamura, P., et al., 1989. Unilateral pigmentary retinopathy associated with ocular toxoplasmosis. Am. J. Ophthalmol. 107, 682 684. Silveira, C., Belfort Jr, R., Muccioli, C., Holland, G.N., Victora, C.G., et al., 2002. The effect of long-term intermittent trimethoprim/sulfamethoxazole treatment on

recurrences of toxoplasmic retinochoroiditis. Am. J. Ophthalmol. 134, 41 46. Silveira, C., Ferreira, R., Muccioli, C., Nussenblatt, R., Belfort Jr, R., 2003. Toxoplamosis transmitted to a newborn from the mother infected 20 years earlier. Am. J. Ophthalmol. 136, 370 371. Silveira, C., Muccioli, C., Holland, G.N., Jones, J.L., Yu, F., de Paulo, A., et al., 2015a. Ocular involvement following an epidemic of Toxoplasma gondii infection in Santa Isabel do Ivaı´, Brazil. Am. J. Ophthalmol. 159, 1013 1021.e3. Available from: https://doi.org/ 10.1016/j.ajo.2015.02.017. Silveira, C., Muccioli, C., Nussenblatt, R., Belfort Jr, R., 2015b. The effect of long-term intermittent trimethoprim/sulfamethoxazole treatment on recurrences of toxoplasmic retinochoroiditis: 10 years of follow-up. Ocul. Immunol. Inflamm. 23, 246 247. Smith, J.R., Cunningham, E.T., 2002. Atypical presentations of ocular toxoplasmosis. Curr. Opin. Ophthalmol. 13, 387 392. Smith, R.E., Ganley, J.P., 1972. Ophthalmic survey of a community. 1. Abnormalities of the ocular fundus. Am. J. Ophthalmol. 74, 1126 1130. Smith, J.M.A., Curi, A.L.L., Pavesio, C.E., 2001. Crystalluria with sulphadiazine. Br. J. Ophthalmol. 85, 1260. Sobrin, L., Kump, L.I., Foster, C.S., 2007. Intravitreal clindamycin for toxoplasmic retinochoroiditis. Retina 27, 952 957. Soheilian, M., Sadoughi, M.-M., Ghajarnia, M., Dehghan, M.H., Yazdani, S., Behboudi, H., et al., 2005. Prospective randomized trial of trimethoprim/sulfamethoxazole versus pyrimethamine and sulfadiazine in the treatment of ocular toxoplasmosis. Ophthalmology 112, 1876 1882. Soheilian, M., Ramezani, A., Azimzadeh, A., Sadoughi, M. M., Dehghan, M.H., Shahghadami, R., et al., 2011. Randomized trial of intravitreal clindamycin and dexamethasone versus pyrimethamine, sulfadiazine, and prednisolone in treatment of ocular toxoplasmosis. Ophthalmology 118, 134 141. Song, A., Scott, I.U., Davis, J.L., Lam, B.L., 2002. Atypical anterior optic neuropathy caused by toxoplasmosis. Am. J. Ophthalmol. 133, 162 164. Sousa, S., Ajzenberg, D., Vilanova, M., Costa, J., Darde´, M.L., 2008. Use of GRA6-derived synthetic polymorphic peptides in an immunoenzymatic assay to serotype Toxoplasma gondii in human serum samples collected from three continents. Clin. Vaccine Immunol. 15, 1380 1386. Available from: https://doi.org/10.1128/ CVI.00186-08. Spalter, H.F., 1966. Laser coagulation in retinal inflammatory disease. Int. Ophthalmol. Clin. 6, 359 378.

Toxoplasma Gondii

References

Stanford, M.R., Gilbert, R.E., 2009. Treating ocular toxoplasmosis: current evidence. Mem. Inst. Oswaldo Cruz 104, 312 315. Stanford, M.R., See, S.E., Jones, L.V., Gilbert, R.E., 2003. Antibiotics for toxoplasmic retinochoroiditis: an evidence-based systematic review. Ophthalmology 110, 926 932. Stanford, M.R., Tomlin, E.A., Comyn, O., Holland, K., Pavesio, C., 2005. The visual field in toxoplasmic retinochoroiditis. Br. J. Ophthalmol. 89, 812 814. Streilein, J.W., 1993. Immune privilege as the result of local tissue barriers and immunosuppressive microenvironments. Curr. Opin. Immunol. 5, 428 432. Available from: https://doi.org/10.1016/0952-7915 (93)90064-Y. Streilein, J.W., 2003. Ocular immune privilege: the eye takes a dim but practical view of immunity and inflammation. J. Leukoc. Biol. 74, 179 185. Available from: https://doi.org/10.1189/jlb.1102574. Su, C., Khan, A., Zhou, P., Majumdar, D., Ajzenberg, D., Darde´, M.-L., et al., 2012. Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proc. Natl. Acad. Sci. U.S.A. 109, 5844 5849. Available from: https://doi.org/10.1073/pnas.1203190109. Suhardjo, U.P.T., Agni, A.N., 2003. Clinical manifestations of ocular toxoplasmosis in Yogyakarta, Indonesia: a clinical review of 173 cases. Southeast Asian J. Trop. Med. Pub. Health 34, 291 297. Systematic Review on Congenital Toxoplasmosis (SYROCOT), 2007. Effectiveness of prenatal treatment for congenital toxoplasmosis: a meta-analysis of individual patients’ data. Lancet 369, 115 122. Available from: https://doi.org/10.1016/S0140-6736(07)60072-5. Tabbara, K.F., O’connor, G.R., 1975. Ocular tissue absorption of clindamycin phosphate. Arch. Ophthalmol. 93, 1180 1185. Talabani, H., Asseraf, M., Yera, H., Delair, E., Ancelle, T., Thulliez, P., et al., 2009. Contributions of immunoblotting, real-time PCR, and the Goldmann-Witmer coefficient to diagnosis of atypical toxoplasmic retinochoroiditis. J. Clin. Microbiol. 47, 2131 2135. Available from: https://doi.org/10.1128/JCM.00128-09. Tan, T.G., Mui, E., Cong, H., Witola, W.H., Montpetit, A., Muench, S.P., et al., 2010. Identification of T. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans. Vaccine 28, 3977 3989. Available from: https://doi.org/10.1016/ j.vaccine.2010.03.028. Tedesco, R.C., Smith, R.L., Corte-Real, S., Calabrese, K.S., 2005. Ocular toxoplasmosis in mice: comparison of two routes of infection. Parasitology 131, 303 307.

289

Tizard, I.R., Fish, A., Quinn, J.P., 1976. Some observations on the epidemiology of toxoplasmosis in Canada. J. Hyg. (Lond.) 77, 11 21. Torgerson, P.R., Mastroiacovo, P., 2013. The global burden of congenital toxoplasmosis: a systematic review. Bull. World Health Organ. 91, 501 508. Available from: https://doi.org/10.2471/BLT.12.111732. Torres-Morales, E., Taborda, L., Cardona, N., de-la-Torre, A., Sepulveda-Arias, J.C., Patarroyo, M.A., et al., 2014. Th1 and Th2 immune response to P30 and ROP18 peptides in human toxoplasmosis. Med. Microbiol. Immunol. 203, 315 322. Available from: https://doi. org/10.1007/s00430-014-0339-0. Trivin˜o-Valencia, J., Lora, F., Zuluaga, J.D., Gomez-Marin, J.E., 2016. Detection by PCR of pathogenic protozoa in raw and drinkable water samples in Colombia. Parasitol. Res. 115, 1789 1797. Available from: https:// doi.org/10.1007/s00436-016-4917-5. Turunen, H., Vuorio, K.A., Leinikki, P.O., 1983. Determination of IgG, IgM and IgA antibody responses in human toxoplasmosis by enzyme-linked immunosorbent assay (ELISA). Scand. J. Infect. Dis. 15, 307 311. Uchida, Y., Kakehashi, Y., Kameyama, K., 1978. Juxtapapillary retinochoroiditis with a psychiatric disorder possibly caused by toxoplasma. Am. J. Ophthalmol. 86, 791 793. Van der Lelij, A., Rothova, A., 1997. Diagnostic anterior chamber paracentesis in uveitis: a safe procedure? Br. J. Ophthalmol. 81, 976 979. Van Gelder, R.N., 2003. CME review: polymerase chain reaction diagnostics for posterior segment disease. Retina 23, 445 452. VanWormer, E., Carpenter, T.E., Singh, P., Shapiro, K., Wallender, W.W., Conrad, P.A., et al., 2016. Coastal development and precipitation drive pathogen flow from land to sea: evidence from a Toxoplasma gondii and felid host system. Sci. Rep. 6, 29252. Available from: https://doi.org/10.1038/srep29252. Vasconcelos-Santos, D.V., Azevedo, D.O.M., Campos, W.R., Ore´fice, F., Queiroz-Andrade, G.M., Carellos, E´.V.M., et al., 2009. Congenital toxoplasmosis in southeastern Brazil: results of early ophthalmologic examination of a large cohort of neonates. Ophthalmology 116, 2199 2205. Vasconcelos-Santos, D.V., Dodds, E.M., Ore´fice, F., 2011. Review for disease of the year: differential diagnosis of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 19, 171 179. Vaudaux, J.D., Muccioli, C., James, E.R., Silveira, C., Magargal, S.L., Jung, C., et al., 2010. Identification of an atypical strain of Toxoplasma gondii as the cause of a waterborne outbreak of toxoplasmosis in Santa Isabel

Toxoplasma Gondii

290

5. Ocular disease due to Toxoplasma gondii

do Ivai, Brazil. J. Infect. Dis. 202, 1226 1233. Available from: https://doi.org/10.1086/656397. Villard, O., Filisetti, D., Roch-Deries, F., Garweg, J., Flament, J., Candolfi, E., 2003. Comparison of enzymelinked immunosorbent assay, immunoblotting, and PCR for diagnosis of toxoplasmic chorioretinitis. J. Clin. Microbiol. 41, 3537 3541. Villard, O., Cimon, B., L’Ollivier, C., Fricker-Hidalgo, H., Godineau, N., Houze, S., et al., 2016. Serological diagnosis of Toxoplasma gondii infection: recommendations from the French National Reference Center for Toxoplasmosis. Diagn. Microbiol. Infect. Dis. 84, 22 33. Vutova, K., Peicheva, Z., Popova, A., Markova, V., Mincheva, N., Todorov, T., 2002. Congenital toxoplasmosis: eye manifestations in infants and children. Ann. Trop. Paediatr. 22, 213 218. Wakefield, D., Cunningham Jr, E.T., Pavesio, C., Garweg, J. G., Zierhut, M., 2011. Controversies in ocular toxoplasmosis. Ocul. Immunol. Inflamm. 19, 2 9. Wallon, M., Peyron, F., 2018. Congenital toxoplasmosis: a plea for a neglected disease. Pathogens 7, 25. Wallon, M., Kodjikian, L., Binquet, C., Garweg, J., Fleury, J., Quantin, C., et al., 2004. Long-term ocular prognosis in 327 children with congenital toxoplasmosis. Pediatrics 113, 1567 1572. Available from: https://doi. org/10.1542/peds.113.6.1567. Wallon, M., Garweg, J.G., Abrahamowicz, M., Cornu, C., Vinault, S., Quantin, C., et al., 2014. Ophthalmic outcomes of congenital toxoplasmosis followed until adolescence. Pediatrics 133, e601 e608. Available from: https://doi.org/10.1542/peds.2013-2153. Warren, J., Sabin, A.B., 1942. Effect of certain antiprotozoal drugs on Toxoplasma in vitro and in vivo. Exp. Biol. Med. 51, 15 18. Available from: https://doi.org/ 10.3181/00379727-51-13808. Watts, E., Zhao, Y., Dhara, A., Eller, B., Patwardhan, A., Sinai, A.P., 2015. Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. mBio 6, e01155 15. Weinberger, A.W.A., Lappas, A., Kirschkamp, T., Mazinani, B.A.E., Huth, J.K., Mohammadi, B., et al., 2006. Fundus near infrared fluorescence correlates with fundus near infrared reflectance. Invest. Ophthalmol. Vis. Sci. 47, 3098 3108. Westfall, A.C., Lauer, A.K., Suhler, E.B., Rosenbaum, J.T., 2005. Toxoplasmosis retinochoroiditis and elevated intraocular pressure: a retrospective study. J. Glaucoma 14, 3 10. Wilder, H.C., 1952a. Toxoplasma chorioretinitis in adults: a preliminary study of forty-one cases diagnosed by

microscopic examination. AMA Arch. Ophthalmol. 47, 425. Available from: https://doi.org/10.1001/archopht. 1952.01700030435003. Wilder, H.C., 1952b. Toxoplasma chorioretinitis in adults. AMA Arch. Ophthalmol. 48, 127 136. Williams, N., Miller, N.R., 1996. Neuroretinitis. Ocular Infection and Immunity. Mosby Year Book. Mosby, St. Louis, MO, pp. 601 608. Wilson, C.B., Remington, J.S., 1980. What can be done to prevent congenital toxoplasmosis? Am. J. Obstet. Gynecol. 138, 357 363. Wilson, W.B., Sharpe, J.A., Deck, J.H.N., 1980. Cerebral blindness and oculomotor nerve palsies in toxoplasmosis. Am. J. Ophthalmol. 89, 714 718. Winterhalter, S., Severing, K., Stammen, J., Maier, A.K., Godehardt, E., Joussen, A.M., 2010. Does atovaquone prolong the disease-free interval of toxoplasmic retinochoroiditis? Graefes Arch. Clin. Exp. Ophthalmol 248, 1187 1192. Witola, W.H., Mui, E., Hargrave, A., Liu, S., Hypolite, M., Montpetit, A., et al., 2011. NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells. Infect. Immun. 79, 756 766. Available from: https://doi.org/10.1128/ IAI.00898-10. Witola, W.H., Liu, S.R., Montpetit, A., Welti, R., Hypolite, M., Roth, M., et al., 2014. ALOX12 in human toxoplasmosis. Infect. Immun. 82, 2670 2679. Available from: https://doi.org/10.1128/IAI.01505-13. Wolf, A., Cowen, D., Paige, B., 1939. Human toxoplasmosis: occurrence in infants as an encephalomyelitis verification by transmission to animals. Science 89, 226 227. ´ Wujcicka, W., Gaj, Z., Wilczynski, J., Nowakowska, D., 2015. Contribution of IL6 2174 G . C and IL1B 13954 C . T polymorphisms to congenital infection with Toxoplasma gondii. Eur. J. Clin. Microbiol. Infect. Dis. 34, 2287 2294. Available from: https://doi.org/10.1007/ s10096-015-2481-z. ´ Wujcicka, W., Wilczynski, J., Nowakowska, D., 2017. Genetic alterations within TLR genes in development of Toxoplasma gondii infection among Polish pregnant women. Adv. Med. Sci. 62, 216 222. Available from: https://doi.org/10.1016/j.advms. 2017.02.002. Yahia, S. Ben, Herbort, C.P., Jenzeri, S., Hmidi, K., Attia, S., Messaoud, R., et al., 2008. Intravitreal bevacizumab (Avastin) as primary and rescue treatment for choroidal neovascularization secondary to ocular toxoplasmosis. Int. Ophthalmol. 28, 311 316.

Toxoplasma Gondii

References

Yanoff, M., Sassani, J.W., 2009. Ocular Pathology. Elsevier Health Sciences. Young, J., 2005. Infliximab and reactivation of cerebral toxoplasmosis. N. Engl. J. Med. 353, 1530 1531. Available from: https://doi.org/10.1056/NEJMc051556. Ysasaga, J.E., Davis, J., 1999. Frosted branch angiitis with ocular toxoplasmosis. Arch. Ophthalmol. 117, 1260 1261.

291

Zimmerman, L.E., 1961. Ocular pathology of toxoplasmosis. Surv. Ophthalmol. 6, 832 856. Zuluaga, L.M., Herna´ndez, J.C., Castan˜o, C.F., Donado, J. H., 2017. Effect of antenatal spiramycin treatment on the frequency of retinochoroiditis due to congenital toxoplasmosis in a Colombian cohort. Biome´dica 37, 86 91.

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C H A P T E R

6 Toxoplasmosis in wild and domestic animals David S. Lindsay1 and J.P. Dubey2 1

Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, United States 2Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD, United States

6.1 Introduction

6.2 Toxoplasmosis in wildlife

Toxoplasma gondii is widely distributed in wild and domestic animals. The present chapter reviews toxoplasmosis in wild and domestic animals. Coverage in wild animal species is limited to confirmed cases of toxoplasmosis, cases with parasite isolation, cases with parasite detection by polymerase chain reaction (PCR), and experimental infection studies (Figs. 6.1 6.3). Studies concerning serological prevalence have not been included for the majority of host species. This was done because many serological tests, e.g. latex agglutination (LAT), indirect fluorescent antibody (IFAT), and indirect hemagglutination), have been demonstrated to underestimate the prevalence of T. gondii.

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00006-2

6.2.1 Felids Congenital toxoplasmosis has been reported in bobcats (Felis rufus) kits (Dubey et al., 1987). Toxoplasmic meningoencephalitis has been observed in a 6-month-old bobcat (Smith et al., 1995). T. gondii has been isolated from the tissues of adult bobcats (Lindsay et al., 1997b; Dubey et al., 2004b). Bobcats are important in maintaining T. gondii in wild herbivores in many areas of the United States (Fig. 6.1). Oocysts excreted by cougars (Felis concolor) were thought to be the source of a large water borne outbreak of human toxoplasmosis in Victoria, British Columbia, Canada, and oocysts were isolated from the feces of cougars

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FIGURE 6.1 Fatal toxoplasmic encephalitis in a naturally infected bobcat. H&E stain. (A) Necrosis and inflammation of a blood vessel (arrow). Bar 5 50 µm. (B) Tachyzoites (arrows) in a capillary. Bar 5 10 µm. (C) Vasculitis and suppurative encephalitis. Bar 5 100 µm. (D) An abscess with degenerating neutrophils and tachyzoites (arrows). Bar 5 10 µm.

collected around the water shed (Aramini et al., 1998). Acute toxoplasmosis was reported in a 16week-old juvenile cheetah (Acinonyx jubatus) that was privately owned in Dubai, United Arab Emirates (Lloyd and Stidworthy, 2007). It was housed with three domestic cats and had been with its present owner for 3 weeks and was fed beef and quail. The cub died suddenly

with signs rapidly progressive pyrexia, tachypnea, abdominal effusion, and hepatomegaly (Lloyd and Stidworthy, 2007). T. gondii stages were demonstrated in multiple tissues using immunohistochemistry and PCR. T. gondii has been isolated by bioassay in mice from a jaguarundi (Puma yagouaroundi) (Pena et al., 2011), a Jaguar (Panthera onca) (Demar et al., 2008), Cougar (Puma concolor)

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FIGURE 6.2 Necrosis associated with Toxoplasma gondii in small intestine. H&E stain. (A) Necrosis of lamina propria (arrows) of villi 7 days after feeding oocysts to a mouse. The surface epithelium is not affected. Numerous tachyzoites are present in lesions but are not visible at this magnification. Bar 5 100 µm. (B) Necrosis of the lamina propria cells including blood vessels in a naturally infected Pallas’s cat. Numerous tachyzoites (small arrows) are present. The surface epithelium (large arrow) was not affected. Bar 5 10 µm.

(Dubey et al., 2008a,b), and sand cat (Felis margarita) (Dubey et al., 2010). Experimental infections resulting in oocyst excretion have been demonstrated in jaguarundi (P. yagouaroundi), ocelot (Furcifer pardalis), bobcats (Lynx rufus), and cheetah (A. jubatus) (Jewell et al., 1972; Miller et al., 1972). In general, these felids are not as efficient at producing oocysts as are domestic cats. Congenital toxoplasmosis is a major factor hindering breeding programs for endangered Pallas’s cats (Otocolobus manul) and sand cats (F. margarita) in zoos worldwide (see next).

6.2.2 Canids Acute toxoplasmosis has been reported in arctic foxes (Alopex lagopus) (Sorensen et al., 2005), Fennec foxes (Fennecus zerda) (Kottwitz et al., 2004), gray foxes (Urocyon cinereoargenteus) (Davidson et al., 1992; Dubey and Lin, 1994; Kelly and Sleeman, 2003), red foxes

(Vulpes vulpes) (Reed and Turek, 1985; Dubey et al., 1990; Kelly and Sleeman, 2003), and sand foxes (Vulpes rueppellii) (Pas and Dubey 2008c). Coinfection with canine distemper virus is often associated with clinical toxoplasmosis in gray (Davidson et al., 1992; Kelly and Sleeman, 2003) and red foxes (Reed and Turek, 1985). Clinical toxoplasmosis has not been documented in wolves, coyotes, hyenas, or dingos. T. gondii has been isolated from artic foxes (Dubey et al., 2011b), red foxes (Smith and Frenkel, 1995; Dubey et al., 2004b, 2011b), gray foxes (Dubey et al., 2004b), and coyotes (Lindsay et al., 1997b; Dubey et al., 2004b). Aubert et al. (2010) found modified agglutination test (MAT) antibodies in 14 of 19 (74%) red foxes from France and isolated T. gondii from the hearts of 9 (69%) of 13 seropositive red foxes. The isolates were all genotype Type II. Herrmann et al. (2012) used serology (immunoblot) and PCR to examine the prevalence of T. gondii in red foxes and rodents from the German Federal States of Brandenburg and

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6.2.3 Bears

FIGURE 6.3 Section of liver from a gazelle with toxoplasmosis showing a central area of hepatitis. Note Toxoplasma gondii (arrows) in hepatocytes at the periphery of the lesion. H&E stain. Bar 5 25 µm.

Saxony-Anhalt. They found 152/204 (74.5%) and 149/176 (84.7%) of red foxes in Brandenburg and Saxony-Anhalt were immunoblot positive, respectively, but none of 72 rodents (69 common voles Microtus arvalis, 2 Mediterranean water shrews Neomys anomalus, and 1 striped field mouse Apodemus agrarius) had antibodies to T. gondii. PCR was conducted on heart tissue from seropositive red fox tissues and 28/152 (18%) and 20/149 (13%) of seropositive foxes from Brandenburg and Saxony-Anhalt, respectively, were positive (Herrmann et al., 2012). PCR was done on heart and lung samples from the 72 rodents and none tested positive (Herrmann et al., 2012).

Clinical toxoplasmosis has not been reported from bears. Viable T. gondii has been isolated from black bears (Ursus americanus) (Dubey et al., 1995a) and brown bears (Ursus arctos horribilis) (Dubey et al., 2011b). The prevalence of T. gondii in black bears in the United States is the highest of any hosts for T. gondii worldwide. In a recent survey, T. gondii antibodies were found in 4% of dams and 5% of their nursing cubs while in dens; the study concluded that there is no transplacental transmission of T. gondii in bears and that 50% of bears acquire infection postnatally by their 10 months of age (Dubey et al., 2016). Compared with black bears, the prevalence of T. gondii in brown bears from Alaska is about half (44%) of black bears (Ramey et al., 2019). The prevalence of T. gondii in 527 polar bears (Ursus maritimus) was 3.6% in cubs still with their dam and 21.4% for subadults and adults from Svalbard and the Barents Sea and East Greenland (Oksanen et al., 2009) and 6% of 500 polar bears from the Beaufort and Chukchi sea areas of the Arctic Ocean (Rah et al, 2005). The prevalence in polar bears was 23.9% (33) of 105 animals from southern Beaufort Sea (Atwood et al., 2017) and in grizzly bears (U. arctos) (Chomel et al., 1995). Meat from any species of bear should be considered a potential source of T. gondii.

6.2.4 Raccoons Many serosurveys indicate that T. gondii is highly prevalent in raccoons (Procyon lotor) (reviewed by Hancock et al., 2005). Encysted T. gondii has been isolated from the tissues of naturally infected raccoons (Lindsay et al., 1997b; Dubey et al., 2004c, 2011b). Clinical toxoplasmosis has not been reported from raccoons and they are resistant to experimental infection (Dubey et al., 1993b).

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6.2.5 Squirrels Acute toxoplasmosis has been reported in gray squirrels (Sciurus carolinensis) (Dubey et al., 2006a), eastern fox squirrels (Sciurus niger) (Kumar et al., 2018), American red squirrels (Tamiasciurus hudsonicus) (Bangari et al., 2007), 13-lined ground squirrels (Citellus tridecemlineatus) (van Pelt and Dieterich, 1972), Eurasian red squirrels (Sciurus vulgaris) (Jokelainen and Nylund, 2012), Swinhoe’s striped squirrel (Tamiops swinhoei) (Fayyad et al., 2016), and Korean squirrels (Tanias sibericus) (Carrasco et al., 2006). T. gondii has been isolated from gray squirrels (Smith and Frenkel, 1995) and Formosan giant flying squirrels (Petaurista petaurista grandis) (Cross et al., 1969).

6.2.6 Rabbits and hares Fatal toxoplasmosis has been reported from three domestic (Oryctolagus cuniculus) rabbits from two different sources in the United States (Dubey et al., 1992a). The rabbits died after an acute illness characterized by fever, lethargy, and diarrhea in one rabbit and no clinical signs in the other two rabbits. The most striking lesion in all three rabbits was foci of necrosis of the spleen and liver associated with massive presence of multiplying tachyzoites (Dubey et al., 1992a). Similar findings were present in 2 18-month-old domestic rabbits from 15 flocks in Germany. Necropsy examinations of 49 rabbits revealed lesions of a generalized granulomatous-necrotizing toxoplasmosis within the spleen, liver, lungs, and lymph nodes (Bergmann et al., 1980). Both authors of the current chapter (DSL and JPD) have inoculated domestic rabbits orally and subcutaneously with T. gondii oocysts (usually 10,000/ rabbit) to generate immune serum for immunohistochemistry. All inoculated rabbits have or would have developed fatal toxoplasmosis had they not been euthanized for humane reasons. Viable T. gondii has been isolated from

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domestic rabbits (O. cuniculus) from Brazil (Dubey et al., 2011a). Brown hares (Lepus europaeus) develop fatal toxoplasmosis after experimental infection with as few as 10 oocysts and all inoculated hares died within 8 19 days after ingesting oocysts (Sedlak et al., 2000). The typical pathological finding in hares is hemorrhagic enteritis, enlargement and hyperemia of mesenteric lymph nodes, splenomegaly, and multiple necrotic lesions in the parenchyma of the liver and other organs (Sedlak et al., 2000). Mountain hare (Lepus timidus) experimentally inoculated with 50 T. gondii oocysts and examined 7 days later had gross lesions in the mesenteric lymph nodes and liver (Gustafsson et al., 1997). Histologically, the hares had extensive necrotic areas in the small intestine, mesenteric lymph nodes and liver, and less prominent foci of necrosis in various other organs (Gustafsson et al., 1997). Recent retrospective studies in Finland (Jokelainen et al., 2011) have documented natural toxoplasmosis in hares similar to these experimental reports. Acute generalized toxoplasmosis was demonstrated immunohistochemically, and T. gondii was confirmed as the cause of death in 14 (8%) of 173 European brown hares (L. europaeus) and 4 (3%) of 148 mountain hares (L. timidus) from Finland (Jokelainen et al., 2011). Aubert et al. (2010) demonstrated that 3 (13%) of 23 European brown hares (L. europaeus) from France were positive in the MAT but were not able to isolate T. gondii from the hearts of two seropositive animals.

6.2.7 Skunks and fisher T. gondii genotype III was isolated from three of six asymptomatic striped skunks (Mephitis mephitis) from Mississippi (Dubey et al., 2004d). Two of the three isolated were mouse pathogenic even thought they were molecularly consistent with the mouse avirulent genotype III. Lesions of toxoplasmosis and T. gondii parasites were not observed at necropsy of 37 striped

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skunks from Illinois (Gehrt et al., 2010). This population was serologically 60% positive for exposure to T. gondii (Gehrt et al., 2010). T. gondii was detected by PCR from brain and skeletal muscle of a free-ranging juvenile fisher (Martes pennanti) from Maryland (Gerhold et al., 2005). Clinically this animal had encephalitis, but it was not associated with the T. gondii infection. T. gondii antibodies were found using MAT in 100% of 38 and using IFAT in 71% of 45 fisher from Pennsylvania (Larkin et al., 2011).

6.2.8 Beavers T. gondii has been isolated from beaver (Castor canadensis) tissue (Dubey, 1983; Smith and Frenkel, 1995). Fatal systematic toxoplasmosis was seen in a 5-month-old beaver that was in a rehabilitation center in Connecticut (Forza´n and Frasca, 2004). Histologic lesions contained T. gondii positive stages by immunohistochemistry and consisted of lymphohistiocytic encephalitis, myocarditis, and interstitial pneumonia with multinucleated cells (Forza´n and Frasca, 2004).

6.2.9 Woodchuck and other large rodents Central nervous system toxoplasmosis has been observed in a woodchuck (Marmota monax) (Bangari et al., 2007) from New York. The woodchuck was euthanized because of progressive clinical signs of head tilt, circling, and rapid weight loss. The brain and heart were positive for T. gondii by immunohistochemistry and PCR (Bangari et al., 2007). Clinical toxoplasmosis has not been reported in capybara (Hydrochaeris hydrochaeris) or nutria (Myocastor coypus). However, the parasite has been isolated from capybara from Brazil (Yai et al., 2009) and T. gondii DNA has

been detected by PCR in nutria from Italy (Nardoni et al., 2011).

6.2.10 Insectivores Little is known about toxoplasmosis in insectivores. The prevalence of T. gondii using the Sabin Feldman dye test was ,1% in 578 insectivores from the Czech Republic (Hejlicek et al., 1997). Fatal toxoplasmosis was diagnosed in a juvenile male common mole (Talpa europaea) from Germany (Geisel et al., 1995). None of 70 T. europaea from the Netherlands were serologically positive using the latex agglutination test (LAT), but T. gondii DNA was detected by real-time PCR in the brain of two of these common moles (Krijger et al., 2014). The brains and/or hearts from 3 of 22 whitetoothed shrews (Crocidura russula) from organic pig farms in the Netherlands were positive for T. gondii by PCR (Kijlstra et al., 2008). In another study from organic pig farms from the Netherlands, none of the brains from 9 common shrews (Sorex araneus) and 2 (2%) brains from 102 white-toothed shrews (C. russula) were positive by PCR for T. gondii (Meerburg et al., 2012). None of two Mediterranean water shrews (N. anomalus) from Germany were positive by serology or PCR (Herrmann et al., 2012). T. gondii DNA was detected in the heart of 1 of 578 striped field mice (A. agrarius) from North Korea (Hong et al., 2014).

6.2.11 Bats Acute toxoplasmosis has been observed in a juvenile spectacled flying-fox (Pteropus conspicillatus) and a juvenile little red flying fox (Pteropus scapulatus) from Australia (Sangster et al., 2012). One was a captive born member of a colony, and the other was undergoing rehabilitation at a wildlife hospital. Severe, acute interstitial pneumonia with varying combinations of neutrophils, large foamy macrophages,

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and fibrin present within alveoli were seen in the lungs and T. gondii confirmed using immunohistochemistry. Lesions in the CNS consisted of multiple foci of gliosis, including gemistocytic astrocytes, at all levels of the cerebrum, cerebellum, and brainstem of the bats (Sangster et al., 2012). The bats are arboreal in nature, and it was suggested that the T. gondii infections might have been acquired in captivity by food accidentally contaminated with oocysts (Sangster et al., 2012). Isolation of T. gondii was reported from pipistrelle bats Vespertilio pipistrellus and the red night bat Nyctalus noctula from Alma-Ata, Kazakhstan, USSR (Galuzo et al., 1970). Inoculation of RH T. gondii did not induce clinical disease in red night bats in these studies (Galuzo et al., 1970). T. gondii is widely prevalent in bats, and the seropositivity in noncarnivorous bats is intriguing. In a study from Brazil antibodies to T. gondii were found in 22 species of bats (Cabral et al., 2014).

6.2.12 White-tailed and mule deer T. gondii is prevalent in deer from North America. Consumption of venison has been linked with clinical toxoplasmosis in humans (Sacks et al., 1983; Ross et al., 2001). Clinical toxoplasmosis has not been described from naturally infected deer in North America. T. gondii has been isolated from the tissues of white-tailed deer (Odocoileus virginianus) (Lindsay et al., 1991a,b, 1997b; Dubey et al., 2004b; Gerhold et al., 2017) and mule deer (Odocoileus hemionus) (Dubey, 1982). Viable T. gondii was isolated from 6 of 61 white-tailed deer fetuses from dams, which were in early pregnancy (45 85 days of gestation) from Iowa and 9 of 27 white-tailed deer fetuses from Minnesota dams which were in mid-gestation (130 150 days) of a gestational period of 7 months (Dubey et al., 2008b). The fetuses from T. gondii positive white-tailed and mule deer were negative for T. gondii antibodies in one

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study, suggesting that seropositive dams do not transmit the infection to their fetuses (Lindsay et al., 2006) unless an acute infection occurs during pregnancy. Acute toxoplasmosis and death can occur in mule deer experimentally inoculated with T. gondii oocysts (Dubey et al., 1982).

6.2.13 Other deer Congenital toxoplasmosis has been observed in reindeer (Rangifer tarandus) from a private collection in the United States (Dubey et al., 2002a). Yearling reindeer may develop enteritis and die after experimental oral infection with T. gondii oocysts (Oksanen et al., 1996). Aubert et al. (2010) demonstrated that 36 (60%) of 60 roe deer (Capreolus capreolus) from France were positive in the MAT and obtained 12 isolates from the hearts (38%) of 33 MAT positive roe deer. They also reported that T. gondii antibodies were present in 4 (17%) of 24 red deer (Cervus elaphus) (Aubert et al., 2010). One (25%) of four fallow deer (Dama dama) from France examined by Aubert et al. (2010) was positive (MAT titer 1:25), but attempts to isolate T. gondii by bioassay in mice were not successful.

6.2.14 Other wild ruminants Elk (Cervus canadensis) are resistant to clinical disease following oral infection with oocysts, but T. gondii can be isolated from many of their tissues indicating that elk are a potential source of infection for humans (Dubey et al., 1980). Antibodies to T. gondii were detected in sera of 221 of 317 (69.7%) elk from Pennsylvania collected during 2013 16 hunting season by the MAT and hearts from 2 of 20 elk were positive by bioassay (Dubey et al., 2017). Eighty of 142 (56.3%) elk from Kentucky were seropositive for T. gondii (Cox et al., 2017).

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T. gondii was as isolated by bioassay in mice from one of seven naturally infected moose (Alces alces) from Montana (Dubey, 1981). The isolate was not pathogenic for mice. Antibodies against T. gondii were detected in 8 of 79 (10%) moose from Minnesota tested by MAT (Verma et al., 2016). The parasite was isolate by bioassay of hearts from 3 of 68 of these moose. Two samples were from adults, and one was from a 3-week-old calve. Congenital toxoplasmosis was observed in a child from Alaska whose mother had consumed PCR positive moose meat (Colosimo et al., 2013). T. gondii was been isolated by bioassay in mice from 1 of 21 naturally infected pronghorn antelope (Antilocapra americana) from Montana (Dubey, 1981). The isolate was not pathogenic for mice. Acute toxoplasmosis and death can occur in pronghorn antelopes experimentally inoculated with T. gondii oocysts (Dubey et al., 1982). Toxoplasmic encephalitis has been observed in a 4-month-old Rocky Mountain bighorn sheep (Ovis canadensis canadensis) (Baszler et al., 2000). Aubert et al. (2010) demonstrated MAT antibodies in 7 (23%) of 31 mouflons (Ovis gmelini musimon) from France and isolated the parasite from 1 (25%) of 4 hearts from seropositive mouflons.

6.2.15 Sea otters and other marine mammals Toxoplasmosis was first recognized as a significant cause of mortality in southern sea otters (Enhydra lutris nereis) in the early 1990s (Cole et al., 2000). Encephalitis is the primary cause of T. gondii associated death in these sea otters (Cole et al., 2000). This was unexpected as sea otters do not ingest the usual intermediate hosts of T. gondii and their location in seawater keeps them segregated from cats. Definitive proof that T. gondii was killing the sea otters came when viable T. gondii was

isolated from the tissues of sea otters (Cole et al., 2000; Lindsay et al., 2001a), and isolated parasites from sea otters were shown to retain the ability to make oocysts when fed to cats (Cole et al., 2000). Initial isolates were all type II genotypes of T. gondii (Cole et al., 2000). It has been postulated that T. gondii oocysts excreted in the feces of feral cats living along the Pacific coast enter the marine environment and are ingested by sea otters when they feed on paratenic hosts (Cole et al., 2000), and this is supported by the fact that coastal freshwater runoff is a risk factor for T. gondii infection in southern sea otters (Miller et al., 2002). T. gondii oocysts will sporulate in seawater (Lindsay et al., 2003) and remain infectious for 1.5 years at room temperature and for at least 2 years at 4 C (Lindsay and Dubey, 2009), and viable T. gondii and T. gondii DNA can be recovered from experimentally inoculated bivalves (Lindsay et al., 2001b, 2004; Arkush et al., 2003), further supporting these assumptions. In addition, two species of filter feeding fish, northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax), have been shown to be able to remove T. gondii oocysts from seawater and can potentially serve as biotic vectors for T. gondii within the marine environment (Massie et al., 2010). T. gondii also cause deaths in other marine mammals off the Pacific coast of the United States often in the same areas as the sea otters (Miller et al., 2001), and it has also been isolated from Pacific harbor seal (Phoca vitulina) and California sea lion (Zalophus californianus). Toxoplasmosis is frequently reported from dolphins worldwide. Congenital toxoplasmosis has been reported in bottlenose dolphin (Tursiops aduncus) (Jardine and Dubey, 2002). Disseminated toxoplasmosis with transplacental fetal infection has been seen in a pregnant Risso’s dolphin (Grampus griseus) (Resendes et al., 2002). Acute cases of toxoplasmosis have been seen in humpbacked dolphins (Sousa chinensis) (Bowater et al., 2003), spinner dolphins

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(Stenella longirostris) (Migaki et al., 1990), striped dolphins (Stenella coeruleoalba) (DiGuardo et al., 2010), and Atlantic bottlenosed dolphins (Tursiops truncatus) (Inskeep et al., 1990). T. gondii has been isolated from the hearts of 3 of 52 bottlenose dolphins (T. aduncus) from the eastern United States by mouse bioassay (Dubey et al., 2008a). T. gondii was isolated from the brain of a stranded female striped dolphin (Stenella coeruleoalba) from Costa Rica that died from non T. gondii related causes (Dubey et al., 2007a). Toxoplasmosis has been reported from several additional species of marine mammals such as beluga whales (Delphinapterus leucas) (Mikaelian et al., 2000), Mediterranean fin whale (Balaenoptera physalus) (Mazzariol et al., 2012), California sea lion (Z. californianus) (Migaki et al., 1977), northern fur seal (Callorhinus ursinus) (Holshuh et al., 1985), elephant seal (Mirounga angustirostris) (Dubey et al., 2004a), Hawaiian monk seal (Monachus schauinslandi) (Honnold et al., 2005), Antillean manatee (Trichechus manatus manatus) (Dubey et al., 2003; Bossart et al., 2012), and West Indian manatee (T. manatus) (Buergelt and Bonde, 1983). Experimental infection of gray seals (Halichoerus grypus) with up to 10,000 T. gondii oocysts did not induce overt clinical disease (Gajadhar et al., 2004). Mild behavioral changes were the only adverse effects, and T. gondii was isolated from brain and muscles of the experimentally infected seals.

6.2.16 New world monkeys Toxoplasmosis can be a problem in exhibited new world monkeys (Table 6.1). Many reports of acute disease have come from squirrel monkeys (Saimiri sciureus) (Cedillo-Pela´ez et al., 2011; Epiphanio et al., 2003) and golden lion tamarins (Leontopithecus rosalia) (Dietz et al., 1997; Pertz et al., 1997; Juan-Salles et al., 1998; Epiphanio et al., 2003). Squirrel monkeys and

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TABLE 6.1 Summary of host species reports of clinical toxoplasmosis in New World primates. Cotton-top tamarin (Saguinus oedipus) Yellow-handed marmoset (Saguinus midas midas) Black marmoset (Saguinus midas niger) Emperor marmoset (Saguinus imperator) Red-bellied white-lipped tamarin (Saguinus labiatus) Black lion tamarin (Leontopithecus chrysopygus) Golden-headed lion tamarins (Leontopithecus chrysomelas) Golden lion tamarins (Leontopithecus rosalia) Squirrel monkeys (Saimiri sciureus) Pygmy marmoset (Callithrix pygmaea) Common marmoset (Callithrix jacchus) Black ear-tufted marmoset (Callithrix penicllata) Pale-headed saki (Pithecia pithecia) Night monkey (Aotus trivirgatus) Howler monkey (Alouatta fusca) Woolly monkey (Lagothrix lagotricha) Dietz, H.H., Henriksen, P., Bille-Hansen, V., Henriksen, S.A., 1997. Toxoplasmosis in a colony of New World monkeys. Vet. Parasitol. 68, 299 304; Bouer, A., Werther, K., Catao-Dias, J.L., Nunes, A.L., 1999. Outbreak of toxoplasmosis in Lagothrix lagotricha. Folia Primatol. (Basel) 70, 282 285; Epiphanio, S., Sa, L.R., Teixeira, R.H., Catao-Dias, J.L., 2001. Toxoplasmosis in a wild-caught black lion tamarin (Leontopithecus chrysopygus). Vet. Rec. 149, 627 628; Epiphanio, S., Sinhorini, I.L., Catao-Dias, J.L., 2003. Pathology of toxoplasmosis in captive new world primates. J. Comp. Pathol. 129, 196 204; Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans, second ed. CRC Press, Boca Raton, FL, pp. 1 313; Cedillo-Pela´ez, C., Rico-Torres, C.P., SalesGarrido C.G., Correa, D., 2011. Acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) in Mexico. Vet. Parasitol. 180, 368 371; Pena, H.F.J., Marvulo, M.F.V., Horta, M.C., Silva, M.A., Silva, J.C.R., Siqueira, D.B., et al., 2011. Isolation and genetic characterisation of Toxoplasma gondii from a red-handed howler monkey (Alouatta belzebul), a jaguarundi (Puma yagouaroundi), and a black-eared opossum (Didelphis aurita) from Brazil. J. Parasitol. 175, 377 381.

Panamanian night monkeys (Aotus lemurinus) are highly susceptible to oral tissue cyst inoculation and develop acute fatal disease (Harper et al., 1985; Escajadillo and Frenkel, 1991; Furuta et al., 2001). Pena et al. (2011) isolated T. gondii by bioassay in mice of its heart and brain of a

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young male red-handed howler monkey (Alouatta belzebul) with suspected toxoplasmosis from a zoo in Brazil.

6.2.17 Old world monkeys Toxoplasmosis is reported infrequently in old world monkeys. A case of concurrent central nervous system toxoplasmosis and simian immunodeficiency virus induced AIDS encephalomyelitis was seen in a Barbary macaque (Macaca sylvana) (Sasseville et al., 1995). Rhesus monkeys (Macaca mulatta) and stump-tailed macaques (Macaca arctoides) have been are used as experimental models for human congenital toxoplasmosis (Wong et al., 1979; Schoondermark-Van de Ven et al., 1993), and cynomolgus monkeys (Macaca fascicularis) have been used as a model for recurrent toxoplasmic retinochoroiditis (Holland et al., 1988).

6.2.18 American marsupials Clinical toxoplasmosis has not been reported from marsupials from the Americas. The parasite has been isolated from opossums (Didelphis virginiana) from Georgia (Dubey et al., 2011b) and Kansas (Smith and Frenkel, 1995) in the United States, and black-eared opossums (Didelphis aurita) from Brazil (Pena et al., 2011) have been examined and proven to be positive by bioassay in mice.

6.2.19 Australian marsupials T. gondii infection is usually life ending in marsupials from Australia or New Zealand. Outbreaks of toxoplasmosis often occur in these animals when housed in zoos (see next). These animals evolved in the absence of cats and T. gondii, and this may be why they are so highly susceptible. Canfield et al. (1990) summarized clinical signs, necropsy findings and histopathological

changes are summarized for 43 macropods (species not given), 2 common wombats (Vombatus ursinus), 2 koalas (Phascolarctos cinereus), 6 possums (species not given), 15 dasyurids (species not given), 2 numbats (Myrmecobius fasciatus), 8 bandicoots (species not given), and 1 bilby (Macrotis lagotis). The animals either died suddenly without clinical signs or exhibited signs associated with respiratory, neurological, or enteric disease. At necropsy, many had no visible lesions. Common necropsy findings included pulmonary congestion, edema and consolidation, adrenal enlargement and reddening, hemorrhage and ulceration of stomach and small intestine, and lymphadenomegaly and splenomegaly (Canfield et al., 1990). Congenital toxoplasmosis apparently occurs in black-faced kangaroos (Macropus fuliginosus melanops) based on the finding of T. gondii in the tissues of a 82 dayold joey that died from toxoplsmosis (Dubey et al., 1988b). T. gondii was seen in the heart, kidney, liver, lung, lymph node, spleen, small intestine, and stomach from two koalas (P. cinereus) that died suddenly in a fauna park in Sydney, Australia (Hartley et al., 1990). Experimental studies support the assumption that Australian marsupials are highly susceptible to toxoplasmosis. Eastern barred bandicoots (Perameles gunnii) develop acute toxoplasmosis when fed 100 T. gondii oocysts and died 15 and 17 days postinfection (Bettiol et al., 2000). Lesions consistent with acute toxoplasmosis were present in their tissues. The authors indicated that T. gondii may be a cause for a reduction in wild populations of eastern barred bandicoots. Tammar wallabies (Macropus eugenii) fed 500, 1000, or 10,000 T. gondii oocysts died of acute toxoplasmosis 9 15 days after challenge (Reddacliff et al., 1993). The lesions consisted of foci of necrosis and inflammation in the intestines, lymphoid tissue, adrenal cortex, heart, skeletal muscle and brain, and severe generalized pulmonary congestion and edema.

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6.2.20 African wildlife Surprisingly little is known about T. gondii and toxoplasmosis from African mammals. Clinical disease has not been reported from free-ranging elephants, hippopotamus, rhinos, giraffes, gazelle, wildebeests, impalas, chimpanzees, baboons, orangutans, and gorillas. T. gondii has not been isolated from these species that were naturally infected. Antibodies to T. gondii have been demonstrated in elephants, hippopotamus, rhinos, giraffes, wildebeests, and chimpanzees. Early after the life cycle in cats was discovered, an experimental study was done in two female chimpanzees fed T. gondii oocysts of the Beverly strain (Draper et al., 1971). One chimpanzee (female 1) was Sabin Feldman dye test negative and the other (female 2) was dye test positive (1:250) before the study was initiated. T. gondii was isolated by bioassay in mice from the blood (sampled 1 week PI), inguinal lymph node (sampled 11 weeks PI), and thigh muscle (sampled 11 weeks PI) of female 1 fed 2.5 3 106 T. gondii oocysts [but not cerebral spinal fluid (taken 11 weeks PI) (Draper et al., 1971)]. Clinically, female 1 became slightly anorexic, developed enlarged superficial lymph nodes, and seroconverted in the dye test to 1:8192 at 30 days PI. Female 2 was fed 1.5 3 106 T. gondii oocysts and did not demonstrate clinical signs or an increase in antibody titer nor was T. gondii isolated from blood samples by bioassay in mice (Draper et al., 1971).

6.2.21 Wild rodents A detailed discussion of T. gondii prevalence in wild rodents (mice and rats) is beyond the scope of this chapter. T. gondii has been isolated from the tissues from wild rodents worldwide (Dubey, 2010). Genotypes of these isolates are similar to isolates from other animals in the same geographic area. Dabritz et al. (2008) has recently reviewed what was known about the global serological

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prevalence of T. gondii in wild rodents. Until large-scale studies are conducted using bioassay or molecular detection methods, the role of wild rodents in maintaining T. gondii in the environment will not be fully understood. Properly conducted serological studies usually indicate that few (,10%) wild mice or rats are usually found to be seropositive. For example, 2 (0.8%) of 238 rats (Rattus norvegicus) from Grenada, West Indies, were found to be serologically positive using the MAT (Dubey et al., 2006b). When the brains and hearts of all 238 rats were examined by bioassay in mice, T. gondii was isolated from only 1 of the 238 rats with the positive rat being one of the 2 serologically positive animals. This clearly demonstrates a low prevalence of T. gondii infection in this rat population.

6.2.22 Wild birds Table 6.2 lists the wild avian hosts from which viable T. gondii has been isolated, and Table 6.3 lists the avian species that have been reported to suffer from clinical toxoplasmosis. T. gondii is readily isolated from the hearts and breast muscles of raptors (Lindsay et al., 1993; Dubey et al., 2011b; Table 6.2). Necrotizing myocarditis caused by T. gondii has been observed in a bald eagle (Haliaeetus leucocephalus) from New Hampshire (Szabo et al., 2004). Severe toxoplasmic hepatitis was seen in an adult barred owl (Strix varia) from Quebec, Canada (Mikaelian et al., 1997). No clinical signs were seen in three red-tailed hawks (Buteo jamaicensis) fed T. gondii tissue cysts (Lindsay et al., 1991a,b). T. gondii was isolated from all three red-tailed hawks. No clinical signs were seen in great horned owls (Bubo virginianus), barred owls (S. varia), or screech owls (Asio otus) feed T. gondii tissue cysts (Dubey et al., 1992b), but parasites were isolated from the tissues of the owls at necropsy. T. gondii was not reisolated from a sparrow hawk (Falco sparverius) that had been experimentally infected (Miller et al., 1972).

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TABLE 6.2 Host records for Toxoplasma gondii isolation from wild birds.

Pigeon (Columba livia) Ruddy ground dove (Columbina talpacoti)

Anseriformes Mallards (Anas platyrhynchos) Pochard (Aythya ferrina) Tufted ducks (Aythya fuligula) Pintail (Anas acuta) Gadwall (Anas strepera) Canada goose (Branta canadensis) Accipitriformes Goshawk (Accipiter gentilis) Cooper’s hawk (Accipiter cooperi) Common buzzard (Buteo buteo) Kestrel (Falco tinnunculus) American kestrel (Falco sparverius) Pallid harrier (Circus macrourus) Bald eagle (Haliaeetus leucocephalus) Black vulture (Aegypius monachus) Red-tailed hawk (Buteo jamaicensis) Red-shouldered hawk (Buteo lineatus) Galliformes Partridge (Perdix perdix) Pheasant (Phasianus colchicus) Wild turkey (Meleagris gallopavo) Gruiformes Coot (Fulica atra) Charadriformes Blackheaded gull (Larus ridibundus)

Strigiformes Ferruginous pygmy owl (Glaucidium brasilianum) Little owl (Athene noctua) Great horned owl (Bubo virginianus) Barred owl (Strix varia) Passeriformes Great gray shrike (Lanius excubitor) Yellowhammer (Emberiza citrinella) Chaffinch (Fringilla coelebs) House sparrow (Passer domesticus) Tree sparrow (Passer montanus) Jay (Garrulus glandarius) Starling (Sturnus vulgaris) Palm tanager (Thraupis palmarum) Blackbird (Turdus merula) Mistle thrush (Turdus viscivorus) Song thrush (Turdus philomelos) Robin (Erithacus rubecula) Great tit (Parus major) Nuthatch (Sitta europaea) Treecreeper (Certhia familiaris) Greenfinch (Chloris chloris) American crow (Corvus brachyrhynchos) Carrion crow (Corvus corone) Jackdaw (Corvus monedula) Rook (Corvus frugilegus)

Common tern (Sterna hirundo) Columbiformes Collared dove (Streptopelia decaocto) Laughing dove (Streptopelia senegalensis)

Dubey, J.P., 2002. A review of toxoplasmosis in wild birds. Vet. Parasitol. 106, 121 153; Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans, second ed. CRC Press, Boca Raton, FL, pp. 1 313; Lindsay, D. S., Smith, P.C., Hoerr, F.J., Blagburn, B.L., 1993. Prevalence of encysted Toxoplasma gondii in raptors from Alabama. J. Parasitol. 79, 870 873.

Woodpigeon (Columba palumbus)

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6.3 Toxoplasmosis in zoos

TABLE 6.3 List of avian species in which clinical toxoplasmosis has been reported.

Galliformes Wild turkeys (Meleagris gallapavo)

Columbiformes Common pigeon (Columba livia)

Partridges (Perdix perdix)

Crown pigeons (Goura sp.)

Capercaillie (Tetrao urogallus)

Torres Strait pigeon (Ducula spilorrhoa)

Erckel’s francolin (Francolinus erckelii)

Wonga pigeon (Leucosarcia melanoleuca)

Guinea fowl (Numida meleagris) Anseriformes

Bleeding-heart dove (Gallicolumba luzonica) Nicobar pigeon (Caloenas nicobarica)

Magpie geese (Anseranas semipalmata)

Luzon bleeding-heart pigeon (Gallicolumba luzonica)

Hawaiian nene goose (Nesochen sandicensis) Sphenisciformes

Orange-breasted green pigeon (Treron bicinta) Crested wood partridge (Rolulus roul roul)

Humboldt penguin (Spheniscus humboldti)

Yellow-headed rockfowl (Picathartes gymnocephaus)

Megellanic penguin (Spheniscus magellanicus)

Passeriformes

Black-footed penguin (Spheniscus demersus)

Canaries (Serinus canarius)

Little penguin (Eudyptula minor)

Greenfinches (Carduelis chloris)

Indian pangolin (Manis crassicaudato)

Goldfinches (Carduelis carduelis) Sirkins (Carduelis spinus) Linnets (Carduelis cannabina) Bullfinches (Pyrrhula pyrrhula) Hawaiian crow (Corvus hawaiiensis) Satin bowerbird (Ptilornorhyncus violaceus)

Pelecaniformes Red-footed booby (Sula sula) Dubey, J.P., Lewis, B., Beam, K., Abbitt, B., 2002a. Transplacental toxoplasmosis in a reindeer (Rangifer tarandus) fetus. Vet. Parasitol. 110, 131 135; Dubey, J.P., Tocidlowski, M.E., Abbitt, B., Llizo, S.Y., 2002b. Acute visceral toxoplasmosis in captive dik-dik (Madoqua guentheri smithi). J. Parasitol. 88, 638 641; Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans, second ed. CRC Press, Boca Raton, FL, pp. 1 313.

Regent bowerbird (Sericulus chrysocephalus) Red-whiskered bulbul (Pycnonotus jocosus) Psittaciformes Budgerigars (Melopsittacus undulatus) Regent parrot (Polytelis anthopeplus) Superb parrot (Polytelis swansonii)

Viable T. gondii was isolated from the hearts of 8 of 16 wild turkeys (Meleagris gallopavo) from Alabama (Lindsay et al., 1994). Fatal systemic toxoplasmosis has been seen in wild turkeys from Georgia (Howerth and Rodenroth, 1985) and West Virginia (Quist et al., 1995).

Red lory (Eos bornea) Swainson’s lorikeet (Trichologlossus moluccanus)

6.3 Toxoplasmosis in zoos

Crimson rosella (Platycercus elegans) Strigiformes barred owl (S. varia)

Toxoplasmosis is a zoo management problem because wild felids can excrete T. gondii oocysts in their feces (Jewell et al., 1972; Miller et al., 1972; Lukesova and Literak, 1998) and

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feral cats occur in zoos (Gorman et al., 1986). Oocysts excreted by these felids can make their way into highly susceptible species. Mammalian species that frequently develop toxoplasmosis in zoos include Australian marsupials (Portas, 2010), New World and arborial monkeys (Dietz et al., 1997; Pertz et al., 1997; Juan-Salles et al., 1998; Epiphanio et al., 2000), lemurs (Dubey et al., 1985), and Pallas’s cats (O. manul) (Riemann et al., 1974; Dubey et al., 1988a, 2002a,b; Basso et al., 2005) (Fig. 6.2). Lesions in these animals are consistent with acute toxoplasmosis and are usually most severe in visceral tissues such as the lungs, liver, and spleen. Toxoplasmosis is common in lemurs exhibited in zoo worldwide (Dubey et al., 1985). A female ring-tailed lemur (Lemur catta) died of toxoplasmosis in a zoo in Spain 1 week after the delivery of 4 stillborn offspring which all had disseminated toxoplasmosis (Juan-Salle´s et al., 2011). T. gondii was isolated from the tissues of a 3-year-old secundiparous female ring-tailed lemur from a zoo in Alabama that died of acute toxoplasmosis (Spencer et al., 2004). The isolate was not pathogenic for mice and was genetically a Type II isolate. This case points out the difficulty in preventing toxoplasmosis in highly susceptible animals because this lemur was housed in a group on an island in the zoo (Spencer et al., 2004), making it easier to prevent contact with feral cats. Oocysts on the lemur’s food (fruit etc.) or carried in by black birds were considered likely sources of infection in this case (Spencer et al., 2004). Sporadic cases of acute toxoplasmosis have been reported in exhibited dik-dik (Madoqua guentheri smithi) (Dubey et al., 2002b), slendertailed meerkats (Suricata suricatta) (Juan-Salles et al., 1997), African crested porcupines (Hystrix cristata) (Harrison et al., 2007), New World porcupines (Erethizontidae sp.) (Fayyad et al., 2016), and Brazilian prehensile-tailed porcupines (Coendou mexicanus) (Morales et al., 1996). Fatal disseminated toxoplasmosis in three captive slender-tailed meerkats (S. suricatta) in a

zoo in La Plata, Argentina, was found to be caused by the normally nonpathogenic genotype Type III isolate of the parasite suggesting that meerkats are highly susceptible to infection (Basso et al., 2009). A case of abortion due to T. gondii has been reported in a Greenland muskox (Ovibos moshatus wardi) (Crawford et al., 2000). Fatal toxoplasmosis was reported in a 7-yearold giant panda (Ailuropoda melanoleuca) in a zoo in China (Ma et al., 2015). The animal had acute gastrointestinal and respiratory signs and T. gondii was seen in lung lesions. It was anorexic and lethargic and died 2 days after it signs developed despite supportive treatment with intramuscular cephalosporin and intravenous infusion of glucose solution. Parasite DNA was detected in the liver, spleen, lung, kidney, and intestines using PCR. Antibodies to T. gondii were detected in sera from 7 of 19 giant pandas in the breeding program at the Chengdu Research Base of Giant Panda Breeding in Sichuan, China (Loeffler et al., 2007). Abortion and neonatal death have been observed in captive nilgais (Boselaphus tragocamelus). T. gondii DNA was demonstrated in the tissues of the nilgais using PCR (Sedlak et al., 2004). Fatal toxoplasmosis was diagnosed in a captive, adult female saiga antelope (Saiga tatarica). T. gondii was detected in the liver, lung, spleen, kidney, and intestine and confirmed by PCR (Sedlak et al., 2004). Acute toxoplasmois has been seen in captive Cuvier’s gazelle (Gazella cuvieri), slender-horned gazelle (Gazella leptoceros), dama gazelle (Gazella dama), and gerenuk (Litocranius walleri) housed in North American Zoos (Stover et al., 1990; Junge et al., 1992). These infections are disseminated and most lesions are in the liver (Fig. 6.3), lungs, lymph nodes, adrenal glands, spleen, intestines, and brain. Outbreaks of toxoplasmosis also occur in avian species exhibited in zoos (Poelma and Zwart, 1972; Hubbard et al., 1986). Toxoplasmosis in canaries has been reported from aviaries worldwide (reviewed by Dubey, 2002). T. gondii

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genotype III was isolated from five of five blackwinged lorys (Eos cyanogenia) from an acute toxoplasmosis outbreak in an aviary in South Carolina (Dubey et al., 2004d). Acute systemic toxoplasmosis was reported to be the cause of death of 3 of 10 Nicobar pigeons (Caloenas nicobarica) in an aviary collection in South Africa (Las and Shivaprasad, 2008). Feral cats were a known management problem and lesions were consistent with oocyst-acquired infection. Three 1 3month-old black-footed penguin chicks (Spheniscus demersus) died from acute toxoplasmosis within 24 hours of showing central nervous signs (Ploeg et al., 2011). The birds were housed in a baby penguin cre`che in a zoo in the Netherlands. A cat with a litter of kittens had recently been observed feeding on fish intended for the penguins in the zoo, and the cat was suspected as the source of infection (Ploeg et al., 2011). Management and husbandry programs can be designed to help achieve prevention of toxoplasmosis in highly susceptible species in zoos and aviaries. Felids should never be fed fresh unfrozen meats because of the possibility contamination with T. gondii tissue cysts. Meat that has been frozen solid and then thawed can be safely fed because freezing kills T. gondii tissue cysts (Kotula et al., 1991). Feral cats should be actively controlled in zoos to prevent them from shedding oocysts. Highly susceptible species should not be housed near felids. Outdoor aviaries are at risk because of oocysts excreted by domestic cats. Aviaries should be designed to exclude cat feces and transport hosts (flies, roaches, etc.) that may bring in T. gondii on or in their bodies.

6.4 Toxoplasma gondii and endangered species Toxoplasmosis can adversely affect endangered avian and mammalian species. The ‘Alala (Hawaiian crow, Corvus hawaiiensis) is an

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endangered species, and only about 25 were left in captivity and the wild in 2000 (Work et al., 2000). Tragically, these birds are highly susceptible to fatal toxoplasmosis and develop disease after being introduced back in to the wild. Toxoplasmosis appears to pose a significant threat and management challenge to reintroduction programs for ‘Alala in Hawaii (Work et al., 2000). Captive breeding groups of golden lion tamarins (L. rosalia) have developed acute toxoplasmosis and suffered many fatalities both in North American and European zoos (Pertz et al., 1997; Juan-Salles et al., 1998). These arboreal monkeys are endangered and attempts to breed them in captivity for eventual release in the wild are hammered, because it is difficult to keep them from being exposed to T. gondii. Repeated transplacental transmission of T. gondii by Pallas’s cats maybe responsible for the high rate of impact of this disease on the Pallas’s cat population in zoos. Efforts by North American zoos to establish genetically viable captive populations of Pallas’s cats (O. manul) have been compromised by high newborn kitten mortality due to toxoplasmosis (Brown et al., 2005). In their natural environment, Pallas’s cats generally have little exposure to T. gondii, and it is believed that they acquire T. gondii infection after captivity (Brown et al., 2005). The mortality rate for toxoplasmosis of Pallas’s cat kittens born in Zoos in the United States is 35% 60% (Kenny et al., 2002; Brown et al., 2005). Sand cats (F. margarita) housed at the Breeding Centre for Endangered Arabian Wildlife in the United Arab Emirates and Al Wabra Wildlife Preservation, Qatar, have been reported to suffer from congenital (Pas and Dubey 2008a) and acquired toxoplasmosis (Dubey et al., 2010). Serological examination of endangered Gordon’s wildcat (Felis silvestris gordoni) kept at the same institution (Pas and Dubey 2008b) indicated that seropositive Gordon’s wildcats were present but no clinical

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history consistent with toxoplasmosis has been reported in these animals. Unlike domestic cats, Sand cat (F. margarita) queens will repeatedly infect litters of kittens making it very difficult to keep up numbers of healthy kittens in breeding programs. Fortunately, Gordon’s wildcats appear to behave like domestic cats in their responses to T. gondii infection.

6.5 Toxoplasmosis in pets 6.5.1 Cats Most cats are asymptomatic during a primary T. gondii infection. Fever (40.0 C 41.7 C) is present in many cats with clinical toxoplasmosis. Clinical signs of dyspnea, polypnea, icterus, and signs of abdominal discomfort were the most frequent findings in 100 cats with histologically confirmed toxoplasmosis (Dubey and Carpenter, 1993). Uveitis and retinochoroiditis are also common clinical signs in cats with toxoplasmosis. Gross and microscopic lesions are found in many organs but are most common in the lungs. Gross lesions in the lungs consist of edema and congestion, failure to collapse, and multifocal areas of firm, white to yellow, discoloration. Pericardial and abdominal effusions may be present. The liver is the most frequently affected abdominal organ and diffuse necrotizing hepatitis may be visible grossly. Gross lesions associated with necrosis can also be observed in the mesenteric lymph nodes and pancreas. All ages, sexes, and breeds of domestic cats are susceptible to T. gondii infection (Dubey et al., 1977). Transplacentally or lactogenically infected kittens will excrete oocysts, but the prepatent period is usually 3 weeks or more, because the kittens are infected with tachyzoites (Dubey et al., 1995b). Domestic cats under 1 year of age produce the most numbers of T. gondii oocysts. Cats that are born and raised outdoors usually become infected with

T. gondii shortly after they are weaned and begin to hunt. T. gondii naive adult domestic cats will excrete oocysts if fed tissue cysts, but they usually will excrete fewer numbers of oocysts and excrete oocysts for a shorter period of time than recently weaned kittens. Intestinal immunity to T. gondii is strong in cats that have excreted oocysts (Dubey, 1995). Primary T. gondii infection in cats does not cause immunosuppression (Lappin et al., 1992; Davis and Dubey, 1995). Serum antibody does not play a significant role in resistance to intestinal infection and intestinal immunity is most likely cell mediated. Oocysts begin to be excreted in the feces before IgM, IgG, or IgA antibodies are present in the serum (Lappin et al., 1989). Partial development of the enteroepithelial stages occur in the intestines of immune cats, but oocyst production is prevented (Davis and Dubey, 1995). Most cats that have excreted oocysts once do not reexcrete oocysts if challenged within 6 months to 1 year. Intestinal immunity will last up to 6 years in about 55% of cats (Dubey, 1995). Vaccination of cats against intestinal T. gondii infection has been successfully achieved using a mutant strain (T-263) of the parasite (Frenkel et al., 1991; Freyre et al., 1993). Oral administration of strain T-263 bradyzoites results in intestinal infection but does not result in oocyst production in cats. These vaccinated cats do not excrete oocysts when challenged with oocyst producing strains of T. gondii. The T-263 strain is safe to use in healthy cats. It is not recommended for use in pregnant cats or FeLV positive cats or immunocompromised cats (Choromanski et al., 1994, 1995). It has only limited ability to persist in the tissues of cats and cannot survive more than 3 back-passages in cats. No reversion to oocyst excretion or increase in virulence has been observed in over 200 inoculated cats. The T-263 strain is rapidly cleared from the mouth of inoculated cats. It is logical to assume that cat owners and veterinarians would be at a greater risk for

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developing toxoplasmosis; however, serological studies do not confirm this assumption. In one study in AIDS patients, it was conclusively shown that owning cats did not increase the risk of developing toxoplasmosis (Wallace et al., 1993). The role of cat ownership and exposure to T. gondii is, however, not completely clear at present. Many studies have been conducted to determine the association between cat ownership or cat exposure and the prevalence of T. gondii infection in humans. Many studies do not find a positive relationship while many find a positive relationship. It must be stressed that preventing exposure to cats is not the same as preventing exposure to T. gondii oocysts. Pregnant women or immunocompromised individuals should not change the cat’s litter box. If feces are removed daily, this will also help prevent exposure by removing oocysts before they can sporulate. T. gondii oocyst can survive in the soil for years and can be disseminated from the original site of deposition by erosion, other mechanical means, and by phoretic vectors. Inhalation of oocysts stirred up in the dust by horses has been associated with an outbreak of human toxoplasmosis at a riding stable (Teutsch et al., 1979). Oocysts are not likely to remain in the air for extended periods of time. Washing fruits and vegetables and wearing gloves while gardening are means of preventing exposure to oocysts. T. gondii oocysts were not isolated from the fur of oocyst-excreting cats (Dubey, 1995). Therefore it is unlikely that infection can be obtained by petting a cat. Tachyzoites are not likely to be present in the oral cavity of cats with active T. gondii infection, and none would be in a chronic infection; therefore it is unlikely that a cat bite would transmit T. gondii infection. Cat scratches are also unlikely to transmit T. gondii infection. T. gondii has been isolated from the tissues from domestic cats worldwide (Dubey, 2010). Genotypes of feline isolates are similar to

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isolates from other animals in the same geographic area.

6.5.2 Dogs T. gondii was once confused with Neospora caninum as a cause of disease in dogs, and many reports of toxoplasmosis in dogs are actually neosporosis (Dubey and Lindsay, 1996; Lindsay and Dubey, 2000). True toxoplasmosis does occur in dogs (Dubey et al., 1989). Clinical toxoplasmosis in dogs is often associated with immunosuppression induced by canine distemper virus infection. Clinical signs are usually most apparent in the respiratory and hepatic systems and probably result from reactivation of latent infections (Dubey et al., 1989). Transplacental infections have not yet been confirmed in naturally infected dogs. Dogs are resistant to experimental infection with tissue cysts and oocysts (Lindsay et al., 1996, 1997a). A role for dogs in the transmission of T. gondii to humans has been postulated based on serological surveys and observations that dogs ingest cat feces and often role in cat feces and other foul smelling substances (Frenkel et al., 2003). It is believed that dogs can bring oocysts to a home after ingesting them and deposit them in or around the home when they defecate. Experimentally infective T. gondii oocysts can be found in dog feces for up to 2 days after they ingest oocysts (Lindsay et al., 1997a). T. gondii oocysts will not sporulate when placed on dog fur (Lindsay et al., 1997a). Schares et al. (2005) found viable T. gondii oocysts in 2 of 24,089 dogs in Germany. The role of dogs as potential transport hosts for T. gondii needs further examination. T. gondii has been isolated from the tissues from domestic dogs worldwide (Dubey, 2010). Genotypes of feline isolates are similar to isolates from other animals in the same geographic area. A related

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Apicomplexan parasitic protozoa N. caninum is present in dogs.

6.5.3 Ferrets Congenital toxoplasmosis has been observed in farmed razed ferrets (Mustela putorius furo) from New Zealand (Thornton and Cook, 1986). Thirty percent of the kits on the farm died acutely and had lesions of disseminated toxoplasmosis. An epizootic of toxoplasmosis occurred among a population of endangered black-footed ferrets (Mustela nigripes) at a zoo in the United States (Burns et al., 2003). Twenty-two adults and 30 kits died from acute toxoplasmosis and an additional 13 adults died from chronic toxoplasmosis after the initial outbreak.

6.6 Domestic farm animals 6.6.1 Mink Acute toxoplasmosis with abortions has been reported in farmed mink (Mustela vison) from Europe and the United States (Frank, 2001; Smielewska-Los and Turniak, 2004). The practice of feeding nonfrozen slaughter offal was blamed for acute toxoplasmosis in one report (Smielewska-Los and Turniak, 2004). Toxoplasmosis was diagnosed using PCR and immunohistochemistry in a young free-ranging mink (M. vison) that had signs of left hind limb lameness, ataxia, head tremors, and bilateral blindness and was found on a college campus in Michigan (Jones et al., 2006). T. gondii has been isolated from wild mink from the United States (Smith and Frenkel, 1995).

6.6.2 Horses Horses are resistant to experimental infection with 1 3 104 or 1 3 105 oocysts. T. gondii can persist in edible tissues of horses for up to

476 days (Dubey, 1985). Although T. gondii has been isolated from tissues of horses, there is no confirmed report of clinical toxoplasmosis in horses (Al-Khalidi and Dubey, 1979). A related Apicomplexan parasitic protozoa Neospora hughesi is present in horses.

6.6.3 Swine Abortion in sows is the most common sign of toxoplasmosis in swine. Sows only abort once. Abortions are rare in most pork producing regions of the world with the exception of Taiwan. Pigs raised on dirt are more likely to have T. gondii in their tissues. Diagnosis of T. gondii abortion in sows is best done by examining fetal fluids for antibodies using the modified agglutination test. Undercooked pork is a source of human infection, and viable tissue cysts can remain in pork for up to 865 days (Dubey, 1988). T. gondii has been isolated from the tissues from domestic pigs worldwide (Dubey, 2010). Genotypes of pig isolates are similar to isolates from other animals in the same geographic area.

6.6.4 Cattle Clinical toxoplasmosis in cattle is rare, and abortions are uncommon. Many reports of bovine abortion due to T. gondii are actually due to N. caninum (Dubey and Lindsay, 1996). Attempts to isolate T. gondii from seropositive cattle are often unsuccessful indicating that beef may not be a significant source of human infection in the United States (Dubey et al., 2005). For example, no T. gondii was isolated from 2094 samples of beef obtained from retail markets in the United States (Dubey et al., 2005). However, viable tissue cysts can remain in cattle for up to 1191 days (Dubey and Thulliez, 1993). Additional studies are needed to full document these experimental findings.

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6.6.5 Sheep T. gondii is a common cause of abortion in ewes and an important production problem. Multiple abortions can occur in a flock indicating a common oocyst source for ewes. Ewes develop solid immunity after aborting T. gondii infected fetuses. A vaccine to prevent abortion in ewes is available in several countries (Buxton and Innes, 1995). Diagnosis of T. gondii abortion in ewes is best done by examining fetal fluids for antibodies using the modified agglutination test. Undercooked lamb and mutton is a source of human infection. T. gondii has been isolated from the tissues from domestic sheep worldwide (Dubey, 2010). Genotypes of sheep isolates are similar to isolates from other animals in the same geographic area.

6.6.6 Goats T. gondii is a common cause of abortion in does. Multiple abortions can occur in a flock indicating a common oocyst source for does. Does develop immunity after aborting T. gondii infected fetuses but repeat abort can occur. Diagnosis of T. gondii abortion in goats is best done by examining fetal fluids for antibodies using the modified agglutination test. Undercooked goat meat is a source of human infection. T. gondii has been isolated from the tissues from domestic goat worldwide (Dubey, 2010). Genotypes of goat isolates are similar to isolates from other animals in the same geographic area. Drinking raw not pasteurized goats milk is a potential source of T. gondii for humans and the parasite can survive for up to 7 days in refrigerated goat milk (Walsh et al., 1999).

6.6.7 Buffalos Naturally occurring clinical toxoplasmois has not been observed in buffalos (Bison bison, Bubalus bubalis, Syncerus caffer) and viable

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T. gondii has not been isolated from buffaloes. Serological surveys indicate that buffalos are exposed to the parasite.

6.6.8 Camels Acute toxoplasmosis was observed in a 6-year-old camel (Camelus dromedarius) (Hagemoser et al., 1990). Dyspnea was the main clinical sign and many tachyzoites were found in its lungs and plural exudates. T. gondii has been isolated from camel meat using cat bioassays (Hilali et al., 1995).

6.6.9 Llamas, alpaca, and vicunas Experimental studies indicate that llamas (Lama glama) are resistant to clinical toxoplasmosis even if challenged during pregnancy (Jarvinen et al., 1999). Naturally occurring toxoplasmosis has not been reported in llamas, alpacas (Lama pacos), or vicunas (Lama vicugna).

6.6.10 Chickens Chickens (Gallus domesticus) that are raised on the ground are a potential source of T. gondii due to high level of exposure to oocysts. Chickens usually do not develop clinical signs even after oral inoculation of large numbers of oocysts (Dubey et al., 1993c; Kaneto et al., 1997). Egg production may adversely be affected in laying hens fed large numbers of oocysts, but T. gondii is not readily transmitted to the eggs of these hens (Biancifiori et al., 1986). Clinical toxoplasmosis does not occur on modern chicken farms where birds are raised indoors. Chickens raised in modern production facilities in confinement indoors are not likely to have viable T. gondii in their tissues. None of 2094 samples from commercial chickens in retail markets from the United States contained viable T. gondii in a survey from the United States (Dubey et al., 2005).

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However, T. gondii has been isolated from the tissues from chickens worldwide (Dubey, 2010). Genotypes of chicken isolates are similar to isolates from other animals in the same geographic area. The prevalence of isolation is dependent on the methods used to raze the chickens with chickens razed outside having a higher prevalence of infection.

6.6.11 Turkeys Domestic turkeys (M. gallopavo) fed T. gondii oocysts remained clinically normal except a few that develop pneumonia associated with Aspergillus-like fungi (Dubey et al., 1993a). Tissue cysts are present in breast and leg muscles of inoculated turkeys. Clinical toxoplasmosis does not occur on modern turkey farms. T. gondii has been isolated from the tissues from turkeys worldwide (Dubey, 2010). Genotypes of turkey isolates are similar to isolates from other animals in the same geographic area. The prevalence of isolation is dependent on the methods used to raze the turkeys with turkeys razed outside having a higher prevalence of infection.

6.6.12 Ducks and geese Domestic ducks (Anas platyrhynchos) fed T. gondii oocysts do not develop clinical toxoplasmosis (Sedlak et al., 2004). Viable T. gondii has been isolated from the tissues of a naturally infected domestic ducks (Dubey et al., 2003) and from a domestic goose (Anser anser) (Dubey et al., 2007b).

6.7 Fish, reptiles, and amphibians Toxoplasmosis does not occur in fish, reptiles, or amphibians. Reports of natural infections in these animals in nature are erroneous. Fish and reptiles can be manipulated to make

them susceptible to T. gondii, but they have to be experimentally infected and kept at temperatures of around 37 C 40 C. For example, zebrafish (Danio rerio) adapted to 37 C from 28 C were able to be infected intraperitoneally with 10 tissue cysts of the Me49 (genotype II) or VEG (genotype III) strains of T. gondii. Clinical signs in zebrafish included bilateral exopthalmia, swollen abdomens, whirling swimming behavior, and generalized subdermal hemorrhaging. Tachyzoites were present in tissue sections of parasites developing in muscle, heart, liver, spleen, kidney, pancreas, reproductive organs, eyes, and brain (Sanders et al., 2015).

References Al-Khalidi, N.W., Dubey, J.P., 1979. Prevalence of Toxoplasma gondii infection in horses. J. Parasitol. 65, 331 334. Aramini, J.J., Stephen, C., Dubey, J.P., 1998. Toxoplasma gondii in Vancouver Island cougars (Felis concolor vancouverensis): serology and oocyst shedding. J. Parasitol. 84, 438 440. Arkush, K.D., Miller, M.A., Leutenegger, C.M., Gardner, I. A., Packham, A.E., Heckeroth, A.R., et al., 2003. Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis). Int. J. Parasitol. 33, 1087 1097. Atwood, T.C., Duncan, C., Patyk, K.A., Nol, P., Rhyan, J., McCollum, M., et al., 2017. Environmental and behavioral changes may influence the exposure of an Arctic apex predator to pathogens and contaminants. Sci. Rep. 7, 13193. Available from: https://doi.org/10.1038/ s41598-017-13496-9. Aubert, D., Ajzenberg, D., Richomme, C., Gilot-Fromont, E., Terrier, M.E., de Gevigney, C., et al., 2010. Molecular and biological characteristics of Toxoplasma gondii isolates from wildlife in France. Vet. Parasitol. 171, 346 349. Bangari, D.S., Mouser, P., Miller, M.A., Stevenson, G.W., Vemulapalli, R., Thacker, H.L., 2007. Toxoplasmosis in a woodchuck (Marmota monax) and two American red squirrels (Tamiasciurus hudsonicus). J. Vet. Diagn. Invest. 19, 705 709. Basso, W., Edelhofer, R., Zenker, W., Mostl, K., KubberHeiss, A., Prosl, H., 2005. Toxoplasmosis in Pallas’ cats (Otocolobus manul) raised in captivity. Parasitology 130, 293 299.

Toxoplasma Gondii

References

Basso, W., More´, G., Quiroga, M.A., Pardini, L., Bacigalupe, D., Venturini, L., et al., 2009. Isolation and molecular characterization of Toxoplasma gondii from captive slender-tailed meerkats (Suricata suricatta) with fatal toxoplasmosis in Argentina. Vet. Parasitol. 161, 201 206. Baszler, T.V., Dubey, J.P., Lohr, C.V., Foreyt, W.J., 2000. Toxoplasmic encephalitis in a free-ranging Rocky Mountain bighorn sheep from Washington. J. Wildl. Dis. 36, 752 754. Bergmann, V., Heidrich, R., Kiupel, H., 1980. Acute toxoplasmosis outbreak in rabbit flocks. Angew. Parasitol. 21, 1 6. Bettiol, S.S., Obendorf, D.L., Nowarkowski, M., Goldsmid, J.M., 2000. Pathology of experimental toxoplasmosis in eastern barred bandicoots in Tasmania. J. Wildl. Dis. 36, 141 144. Biancifiori, F., Rondini, C., Grelloni, V., Frescura, T., 1986. Avian toxoplasmosis: experimental infection of chicken and pigeon. Comp. Immunol. Microbiol. Infect. Dis. 9, 337 346. Bossart, G.D., Mignucci-Giannoni, A.A., Rivera-Guzman, A.L., Jimenez-Marrero, N.M., Camus, A., Bonde, R., et al., 2012. Disseminated toxoplasmosis in Antillean manatees (Trichechus manatus manatus) from Puerto Rico. Dis. Aqua. Org. 101, 139 144 . Bouer, A., Werther, K., Catao-Dias, J.L., Nunes, A.L., 1999. Outbreak of toxoplasmosis in Lagothrix lagotricha. Folia Primatol. (Basel). 70, 282 285. Bowater, R.O., Norton, J., Johnson, S., Hill, B., O’Donoghue, P., Prior, H., 2003. Toxoplasmosis in IndoPacific humpbacked dolphins (Sousa chinensis), from Queensland. Aust. Vet J. 81, 627 632. Brown, M., Lappin, M.R., Brown, J.L., Munkhtsog, B., Swanson, W.F., 2005. Exploring the ecologic basis for extreme susceptibility of Pallas’ cats (Otocolobus manul) to fatal toxoplasmosis. J. Wildl. Dis. 41, 691 700. Buergelt, C.D., Bonde, R.K., 1983. Toxoplasmic meningoencephalitis in a West Indian manatee. J. Am. Vet. Med. Assoc. 183, 1294 1296. Burns, R., Williams, E.S., O’Toole, D., Dubey, J.P., 2003. Toxoplasma gondii infections in captive black-footed ferrets (Mustela nigripes), 1992-1998: clinical signs, serology, pathology, and prevention. J. Wildl. Dis. 39, 787 797. Buxton, D., Innes, E.A., 1995. A commercial vaccine for ovine toxoplasmosis. Parasitology 110, S11 16. Cabral, A.D., D’Auria, S.R.N., Camargo, M.C.G.O., Rosa, A. R., Sodre´, M.M., Galva˜o-Dias, M.A., et al., 2014. Seroepidemiology of Toxoplasma gondii infection in bats from Sa˜o Paulo city, Brazil. Vet. Parasitol. 206, 293 296. Canfield, P.J., Hartley, W.J., Dubey, J.P., 1990. Lesions of toxoplasmosis in Australian marsupials. J. Comp. Pathol. 103, 159 167. Carrasco, L., Raya, A.I., Nu´n˜ez, A., Go´mez-Laguna, J., Herna´ndez, S., Dubey, J.P., 2006. Fatal toxoplasmosis

313

and concurrent (Calodium hepaticum) infection in Korean squirrels (Tanias sibericus). Vet. Parasitol. 137, 180 183. Cedillo-Pela´ez, C., Rico-Torres, C.P., Sales-Garrido, C.G., Correa, D., 2011. Acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) in Mexico. Vet. Parasitol. 180, 368 371. Chomel, B.B., Zarnke, R.L., Kasten, R.W., Kass, P.H., Mendes, E., 1995. Serologic survey of Toxoplasma gondii in grizzly bears (Ursus arctos) and black bears (Ursus americanus), from Alaska, 1988 to 1991. J. Wildl. Dis. 31, 472 479. Choromanski, L., Freyre, A., Brown, K., Popiel, I., Shibley, G., 1994. Safety aspects of a vaccine for cats containing a Toxoplasma gondii mutant strain. J. Eukaryot. Microbiol. 41, 8S. Choromanski, L., Freyre, A., Popiel, R., Brown, K., Grieve, R., Shibley, G., 1995. Safety and efficacy of modified live feline Toxoplasma gondii vaccine. Dev. Biol. Stand. 84, 269 281. Cole, R.A., Lindsay, D.S., Howe, D.K., Roderick, C.L., Dubey, J.P., Thomas, N.J., et al., 2000. Biological and molecular characterizations of Toxoplasma gondii strains obtained from southern sea otters (Enhydra lutris nereis). J. Parasitol. 86, 526 530. Colosimo, S.M., Montoya, J.G., Westley, B.P., Jacob, J., Isada, N.B., 2013. Congenital toxoplasmosis presenting with fetal atrial flutter after maternal ingestion of infected moose meat. Alaska Med. 54, 27 31. Cox, J.J., Slabach, B., Hast, J.T., Murphy, S.M., Kwok, O.C., Dubey, J.P., 2017. High seroprevalence of Toxoplasma gondii in elk (Cervus canadensis) of the central Appalachians, USA. Parasitol. Res. 116, 1079 1083. Crawford, G.C., Dunker, F.H., Dubey, J.P., 2000. Toxoplasmosis as a suspected cause of abortion in a Greenland muskox (Ovibos moshatus wardi). J. Zoo Wildl. Med. 31, 247 250. Cross, J., Lein, J., Hsu, M., 1969. Toxoplasma isolated from the Formosan giant flying squirrel. Taiwan Yi Xue Hui Za Zhi 68, 678 683. Dabritz, H.A., Miller, M.M., Gardner, I.A., Packham, A.E., Atwill, E.R., Conrad, P.A., 2008. Risk factors for Toxoplasma gondii infection in wild rodents from Central Coastal California and a review of T. gondii prevalence in rodents. J. Parasitol. 94, 675 683. Davidson, W.R., Nettles, V.F., Hayes, L.E., Howerth, E.W., Couvillion, C.E., 1992. Diseases diagnosed in gray foxes (Urocyon cinereoargenteus) from the southeastern United States. J. Wildl. Dis. 28, 28 33. Davis, S.W., Dubey, J.P., 1995. Mediation of immunity to Toxoplasma gondii oocyst shedding in cats. J. Parasitol. 81, 882 886. Demar, M., Ajzenberg, D., Serrurier, B., Darde´, M.L., Carme, B., 2008. Case Report: Atypical Toxoplasma gondii strain from a free-living Jaguar (Panthera onca) in French Guiana. Am. J. Trop. Med. Hyg. 78, 195 197.

Toxoplasma Gondii

314

6. Toxoplasmosis in wild and domestic animals

Dietz, H.H., Henriksen, P., Bille-Hansen, V., Henriksen, S. A., 1997. Toxoplasmosis in a colony of New World monkeys. Vet. Parasitol. 68, 299 304. DiGuardo, G., Proietto, U., Di Francesco, C.E., Marsilio, F., Zaccaroni, A., Scaravelli, D., et al., 2010. Cerebral toxoplasmosis in striped dolphins (Stenella coeruleoalba) stranded along the Ligurian Sea Coast of Italy. Vet. Pathol. 47, 245 253. Draper, C.C., Killick-Kendrick, R., Hutchison, W.M., Siim, J.C., Garnham, P.C.C., 1971. Experimental toxoplasmosis in chimpanzees. Br. Med. J. 2, 375 378. Dubey, J.P., 1981. Isolation of encysted Toxoplasma gondii from musculature of moose and pronghorn in Montana. Am. J. Vet. Res. 42, 126 127. Dubey, J.P., 1982. Isolation of encysted Toxoplasma gondii from muscles of mule deer in Montana. J. Am. Vet. Med. Assoc. 181, 1535. Dubey, J.P., 1983. Toxoplasma gondii infection in rodents and insectivores from Montana. J. Wildl. Dis. 19, 149 150. Dubey, J.P., 1985. Persistence of encysted Toxoplasma gondii in tissues of equids fed oocysts. Am. J. Vet. Res. 46, 1753 1754. Dubey, J.P., 1988. Long-term persistence of Toxoplasma gondii in tissues of pigs inoculated with T. gondii oocysts and effect of freezing on viability of tissue cysts in pork. Am. J. Vet. Res. 49, 910 913. Dubey, J.P., 1995. Duration of immunity to shedding of Toxoplasma gondii oocysts by cats. J. Parasitol. 81, 410 415. Dubey, J.P., 2002. A review of toxoplasmosis in wild birds. Vet. Parasitol. 106, 121 153. Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans, second ed. CRC Press, Boca Raton, FL, pp. 1 313. Dubey, J.P., Carpenter, J.L., 1993. Histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952 1990). J. Am. Vet. Med. Assoc. 203, 1556 1566. Dubey, J.P., Lin, T.L., 1994. Acute toxoplasmosis in a gray fox (Urocyon cinereoargenteus). Vet. Parasitol. 51, 321 325. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet Parasitol. 67, 1 59. Dubey, J.P., Thulliez, P., 1993. Persistence of tissue cysts in edible tissues of cattle fed Toxoplasma gondii oocysts. Am. J. Vet. Res. 54, 270 273. Dubey, J.P., Hoover, E.A., Walls, K.W., 1977. Effect of age and sex on the acquisition of immunity to toxoplasmosis in cats. J. Protozool. 24, 184 186. Dubey, J.P., Thorne, E.T., Sharma, S.P., 1980. Experimental toxoplasmosis in elk (Cervus canadensis). Am. J. Vet. Res. 41, 792 793. Dubey, J.P., Thorne, E.T., Williams, E.S., 1982. Induced toxoplasmosis in pronghorns and mule deer. J. Am. Vet. Med. Assoc. 181, 1263 1267.

Dubey, J.P., Kramer, L.W., Weisbrode, S.E., 1985. Acute death associated with Toxoplasma gondii in ring-tailed lemurs. J. Am. Vet. Med. Assoc. 187, 1272 1273. Dubey, J.P., Quinn, W.J., Weinandy, D., 1987. Fatal neonatal toxoplasmosis in a bobcat (Lynx rufus). J. Wildl. Dis. 23, 324 327. Dubey, J.P., Gendron-Fitzpatrick, A.P., Lenhard, A.L., Bowman, D., 1988a. Fatal toxoplasmosis and enteroepithelial stages of Toxoplasma gondii in a Pallas cat (Felis manul). J. Protozool. 35, 528 530. Dubey, J.P., Ott-Joslin, J., Torgerson, R.W., Topper, M.J., Sundberg, J.P., 1988b. Toxoplasmosis in black-faced kangaroos (Macropus fuliginosus melanops). Vet. Parasitol. 30, 97 105. Dubey, J.P., Carpenter, J.L., Topper, M.L., Uggla, A., 1989. Fatal toxoplasmosis in dogs. J. Am. Anim. Hosp. Assoc. 25, 659 664. Dubey, J.P., Hamir, A.N., Rupprecht, C.E., 1990. Acute disseminated toxoplasmosis in a red fox (Vulpes vulpes). J. Wildl. Dis. 26, 286 290. Dubey, J.P., Brown, C.A., Carpenter, J.L., Moore III, J.J., 1992a. Fatal toxoplasmosis in domestic rabbits in the USA. Vet. Parasitol. 44, 305 309. Dubey, J.P., Porteer, S.I., Tseng, F., Shen, S.K., Thulliez, P., 1992b. Induced toxoplasmosis in owls. J. Zoo Wildl. Med. 23, 98 102. Dubey, J.P., Camargo, M.E., Ruff, M.D., Wilkins, G.C., Shen, S.K., Kwok, O.C., et al., 1993a. Experimental toxoplasmosis in turkeys. J. Parasitol. 79, 949 952. Dubey, J.P., Hamir, A.N., Shen, S.K., Thulliez, P., Rupprecht, C.E., 1993b. Experimental Toxoplasma gondii infection in raccoons (Procyon lotor). J. Parasitol. 79, 548 552. Dubey, J.P., Ruff, M.D., Camargo, M.E., Shen, S.K., Wilkins, G.L., Kwok, O.C., et al., 1993c. Serologic and parasitologic responses of domestic chickens after oral inoculation with Toxoplasma gondii oocysts. Am. J. Vet. Res. 54, 1668 1672. Dubey, J.P., Humphreys, J.G., Thulliez, P., 1995a. Prevalence of viable Toxoplasma gondii tissue cysts and antibodies to T. gondii by various serologic tests in black bears (Ursus americanus) from Pennsylvania. J. Parasitol. 81, 109 112. Dubey, J.P., Lappin, M.R., Thulliez, P., 1995b. Diagnosis of induced toxoplasmosis in neonatal cats. J. Am. Vet. Med. Assoc. 207, 179 185. Dubey, J.P., Lewis, B., Beam, K., Abbitt, B., 2002a. Transplacental toxoplasmosis in a reindeer (Rangifer tarandus) fetus. Vet. Parasitol. 110, 131 135. Dubey, J.P., Tocidlowski, M.E., Abbitt, B., Llizo, S.Y., 2002b. Acute visceral toxoplasmosis in captive dik-dik (Madoqua guentheri smithi). J. Parasitol. 88, 638 641. Dubey, J.P., Zarnke, R., Thomas, N.J., Wong, S.K., Van Bonn, W., Briggs, M., et al., 2003. Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and Sarcocystis

Toxoplasma Gondii

References

canis-like infections in marine mammals. Vet. Parasitol. 116, 275 296. Dubey, J.P., Lipscomb, T.P., Mense, M., 2004a. Toxoplasmosis in an elephant seal (Mirounga angustirostris). J. Parasitol. 90, 410 411. Dubey, J.P., Graham, D.H., De Young, R.W., Dahl, E., Eberhard, M.L., Nace, E.K., et al., 2004b. Molecular and biologic characteristics of Toxoplasma gondii isolates from wildlife in the United States. J. Parasitol. 90, 67 71. Dubey, J.P., Navarro, I.T., Sreekumar, C., Dahl, E., Freire, R.L., Kawabata, H.H., et al., 2004c. Toxoplasma gondii infections in cats from Parana, Brazil: seroprevalence, tissue distribution, and biologic and genetic characterization of isolates. J. Parasitol. 90, 721 726. Dubey, J.P., Parnell, P.G., Sreekumar, C., Vianna, M.C., De Young, R.W., Dahl, E., et al., 2004d. Biologic and molecular characteristics of Toxoplasma gondii isolates from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), black-winged lory (Eos cyanogenia), and cats (Felis catus). J. Parasitol. 90, 1171 1174. Dubey, J.P., Hill, D.E., Jones, J.L., Hightower, A.W., Kirkland, E., Roberts, J.M., et al., 2005. Prevalence of viable Toxoplasma gondii in beef, chicken and pork from retail meat stores in the United States; risk assessment to consumers. J. Parasitol. 91, 1082 1093. Dubey, J.P., Hodgin, E.C., Hamir, A.N., 2006a. Acute fatal toxoplasmosis in squirrels (Sciurus carolensis) with bradyzoites in visceral tissues. J. Parasitol. 92, 658 659. Dubey, J.P., Bhaiyat, M.I., Macpherson, C.N.L., de Allie, C., Chikweto, A., Kwok, O.C.H., et al., 2006b. Prevalence of Toxoplasma gondii in rats (Rattus norvegicus) in Grenada, West Indies. J. Parasitol. 92, 1107 1108. Dubey, J.P., Morales, J.A., Sundar, N., Velmurugan, G.V., Gonza´lez-Barrientos, C.R., Herna´ndez-Mora, G., et al., 2007a. Isolation and genetic characterization of Toxoplasma gondii from striped dolphin (Stenella coeruleoalba) from Costa Rica. J. Parasitol. 93, 710 711. Dubey, J.P., Webb, D.M., Sundar, N., Velmurugan, G.V., Bandini, L.A., Kwok, O.C.H., et al., 2007b. Endemic avian toxoplasmosis on a farm in Illinois: Clinical disease, diagnosis, biologic and genetic characteristics of Toxoplasma gondii isolates from chickens (Gallus domesticus), and a goose (Anser anser). Vet. Parasitol. 148, 207 212. Dubey, J.P., Fair, P.A., Sundar, N., Velmurugan, G., Kwok, O.C.H., McFee, W.E., et al., 2008a. Isolation of Toxoplasma gondii from bottlenose dolphins (Tursiops truncatus). J. Parasitol. 94, 821 823. Dubey, J.P., Velmurugan, G.V., Ulrich, V., Gill, J., Carstensen, M., Sundar, N., et al., 2008b. Transplacental toxoplasmosis in naturally-infected white-tailed deer: isolation and genetic characterisation of Toxoplasma gondii from foetuses of different gestational ages. Int. J. Parasitol. 38, 1057 1063.

315

Dubey, J.P., Pas, A., Rajendran, C., Kwok, O.C.H., Ferreira, L.R., Martinc, J., et al., 2010. Toxoplasmosis in Sand cats (Felis margarita) and other animals in the Breeding Centre for Endangered Arabian Wildlife in the United Arab Emirates and Al Wabra Wildlife Preservation, the State of Qatar. Vet. Parasitol. 172, 195 203. Dubey, J.P., Passos, I.M.F., Rajendran, C., Ferreira, I.R., Gennari, S.M., Su, C., 2011a. Isolation of viable Toxoplasma gondii from feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctolagus cuniculus) from Brazil. J. Parasitol. 97, 842 845. Dubey, J.P., Velmurugan, G.V., Rajendran, C., Yabsley, M. J., Thomas, N.J., Beckmen, K.B., et al., 2011b. Genetic characterisation of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type. Int J Parasitol. 41, 1139 1147. Dubey, J.P., Brown, J., Ternent, M., Verma, S.K., Hill, D.E., Cerqueira-Ce´zar, C.K., et al., 2016. Seroepidemiologic study on the prevalence of Toxoplasma gondii and Trichinella spp. infections in black bears (Ursus americanus) in Pennsylvania, USA. Vet. Parasitol. 229, 76 80. Dubey, J.P., Brown, J., Verma, S.K., Cerqueira-Cerar, C.K., Banfield, J., Kowk, O.C.H., et al., 2017. Isolation of viable Toxoplasma gondii, molecular characterization, and seroprevalence in elk (Cervus canadensis) in Pennsylvania, USA. Vet. Parasitol. 243, 1 5. Epiphanio, S., Guimaraes, M.A., Fedullo, D.L., Correa, S.H., Catao-Dias, J.L., 2000. Toxoplasmosis in golden-headed lion tamarins (Leontopithecus chrysomelas) and emperor marmosets (Saguinus imperator) in captivity. J. Zoo Wildl. Med. 31, 231 235. Epiphanio, S., Sa, L.R., Teixeira, R.H., Catao-Dias, J.L., 2001. Toxoplasmosis in a wild-caught black lion tamarin (Leontopithecus chrysopygus). Vet. Rec. 149, 627 628. Epiphanio, S., Sinhorini, I.L., Catao-Dias, J.L., 2003. Pathology of toxoplasmosis in captive new world primates. J. Comp. Pathol. 129, 196 204. Escajadillo, A., Frenkel, J.K., 1991. Experimental toxoplasmosis and vaccine tests in Aotus monkeys. Am. J. Trop. Med. Hyg. 44, 382 389. Fayyad, A., Kummerfeld, M., Davina, I., Wohlsein, P., Beineke, A., Baumga¨rtner, W., et al., 2016. ). Fatal systemic Toxoplasma gondii infection in a red squirrel (Sciurus vulgaris), a swinhoe’s striped squirrel (Tamiops swinhoei) and a New World Porcupine (Erethizontidae sp.). J. Comp Pathol. 154, 263 267. Forza´n, M.J., Frasca, S., 2004. Systemic toxoplasmosis in a five-month-old beaver, (Castor canadensis). J. Zoo Wildl. Med. 35, 113 115. Frank, R.K., 2001. An outbreak of toxoplasmosis in farmed mink (Mustela vison S.). J. Vet. Diagn. Invest. 13, 245 249. Frenkel, J.K., Pfefferkorn, E.R., Smith, D.D., Fishback, J.L., 1991. Prospective vaccine prepared from a new mutant

Toxoplasma Gondii

316

6. Toxoplasmosis in wild and domestic animals

of Toxoplasma gondii for use in cats. Am. J. Vet. Res. 52, 759 763. Frenkel, J.K., Lindsay, D.S., Parker, B.B., Dobesh, M., 2003. Dogs as possible mechanical carriers of Toxoplasma and dog fur as source of infection for young children. Int. J. Infect. Dis. 7, 292 293. Freyre, A., Choromanski, L., Fishback, J.L., Popiel, I., 1993. Immunization of cats with tissue cysts, bradyzoites, and tachyzoites of the T-263 strain of Toxoplasma gondii. J. Parasitol 79, 716 719. Furuta, T., Une, Y., Omura, M., Matsutani, N., Nomura, Y., Kikuchi, T., et al., 2001. Horizontal transmission of Toxoplasma gondii in squirrel monkeys (Saimiri sciureus). Exp. Anim. 50, 299 306. Gajadhar, A.A., Measures, L., Forbes, L.B., Kapel, C., Dubey, J.P., 2004. Experimental Toxoplasma gondii infection in grey seals (Halichoerus grypus). J. Parasitol. 90, 255 259. Galuzo, I.G., Vysokova, L.A., Krivkova, A.M., 1970. Toxoplasmosis in bats. In: Fitzgerald, P.R. (Ed.), Toxoplasmosis in Animals. University of Illinois, College of Veterinary Medicine, Urbana, IL, pp. 190 194. Gehrt, S.D., Michael, J., Kinsel, M.J., Anchor, C., 2010. Pathogen dynamics and morbidity of striped skunks in the absence of rabies. J. Wildl. Dis. 46, 335 347. Geisel, O., Breuer, W., Minkus, G., Hermanns, W., 1995. Toxoplasmosis causing death in a mole (Talpa europaea). Berl. Munch. Tierarztl. Wochenschr. 108, 241 243. Gerhold, R.W., Howerth, E.W., Lindsay, D.S., 2005. Sarcocystis neurona-associated meningoencephalitis and description of intramuscular sarcocysts in a fisher (Martes pennanti). J. Wildl. Dis. 41, 224 230. Gerhold, R.W., Saraf, P., Chapman, A., Zou, X., Hickling, G., Stiver, W.H., et al., 2017. Toxoplasma gondii seroprevalence and genotype diversity in select wildlife species from the southeastern United States. Para. Vec. 10, 508. Gorman, T.R., Riveros, V., Alcaino, H.A., Salas, D.R., Thiermann, E.R., 1986. Helminthiasis and toxoplasmosis among exotic mammals at the Santiago National Zoo. J. Am. Vet. Med. Assoc. 189, 1068 1070. Gustafsson, K., Uggla, A., Jarplid, B., 1997. Toxoplasma gondii infection in the mountain hare (Lepus timidus) and domestic rabbit (Oryctolagus cuniculus). I. Pathology. J. Comp. Pathol. 117, 351 360. Hagemoser, W.A., Dubey, J.P., Thompson, J.R., 1990. Acute toxoplasmosis in a camel. J. Am. Vet. Med. Assoc. 196, 347. Hancock, K., Thiele, L.A., Zajac, A.M., Elvinger, F., Lindsay, D.S., 2005. Prevalence of antibodies to Toxoplasma gondii in raccoons (Procyon lotor) from an urban area of Northern Virginia. J. Parasitol. 91, 694 695. Harper III, J.S., London, W.T., Sever, J.L., 1985. Five drug regimens for treatment of acute toxoplasmosis in squirrel monkeys. Am. J. Trop. Med. Hyg. 34, 50 57.

Harrison, M., Moorman, J.B., Bolin, S.R., Grosjean, N.L., Lim, A., Fitzgerald, S.D., 2007. Toxoplasma gondii in an African crested porcupine (Hystrix cristata). J. Vet. Diagn. Invest. 19, 191 194. Hartley, W.J., Dubey, J.P., Spielman, D.S., 1990. Fatal toxoplasmosis in koalas (Phascolarctos cinereus). J. Parasitol. 76, 271 272. Hejlicek, K., Literak, I., Nezval, J., 1997. Toxoplasmosis in wild mammals from the Czech Republic. J. Wildl. Dis. 33, 480 485. Herrmann, D.C., Maksimov, P., Maksimov, A., Sutor, A., Schwarz, S., Jaschke, W., et al., 2012. Toxoplasma gondii in foxes and rodents from the German Federal States of Brandenburg and Saxony-Anhalt: seroprevalence and genotypes. Vet. Parasitol. 185, 78 85. Hilali, M., Fatani, A., al-Atiya, S., 1995. Isolation of tissue cysts of Toxoplasma, Isospora, Hammondia and Sarcocystis from camel (Camelus dromedarius) meat in Saudi Arabia. Vet. Parasitol. 58, 353 356. Holland, G.N., O’Connor, G.R., Diaz, R.F., Minasi, P., Wara, W.M., 1988. Ocular toxoplasmosis in immunosuppressed nonhuman primates. Invest. Ophthalmol. Vis. Sci. 29, 835 842. Holshuh, H.J., Sherrod, A.E., Taylor, C.R., Andrews, B.F., Howard, E.B., 1985. Toxoplasmosis in a feral northern fur seal. J. Am. Vet. Med. Assoc. 187, 1229 1230. Hong, S.H., Lee, S.E., Jeong, Y.I., Kim, H.C., Chong, S.T., Klein, T.A., et al., 2014. Prevalence and molecular characterizations of Toxoplasma gondii and Babesia microti from small mammals captured in Gyeonggi and Gangwon Provinces, Republic of Korea. Vet Parasitol. 205, 512 517. Honnold, S.P., Braun, R., Scott, D.P., Sreekumar, C., Dubey, J.P., 2005. Toxoplasmosis in a Hawaiian monk seal (Monachus schauinslandi). J. Parasitol. 91, 695 697. Howerth, E.W., Rodenroth, N., 1985. Fatal systemic toxoplasmosis in a wild turkey. J. Wildl. Dis. 21, 446 449. Hubbard, G., Witt, W., Healy, M., Schmidt, R., 1986. An outbreak of toxoplasmosis in zoo birds. Vet. Pathol. 23, 639 641. Inskeep II, W., Gardiner, C.H., Harris, R.K., Dubey, J.P., Goldston, R.T., 1990. Toxoplasmosis in Atlantic bottlenosed dolphins (Tursiops truncatus). J. Wildl. Dis. 26, 377 382. Jardine, J.E., Dubey, J.P., 2002. Congenital toxoplasmosis in a Indo-Pacific bottlenose dolphin (Tursiops aduncus). J. Parasitol. 88, 197 199. Jarvinen, J.A., Dubey, J.P., Althouse, G.,C., 1999. Clinical and serologic evaluation of two llamas (Lama glama) infected with Toxoplasma gondii during gestation. J. Parasitol. 85, 142 144. Jewell, M.L., Frenkel, J.K., Johnson, K.M., Reed, V., Ruiz, A., 1972. Development of Toxoplasma oocysts in neotropical felidae. Am. J. Trop. Med. Hyg. 21, 512 517.

Toxoplasma Gondii

References

Jokelainen, P., Nylund, M., 2012. Acute fatal toxoplasmosis in three Eurasian Red Squirrels (Sciurus vulgaris) caused by genotype II of Toxoplasma gondii. J. Wildl. Dis 48, 454 457. Jokelainen, P., Isomursu, M., Na¨reaho, A., Oksanen, A., 2011. Natural Toxoplasma gondii infections in European brown hares and mountain hares in Finland: proportional mortality rate, antibody prevalence, and genetic characterization. J. Wildl. Dis. 47, 154 163. Jones, Y.L., Fitzgerald, S.D., Sikarske, J.G., Murphy, A., Grosjean, N., Kiupel, M., 2006. Toxoplasmosis in a freeranging mink. J. Wildl. Dis. 42, 865 869. Juan-Salles, C., Prats, N., Lopez, S., Domingo, M., Marco, A.J., Moran, J.F., 1997. Epizootic disseminated toxoplasmosis in captive slender-tailed meerkats (Suricata suricatta). Vet. Pathol. 34, 1 7. Juan-Salles, C., Prats, N., Marco, A.J., Ramos-Vara, J.A., Borras, D., Fernandez, J., 1998. Fatal acute toxoplasmosis in three golden lion tamarins (Leontopithecus rosalia). J. Zoo Wildl. Med. 29, 55 60. Juan-Salle´s, C., Mainez, M., Marco, A., Sanchı´s, A.M.M., 2011. Localized toxoplasmosis in a ring-tailed lemur (Lemur catta) causing placentitis, stillbirths, and disseminated fetal infection. J. Vet. Diag. Invest. 23, 1041 1045. Junge, R.E., Fischer, J.R., Dubey, J.P., 1992. Fatal disseminated toxoplasmosis in a captive Cuvier’s gazelle (Gazella cuvieri). J. Zoo Wildl. Med. 23, 342 345. Kaneto, C.N., Costa, A.J., Paulillo, A.C., Moraes, F.R., Murakami, T.O., Meireles, M.V., 1997. Experimental toxoplasmosis in broiler chicks. Vet. Parasitol. 69, 203 210. Kelly, T.R., Sleeman, J.M., 2003. Morbidity and mortality of red foxes (Vulpes vulpes) and gray foxes (Urocyon cinereoargenteus) admitted to the Wildlife Center of Virginia, 1993-2001. J. Wildl. Dis. 39, 467 469. Kenny, D.E., Lappin, M.R., Knightly, F., Baler, J., Brewer, M., Getzy, D.M., 2002. Toxoplasmosis in Pallas’ cats (Otocolobus felis manul) at the Denver Zoological Gardens. J, Zoo Wildl, Med. 33, 131 138. Kijlstra, A., Meerburg, B., Cornelissen, J., De Craeye, S., Vereijken, P., Jongert, E., 2008. The role of rodents and shrews in the transmission of Toxoplasma gondii to pigs. Vet. Parasitol. 156, 183 190. Kottwitz, J.J., Preziosi, D.E., Miller, M.A., Ramos-Vara, J.A., Maggs, D.J., Bonagura, J.D., 2004. Heart failure caused by toxoplasmosis in a Fennec fox (Fennecus zerda). J. Am. Anim. Hosp. Assoc. 40, 501 507. Kotula, A.W., Dubey, J.P., Sharar, A.K., Andrews, C.D., Shen, S.K., Lindsay, D.S., 1991. Effect of freezing on infectivity of Toxoplasma gondii tissue cysts in pork. J. Food Protect. 54, 687 690. Krijger, I.M., Cornelissen, J.B.W.J., Wisselink, H.J., Meerburg, B.G., 2014. Prevalence of Toxoplasma gondii in common moles (Talpa europaea). Acta Vet. Scand. 56, 48. Available from: https://doi.org/10.1186/s13028-014-0048-0.

317

Kumar, A., Melotti, J.R., Cooley, T.M., Fitzgerald, S.D., 2018. Mortality due to toxoplasmosis in suburban eastern fox squirrels (Sciurus niger) in Michigan, USA. J Wildl Dis. 55, 213 217. Lappin, M.R., Greene, C.E., Prestwood, A.K., Dawe, D.L., Tarleton, R.L., 1989. Diagnosis of recent Toxoplasma gondii infection in cats by use of an enzyme-linked immunosorbent assay for immunoglobulin M. Am. J. Vet. Res. 50, 1580 1585. Lappin, M.R., Dawe, D.L., Lindl, P., Greene, C.E., Prestwood, A.K., 1992. Mitogen and antigen-specific induction of lymphoblast transformation in cats with subclinical toxoplasmosis. Vet. Immunol. Immunopathol. 30, 207 220. Larkin, J.L., Gabriel, M., Gerhold, R.W., Yabsley, M.J., Wester, J.C., Humphreys, J.G., et al., 2011. Prevalence to Toxoplasma gondii and Sarcocystis spp. in a reintroduced fisher (Martes pennanti) population in Pennsylvania. J. Parasitol. 97, 425 429. Las, R.D., Shivaprasad, H.L., 2008. An outbreak of toxoplasmosis in an aviary collection of Nicobar pigeons (Caloenas nicobarica). J. S. Afr. Vet. Assoc. 79, 149 152. Lindsay, D.S., Dubey, J.P., 2000. Canine neosporsis. J. Vet. Parasitol. 14, 4 14. Lindsay, D.S., Dubey, J.P., 2009. Long-term survival of Toxoplasma gondii sporulated oocysts in seawater. J. Parasitol. 95, 1019 1020. Lindsay, D.S., Blagburn, B.L., Dubey, J.P., Mason, W.H., 1991a. Prevalence and isolation of Toxoplasma gondii from white-tailed deer in Alabama. J. Parasitol. 77, 62 64. Lindsay, D.S., Dubey, J.P., Blagburn, B.L., 1991b. Toxoplasma gondii infections in red-tailed hawks inoculated orally with tissue cysts. J. Parasitol. 77, 322 325. Lindsay, D.S., Smith, P.C., Hoerr, F.J., Blagburn, B.L., 1993. Prevalence of encysted Toxoplasma gondii in raptors from Alabama. J. Parasitol. 79, 870 873. Lindsay, D.S., Smith, P.C., Blagburn, B.L., 1994. Prevalence and isolation of Toxoplasma gondii from wild turkeys in Alabama. J. Helminthol. Soc. Wash. 61, 115 117. Lindsay, D.S., Dubey, J.P., Butler, J.M., Blagburn, B.L., 1996. Experimental tissue cyst induced Toxoplasma gondii infections in dogs. J. Eukaryot. Microbiol. 43, 113S. Lindsay, D.S., Dubey, J.P., Butler, J.M., Blagburn, B.L., 1997a. Mechanical transmission of Toxoplasma gondii oocysts by dogs. Vet. Parasitol. 73, 27 33. Lindsay, D.S., Sundermann, C.A., Dubey, J.P., Blagburn, B. L., 1997b. Update on Toxoplasma gondii infections in wildlife and exotic animals from Alabama. J. Alabama Acad. Sci. 68, 246 254. Lindsay, D.S., Thomas, N.J., Rosypal, A.C., Dubey, J.P., 2001a. Dual Sarcocystis neurona and Toxoplasma gondii infection in a Northern sea otter from Washington state, USA. Vet. Parasitol. 97, 319 327.

Toxoplasma Gondii

318

6. Toxoplasmosis in wild and domestic animals

Lindsay, D.S., Phelps, K.K., Smith, S.A., Flick, G., Sumner, S.S., Dubey, J.P., 2001b. Removal of Toxoplasma gondii oocysts from sea water by eastern oysters (Crassostrea virginica). J. Eukaryot. Microbiol. (Suppl), 197S 198S. Lindsay, D.S., Collins, M.V., Mitchell, S.M., Cole, R.A., Flick, G.J., Wetch, C.N., et al., 2003. Sporulation and survival of Toxoplasma gondii oocysts in seawater. J. Eukaryot. Microbiol. 50 (Suppl), 687 688. Lindsay, D.S., Collins, M.V., Mitchell, S.M., Wetch, C.N., Rosypal, A.C., Flick, G.J., et al., 2004. Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica). J. Parasitol. 90, 1054 1057. Lindsay, D.S., McKown, R.D., DiCristina, J., Jordan, C.N., Mitchell, S.M., Oates, D.W., et al., 2006. Prevalence of agglutinating antibodies to Toxoplasma gondii in adult and fetal mule deer (Odocoileus hemionus) from Nebraska. J. Parasitol. 91, 475 478. Lloyd, C., Stidworthy, M.F., 2007. Acute disseminated toxoplasmosis in a juvenile cheetah (Acinonyx jubatus). J. Zoo Wildl. Med. 38, 475 478. Loeffler, I.K., Howard, J., Montali, R.J., Hayek, L.A., Dubovi, E., Zhang, Z., et al., 2007. Serosurvey of ex situ giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China with implications for species conservation. J. Zoo Wildl. Med. 38, 559 566. Lukesova, D., Literak, I., 1998. Shedding of Toxoplasma gondii oocysts by Felidae in zoos in the Czech Republic. Vet. Parasitol. 74, 1 7. Ma, H., Wang, Z., Wang, C., Li, C., Wei, F., Liu, Q., 2015. Fatal Toxoplasma gondii infection in the giant panda. Parasite 22, 30. Available from: https://doi.org/ 10.1051/parasite/2015030. Massie, G.N., Ware, M.N., Villegas, E.N., Black, M.W., 2010. Uptake and transmission of Toxoplasma gondii oocysts by migratory, filter-feeding fish. Vet. Parasitol. 169, 296 303. Mazzariol, S., Federica Marcer, F., Mignone, W., 2012. Toxoplasma gondii coinfection in a Mediterranean fin whale (Balaenoptera physalus). BMC Vet. Res. 8, 20. Meerburg, B.G., De Craeye, S., Dierick, K.A., Kijlstra, A., 2012. Neospora caninum and Toxoplasma gondii in brain tissue of feral rodents and insectivores caught on farms in the Netherlands. Vet. Parasitol. 184, 317 320. Migaki, G., Allen, J.F., Casey, H.W., 1977. Toxoplasmosis in a California sea lion (Zalophus californianus). Am. J. Vet. Res. 38, 135 136. Migaki, G., Sawa, T.R., Dubey, J.P., 1990. Fatal disseminated toxoplasmosis in a spinner dolphin (Stenella longirostris). Vet. Pathol. 27, 463 464. Mikaelian, I., Dubey, J.P., Martineau, D., 1997. Severe hepatitis resulting from toxoplasmosis in a barred owl (Strix varia) from Quebec, Canada. Avian Dis. 41, 738 740.

Mikaelian, I., Boisclair, J., Dubey, J.P., Kennedy, S., Martineau, D., 2000. Toxoplasmosis in beluga whales (Delphinapterus leucas) from the St Lawrence estuary: two case reports and a serological survey. J. Comp. Pathol. 122, 73 76. Miller, N.L., Frenkel, J.K., Dubey, J.P., 1972. Oral infections with Toxoplasma cysts and oocysts in felines, other mammals, and in birds. J. Parasitol. 58, 928 937. Miller, M.A., Sverlow, K., Crosbie, P.R., Barr, B.C., Lowenstine, L.J., Gulland, F.M., et al., 2001. Isolation and characterization of two parasitic protozoa from a Pacific harbor seal (Phoca vitulina richardsi) with meningoencephalomyelitis. J. Parasitol. 87, 816 822. Miller, M.A., Gardner, I.A., Kreuder, C., Paradies, D.M., Worcester, K.R., Jessup, D.A., et al., 2002. Coastal freshwater runoff is a risk factor for Toxoplasma gondii infection of southern sea otters (Enhydra lutris nereis). Int. J. Parasitol. 32, 997 1006. Morales, J.A., Pena, M.A., Dubey, J.P., 1996. Disseminated toxoplasmosis in a captive porcupine (Coendou mexicanus) from Costa Rica. J. Parasitol. 82, 185 186. Nardoni, S., Angelici, M.C., Mugnaini, L., Mancianti, F., 2011. Prevalence of Toxoplasma gondii infection in Myocastor coypus in a protected Italian wetland. Parasit. Vect. 4, 240. Oksanen, A., Gustafsson, K., Lunden, A., Dubey, J.P., Thulliez, P., Uggla, A., 1996. Experimental Toxoplasma gondii infection leading to fatal enteritis in reindeer (Rangifer tarandus). J. Parasitol. 82, 843 845. Oksanen, A., Asbakk, K., Prestrud, K.W., Aars, J., Derocher, A.E., Tryland, M., et al., 2009. Prevalence of antibodies against Toxoplasma gondii in polar bears (Ursus maritimus) from Svalbard and East Greenland. J. Parasitol. 95, 89 94. Pas, A., Dubey, J.P., 2008a. Fatal toxoplasmosis in sand cats (Felis margarita). J. Zoo Wildl. Med. 39, 362 369. Pas, A., Dubey, J.P., 2008b. Seroprevalence of antibodies to Toxoplasma gondii in Gordon’s wild cat (Felis silvestris gordoni) in the Middle East. J. Parasitol. 94, 1169. Pas, A., Dubey, J.P., 2008c. Toxoplasmosis in Sand fox (Vulpes rueppellii). J. Parasitol. 94, 976 977. Pena, H.F.J., Marvulo, M.F.V., Horta, M.C., Silva, M.A., Silva, J.C.R., Siqueira, D.B., et al., 2011. Isolation and genetic characterisation of Toxoplasma gondii from a red-handed howler monkey (Alouatta belzebul), a jaguarundi (Puma yagouaroundi), and a black-eared opossum (Didelphis aurita) from Brazil. J. Parasitol. 175, 377 381. Pertz, C., Dubielzig, R.R., Lindsay, D.S., 1997. Fatal Toxoplasma gondii infection in golden lion tamarins (Leontopithecus rosalia rosalia). J. Zoo Wildl. Med. 28, 491 493.

Toxoplasma Gondii

References

Ploeg, M., Ultee, T., Marja Kik, M., 2011. Disseminated toxoplasmosis in black-footed penguins (Spheniscus demersus). Avian Dis. 55, 701 703. Poelma, F.,G., Zwart, P., 1972. Toxoplasmosis in crowned pigeons and other birds at the Royal Zoo, Blijdorp, in Rotterdam. Acta Zool. Pathol. Antverp. 55, 29 40. Portas, T.J., 2010. Toxoplasmosis in Macropodids: a review. J. Zoo Wildl. Med. 41, 1 6. Quist, C.F., Dubey, J.P., Luttrell, M.P., Davidson, W.R., 1995. Toxoplasmosis in wild turkeys: a case report and serologic survey. J. Wildl. Dis. 31, 255 258. Rah, H., Chomel, B.B., Follmann, E.H., Kasten, R.W., Hew, C.H., Farver, T.B., et al., 2005. Serosurvey of selected zoonotic agents in polar bears (Ursus maritimus). Vet, Rec. 156, 7 13. Ramey, A.M., Cleveland, C.A., Hilderbrand, G.V., Joly, K., Gustine, D.D., Mangipane, B., et al., 2019. Evidence that exposure of Alaska brown bears (Ursus arctos) to bacterial, viral, and parasitic agents varies spatiotemporally and is influenced by demography. J. Wildl. Dis. 55, 576 588. Reddacliff, G.L., Hartley, W.J., Dubey, J.P., Cooper, D.W., 1993. Pathology of experimentally-induced, acute toxoplasmosis in macropods. Aust. Vet J. 70, 4 6. Reed, W.M., Turek, J.J., 1985. Concurrent distemper and disseminated toxoplasmosis in a red fox. J. Am. Vet. Med. Assoc. 187, 1264 1265. Resendes, A.R., Almeria, S., Dubey, J.P., Obon, E., JuanSalles, C., Degollada, E., et al., 2002. Disseminated toxoplasmosis in a Mediterranean pregnant Risso’s dolphin (Grampus griseus) with transplacental fetal infection. J. Parasitol. 88, 1029 1032. Riemann, H.P., Fowler, M.E., Schulz, T., Lock, A., Thilsted, J., Pulley, L.T., et al., 1974. Toxoplasmosis in pallas cats. J. Wildl. Dis. 10, 471 477. Ross, R.D., Stec, L.A., Werner, J.C., Blumenkranz, M.S., Glazer, L., Williams, G.A., 2001. Presumed acquired ocular toxoplasmosis in deer hunters. Retina 21, 226 229. Sacks, J.J., Delgado, D.G., Lobel, H.O., Parker, R.L., 1983. Toxoplasmosis infection associated with eating undercooked venison. Am. J. Epidemiol. 118, 832 838. Sanders, J.L., Zhou, Y., Moulton, H., Moulton, Z., McLeod, R., Dube, J.P., et al., 2015. The zebrafish, Danio rerio, as a model for Toxoplasma gondii: an initial description of infection in fish. J Fish Dis. 38, 675 679. Available from: https://doi.org/10.1111/jfd.12393. Sangster, C.R., Gordon, A.N., Hayes, D., 2012. Systemic toxoplasmosis in captive flying-foxes. Aust. Vet. J. 90, 140 142. Sasseville, V.G., Pauley, D.R., MacKey, J.J., Simon, M.A., 1995. Concurrent central nervous system toxoplasmosis and simian immunodeficiency virus-induced AIDS encephalomyelitis in a Barbary macaque (Macaca sylvana). Vet. Pathol. 32, 81 83.

319

Schares, G., Pantchev, N., Barutzki, D., Heydorn, A.O., Bauer, C., Conraths, F.J., 2005. Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondi in faeces collected from dogs in Germany. Int. J. Parasitol. 35, 1525 1537. Schoondermark-Van de Ven, E., Melchers, W., Galama, J., Camps, W., Eskes, T., Meuwissen, J., 1993. Congenital toxoplasmosis: an experimental study in rhesus monkeys for transmission and prenatal diagnosis. Exp. Parasitol. 77, 200 211. Sedlak, K., Literak, I., Faldyna, M., Toman, M., Benak, J., 2000. Fatal toxoplasmosis in brown hares (Lepus europaeus): possible reasons of their high susceptibility to the infection. Vet. Parasitol. 93, 13 28. Sedlak, K., Bartova, E., Literak, I., Vodicka, R., Dubey, J.P., 2004. Toxoplasmosis in nilgais (Boselaphus tragocamelus) and a saiga antelope (Saiga tatarica). J. Zoo Wildl. Med. 35, 530 533. Smielewska-Los, E., Turniak, W., 2004. Toxoplasma gondii infection in Polish farmed mink. Vet. Parasitol. 122, 201 206. Smith, D.D., Frenkel, J.K., 1995. Prevalence of antibodies to Toxoplasma gondii in wild mammals of Missouri and east central Kansas: biologic and ecologic considerations of transmission. J. Wildl. Dis. 31, 15 21. Smith, K.E., Fisher, J.R., Dubey, J.P., 1995. Toxoplasmosis in a bobcat (Felis rufus). J. Wildl. Dis. 31, 555 557. Sorensen, K.K., Mork, T., Sigurdardottir, O.G., Asbakk, K., Akerstedt, J., Bergsjo, B., et al., 2005. Acute toxoplasmosis in three wild arctic foxes (Alopex lagopus) from Svalbard; one with co-infections of Salmonella Enteritidis PT1 and Yersinia pseudotuberculosis serotype 2b. Res. Vet. Sci. 78, 161 167. Spencer, J.A., Joiner, K.S., Hilton, C.D., Dubey, J.P., ToivioKinnucan, M., Minc, J.K., et al., 2004. Disseminated toxoplasmosis in a captive ring-tailed lemur (Lemur catta). J. Parasitol. 90, 904 906. Stover, J.E., Jacobson, E.R., Lukas, J., Lappin, M.R., Buergelt, C.D., 1990. Toxoplasma gondii in a collection of nondomestic ruminants. J. Zoo Anim. Med. 21, 295 301. Szabo, K.A., Mense, M.G., Lipscomb, T.P., Felix, K.J., Dubey, J.P., 2004. Fatal toxoplasmosis in a bald eagle (Haliaeetus leucocephalus). J. Parasitol. 90, 907 908. Teutsch, S.M., Juranek, D.D., Sulzer, A., Dubey, J.P., Sikes, R.K., 1979. Epidemic toxoplasmosis associated with infected cats. N. Engl. J. Med. 300, 695 699. Thornton, R.N., Cook, T.G., 1986. A congenital Toxoplasmalike disease in ferrets (Mustela putorius furo). NZ. Vet. J. 34, 31 33. van Pelt, R.W., Dieterich, R.A., 1972. Toxoplasmosis in thirteen-lined ground squirrels. J. Am. Vet. Med. Assoc. 161, 643 647. Verma, S.K., Carstensen, M., Calero-Bernal, R., Moore, S.A., Jiang, T., Su, C., et al., 2016. Seroprevalence, isolation, first genetic characterization of Toxoplasma gondii, and

Toxoplasma Gondii

320

6. Toxoplasmosis in wild and domestic animals

possible congenital transmission in wild moose from Minnesota, USA. Parasitol. Res. 115, 687 690. Wallace, M.R., Rossetti, R.J., Olson, P.E., 1993. Cats and toxoplasmosis risk in HIV-infected adults. J. Am. Med. Assoc. 269, 76 77. Walsh, C.P., Hammond, S.E., Zajac, A.M., Lindsay, D.S., 1999. Survival of Toxoplasma gondii tachyzoites in goat milk: potential source of human toxoplasmosis. J. Eukart. Microbiol. 73s 74s. Wong, M.M., Kozek, W.J., Karr Jr., S.L., Brayton, M.A., Theis, J.H., Hendrickx, A.G., 1979. Experimental congenital infection of Toxoplasma gondii in Macaca arctoides. Asian J. Infect. Dis. 3, 61 67. Work, T.M., Massey, J.G., Rideout, B.A., Gardiner, C.H., Ledig, D.B., Kwok, O.C., et al., 2000. Fatal toxoplasmosis in free-ranging endangered ‘Alala from Hawaii. J. Wildl. Dis. 36, 205 212.

Yai, L.E.O., Ragozo, A.M.A., Soares, R.M., Pena, H.F.J., Su, C., Gennari, S.M., 2009. Genetic diversity among capybara (Hydrochaeris hydrochaeris) isolates of Toxoplasma gondii from Brazil. Vet. Parasitol. 162, 332 337.

Further reading DiGuardo, G., Agrimi, U., Morelli, L., Cardeti, G., Terracciano, G., Kennedy, S., 1995. Post mortem investigations on cetaceans found stranded on the coasts of Italy between 1990 and 1993. Vet. Rec. 136, 439 442. Sreekumar, C., Graham, D.H., Dahl, E., Lehmann, T., Raman, M., Bhalerao, D.P., et al., 2003. Genotyping of Toxoplasma gondii isolates from chickens from India. Vet. Parasitol. 118, 187 194.

Toxoplasma Gondii

C H A P T E R

7 Toxoplasma animal models and therapeutics Carsten G.K. Lu¨der1, Utz Reichard1,2 and Uwe Groß1 1

Institute for Medical Microbiology, University Medical Center, University of Go¨ttingen, Go¨ttingen, Germany 2Amedes MVZ Wagnerstibbe for Medical Microbiology, Infectious Diseases, Hygiene and Tropical Medicine, Go¨ttingen, Germany

7.1 Introduction This chapter will discuss animal models on toxoplasmosis, with special regard to pharmacological applications, and thereby update existing reviews (Darcy and Zenner, 1993; Derouin et al., 1995; Lu¨der et al., 2014; Dunay et al., 2018). Virtually, all mammals can be infected with Toxoplasma gondii. However, different animal species differ markedly in their resistance to Toxoplasma infection. For example, rats are usually resistant against symptomatic toxoplasmosis, but most mouse strains in general are susceptible (Fujii et al., 1983; Dubey and Frenkel, 1998; Zenner et al., 1998; Gao et al., 2015; Dubey et al., 2016). In addition, the outcome of infection is not only dependent on the animal species but also on the animal strain. The genetic background seems to be of importance since, after infection with Toxoplasma, striking differences in susceptibility of

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00007-4

various strains of inbred and outbred mice and rats were observed (McLeod et al., 1984, 1989; Araujo et al., 1976; Williams et al., 1978; Suzuki et al., 1991, 1994; Brown et al., 1995; Kempf et al., 1999; Sergent et al., 2005; Gao et al., 2015) (Fig. 7.1). We know today that, at least in part, such animal strain-dependent variation in susceptibility may be attributed to certain chromosomal regions and even to the presence of certain single genes that may influence the parasite burden dramatically (Brown and McLeod, 1990; Deckert-Schlu¨ter et al., 1994; Brown et al., 1995; Johnson et al., 2002; Cavailles et al., 2006, 2014; Hunn et al., 2011; Lilue et al., 2013). Nevertheless, many questions in this respect still remain to be solved, which may be exemplarily underlined by the intriguing finding that the control of host genetic resistance against acute infection itself differs according to virulence and genotype of the T. gondii strain used (Suzuki et al., 1995; Lilue et al., 2013).

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7. Toxoplasma animal models and therapeutics

The situation becomes even more complex since these differences are not uniform with respect to the strains but are also a function of the mode of inoculation (Fig. 7.1). This means that, for instance, a mouse strain that is highly susceptible to intraperitoneal (i.p.) infection compared to another mouse strain may not necessarily be highly susceptible to oral infection and vice versa. Indeed, almost mirrorimage susceptibility between oral and i.p. challenge has been found with inbred mouse strains (Johnson, 1984), suggesting at least partly independent modes of the defense involved in each infection route. In this context, one has to be aware that, mostly, two infection modes are used: (1) injection of tachyzoites grown in culture or in mice i.p., s.c., or i. v. and (2) oral administration of tissue cysts of T. gondii obtained mostly from brains of chronically infected mice (McLeod et al., 1984). Since oocysts are only shed by the definitive host, that is, the cat, and their production is more difficult, they are less frequently used for oral infection, although this mode of infection represents one of the two major natural routes of parasite transmission (Dubey et al., 2016). Oral administration of tissue cysts is inherently less reproducible due to variable size and parasite content of the cysts but has the advantage that it is a natural route of infection and that tissue cysts can be easily isolated from chronically infected mice. As tachyzoites are not resistant to the acidic pH in the stomach, they are only less infective when given orally (Dubey, 1998). In addition to host factors, the outcome of a challenge with Toxoplasma is largely influenced by the nature of the infectious agent itself and one of the most common characteristics of many Toxoplasma strains is their variation in virulence (Fig. 7.1). Depending on the parasite dose which is lethal in mice, or on the time before animals succumb to infection, or on the percentage of animals that do succumb, highly virulent, moderately virulent and less virulent

strains have been characterized (Kaufman et al., 1958; Sibley and Boothroyd, 1992; Sibley and Ajioka, 2008). A growing number of excreted/secreted effector molecules including rhoptry and dense granule proteins have been identified as key determinants of virulence in mice (Taylor et al., 2006; Saeij et al., 2006; Behnke et al., 2011; Rosowski et al., 2011; Hakimi et al., 2017). However, straindependent virulence does not appear as a static feature as it can be enhanced by the continuous passage of the parasite in laboratory animals. For example, when the RH strain was initially isolated, mice succumbed to infection 17
21 days in the first passage, 7
8 days in the second, and 3
5 days in the third passage, and thereafter (Sabin, 1941). In any case the virulence of T. gondii strains is commonly assessed according to the outcome of a systemic i.p. infection in mice and studies of the population genetic structure have defined that the most commonly used laboratory strains of T. gondii belong to three clonal lineages, of which type 1 includes mouse-virulent strains, and types 2 and 3, mouse-avirulent strains (Sibley and Boothroyd, 1992; Howe and Sibley, 1995; Sibley and Ajioka, 2008). In fact, type 1 strains may be lethal in mice when a single infectious organism is injected (Howe et al., 1996).

7.2 Congenital toxoplasmosis An ideal animal model for congenital toxoplasmosis, useful for drug studies as well as for immunological studies and vaccine design, would be one that mimics the conditions of the infection in the human host. For example, the risk of vertical transmission in humans increases, whereas the occurrence of clinical symptoms decreases with time of pregnancy (Desmonts and Couvreur, 1984). In addition, the outcome of the infection should be clearly assessable. In practice, the fulfillment of these requirements seems to be dependent on several

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FIGURE 7.1 Factors which influence the outcome of Toxoplasma animal infection.

conditions: (1) the nature of the animal placenta as the organ where transmission takes place, (2) the duration of pregnancy, (3) the duration of parasitemia, and (4) the size of the animal (for review see Derouin et al., 1995). The latter point is not as critical as it used to be since technical developments during the last few decades, such as quantitative real-time PCR and advances in imaging techniques, have greatly enhanced the sensitivity of assessment of infection in small animal models (see, e.g., Flori et al., 2002, 2003). Importantly, an extensive literature search recently suggested that animal models only partially mimic the vertical transmission rates and the fetal damage rates along gestation as observed in humans (Vargas-Villavicencio et al., 2016a). Unlike most other organs, the placenta shows a wide variation in structure among different mammalian species and may be classified according to the number of maternal and fetal cell layers into different classes (Loke, 1982). While the placentas of carnivores and ruminants in general have four to six layers, the placenta of humans consists of only three— a fetal trophoblastic, mesenchymal and endothelial cell layer. Such a relatively thin interface which may facilitate parasite transmission is called a hemochorial placenta type, and this

type is also present in primates and rodents (Loke, 1982; Darcy and Zenner, 1993). Indeed, most studies on congenital toxoplasmosis were conducted using rodents. Of course, they were mostly not chosen because of the nature of their placenta, but rather for practical reasons connected with the ease of keeping and handling small laboratory animals. However, except for simple models for congenital chorioretinitis, studies involving a detailed anatomical assessment of the fetal infection often require larger animals, such as pigs, sheep or even primates. In the following, different animal species and their use in models of congenital toxoplasmosis are discussed. A summary is provided in Tables 7.1 and 7.2.

7.2.1 Mouse As indicated by numerous articles that have appeared during the recent decades, mice are well-studied animals in congenital toxoplasmosis (Beverley, 1959; Remington et al., 1961; Beverley and Henry, 1970; Hay et al., 1981, 1984; Roberts and Alexander, 1992; Roberts et al., 1994; Wang et al., 2011; Mu¨ller et al., 2017). This may be somewhat astonishing as early studies indicated that under natural

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TABLE 7.1 Pharmacological studies on congenital toxoplasmosis. Pharmacological studies (literature examples)

Animal species

Specific comments

Mouse

Hemotrichorial placenta; vertical transmission during chronic infection possible (depending on mouse strain); vertical transmission in inbred BALB mice during acute infection common

Araujo and Remington (1974), Nguyen and Stadtsbaeder (1985), Fux et al. (2000), Mu¨ller et al. (2017)

Rat

Hemotrichorial placenta; rare vertical transmission during chronic infection; strain-dependent variation in transmission rates

Usmanova (1965)

Calomys callosus

Hemotrichorial placenta; transmission rates during acute infection higher than in humans; no vertical transmission during chronic infection

Costa et al. (2009)

Guinea pig

Hemomonochorial placenta; vertical transmission during chronic Youssef et al. (1985) infection observed; threefold longer gestation time than mice and rats

Primate

Hemomonochorial placenta; transmission rates resemble those in humans

conditions, vertical transmission occurs during chronic infection and through successive generations of mice (Remington et al., 1961; Beverley, 1959). Thus at first glance, the mouse model may not be best suited to mimic the situation in humans, where vertical transmission usually occurs only during primary infection in pregnancy. However, other findings prove that whether transmission to the fetus during a chronic infection of the mother occurs is largely dependent on the parasite strain as well as on the mouse strain used; for example, in mice that were latently infected with 11 different Toxoplasma strains, placental transmission succeeded with only six strains (Werner et al., 1977). Furthermore, chronically infected BALB/c mice normally do not allow vertical transmission, whereas acutely infected BALB/ c mice transmit the parasite to approximately 50% of the littermates (Roberts and Alexander, 1992). In addition, no congenital transmission was detected in the litters of chronically infected BALB/K mice, although the mothers themselves were found to have extremely high

Schoondermark-van de Ven et al. (1994a,b, 1995)

cyst counts (Roberts and Alexander, 1992). The explanation for this contradictive outcome of vertical transmission of chronically infected mice most likely lies in their genetic background (as known for low susceptibility of BALB/c mice in general), and this view may be supported by the fact that in contrast to the inbred BALB mice, most earlier studies used outbred animals or NMRI mice. Another critical factor that can impact vertical transmission of T. gondii is whether chronically infected hosts are reinfected during pregnancy and which parasite strain is used for reinfection (ElbezRubinstein et al., 2009). At least in BALB/c mice, a chronic infection seems to protect against vertical transmission after reinfection with a homologous or even a heterologous parasite strain (Freyre et al., 2006a; Pezerico et al., 2009). Since, in principle, reinfection of mice with heterologous Toxoplasma strains is possible (Elbez-Rubinstein et al., 2009), further studies using other mouse and parasite strains are required in order to verify this issue in the congenital infection model. Recent investigations

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TABLE 7.2

Models of congenital Toxoplasmosis.

Toxoplasma Toxoplasma stage and route of Species and strain strain of animal useda administereda infection

Outcome of animal infection II (with Outcome of animal regard to Administration time infection I (with regard transmission in point and Toxoplasma to fetal transmission in chronically infected dose in relation to acutely infected animals or after pregnancy animals) treatment) Transmission of chronically infected mice occurred, most frequently with the Beverley strain

Remington et al. (1961)

Five cysts on day 5, 10, Overall transmission or 15 of gestation rate greater than 90%; clinical outcome in offspring more severe if infected early during pregnancy

Not determined

Wang et al. (2011)

Cysts i.p. prior to pregnancy; cysts orally during pregnancy only in a primary infection trial with Toxoplasma ALT strain

20 cysts 4
8 weeks before mating; 20 cysts on day 10 of pregnancy

Primary infection during pregnancy (ALT strain only): Transmission rate 28%

Infection prior to Werner et al. pregnancy: S93, K8, (1977) 558/72, Witting, Gail: no transmission; KSU, 1070, 162/74, 248/70, MO, ALT: transmission rate 1%
3%

Cysts s.c.

Undefined number of cysts 5
8 days after mating

75% offspring with positive serology; offspring survival 40%; number of offspring approximately 20% compared to noninfected control; 30% mortality of infected mothers during gestation

Cotrimoxazole treatment during pregnancy almost normalized offspring number and survival

Mouse: NIH strain RH, 113-CE, Beverley, M7727

Tachyzoites (RH) and cysts (113-CE, Beverley, M7727) i. p. or s.c.

Undefined numbers for acute and chronic infections; acute (RH) on day 16 of pregnancy; chronic (113-CE, Beverley, M7727) 2
15 months prior delivery

Mouse: NIH strain Prugniaud

Cysts orally

Mouse: NMRI

S93, K8, 558/ 72, Witting, Gail, KSU, 1070, 162/74, 248/70, MO, ALT

Mouse: NMRI

Beverley

RH: No infection of young if delivery before the fifth day after infection; 56% infection on day 7 after i.p. inoculation

Publicationb

Nguyen and Stadtsbaeder (1985)

(Continued)

TABLE 7.2

(Continued)

Toxoplasma Toxoplasma stage and route of Species and strain strain of animal useda administereda infection

Outcome of animal infection II (with Outcome of animal regard to Administration time infection I (with regard transmission in point and Toxoplasma to fetal transmission in chronically infected dose in relation to acutely infected animals or after pregnancy animals) treatment)

Publicationb

Mouse: Swiss Webster

RH

Tachyzoites i.p.

1 3 105 at day 11
14 of Diaplacental gestation transmission in 9 out of 11 dams

No diaplacental Araujo and transmission after Remington clindamycin treatment (1974)

Mouse: CD1

ME49 (PTG)

Tachyzoites i.p.

50
2400 (600 for chemotherapy groups) during 2nd trimester of gestation

Severe clinical symptoms including abortion, early birth or still birth

Partial protection after Oz and Tobin treatment with (2012, 2014) atovaquone or diclazuril

Mouse: CD1

ME49

Oocysts orally

5
2000 oocysts (20 oocysts for chemotherapy groups) at day 7 after mating

Transmission rate up to 100% irrespective of infection dose; survival of pups 0%
31%

Transmission rate only Mu¨ller et al. 7% (BKI-1294) and (2017) 22% (Buparvaquone)

Mouse: BALB/c, BALB/K (inbred)

Beverley

Cysts orally

8 weeks before mating 5 cysts; day 12 of pregnancy 20 cysts

Transmission rate of approximately 50% (cysts for first time in pregnancy only)

Congenital infection occurred only if the mother was infected for the first time during pregnancy

Roberts and Alexander (1992)

Mouse: BALB/c (inbred)

P

Cysts orally

Two cysts between days 6
15 of pregnancy

Fetal transmission rate between 50% and 60%

Minocycline-treated mice showed transmission in only 3.6%

Fux et al. (2000)

Mouse: BALB/c (inbred)

ME49, Prugniaud, M7741, M3

Bradyzoites and oocysts orally

Chronically infected mice, reinfection during pregnancy

Cross-protection of chronic infection against homologous and heterologous reinfection

Transmission in 2 of 10 chronically infected mice or no transmission

Freyre et al. (2006a), Pezerico et al. (2009)

Mouse: BALB/c (inbred)

ME49

Tachyzoites i.v.

2.5 3 106 and 10 3 106 tachyzoites at day 10 of gestation

Transmission rate 18%
Not done 78%

VargasVillavicencio et al. (2016b)

Rat: Sprague
Dawley

RH

Tachyzoites i.p.

1 3 107 and 2 3 107 tachyzoites 6
8 weeks before mating

Virtually no fetal transmission (three offspring from 140 in total, all in a single litter)

Remington et al. (1958)

Rat: Sprague
Dawley, Osborne
Mendel, Black rat, Holtzman rat

RH, S-6, Beverley

Tachyzoites (RH, S-6) and cysts (Beverley) i.p.

1 3 104 to 1 3 107 tachyzoites and undefined number of cysts 2
8 months prior gestation

No fetal transmission from chronically infected rats with RH or S-6; Beverley: 5% transmission

Remington et al. (1961)

Rat: Sprague
Dawley

CT-1

Oocysts orally or s. 1 3 104 oocysts at 7
15 c.; Bradyzoites s.c. days of pregnancy; 1 3 104 bradyzoites at 10
14 days of pregnancy

Transmission rate of No transmission in 82.1% (oocysts orally), chronically infected 90.9% (oocysts s.c.), 43.8 rats (bradyzoites s.c.)

Dubey and Shen (1991)

Rat: Fischer

RH, 76K, Prugniaud

RH: tachyzoites i. p.; Prugniaud and 76K: cysts orally

Between days 8
12 of pregnancy: 8 3 106 RH tachyzoites; 1200 Prugniaud or 76K cysts

Transmission rates of 58% (RH), 63% (Prugniaud), 35% (76K)

Zenner et al. (1993, 1999a)

Rat: Sprague
Dawley

VEG

Oocysts orally

1 3 104 oocysts on day 6, 9, 12, or 15 of gestation

Transmission rates: 33% Virtually no (day 6), 55% (day 9), transmission to the 83% (day 12), and 57% next generation (day 15)

Rat: Wistar and Long Evans

12 different strains of low to high pathogenicity for mice (e.g., M7741, Beverley, M49)

Cysts orally

2 3 102 to 3.4 3 103 cysts at 6
8 and 15 days of pregnancy

Overall transmission of 44% with a range of 0%
90% attributed to genetically based susceptibility of outbred Wistar rats; frequency of transmission not affected by the strain or dose of Toxoplasma nor by the time point of infection

No transmission to fetuses of chronically infected rats, even if rats were reinfected during pregnancy

Transmission more frequent in Long Evans than in Wistar rats

Dubey et al. (1997)

Freyre et al. (2001a)

(Continued)

TABLE 7.2

(Continued)

Toxoplasma Toxoplasma stage and route of Species and strain strain of animal useda administereda infection

Outcome of animal infection II (with Outcome of animal regard to Administration time infection I (with regard transmission in point and Toxoplasma to fetal transmission in chronically infected dose in relation to acutely infected animals or after pregnancy animals) treatment)

Publicationb Freyre et al. (2003b)

Rat: Wistar

Six different strains of low to high pathogenicity for mice

Oocysts orally

1 3 104 oocysts at 15 days of pregnancy

Transmission rates of 10%
80%; higher transmission with strains more pathogenic for mice

Rat: Sprague
Dawley, Fischer

RH, Prugniaud, M3

Tachyzoites (RH), cysts and oocysts orally

Chronic infection with RH, Prugniaud or M3 2 months before gestation, reinfection during pregnancy

Complete protection Not determined against vertical transmission after reinfection with same strain and stage; partial protection after heterologous reinfection

Freyre et al. (2006b)

Rat: Fischer

Prugniaud

Bradyzoites and oocysts orally

1 3 104 bradyzoites or 100 oocysts

Transmission rate greater than 50%

Freyre et al. (2008)

Calomys callosus: Canabrava strain

ME49

Cysts orally

20 cysts at days 250, 230, 210 of pregnancy or during pregnancy

Transmission rate 100% No transmission if infected at day 210 or during chronic during pregnancy infection (infection 50
30 days before pregnancy)

Pereira Mde et al. (1999), Barbosa et al. (2007), Costa et al. (2009)

Hamster

ME49 for chronic infection; Prugniaud, M7741, M3 for acute infection

Bradyzoites orally, oocysts orally

103
104 bradyzoites; 102
104 oocysts; time of administration not specified

Transmission rates of 25%
100%

Freyre et al. (2009, 2012)

No statistically significant differences in the rate of transmission in rats fed with cysts (previous work)

Not determined

Transmission rate of 9%

Guinea pig: not specified

Beverley

Cysts i.p.

60 or 100 cysts at 4
54 days of pregnancy; 100
200 cysts 2
6 months before mating

Transmission rate of Transmission rate of Wright (1972) 100% if infection during 30% if infection before pregnancy pregnancy; overall high number of stillborn or severely ill pups

Guinea pig: Dunkin
Hartley

C56

Tachyzoites intradermally

5 3 105 tachyzoites after 7 weeks of gestation

Transmission in more than 80%

SAG1-immunized animals with lower transmission rates

Haumont et al. (2000)

Guinea pig: Dunkin
Hartley

RH, 76K, Prugniaud

Tachyzoites (RH) i. p.; cysts (76K, Prugniaud) i.p. or orally

Various time points from 90 days before pregnancy to day 40 of pregnancy: 100 tachyzoites (i.p.); 100 cysts (i.p. or orally)

Transmission rates if infection during pregnancy: 54% (RH); 84% (Prugniaud); 86% (76K)

Transmission rate of 17% if infection 30
90 days before mating; overall high number of stillborn and nonviable fetuses

Flori et al. (2002)

Guinea pig: Dunkin
Hartley

76K

Cysts orally

100 cysts at days 20 or 40 of pregnancy

Transmission rates of 84.6% and 100% after inoculation on days 20 and 40, respectively

Infection assessed by real time PCR

Flori et al. (2003)

Rabbit: not specified

Witting/ALT

Cysts i.p. prior pregnancy; cysts orally during pregnancy

200 cysts 267
19 days before mating; 200 cysts during the first or second trimester to preinfected or nonpreinfected animals

Fetal transmission rate up to 79%

No congenital transmission when first infection was placed at least 35 days prior mating

Werner et al. (1977)

Primate: Macaca mulatta (rhesus)

RH

Tachyzoites i.v.

5 3 106 tachyzoites on days 90 or 130 of pregnancy

Overall transmission rate of 61% which is comparable to that found in humans

a

For definition of animal or Toxoplasma strains, please see respective articles. Due to numerous publications on this topic, only few were chosen exemplarily for this table. BKI, Bumped kinase inhibitor; i.p., intraperitoneal. b

Schoondermarkvan de Ven et al. (1993)

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7. Toxoplasma animal models and therapeutics

on congenital toxoplasmosis tend to use a BALB/c mouse model (Thouvenin et al., 1997; Fux et al., 2000; Elsaid et al., 2001; Abou-Bacar et al., 2004a, 2004b; Beghetto et al., 2005; Freyre et al., 2006a; Vargas-Villavicencio et al., 2016b), but Swiss Webster and CD1 outbred mouse strains have also been successfully used (Araujo and Remington, 1974; Oliveira et al., 2016; Mu¨ller et al., 2017). The mouse model was also used as a model for evaluating therapeutics (Araujo and Remington, 1974; Nguyen and Stadtsbaeder, 1985; Fux et al., 2000; Oz and Tobin, 2012, 2014; Mu¨ller et al., 2017) but even more commonly as a model for studying pathogenetic mechanisms and, more recently, for an evaluation of vaccine approaches (McLeod et al., 1988; Roberts et al., 1994; Elsaid et al., 2001; Couper et al., 2003; Letscher-Bru et al., 2003; Ali et al., 2003; Mevelec et al., 2005; Ismael et al., 2006). However, Fux et al. (2000) were able to show positive effects of a minoxycycline treatment on congenital Toxoplasma transmission. Using the bumped kinase inhibitor 1294, survival of pups from infected CD1 dams could even be increased to 100% and only 7% of them were T. gondii-positive as compared to 31% and 84%, respectively, in the control group (Mu¨ller et al., 2017). Considering the advantages of easy handling of mice as laboratory animals and the availability of new sensitive PCR-based detection methods that allow better diagnostics also in small animals, a mouse model may thus have its place as the first-line screening model for testing new chemotherapeutic agents against congenital toxoplasmosis.

7.2.2 Rat Like humans and primates, rats are relatively resistant to T. gondii with respect to a clinical manifestation of the infection. While transmission during acute infection of maternal rats induced by intracerebral or i.v. infection was first reported during the early 1950s

(Schultz and Bauer, 1952; Hellbru¨gge and Dahme, 1953; Hellbru¨gge 1955; Hellbru¨gge et al., 1953), it has been shown more recently that also oral ingestion of oocysts or tissue cysts leads to fetal transmission (Dubey and Shen, 1991; Zenner et al., 1993; Freyre et al., 2001a, 2003b, 2008). In general, transmission rates seem to be high and were reported to lie mostly between 30% and 90%. However, there was great variation attributed to the Toxoplasma strains and to the rat strains used (Zenner et al., 1993; Freyre et al., 2003b). Indeed, also a wide variability for the formation of Toxoplasma cysts in rats of the same outbred strain and age, inoculated with the same strain, stage and dose of Toxoplasma, using the same infection route was observed (Freyre et al., 2001a). Such an individual resistance of rats belonging to the same outbred strain may be attributed to the individual genetic background of the infected rat (Freyre et al., 2001a; Cavailles et al., 2006). In an experimental study, design for drug-efficacy testings, for example, such a lack of individual reproducibility may be overcome either by a comparatively high number of animals per group or by the use of inbred animals. Except in rare instances, when unnaturally high doses of several million organisms were used for infection (Hellbru¨gge, 1955), T. gondii parasites were either not transmitted at all or extremely rarely from chronically infected rats to their littermates, irrespective of the route of inoculation, stage, strain, or size of inoculum (Remington et al., 1958, 1961; Dubey and Shen, 1991; Zenner et al., 1993; Dubey et al., 1997). Partial protection of chronically infected rats against vertical transmission after reinfection with parasites of a different clonotype during pregnancy was reported (Freyre et al., 2006b). In contrast to the situation in some, but not all mouse strains in which the organism is transmitted repeatedly during chronic infection, vertical transmission of chronically infected rats requires reinfection with a heterologous

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7.2 Congenital toxoplasmosis

parasite strain during pregnancy (Freyre et al., 2006b), as also reported for humans (ElbezRubinstein et al., 2009). Thus with respect to clinical course and in utero transmission, toxoplasmosis in rats and humans is similar and the infection in rats may serve as a proper model especially for human congenital toxoplasmosis (for review, see also Dubey and Frenkel, 1998). In spite of the obvious analogies concerning transmission, transmission rates and rates of clinical manifestation, rat models, with rare exceptions (Usmanova, 1965), have so far not been used for drug testing in congenital toxoplasmosis. This may be due to the fact that infected litters usually appear healthy. However, regarding the T. gondii strains, stage and routes of inoculation and probably also the rat strains, rats may serve as excellent models, especially when emphasis lies on placental transmission. In any case, as total protection against congenital toxoplasmosis can be achieved regardless of the Toxoplasma strain, rats may be attractive models for evaluating future vaccine candidates against the disease (Zenner et al., 1993, 1999a).

7.2.3 Calomys callosus More recently, Calomys callosus, a cricetid rodent from South America, has been evaluated as a model for human congenital toxoplasmosis (Pereira Mde et al., 1999; Ferro et al., 2002; Franco et al., 2011). The results show that in C. callosus, T. gondii is efficiently transmitted during acute infection to their fetuses with rates up to 100% (Pereira Mde et al., 1999; Costa et al., 2009). In contrast, vertical transmission does not seem to occur during chronic infection or even after reinfection with a heterologous Toxoplasma strain during pregnancy (Barbosa et al., 2007; Franco et al., 2011). Due to the high vertical transmission rates and the obviously low variability, C. callosus may

331

represent a valuable model to test the efficacy of novel drug and vaccine regimes during congenital toxoplasmosis. A drawback certainly is represented by the limited availability of immunological reagents in order to investigate pathogenic issues. However, using this model, a high efficacy of azithromycin as compared to spiramycin, a combination of sulfadiazine, pyrimethamine and folinic acid, or Artemisia annua infusion in preventing vertical transmission has been reported (Costa et al., 2009).

7.2.4 Hamster Although it is known for several decades that the hamster supports vertical transmission of Toxoplasma (De Roever-Bonnet, 1960), it has only recently been reevaluated as another model for congenital toxoplasmosis (Freyre et al., 2009, 2012). Depending on the parasite strain and stage, the rate of vertical transmission during acute infection varies between 25% and 100% (Freyre et al., 2009). Female hamsters also transmit T. gondii to their fetuses during chronic infection, although with lower frequency (less than 10%) than during acute infection (Freyre et al., 2009). Chemotherapeutic drugs have not yet been evaluated using the hamster model.

7.2.5 Guinea pig A guinea pig model for congenital toxoplasmosis has been described in various studies (Adams et al., 1949; Huldt, 1960; Wright, 1972; Haumont et al., 2000; Flori et al., 2002, 2003; Berard-Badier et al., 1968). As in humans, the guinea pig placenta is of the hemomonochorial type (Darcy and Zenner, 1993) suggestive of similar modes of transmission. However, transmission rate and sensitivity of guinea pigs to T. gondii after i.p. or oral infection are much higher and about halfway between the rat and mouse. In addition, congenital transmission

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during pregnancy of chronically infected females was observed (Wright, 1972; Flori et al., 2002). As a possible advantage in comparison to mice and rats, guinea pigs have approximately threefold longer gestation periods (with a duration of 65 days), which are long enough to enable comparative studies with different inoculation times and comparative chemotherapy studies (Flori et al., 2002). For this application the guinea pig model may be best suited; however, except for rare instances (Youssef et al., 1985), in spite of its potential advantages, a guinea pig model for congenital toxoplasmosis has not yet been employed for drug testing.

7.2.6 Primate With respect to hemochorial placentation (Ramsey and Harris, 1966; Darcy and Zenner, 1993), fetal blood sampling and assessment of fetal infection, a primate model should actually best meet the requirements for the study of the effect of medication in the infected fetus. One of the first such studies was conducted with Macaca arctoides as a model for primates (Wong et al., 1979). Data obtained with this model suggested that although certain developmental stages of the Toxoplasma organism and of the fetus may favor the occurrence of congenital infection, transmission rate in general seems to be low and very little neonatal disease results (Wong et al., 1979). In contrast, a more applicable model was established by Schoondermarkvan de Ven et al. (1993) with rhesus monkeys (Macaca mulatto). Herein, the frequencies of transmission which were found in the rhesus monkey after maternal infection in the second and third trimester of gestation equal those observed in humans (Schoondermark-van de Ven et al., 1993). The rhesus model was then used to prove the efficacy of spiramycin or the combination of pyrimethamine and sulfadiazine for the treatment of congenital T. gondii

infection (Schoondermark-van de Ven et al., 1994b, 1995). The results showed that both regimens were clearly effective in reducing the number of parasites in the infected fetus as proven by PCR and mouse inoculation with amniotic fluid. However, spiramycin was less active (Schoondermark-van de Ven et al., 1994b) and was not found anywhere in the fetal brain (Schoondermark-van de Ven et al., 1994a). The rhesus monkey model is perhaps theoretically the best animal model to prove the efficacy of a drug against human congenital toxoplasmosis, especially with regard to placental transmission. However, housing and handling of monkeys require special facilities and particularly well trained employees. It is also time-consuming and expensive which may limit the numbers of animals used for the studies. Considering this fact and considering that the outcome of the congenital infection, as in humans, often seems to be subclinical or at least shows a high degree of variation (Schoondermark-van de Ven et al., 1993), no direct drug evaluation concerned with its influence on the clinical course of a congenital toxoplasmosis seems practical so far. Thus drug efficacy must be extrapolated indirectly from the demonstration of the parasite or parasite DNA in the amniotic fluid or fetal tissue and by pharmacokinetic data of the drug that may also be obtained from the monkey fetus. In summary, the rhesus monkey model might have its place as a last evaluation step for a new drug before it is admitted to clinical trials.

7.2.7 Rabbit Surprisingly, rabbit congenital toxoplasmosis has not been extensively studied, although rabbits are widely used laboratory animals, and transmission from the mother to the fetus has been demonstrated (Uhlikova and Hubner, 1973). In addition, fetuses from chronically

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infected mothers were protected, thus a rabbit model would share common features with the infection in humans (Werner et al., 1977). A rabbit model may be of particular interest when the small size of other common laboratory animals such as mice and rats hampers experiments, for instance, when larger volumes or subsequent blood samples are needed for examination of an antibody response (Araujo and Remington, 1975). To our knowledge no pharmaceutical studies on congenital toxoplasmosis have been conducted using a rabbit model.

7.2.8 Other animals Various other animals have been suggested as a model for congenital toxoplasmosis but have never been broadly used. As an example, although pigs are well known to acquire toxoplasmosis and to play a decisive role in transmitting the disease to humans via their meat (for review see Tenter et al., 2000; Montoya et al., 2004), their use as animal models has never been thoroughly investigated. However, when infecting pregnant miniature pigs with strains of different virulence, even a strain that was considered apathogenic to both, pigs and mice, resulted in significant numbers of congenitally infected piglets (Jungersen et al., 2001). Such a pig model of congenital toxoplasmosis may therefore be of value for situations where big animals are needed and they may be relevant animal models for certain Toxoplasma strains with low virulence that are obviously transmitted to the fetus (Jungersen et al., 2001). Congenital disease due to T. gondii is a major cause of abortion and neonatal mortality in sheep. In addition, undercooked meat from infected sheep is an important source of infection for humans. Although congenital transmission is well described and, in fact, is known as a major cause of abortion in this species (Dubey and Rommel, 1992; Anderson et al.,

1994), an animal model mimicking congenital infection in humans has, to our knowledge, not been established. Recent studies with sheep indicate that reactivation of chronic infection during pregnancy may be a major cause for the congenital infection (Duncanson et al., 2001; Williams et al., 2005). In this respect, congenital toxoplasmosis in sheep seems more to resemble a common mode of congenital transmission in mice than in humans. Que et al. (2004) reported a novel chicken embryo model which had been adopted from a chicken model that had been developed for the study of metastatic diseases. Basically, tachyzoites were injected directly into the chorioallantoic veins of 12 days old chickens and after an incubation period of 3
6 days, the degree of infection was assessed by histopathological examination and quantitative real-time PCR on the Toxoplasma DNA. As this model also provides the possibility of injecting drugs via the chorioallantoic vein, it may prove precious for an initial drug screening setup with a course of infection that is shorter than that in mice (Que et al., 2004).

7.3 Ocular toxoplasmosis Ocular toxoplasmosis may result from an in utero infection of a fetus via a mother whose primary infection was acquired during pregnancy or from postnatal infection. Nowadays, postnatally acquired infection is considered to account for the majority of cases of human ocular toxoplasmosis. The number of cases and severity of disease are higher in South and Central America, the Caribbean, and parts of tropical Africa than in Europe and North America; this may relate to the high prevalence of atypical genotypes of the parasite (for review, see Holland, 2003; Petersen et al., 2012). Different modes of infection and pathogenesis may lead to various outcomes in ocular toxoplasmosis as recently suggested by the

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TABLE 7.3 Pharmacological studies on ocular toxoplasmosis. Infection model

Pharmacological studies (literature examples)

Specific comments

Animal species

Localized eye infection models

Best for confining infection to eyes but with the principal drawback of tissue needle damage

Rabbit, primate, mouse, guinea pig

Beverley (1958), Kaufman (1960), Jacobs et al. (1964), Tabbara et al. (1974), Rollins et al. (1982), Jacobs et al. (1964)

Infection via the carotid artery

Cat is definitive natural host which may influence, for example, immunoreactions

Cat

Davidson et al. (1996)

Systemic infection models

Particularly hamsters and Calomys callosus produce consistent ocular disease following i.p. or oral infection

Mouse, Hamster, C. callosus

Olle et al. (1996), Norose et al. (2006), Gormley et al. (1998), Lopes et al. (2009)

i.p., intraperitoneal.

more pronounced lesions in the retinae of mice which acquired the infection congenitally as compared to those with postnatal infection (Ashour et al., 2018). The majority of animal models for human ocular toxoplasmosis have been developed to mimic a primary infection of adults, although there have been also efforts which have successfully established an ocular disease as a consequence of a transplacental Toxoplasma transmission (Lee et al., 1983; Hay et al., 1984; Dutton et al., 1986; Hutchison et al., 1982; Hay and Dutton, 1996; Lopes et al., 2009; Lahmar et al., 2010). In patients with underlying immunosuppression or immune defects such as bone marrow transplantation or HIV, toxoplasmic chorioretinitis is often associated with concurrent toxoplasmic encephalitis (TE) or disseminated infection (for review, see Montoya et al., 2004). However, even in individuals with AIDS, toxoplasmic chorioretinitis is encountered relatively infrequently (Holland et al., 1988a), so that most of the human cases of ocular toxoplasmosis are found in immunocompetent patients. Indeed, T. gondii is one of the most frequently identified cause of uveitis and the most commonly identified pathogen infecting the retina of otherwise immunocompetent

individuals (Holland, 1999). Regardless of whether ocular toxoplasmosis is due to a reactivated congenital infection or to an infection that is acquired after birth, it usually presents in the immunocompetent host as a more or less localized eye disease (Montoya et al., 2004). An animal model, particularly one that may be suited to the evaluation of the efficacy of a given drug, should ideally be characterized by a localized eye infection or at least predominantly by a localized eye infection, rather than by a generalized or CNS infection where the eye is just one disease location among others— an experimental challenge that is not easy to fulfill. Current models of postnatally acquired ocular toxoplasmosis are either based on primary local inoculation of T. gondii into the animal’s eye, on a semilocal infection via the carotid artery, or on a primary systemic infection which then affects the eyes as their predominant organ of manifestation (see Tables 7.3 and 7.4).

7.3.1 Models based on local eye infection In order to meet the abovementioned requirements the localized infection of animal eyes was the first choice which, for technical

Toxoplasma Gondii

TABLE 7.4 Models of ocular toxoplasmosis. Species and strain of animal useda

Toxoplasma strain Toxoplasma dose, stage, and administereda route of infection Outcome of animal infection

Remarks

Publicationb Hutchison et al. (1982), Dutton et al. (1986)

Mouse: strain A albino Beverley

10 cysts s.c. on day 12 of pregnancy

Approximately 50% of offspring infected, 5.3% of these developed cataract; acute uveitis in a small proportion of eyes

Model of congenital ocular toxoplasmosis

Mouse: C57BL/6

ME49

10
20 cysts i.p.

Mild uveitis and retinal vasculitis in all infected animals at 15 days postinfection

In most ocular lesions, the parasite Gazzinelli could not be demonstrated even et al. (1994) with PCR

Mouse: Swiss Webster

Beverley

Cysts (undefined number) i. p.; immunosuppression by injecting polyclonal antibodies against γ-interferon

26% of mice developed chorioretinitis on days 13
15

Toxoplasma gondii was revealed by routine cellular cultures in all immunosuppressed mice with ocular lesions

Mouse: Swiss Webster

Prugniaud (PRU)

10 cysts orally on day 12 of pregnancy

Mortality in littermates 60%; ocular Congenital ocular toxoplasmosis lesions in eyes from less than 20% of model surviving littermates until 4 weeks after birth; T. gondii DNA in 75% of eyes from surviving mice

Lahmar et al. (2010)

Mouse: Swiss Webster

PRU

Five cysts s.c. on day 7 after birth

100% of mice developed retinitis and retinal vasculitis until 4 weeks p.i.; T. gondii DNA present in all eyes

Neonatal ocular toxoplasmosis model

Lahmar et al. (2010, 2014)

Mouse: C57BL/6, MRL
MpJ

PLK (derived from ME49)

50
5 3 104 tachyzoites injected into the anterior chamber

Dose-dependent intraocular inflammation: 50 (none), 5 3 102 and 5 3 103 (moderate to severe), 5 3 104 tachyzoites (severe, early mortality)

Some protection if mice were preinfected before challenge

Hu et al. (1999a)

Mouse: 129/SVJ (WT), Beverley IL-6-deficient strain with same genetic background

10 cysts i.p.

Regular mild to moderate retinochoroiditis 4 weeks after challenge, severe inflammation in IL-6 deficient mice

Cytokine expression study

Lyons et al. (2001)

Mouse: C57BL/6, ME49 B6MRL/Ipr and B6MRL/gld (defective Fas or FasL expression, respectively)

20
30 cysts i.p.

Regular mild retinochoroiditis and moderate encephalitis after 14 days, becoming worse after 28 days

No significant difference in the degree of ocular inflammation between WT and Fas or FasL mutant mice

Shen et al. (2001)

Olle et al. (1994)

(Continued)

TABLE 7.4 (Continued) Species and strain of animal useda

Toxoplasma strain Toxoplasma dose, stage, and administereda route of infection Outcome of animal infection

Remarks

Publicationb

Mouse: C57BL/6, BALB/c and IFN-γ knockout mice (GKO) of both WT backgrounds

Fukaya

5 cysts perorally

Evidence of eye inflammation in GKO mice only; assessment via PCR, histopathology and fluorescein angiography

Toxoplasmic eye vasculitis model for GKO mice; GKO mice died 11
12 days after infection; improved outcome after sulfamethoxazole treatment

Norose et al. (2003, 2006)

Mouse: C57BL/6, NMRI

ME49

5
20 tissue cysts orally (C57BL/6), 20 or 100 tissue cysts (NMRI)

Ocular lesions in both mouse strains common; cysts regularly present in C57BL/6, but only occasionally detected in NMRI

Postnatally acquired ocular toxoplasmosis model

Dukaczewska et al. (2015)

Mouse: C57BL/6

ME49

5 3 103 bradyzoites injected intravitreally or via conjunctival instillation

Regular retinochoroiditis with both infection routes 7 days after infection

Additional eye damage caused by the intravitreal injection: instillation route preferable

Tedesco et al. (2005)

Mouse: C57BL/6, BALB/c, CBA/J

RH, PLK, SAG1 (P30)deficient RH derived mutant strain

100 tachyzoites injected into the anterior chamber (Lu et al., 2005); 103
104 tachyzoites intravitreally (Charles et al., 2007)

C57BL/6: severe eye inflammation and 100% mortality with all T. gondii strains; BALB/c and CBA/J: mild to medium eye inflammation most pronounced with RH (all mice survived)

All strains of mice were protected after i.p. vaccination with temperature-sensitive mutant tachyzoites (ts-4)

Lu et al. (2005), Charles et al. (2007)

Mouse: CD4-, CD8-, B cell-, IL-10-deficient C57BL/6

Temperaturesensitive strain ts-4 (RH background), RH

100 tachyzoites injected into the anterior chamber

Ocular lesions but no host death, most Partial protection by intraocular ts- Lu et al. severe eye lesions in CD8 KO and IL-10 4 immunization against intraocular (2009) KO RH challenge

Rat: Sprague Dawley

11 strains of various genotypes

10
105 (mouse infective units) oocysts perorally

Ocular lesions in 23/92 eyes of rats euthanized 2 months p.i.

Rabbit: not specified

113-CE

5 3 103 or 1 3 104 tachyzoites Uveitis developed after a few days into the anterior chamber

Pyrimethamine
sulfadiazine treatment

Rabbit: pigmented Dutch rabbits, New Zealand white rabbits, pigmented California rabbits

RH, Beverley

Transscleral inoculation of 1000
2000 tachyzoites into the suprachoroidal space

California rabbits best for technical Nozik and handling O’Connor (1968)

RH: animal death from encephalitis; Beverley: retinochoroiditis in most animals after 7 days, resolving after 3 weeks

Mostly focal inflammation in the Dubey et al. retinae; tissue cysts detected in 20/ (2016) 23 diseased eyes Jacobs et al. (1964)

Rabbit: pigmented California rabbits

Beverley

Transscleral inoculation of 400 tachyzoites into the suprachoroidal space

Constant induction of retinochoroiditis

Clinical improvement of retinochoroiditis in clindamycin treated rabbits

Rabbit: pigmented California rabbits

RH

Transscleral inoculation of 400 tachyzoites into the suprachoroidal space

Nontreated animals developed retinitis but all died from toxoplasmic encephalitis

Minocycline prevented death from Rollins et al. toxoplasmic encephalitis in 75% of (1982) animals

Rabbit: Burgundy

BK

Injection of 5 3 103 tachyzoites across the vitreous cavity into the superficial part of the retina

All animals developed retinochoroiditis Primed animals also with high after 7 days; 22% of naive rabbits died incidence of retinochoroiditis from generalized infection (greater than 90%); Model used to monitor humoral response intraocularly

Garweg et al. (1998), Garweg and Boehnke (2006)

Syrian Golden Hamster

ME49

10
25 cysts i.p.

All animals developed bilateral eye disease peaking after 4
5 weeks

No animal developed signs of systemic disease

Pavesio et al. (1995)

Syrian Golden Hamster

ME49

100 cysts orally

All animals developed unilateral or bilateral eye disease 4
8 weeks after infection

No animal developed signs of systemic disease

Gormley et al. (1999)

Guinea pig: not specified

RH

5 3 103 tachyzoites injected into the posterior chamber

Most animals developed acute chorioretinitis within 1
3 weeks

Recovery of Toxoplasma from eyes even after 10 months

Hogan et al. (1956)

Guinea pig: not specified

RH

5 3 103 tachyzoites injected into the posterior chamber

Not specified

Pyrimethamine
sulfadiazine treatment

Jacobs et al. (1964)

Calomys callosus: Canabrava strain

ME49

20 cysts orally at days 5
7 of 40% of fetuses presented ocular lesions; Model for congenital as well as for pregnant or nonpregnant 50%
75% of adult animals presented acquired ocular toxoplasmosis; animals unilateral ocular cysts Congenital ocular toxoplasmosis prevented by azithromycin

Pereira Mde et al. (1999), Lopes et al. (2009)

Pig

M4 (type II), S48 (type I)

103 M4 oocysts or tissue cysts, 1.2 3 105 S48 tachyzoites

DNA detected in the eyes of 47% of pigs; positive Goldmann
Witmer antibody index in 40% of pigs

Model of postnatally acquired ocular toxoplasmosis

Garcia et al. (2017)

Domestic cat

ME49

5 3 103 tachyzoites into the common carotid artery

Progressive multifocal retinal and choroidal inflammatory foci (mostly bilateral) beginning 5
8 days postinoculation in all cats tested

Minimal to no clinical signs of generalized toxoplasmosis; resolution of lesions 21
70 days postinoculation

Davidson et al. (1993)

Primate: Macaca fascicularis, Cercopithecus aethiops, Macaca mulatta (rhesus)

RH

Injection of 5 3 103 to 1 3 105 tachyzoites across the vitreous cavity into the superficial part of the retina

Retinitis was reliably produced in all monkeys’ eyes injected with 1 3 104 or more living tachyzoites

After 20 days the lesions began to resolve

Culbertson et al. (1982)

a

For definition of animal or Toxoplasma strains, please see respective articles. Due to numerous publications on this topic, only few were chosen exemplarily for this table. IFN, Interferon; i.p., intraperitoneal.

b

Tabbara et al. (1974)

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7. Toxoplasma animal models and therapeutics

reasons, required the use of larger animals. Thus until 1982, the rabbit served as the most important experimental model for ocular toxoplasmosis and morphological lesions of acute experimental Toxoplasma chorioretinitis were produced by injection of parasites intravitreally (Kaufman, 1960), into the anterior chamber (Beverley et al., 1954; Beverley, 1958, 1961; Jacobs et al., 1964) or by transscleral inoculation into the suprachoroidal space (Nozik and O’Connor, 1968; Tabbara et al., 1974; Rollins et al., 1982). In fact, the anterior chamber model was used to show efficacy of pyrimethamine and sulfadiazine (Jacobs et al., 1964), whereas the latter model was used to demonstrate the efficacy of clindamycin and minocycline on toxoplasmic chorioretinitis (Tabbara et al., 1974; Rollins et al., 1982). Intraocular injection of T. gondii tachyzoites in naive or chronically infected rabbits has recently been employed to monitor antibody levels in serum and aqueous humor (Garweg and Boehnke, 2006). In 1982 a primate model was established that reliably produced acute toxoplasmic chorioretinitis by injection of viable RH strain Toxoplasma organisms (Culbertson et al., 1982; Newman et al., 1982). Whereas in the rabbit model the injections were made transsclerally into the suprachoroidal space at the posterior pole, in monkeys it was not possible to expose the posterior part of the sclera for direct injection. Therefore the retinal injections were made through the pars plana across the vitreous cavity, directly into the superficial part of the retina at the posterior pole (Culbertson et al., 1982). Nonhuman primates as well as rabbits infected via a transvitreal approach were later also used in other studies (Webb et al., 1984; Holland et al., 1988b; Garweg et al., 1998). These transvitreal inoculation models have the principal drawback that the integrity of the vitreous cavity is disrupted and that they produce some mechanical damage to the retina (Culbertson et al., 1982), but on the other hand, the blood
retinal barrier may be better

maintained than by using the suprachoroidal approach, at least during the initial phase of the infection (Garweg et al., 1998). Hence, the disease profile in the suprachoroidal model involves an initial local infection followed by a very early systemic one and, as such, the situation is not immunologically comparable with that evinced in humans (Friedrich and Mu¨ller, 1989). In any case, apart from the shorter time course, the transvitreal inoculation primate model represents clinical and histopathological conditions resembling those of the natural disease in humans (Culbertson et al., 1982). It may also circumvent a disadvantage of the rabbit model which is the anatomic dissimilarity of the retina compared to humans (O’Connor, 1984). However, to our knowledge, this model has not yet been used for assessing drugs but rather to elucidate the pathogenesis of ocular toxoplasmosis. In addition to the local eye infection in larger animals, a small animal model uses C57BL/6 as well as MRL mice and injection of T. gondii (PLK strain, a clone of ME49) into the anterior eye chamber (Hu et al., 1999a,b). Pathological and histopathological features of this model resemble in part acute ocular toxoplasmosis in humans, particularly when mice have been primed (preinfected perorally) (Hu et al., 1999a). As local infection models with larger animals and especially primates are rather difficult and costly, this mouse model may offer a rational alternative, at least for larger scaled controlled studies with therapeutics to be screened. In addition, the disadvantage of the small infection area that has to be investigated may be overcome partly by the use of current, highly sensitive detection methods such as PCR or quantitative real-time PCR. The other disadvantage of potentially extensive needle damage, particularly when such small animals as mice are used, may perhaps be circumvented when instead of intraocular injection, local instillation of the parasite is used. An investigation has shown that this, in

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7.3 Ocular toxoplasmosis

principle, leads to infection of the retinal vessels with glial reactions (Tedesco et al., 2005). However, to our knowledge, local eye infection models in mice have not yet been used for pharmacological drug testing but rather for immunological and pathogenetic studies (Hu et al., 1999b, 2001; Lu et al., 2004, 2005, 2009; Charles et al., 2007). Local infection of guinea pig eyes was also reliably achieved as early as in 1956 by injecting the RH strain into the vitreous humor (Hogan et al., 1956). This animal was selected because of its relative resistance to toxoplasmic infection and because the size of the eye did allow ophthalmoscopic examination. In fact, as early as in 1964, a guinea pig model with posterior chamber inoculation of a low virulent T. gondii strain, together with a rabbit model mentioned above, was successfully used to show a therapeutic effect of sulfadiazine and pyrimethamine in the treatment of ocular eye disease (Jacobs et al., 1964). However, to our knowledge, local infection models using guinea pigs have not been used for pharmacological studies during the last few decades.

7.3.2 Models based on infection via the carotid artery An intermediate model situated between the localized eye infection and the eye infection as a consequence of a generalized challenge was established in cats by Davidson et al. (1993) who used intracarotid inoculation to direct the parasites to ocular tissues to gain more predictable experimental ocular lesions with fewer systemic side effects. Indeed, all eight cats infected with a relatively small number of the ME49 strain developed the ocular disease but showed no signs or only mild signs of a generalized infection (three cats developed an increase in temperature). The multifocal areas of choroidal and retinal inflammation exhibited many similarities to ocular toxoplasmosis in

339

humans; however, it differed from human ocular toxoplasmosis in its primary choroidal versus retinal nature (Davidson et al., 1993). The cat model has been used once to examine the effect of clindamycin in the treatment of ocular toxoplasmosis (Davidson et al., 1996). Paradoxically, clindamycin administration was associated with increased morbidity and mortality from hepatitis and interstitial pneumonia, which are characteristic of generalized toxoplasmosis. The reasons for this outcome were unclear and may have been due to various aspects of the experimental setting (Davidson et al., 1996). As the natural definitive host, the cat may also differ from humans in some undefined manner with regard to its immunologic response to the parasite, which leads us to believe that it is not the ideal laboratory animal for drug testing.

7.3.3 Models based on systemic infection The most frequently used animal for systemic infection models is the mouse. Essentially, two different methods have been employed to establish the disease: (1) infection of pregnant mice to induce the development of ocular lesions in the pups (Hay et al., 1981, 1984; Lee et al., 1983; Dutton et al., 1986; Hutchison et al., 1982; Hay and Dutton, 1996) and (2) systemic infection of mice which then predominantly develop ocular manifestations (Gazzinelli et al., 1994; Olle et al., 1994, 1996; Lyons et al., 2001; Shen et al., 2001, Norose et al., 2003, 2005). Lahmar et al. (2010, 2014) have proposed an alternative model which may be viewed as an intermediate model of congenital and postnatally acquired ocular toxoplasmosis: the neonatal systemic infection of mice. Such an infection leads to a similar pathophysiology as compared to the congenital ocular toxoplasmosis model but at higher and more reproducible rates of ocular involvement.

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7. Toxoplasma animal models and therapeutics

In the congenital model the infection is established via infection of gestating female mice, for example, with the Beverley strain (Hay et al., 1981; Hay and Kerrigan, 1982; Hutchison et al., 1982). This model has the advantage that its etiology is probably analogous to a substantial extent to human ocular toxoplasmosis in that the fetus becomes infected in utero via a mother whose primary infection is acquired during pregnancy (Hay et al., 1984). Interestingly, it was found that the ocular lesions in this model resemble those that have been described in experimental allergic uveitis (Lee et al., 1983; Hay et al., 1984; Dutton et al., 1986) and, in fact, a mouse model (the adult, not the congenital) was thereafter used to further clarify the pathogenesis and particularly the nature and influence of the immune response involved in the ocular disease (Gazzinelli et al., 1994; Olle et al., 1996). However, for pharmacological screening studies the congenital model as described does not seem to be appropriate because, in spite of low postnatal mortality, it has the disadvantage that ocular morbidity, discovered by cataract manifestation, is only present in 5% of the pups (Hutchison et al., 1982). Nevertheless, the use of different mouse and Toxoplasma strains as well as new sensitive screening methods may further develop such a model to be also suitable for drug testing. Alternatively, the neonatal infection model (as explained previously; Lahmar et al., 2010) may lead to ocular pathology in a sufficiently high percentage of animals that warrants evaluating novel drug regimes. Gazzinelli et al. (1994) reported that C57BL/ 6 (B6) mice develop mild intraocular inflammation commonly observed 15 days after intraperitoneal injection of cysts of the ME49 strain, demonstrating the possible usefulness of adult mice for an eye model. In most of the ocular lesions the presence of the parasite could not be demonstrated even with the PCR technique, but parasite load did increase after treatment

of mice with antibodies directed against lymphocytes or cytokines [gamma-interferon (IFNγ) or TNF-α] (Gazzinelli et al., 1994). Treatment with anti-IFN-γ also ended with clinical eye lesions, including single foci of chorioretinitis, and multifocal lesions of diffuse areas of retinal necrosis in an experimental model of chronically infected Swiss Webster mice (avirulent Beverley strain) (Olle et al., 1996). Using also the T. gondii Beverley strain for i.p. infection, Lyons et al. (2001) found retinal inflammation most marked in the inner retinal layers of wild type (WT) 129/SVJ mice and more severe in corresponding IL-6 knockout mice. Norose et al. (2003) established a mouse model for the ocular disease that followed the natural peroral route of infection with five cysts of a T. gondii avirulent strain. The model was established for immune-competent mice using resistant BALB/c or susceptible C57BL/ 6 and for the immune deficient mice using IFN-γ knockout (GKO) mice. Whereas all GKO mice died after 11
12 days demonstrating disseminated infection, both strains of WT mice survived for more than 1 month. In contrast to the knockout mice, there was no histopathological evidence for inflammation in the eyes and brains of WT mice, and no characteristic findings using fluorescein angiography and documentation with a fundus camera (Norose et al., 2003). Electroretinograms, as shown later, were also only changed in GKO mice (Norose et al., 2005). However, the authors were able to show differential parasite distribution in the eyes of WT mice using a quantitative competitive polymerase chain reaction (QC
PCR). The GKO ocular toxoplasmosis model has recently been used to show a beneficial effect of sulfamethoxazole on the parasite load in different ocular tissues (Norose et al., 2006). However, due to the severe immune defect the improvement in the outcome of infection was not specific to the eyes, but rather a general decrease in systemic infection. In addition, the authors observed an increased percentage of

Toxoplasma Gondii

7.3 Ocular toxoplasmosis

bradyzoites indicating that the drug, besides killing the parasite, may also favor stage conversion from the fast replicating tachyzoite to the latent bradyzoite stage. Altogether, this suggests that the GKO model may not be best suited to mimic a localized eye infection that is commonly observed in human ocular toxoplasmosis. Nevertheless, it may have its place in evaluating ocular manifestations and their therapeutic prevention in immunocompromised individuals. In contrast to Norose et al. (2003), Dukaczewska et al. (2015) found substantial inflammatory lesions in the eyes of C57BL/6 WT mice after peroral infection with a type 2 avirulent T. gondii strain (ME49). This included vitritis, perivascular infiltrates, migration of the retinal pigment epithelium (RPE) and inflammatory cells particularly in the ganglion cell layer. Furthermore, ocular T. gondii tissue cysts, mostly within the ganglion cell layer, were detected in 50%
80% of the mice depending on the size of the inoculum (Dukaczewska et al., 2015). The different Toxoplasma strains might explain the differential ocular manifestation as observed by Norose et al. (2003) and Dukaczewska et al. (2015), since even a low dose of 5 cysts of the ME49 strain used in the latter study led already to a mortality of 20%. Infection of NMRI mice with tissue cysts of the same ME49 parasite line led to inflammatory retinal lesions as observed in C57BL/6 mice but cysts were only occasionally detected (Dukaczewska et al., 2015). The relatively low ocular cyst burden is consistent with the higher resistance of NMRI mice to T. gondii infection as compared to C57BL/6 mice. In summary, adult mouse models may provide reasonable tools for investigation of various pathological or pathogenetic aspects of ocular toxoplasmosis; however, mouse and particularly parasite strains as well as the dose and route of infection are critical in order to achieve sufficient rates of ocular toxoplasmosis

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without extensive generalized disease and possibly host death. They may also provide a system for screening drugs particularly in the immunocompromised host when using various knockout mice as, for example, IFN-γ knockouts (Belal et al., 2004; Norose et al., 2006). At any rate, QC
PCR as described before (Kobayashi et al., 1999) combined with DNA extraction from different eye parts (cornea, iris/ciliary body, lens, posterior retina, peripheral retina, choroids, sclera, optical nerve, and brain) (Norose et al., 2003) allows measurement of the parasite load in the eyes of small animals such as mice and may prove valuable in other models of ocular toxoplasmosis. Alternatively, visual inspection of flatmounted retinae from mice infected with a recombinant avirulent T. gondii strain expressing the Escherichia coli β-galactosidase reporter has enabled detection of single cysts in B40% of mice within 28 days of infection (Escoffier et al., 2010). Recently, ocular lesions were found in 25% of the eyes of asymptomatic female Sprague Dawley rats orally infected with oocysts of different T. gondii strains (Dubey et al., 2016). Rats are generally considered more resistant to symptomatic T. gondii infection than mice (Fujii et al., 1983; Dubey and Frenkel, 1998; Zenner et al., 1998) and may thus mimic the course of infection as observed in humans. Although percentages of rats with ocular lesions ranged from 0% to 60% depending on the parasite strain used, eye manifestations were observed after infection with a variety of different T. gondii genotypes (Dubey et al., 2016). Ocular lesions were most common in rats infected with strains TgBbUS1 (atypical strain; 60% of rats), VEG (type 3, 40%), and TgRabbitBr1 (atypical; 30%). As in humans, the retina was most commonly affected, and tissue cysts were regularly detected in the affected eyes. Thus the rat model may be well suited to evaluate in the future the efficacy of drugs in the treatment of ocular toxoplasmosis.

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Based on the observations of Frenkel (1953, 1955), who found frequent but sporadic ocular disease in Syrian golden hamsters several months after inoculation with the RH or CJ strains of Toxoplasma, reliable models with this animal and the ME49 strain were developed that show ocular disease following the i.p. or oral infection route (Pavesio et al., 1995; Gormley et al., 1999). The main advantage of these models is that they consistently produced ocular disease with a short incubation time but without artificial breaching of the ocular barrier and also without causing any clinical signs of systemic disease. In addition, hamster eyes are large enough to allow fundus photography to document the progression of the infection and, as usually encountered in humans when immunity is not impaired, the disease resolves spontaneously with time, without treatment (Pavesio et al., 1995; Gormley et al., 1999). There are, however, marked differences to the human disease as, for example, vitritis, which in hamsters was usually not significant (Pavesio et al., 1995). In conclusion, the hamster model may be an option as a drug screening model mainly because of its good reproducibility and monitoring possibilities. However, one study compared conventional therapies (pyrimethamine plus sulfadiazine, clindamycin, spiramycin) with atovaquone and did not show any drug effects on the acute disease but only on the number of cerebral Toxoplasma cysts (Gormley et al., 1998). In addition to conventional laboratory animals, an acquired, as well as a congenital, model of ocular toxoplasmosis has been established in C. callosus (Pereira Mde et al., 1999). Following oral infection with the ME49 strain, 40% of fetuses presented ocular lesions (examined after laparotomic removal of the fetus), while 75% of females and 50% of males presented ocular lesions in the acquired study setting. Adult animals survived the infection for several months without treatment and demonstrated no clinical signs of systemic disease

(Pereira Mde et al., 1999). Treatment of acutely infected pregnant females with azithromycin (300 mg/kg) has been shown to efficiently prevent ocular toxoplasmosis in the littermates and to be superior to a treatment with pyrimethamine, sulfadiazine, and folinic acid (Lopes et al., 2009). Whether azithromycin also reduces acquired ocular toxoplasmosis in adult C. callosus remains to be established. Pigs have recently been evaluated as a model for ocular toxoplasmosis (Garcia et al., 2017). Animals were infected either subcutaneously with T. gondii tachyzoites or orally with tissue cysts or oocysts. Parasite DNA could be detected in the eyes of 47% of all infected pigs, indicating a substantial rate of ocular infection (Garcia et al., 2017). Specific antibodies were detected in aqueous or vitreous humors of 73% of pigs; however, in only 40% of pigs, antibodies were produced intraocularly as indicated by the Goldmann
Witmer antibody index (Goldmann and Witmer, 1954; Desmonts, 1966). Both, detection of parasite DNA and the Goldmann
Witmer index indicated that infection with tissue cysts led to the highest rate of eye infection (80% of pigs; Garcia et al., 2017). Although it is yet unknown whether the course of ocular toxoplasmosis in pigs resembles that observed in humans, pigs after oral infection with tissue cysts may thus represent a valuable animal model for future pathogenetic studies or drug efficacy testing.

7.4 Cerebral toxoplasmosis This section will discuss animal models available for TE as the predominant manifestation of the disease in the immunocompromised host. It will also include acute systemic models where disseminated infection is prominent and where the brain is usually involved as a part of the systematic infection. The most often used animal for acute or cerebral toxoplasmosis, particularly with respect to pharmacological

Toxoplasma Gondii

7.4 Cerebral toxoplasmosis

testing, is the mouse (see, e.g., Perea and Daza, 1976; Grossman and Remington, 1979; Hofflin and Remington, 1987a,b; Chang and Pechere, 1987; Chang et al., 1988, 1991, 1994; Israelski and Remington, 1990; Derouin et al., 1991, 1992; Araujo et al., 1991b, 1992a,b, 1996, 1998; Weiss et al., 1992; Romand et al., 1993, 1995, 1996; Rodriguez-Diaz et al., 1993; Dumas et al., 1994, 1999; Olliaro et al., 1994; Alder et al., 1994; Martinez et al., 1996; Khan et al., 1996, 2000; Aguirre-Cruz and Sotelo, 1998; Sordet et al., 1998; Aguirre-Cruz et al., 1998; Djurkovic-Djakovic et al., 1999, 2002, 2005; Moshkani and Dalimi, 2000; Scho¨ler et al., 2001; Ferreira et al., 2002; Degerli et al., 2003; Belal et al., 2004; Dunay et al., 2004, 2009; Lescano et al., 2004; Grujic et al., 2005; Ling et al., 2005; Mitchell et al., 2006; Jost et al., 2007; Shubar et al., 2008, 2009, 2011; Bajohr et al., 2010; Martins-Duarte et al., 2010, 2015; Doggett et al., 2012, 2014; Schultz et al., 2014; Benmerzouga et al., 2015; Vidadala et al., 2016; Mokua Mose et al., 2017; Azami et al., 2018; McConnell et al., 2018). For some purposes, hamsters (Frenkel et al., 1975; Gormley et al., 1998) or rats (Foulet et al., 1994; Dubey, 1996; De Champs et al., 1997; Kempf et al., 1999; Zenner et al., 1999b; Freyre et al., 2001b, 2003a, 2004; Kannan and Pletnikov, 2012; Gao et al., 2015; Dubey et al., 2016) have been used but not usually for drug evaluation. Recently, cats have been experimentally infected with T. gondii and proposed for evaluating the efficacy of drugs or vaccines (Cornelissen et al., 2014). However, chemotherapy of T. gondii
infected cats was not designed as a model for treating human toxoplasmosis but rather to evaluate the reduction of shed oocysts, that is, one of the major stages that are regularly transmitted to humans. Generally, experimental models are based either on a primary acute infection of the animals, on direct inoculation of the parasite into the animal brain with or without immune suppression, or on a chronic infection of the animal that may or may not be immune

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suppressed (e.g., with immunosuppressive drugs or radiation, antibodies directed against lymphocytes or cytokines, or concomitant infections with viruses that modulate the immune response) (see Tables 7.5 and 7.6). For certain applications, the use of genetically modified mice with various defects in their immune system may also be an option. In general, the acute and strictly localized models have most often been used to evaluate the treatment efficacy of antiparasitic drugs, whereas the chronic infection models have been used to study their influence on cyst formation and/or prevention of a relapsing disease.

7.4.1 Acute infection models Acute infection models are usually associated with a high level of mortality (often up to 100%) of laboratory animals within 8
10 days and survival in particular treatment groups, which are estimated by the Kaplan
Meier or product limit survival analysis. So far they have been the overall preferred models for drug testing mainly because of their consistent reproducibility. Most often, between 50 and 2 3 104 tachyzoites of the virulent RH strain are injected i.p. into female Swiss Webster mice (see, e.g., Khan et al., 1996, 1998; Araujo et al., 1997; Djurkovic-Djakovic et al., 1999; Schultz et al., 2014), female CF-1 mice (Doggett et al., 2012, 2014) or female BALB/c mice (Azami et al., 2018), but sometimes animals are infected i.p. or orally with, for example, 10 cysts of T. gondii ME49 or C56 (McLeod et al., 1989; Araujo et al., 1997; Khan et al., 1998; Yardley et al., 2002; Shubar et al., 2009, 2011; Martins-Duarte et al., 2010) or lethal doses of type 2 strains (e.g., 104 ME49 or 106 Prugniaud (PRU) tachyzoites; Benmerzouga et al., 2015). Mice are then often observed for up to 30 days from the date of infection. Surviving mice are examined for residual infection by microscopy

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TABLE 7.5 Pharmacological studies on cerebral and acute toxoplasmosis. Infection model (mouse)

Specific comments

Pharmacological studies (literature examples)

Acute infection models

Standard models for drug tests; i.p. infection with up to 100% mortality within 8
10 days (type 1 strains); oral infection with cysts of low virulent strains (type 2) and quantification of parasite numbers at different time points

Khan et al. (1996, 1998), Araujo et al. (1997), Mui et al. (2005), Shubar et al. (2009, 2011), Dunay et al. (2009), Doggett et al. (2012, 2014), Schultz et al. (2014), Benmerzouga et al. (2015), Martins-Duarte et al. (2015), Vidadala et al. (2016), McConnell et al. (2018)

Localized brain infection models

Direct inoculation of parasites into the brain; does not follow the natural route of infection

Hofflin and Remington (1987a,b), Arribas et al. (1995)

Progressive Toxoplasma encephalitis models

For example, i.p. or oral infection with brain Araujo et al. (1991a, 1996), Dumas et al. (1994, cysts of type 2 strains; assessment, for example, 1999) via brain cysts counting

Chronic relapsing infection models (reactivated toxoplasmosis)

Infection with type 2 or 3 strains; drug-induced Djurkovic-Djakovic et al. (2002), Scho¨ler et al. immunosuppression of mice or use of (2001), Dunay et al. (2004, 2009), Mitchell et al. (2006) genetically altered immunodeficient mice

Latent infection models

Infection with type 2 strains; chemotherapy after establishment of chronic infection

Doggett et al. (2012), Schultz et al. (2014), Benmerzouga et al. (2015), Vidadala et al. (2016)

i.p., intraperitoneal.

of brain tissue for the presence of T. gondii cysts or by i.p. subinoculation of suspensions of portions of various organs into healthy mice (bioassay). The probability of untreated mice to succumb during acute infection following peroral infection critically depends on the genetic background of the mouse strain used with C3H/HeJ being highly resistant, BALB/c being intermediately resistant, and C57BL/6J being highly susceptible (Araujo et al., 1976; Williams et al., 1978; McLeod et al., 1989). In addition, when choosing a mouse model, one has to consider that the route of infection (e.g., i.p. or oral infection) also largely influences the outcome of infection (Johnson, 1984). In general, acute murine infection models are not very close to most Toxoplasma-induced human diseases of the immunocompromised host, where the infection takes a more clinically localized course often, though not always, confined to the brain. In contrast, acute primary

toxoplasmosis in animal models is a generalized infection, substantially involving organs other than the brain, such as the lungs or the liver. Thus there are inherent difficulties in these models for the projection of drug efficacy deduced from a Kaplan
Meier diagram which is ultimately dependent not only on direct drug action but also on organ-specific pharmacokinetics and drug metabolism. This means, as a consequence, that a drug successfully tested in acute infection models may fail in the treatment of localized brain infection and vice versa. At any rate, this model is the standard model for the first in vivo screening of a new drug. In addition to survival curves the count or titration of cysts of succumbed and/or surviving animal brains, as well as subinoculation into naive mice, may be performed. Alternatively, tissue-culture methods or detection methods such as quantitative PCR from

Toxoplasma Gondii

TABLE 7.6 Models of cerebral toxoplasmosis. Chronic relapsing infection models (reactivated Toxoplasmosis) Species and strain of mouse useda

Toxoplasma Toxoplasma dose, strain stage, and route of administereda infection

Porton

M3

Swiss
Webster ME49

Means for reactivation

Outcome of animal infection

Remarks

Publicationb

30 cysts i.p.

DXM 6 weeks after infection

40% of mice developed clinical signs of toxoplasmosis, most of them with brain inflammation

Brain cysts observed in only 30%
40% of mice with suspected toxoplasmosis

Nicoll et al. (1997)

10 cysts orally

DXM alone or combined with CA 6 weeks after infection

Mortality after 7 weeks with immunosuppression: untreated 0%, DXM 61.1%, DXM 1 CA 85%, uninfected 1 DXM 33%

Mean cyst number 2
9-fold increased compared to untreated control; 14.2% developed clinical TE (both treatment regimens)

DjurkovicDjakovic and Milenkovic (2001) Mokua Mose et al. (2017)

BALB/c

Isolate 15 cysts i.p. obtained from free range chicken from Kenya

DXM 6 weeks after infection

15% and 28.6% mortality rate after treatment with 2.66 or 5.32 mg/kg/day DXM, respectively, over 6 weeks; increased cyst numbers and inflammatory brain lesions after DXM treatment

Nonlethal chronic infection with significant cyst numbers before DXM treatment

B6

C56 (or ME49) 1 3 105 tachyzoites i.p. of C56 followed by 2 weeks treatment with sulfadiazine (20 cysts ME49 i.p. for mice preinfected with virus)

Coinfection with LP
BM5 murine leukemia virus 8 weeks after challenge with C56 (coinfection 12, 8, 4, or 2 weeks before ME49 challenge)

Chronic infection with C56: 30%
40% mortality by 80 days following viral coinfection; mice with encephalitic lesions

No effects if challenged Gazzinelli (ME49) 4 or 2 weeks after et al. (1992) viral infection; all mice died if challenged 12 or 8 weeks after viral infection (pneumonitis, only occasional necrotic areas in brain)

C57BL/6

Fukaya

Coinfection with LP
BM5 murine leukemia virus 6 weeks after Toxoplasma infection

All mice infected with both Toxoplasma gondii and LP
BM5 MuLV died 9
14 weeks after the virus infection due to severe encephalitis

In contrast to other studies with the LP
BM5 virus, in this study other organs than brain seem to be less affected

10 cysts i.p.

Watanabe et al. (1993)

(Continued)

TABLE 7.6 (Continued) Chronic relapsing infection models (reactivated Toxoplasmosis) Species and strain of mouse useda

Toxoplasma Toxoplasma dose, strain stage, and route of administereda infection

C57BL/6

C-strain

C57BL/6

Means for reactivation

Outcome of animal infection

Remarks

Publicationb

10 cysts orally

Coinfection with LP
BM5 murine leukemia virus 30 days before or 20, 30, and 60 days after challenge

None of the animals developed Toxoplasma encephalitis

Increase in Toxoplasma lung counts

Lacroix et al. (1994)

ME49

15 cysts orally

Coinfection with LP
BM5 2 weeks after challenge

70%
80% animals succumbed Transfer of immune CD8 1 T- Khan et al. to disseminated infection cells prevented reactivation (1999) (including brain but also lung, spleen and liver) by 12 weeks after LP
BM5 challenge

SCID (on C.B17/Smn background)

ME49

20 cysts i.p.

Sulfadiazine treatment started 10 days after infection for 18 days, then discontinuation of therapy

All SCID mice died with TE 6
9 days after sulfadiazine treatment was stopped

No other organs except the brain with cysts, tachyzoites, or inflammation foci

SCID

ME49

10 cysts orally

Sulfadiazine treatment started 2 days after infection for 3 weeks, then discontinuation of therapy

Mortality 100% within 2 weeks (TE); splenocyte transfer from immune syngenic donors prevented reactivation

Reactivation started from liver Beaman et al. spreading into other organs (1994) (including the brain)

BALB/c IFNγ2/2 (interferonγ-deficient)

ME49

10 cysts i.p. or orally

Sulfadiazine treatment started 4 days after infection for 3 weeks, then discontinuation of therapy

Mortality due to TE 100% within 1 week independent from infection mode; control WT BALB/c mice survived for more than 3 months

Treatment with recombinant IFN-γ prevented TE

C57BL/6 IFNγ2/2 (interferonγ-deficient)

PRU-Luc-GFP 20 cysts orally (transgenic type 2 strain)

Sulfadiazine treatment from 2 days p.i. onwards for 3 weeks, then discontinuation of therapy

Mortality 100% within 10 days Treatment with artemiside or of discontinuation of artemisone reduced mortality sulfadiazine to 20% and 40%, respectively

Johnson (1992)

Suzuki et al. (2000)

Dunay et al. (2009)

(Continued)

TABLE 7.6 (Continued) Chronic relapsing infection models (reactivated Toxoplasmosis) Species and strain of mouse useda

Toxoplasma Toxoplasma dose, strain stage, and route of administereda infection

BALB/c IFNγ2/2 (interferonγ-deficient)

Type 3 strain isolated from chicken

C57BL/6 ME49 ICSBP/IRF-82/ 2 (interferon regulatory factor 8deficient)

Remarks

Publicationb

Means for reactivation

Outcome of animal infection

10 cysts orally

Sulfadiazine treatment started 4 days after infection for 11 weeks, then discontinuation of therapy

100% mortality within 10 days Treatment with ponazuril after discontinuation of prevented reactivated TE in sulfadiazine treatment five out of seven mice; no mice died

Mitchell et al. (2006)

5
10 cysts orally

Sulfadiazine treatment started 2
7 days after infection for 2
4 weeks, then discontinuation of therapy

Mortality and time to death depend on the time of sulfadiazine treatment; synchronized development of TE

Scho¨ler et al. (2001), Dunay et al. (2004), Jost et al. (2007), Shubar et al. (2009, 2011)

Treatment as well as maintenance therapy studies possible

Models based on localized brain infection instead of reactivation Species and strain of mouse useda

Toxoplasma dose, stage, and Toxoplasma route of strain administereda infection

Swiss Webster

C56

Swiss Webster

Swiss Ico

Outcome of animal infection

Remarks

Publicationb

1 3 104 tachyzoites injected intracerebrally

Normal mice survived but immunosuppressed died from progressive disease (no rates specified); immunosuppression with either cortisone, cyclophosphamide or cyclosporine

Cerebral lesions: inflammation intensity, tachyzoite and cyst number dependent on type of immunosuppression

Hofflin et al. (1987)

C56

1 3 104 tachyzoites injected intracerebrally

Mortality of 40% in normal mice and 100% in cortisonetreated animals

Drug evaluation study (efficacy of clindamycine was shown)

Hofflin and Remington (1987a)

RH

1 3 103 tachyzoites injected intracerebrally

100% of animals died within 5 days after infection due to necrotizing Toxoplasma meningoencephalitis

Pharmaceutical study: evaluation of highly active drugs possible

Arribas et al. (1995)

Progressive Toxoplasma encephalitis models

Species and strain of mouse useda

Toxoplasma Toxoplasma dose, stage, strain and route of administereda infection

Outcome of animal infection

Remarks

Publicationb

A/J, CBA/J, C57BL/6, C3/H, ME49 C57BL/10, DBA/2, BALB/c, different B10 mice

10 cysts i.p. or Mice with H-2b and H-2k haplotype developed 100 cysts TE, mice of H-2d and H-2a haplotype did not orally

Brown and McLeod Histological evaluation of the number of cysts in brain (1990), Suzuki et al. sections and inflammation (1991) in the brain

B10 and BALB congenic mice

DX (type 2)

10 cysts orally Outcome of infection differed between mouse strains: B10 generally more susceptible than BALB, important impact of MHC haplotype; mortality due to TE in some strains

Assessment of mouse survival, number of tachyzoites and cysts in the brain

Deckert-Schlu¨ter et al. (1994)

C57BL/6 WT and various knockout strains including cell type-specific knockouts (cre/lox system)

DX

5 cysts orally

Mainly used for pathogenicity studies; evaluation of parasites/ cysts by histology and PCR

Schlu¨ter et al. (2003), Dro¨gemu¨ller et al. (2008), Ha¨ndel et al. (2012)

CBA/Ca (inbred)

ME49

20 cysts i.p. or Development of a chronic progressive orally encephalitis and mice began to die approximately 6 weeks after infection

Assessment by cyst counting (light microscopy) or brain histopathology scoring

Araujo et al. (1996)

C57BL/6J

PRU

10 cysts i.p.

Assessment by brain histopathology scoring

Dumas et al. (1999)

WT mice generally survived until 40
60 days; thereafter 30%
50% of the mice may have died due to TE depending on the individual experiment

Development of a chronic progressive encephalitis characterized by brain cysts and inflammation with mortality of 60%
80% within the following months

Latent infection models Species and strain of rodent useda

Toxoplasma Toxoplasma dose, strain stage, and route of administereda infection

CBA/J

ME49

18 cysts i.p.

Outcome of Animal Infection

Remarks

Publicationb

Development of a latent chronic infection without high mortality during acute infection; high mean cyst numbers at B9 weeks p.i. (B1500
2500 per brain depending on the study)

Pharmacological studies: reduction of brain cysts by up to 88%

Doggett et al. (2012), Schultz et al. (2014)

Latent infection models Species and strain of rodent useda

Toxoplasma Toxoplasma dose, strain stage, and route of administereda infection

BALB/c

PRU

Rat: Lewis, Wistar, Sprague Dawley, Brown Norway, Fischer 344 Rat: Sprague Dawley

a

Outcome of Animal Infection

Remarks

Publicationb

105 tachyzoites i.p.

Development of a nonlethal chronic infection; B900 cysts per brain at B6.5 weeks p.i.

Pharmacological study: reduction of brain cysts by 69%

Benmerzouga et al. (2015)

PRU

Adults: 200 tissue cysts orally, Neonates: 50 tissue cysts i.p.

Infection of adults: nonlethal chronic infection with high cyst numbers in the brains of Fischer 344 rats (B1250 cysts per brain); Infection of neonates: nonlethal chronic infection with varying cyst numbers in all Fischer 344 and Brown Norway and B50% of Sprague Dawley rats

Infectivity in pups of different rat strains correlated with iNOS/ Arginase ratios

Gao et al. (2015)

11 strains of various genotypes

10
105 (mouse infective units) oocysts orally

Depending on the infection dose and parasite strain rats developed nonlethal chronic infection with 12
900 cysts per brain

Tissue cysts present in all brain regions; inflammatory lesions mostly present

Dubey et al. (2016)

For definition of animal or Toxoplasma strains, please see respective articles. Due to numerous publications on this topic, only few were chosen exemplarily for this table. CA, Cortisone acetate; DXM, dexamethasone; GFP, green fluorescent protein; MHC, major histocompatibility complex; TE, toxoplasmic encephalitis; WT, wild type.

b

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7. Toxoplasma animal models and therapeutics

organs of survivors may be used to assess the residual parasite load (Miedouge et al., 1997; Weiss et al., 1991; Djurkovic-Djakovic et al., 1999; Bajohr et al., 2010; Martins-Duarte et al., 2010). An important development is the use of bioluminescence imaging of mice infected with luciferase (luc)-transgenic T. gondii parasites (Saeij et al., 2005). Luc-transgenic parasites of type 1 and 2 background are available, and after injection of the substrate (e.g., firefly luciferin), bioluminescence intensity can be regularly monitored, for example, every 2 days. This enables one to monitor the course of an acute disease in single living animals quantitatively, although the imaging output is not directly related to the parasite number, but is a relative measurement for the intensity of an infection (Saeij et al., 2005; Dunay et al., 2009). This system has been used to show the efficacy of artemisone and artemiside against an acute i.p. infection of CD1 outbred mice with 106 of a type 2 Toxoplasma strain (PRU background) (Dunay et al., 2009). In addition to primary evaluation of drug efficacy based on a fatal outcome of acute toxoplasmosis, mouse models have been developed that do not have the major disadvantage of the possibly severe suffering of mice before they die from overwhelming T. gondii infection (Samuel et al., 2003; Mui et al., 2005; Shubar et al., 2008, 2009, 2011; Doggett et al., 2012, 2014; Vidadala et al., 2016). In one of these models, mice are inoculated i.p. with the RH strain but on the fourth day after infection, 1.5 mL of phosphate buffered saline (PBS) is injected i.p. and then withdrawn together with all peritoneal fluid. Total numbers of parasites and concentrations of parasites are quantified microscopically as the basis for subsequent statistical analysis (Samuel et al., 2003). In fact, this model has been proven by the addition of sulfadiazine to the drinking water of infected mice, which significantly reduced the parasite burden (Samuel et al., 2003), and thus may well be suited to replacing the survival based acute infection models in the future. A

relatively new and interesting alternative to evaluate the efficacy of a drug regime is the use of transgenic parasites expressing fluorescent proteins, for example, the green fluorescent protein (GFP). It allows determining the number of infected host cells isolated from the peritoneal cavity or other tissues for instance on day 5 of infection using flow cytometry (Shubar et al., 2008). This read-out system avoids the time-consuming and error-prone microscopic histochemical evaluation of the number of parasites and enables gathering of additional information as, for example, the types of the parasite-infected host cells or their viability. Infection of NMRI mice with 105 GFP-expressing type 1 RH parasites has been applied to prove an excellent activity of quinolones and distinct bisphosphonates against T. gondii (Shubar et al., 2008; Bajohr et al., 2010). In another model, mice are orally infected with ME49 tissue cysts, and the outcome of disease is examined 16 days p.i. by evaluating the number of tachyzoites, tissue cysts, and inflammatory foci in sections of the brain (Shubar et al., 2009, 2011). This model was successfully applied to prove a beneficial effect of atovaquone nanosuspensions in acute murine toxoplasmosis (Shubar et al., 2009, 2011).

7.4.2 Localized brain infection models The direct inoculation of tachyzoites into the frontal lobe of mice may give the most reproducible results with lesions histologically resembling those that could be observed in immunocompromised humans (Hofflin et al., 1987). The model was successfully used to demonstrate the efficacy of clindamycin, roxithromycin, and IFN-γ on Toxoplasma encephalitis (Hofflin and Remington, 1987a,b). Subsequently, in another study using a localized brain infection model with the highly virulent RH strain, clindamycin showed no detectable effect; instead pyrimethamine,

Toxoplasma Gondii

7.4 Cerebral toxoplasmosis

sulfadiazine, and their combination were useful in terms of reducing mortality and histopathology (Arribas et al., 1995). The procedure of local brain inoculation requires high technical expertise and thus may not be suitable for investigations on a larger scale in average laboratories. In addition, the main disadvantage of this model is that TE does not follow the natural history of T. gondii infection, as it is not a consequence of recrudescence of a previously established infection. It therefore may be not ideal for pharmacological trials related to these aspects (e.g., for investigating drug interference with mechanisms of recrudescence) but rather for studying certain features of pathogenesis.

7.4.3 Progressive Toxoplasma encephalitis models Subacute or chronic infection models may be suited to evaluating the efficacy of drugs against the cyst form of T. gondii. Indeed, the cyst form is critical within the life cycle of the parasite with regard to pathogenesis of toxoplasmosis in immunocompromised individuals, especially with regard to development of TE (Frenkel and Escajadillo, 1987; Frenkel et al., 1975; Hofflin et al., 1987; Ferguson et al., 1989). Three different types of murine models with prolonged infection times have been widely used, mostly for studying the pathogenesis of chronic toxoplasmosis but also for evaluating drug treatments: (1) progressive TE, (2) reactivation of latent infections, and (3) latent infection. It is important to note that the development of clinical overt disease in the first two models involves recrudescence of the tachyzoite stage of T. gondii. Hence, an improvement in the outcome of the disease after drug treatment cannot be taken as an indication for a microbicidal activity against the cyst form of the parasite. To evaluate a putative efficacy against the tissue cyst, latent infection models have to be employed.

351

In progressive TE models, mice are usually infected i.p. or orally with brain cysts of a mildly virulent typical type 2 Toxoplasma strain such as ME49 or DX. Mice can then develop a chronic progressive TE (type 2 strains such as the ME49 strain tend to give rise to new cyst formation during the first week of infection, presumably preceded by cyst rupture and proliferation of tachyzoites that are then again converted into bradyzoites) and, unless treated, begin to die within the following weeks or months. Morbidity and mortality rates due to progressive TE vary considerably depending on the Toxoplasma strain, the dose of infection, and particularly the mouse strain used (Araujo et al., 1991a; Suzuki et al., 1991; Dumas et al., 1994; Deckert-Schlu¨ter et al., 1994; DjurkovicDjakovic et al., 2002). Genes present within the major histocompatibility complex (MHC) and genes outside of the MHC are decisive for the development of TE and for the time until the majority of mice succumb due to TE (DeckertSchlu¨ter et al., 1994). In order to evaluate drug efficacies in these models, treated mice and untreated controls are usually sacrificed at distinct time points and the activity of drugs is assessed by light microscopic examination and counting of cysts from brains previously ground with a pestle and mortar, homogenized by needle passage or by using glass beads and suspended in a defined volume of PBS (Araujo et al., 1991a; Sarciron et al., 1997; Lescano et al., 2004; Djurkovic-Djakovic et al., 2002). Alternatively, brains of mice may be examined histopathologically by scoring the severity of inflammatory lesions (Araujo et al., 1996) and/ or Kaplan
Meier curves may be obtained (Dumas et al., 1999; Djurkovic-Djakovic et al., 2002). A well-established model of chronic TE is the infection of C57BL/6 WT mice or those with deficiencies in various immunity-related proteins with five cysts of the low virulent type 2 strain DX (see, e.g., Schlu¨ter et al., 2003). Survival of WT mice is 100% until at least 40
50 days p.i. with increasing mortality due

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7. Toxoplasma animal models and therapeutics

to TE thereafter. This model has been extensively used to define immune factors which prevent the development of TE (Schlu¨ter et al., 2003; Dro¨gemu¨ller et al., 2008; Ha¨ndel et al., 2012). It may also be useful for evaluating the efficacy of drug therapies against a mild course of the chronic stage of toxoplasmosis.

7.4.4 Chronic relapsing infection models (reactivated toxoplasmosis) In order to describe and mimic the natural course of reactivation of T. gondii infection in humans, animal models based on reactivation of a chronic infection have been attempted to be developed. It was first observed in 1966 that when infecting splenectomized mice or mice treated with cortisone or 6-mercaptopurine with the T. gondii Beverley strain that the course of the disease was greatly altered in the experimental animals, distinguished by signs of severe neurological involvement and meningoencephalitis (Stahl et al., 1966). Reactivation of an otherwise chronic infection had succeeded in hamsters infected with the RH strain by the administration of cortisone, cyclophosphamide, or whole body irradiation (Frenkel et al., 1975). On the other hand, in mice, only little reactivation using cortisol acetate, azathioprine, or cyclosporine was observed (Sumyuen et al., 1996), but reactivation was obtained with dexamethasone (DMX) (Nicoll et al., 1997; Mokua Mose et al., 2017). However, brain cysts were only demonstrated in a few of the animals. Perhaps, the most promising mouse model based on immunosuppressive drugs is that of Djurkovic-Djakovic et al., (2002) who used a mildly virulent type 2 T. gondii strain (ME49) to assess the efficacy of atovaquone combined with clindamycin treatment. Indeed, type 2 strains are also responsible for most cases of human TE in Europe and North America (Howe and Sibley, 1995). Reactivation was

achieved in those animals that had been previously orally infected with 10 tissue cysts of the ME49 strain by immunosuppression with DXM alone and more efficiently by combined DXM and hydrocortisone-21-acetate [cortisone acetate (CA)] treatment (Djurkovic-Djakovic and Milenkovic, 2001; Djurkovic-Djakovic et al., 2002). In addition to the abovementioned models, reactivation may be induced in dual infection models with T. gondii and viruses such as CMV (Pomeroy et al., 1989) or LP
BM5 murine leukemia virus which is responsible for murine AIDS (Gazzinelli et al., 1992; Watanabe et al., 1993; Lacroix et al., 1994; Khan et al., 1999). Such models are most precious for investigation of the immunopathogenesis of analogous disease in humans; however, due to their complexity and obstacles in reproducibility, they may not be well suited to pharmacological investigations (Lacroix et al., 1994). Another strategy besides suppressing the host immune system of immune-competent mice is based on the use of severely immune compromised mice, such as those with severe combined immunodeficiency (SCID) mice lacking T and B lymphocytes, or athymic (nude mice) which lack functional T cells. An otherwise fatal infection in such mice may be made chronic by administering sulfadiazine treatment, and withdrawal of sulfadiazine then leads to relapse of infection (Johnson, 1992; Beaman et al., 1994). To date, however, such models have been mostly used to investigate the immunopathogenesis of relapsing or acute toxoplasmosis rather than to assess drug regimens for treatment or prevention of the reactivated disease (for review see Denkers and Gazzinelli, 1998). In addition, severely immune compromised animals, such as SCID or athymic mice, are difficult to work with because of their impaired immune systems and the requirement for them to be kept in sterile conditions to prevent opportunistic infections.

Toxoplasma Gondii

7.4 Cerebral toxoplasmosis

A promising alternative to SCID mouse models that has been presented by Suzuki et al. (2000) is BALB/c mice deficient for IFN-γ. BALB/c mice are genetically relatively resistant to T. gondii infection (Suzuki et al., 1991, 1994; Brown et al., 1995). IFN-γ, but not TNF-α or iNOS, is crucial for resistance against the development of TE, and mice deficient for IFN-γ die after an infection with the ME49 T. gondii strain when treatment with sulfadiazine is discontinued (Suzuki et al., 2000). IFN-γ knockout mice treated with sulfadiazine in order to establish a chronic infection were successfully employed to show that ponazuril, that is, an anticoccidial triazine used in the poultry industry, or artemisinin analogues efficiently prevented TE after the discontinuation of the sulfadiazine treatment (Mitchell et al., 2006; Dunay et al., 2009). Based on the findings with the IFN-γ knockout model, reactivated TE in chronically infected mice (T. gondii strain ME49) deficient for the IFN regulatory factor 8 (IFN consensus sequence binding protein ICSBP/IRF-82/2 on a C57BL/6 background) was achieved by withdrawal of sulfadiazine treatment (Scho¨ler et al., 2001). This model, which relies on an impairment of the IL12-dependent IFN-γ production (SchartonKersten et al., 1997; Holtschke et al., 1996), was then used to show the efficacy of different formulations of atovaquone nanosuspensions in the treatment of TE (Scho¨ler et al., 2001; Shubar et al., 2009, 2011) and, thereafter, to prevent the disease from relapsing by an atovaquone maintenance therapy (Dunay et al., 2004). The results obtained with this murine model of reactivated toxoplasmosis mimicked the signs of reactivated toxoplasmosis in immunocompromised patients, including the presence of parasite-associated focal necrotic lesions in the brain parenchyma and meningeal inflammation (Scho¨ler et al., 2001; Dunay et al., 2004). However, it appears to be important to start the sulfadiazine treatment within 2
3 days after infection because later

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treatment increases the mortality rate considerably and leads to TE due to continuous invasion of the brain by tachyzoites rather than by reactivation of latent cysts (Jost et al., 2007). Mice with impaired IFN-γ production, in contrast to SCID or nude mice, may not be as prone to common infection; however, they have to be kept under specific-pathogen-free conditions, reducing the number of facilities where such experiments can be performed. Advantages of these models in immunodeficient mice as compared to those involving reactivation by administration of immunosuppressive drugs are (1) the easiness of reactivation simply by discontinuation of sulfadiazine and (2) a relatively synchronized development of TE within days (Scho¨ler et al., 2001).

7.4.5 Latent infection models Bradyzoites within tissue cysts are generally resistant to those drugs which are commonly used to cure or to avoid overt toxoplasmosis caused by the tachyzoite stage. However, several compounds including distinct endochinlike quinolones, a thiazole derivative of artemisinin or the FDA-approved drug guanabenz have recently been identified which appear to also target the tissue cyst of T. gondii (e.g., Doggett et al., 2012; Schultz et al., 2014; Benmerzouga et al., 2015; Vidadala et al., 2016). In general, their efficacy against tissue cysts has been evaluated by treating mice several weeks (mostly at 5 weeks) after infection, that is, when they have developed a chronic infection. At that time of infection, tachyzoites originating from the acute infection may have been eradicated by the hosts’ immune system. However, a subpopulation of bradyzoites inside tissue cysts may be more dynamic as previously thought (Watts et al., 2015) and could even occasionally reactivate. It is therefore still possible that the recently described drugs target semiactive bradyzoites or even

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tachyzoites that occasionally arise from tissue cysts but not the bona fide dormant bradyzoite stage. Regardless, the proven considerable efficacy of these drugs against brain tissue cysts is an important development in the chemotherapy of toxoplasmosis. Critical to the latent infection model is to reliably achieve a significant number of tissue cysts in the brains of infected mice without a high rate of mortality during acute infection. In this respect, as indicated above, the mouse and parasite strains, the infection route and dose, as well as the parasite stage are most important. High brain cysts numbers have consistently developed after intraperitoneal infection of CBA/J mice with 18 ME49 tissue cysts and, to a lesser extent, after i.p. infection of BALB/c mice with 105 PRU tachyzoites (Doggett et al., 2012; Schultz et al., 2014; Benmerzouga et al., 2015). A recent metaanalysis indicated the lowest variation in brain cyst burdens after infection of BALB/c and CBA mice with ME49 parasites (Watson and Davis, 2019). An alternative to the murine model may be infection of distinct rat strains. Rats are generally considered more resistant to T. gondii infection than mice, and lethal infections are indeed only observed after high dose infections mostly with highly mouse-virulent type 1 or atypical strains (Dubey et al., 2016). Furthermore, high numbers of brain cysts develop after infection of adult Fischer 344 rats or after infection of newborns of Fischer 344 and Brown Norway rats with tissue cysts of the PRU strain (Gao et al., 2015). These models may therefore be well suited for drug efficacy testing during the chronic phase of infection in the future.

References Abou-Bacar, A., Pfaff, A.W., Georges, S., Letscher-Bru, V., Filisetti, D., Villard, O., et al., 2004a. Role of NK cells and gamma interferon in transplacental passage of

Toxoplasma gondii in a mouse model of primary infection. Infect. Immun. 72, 1397
1401. Abou-Bacar, A., Pfaff, A.W., Letscher-Bru, V., Filisetti, D., Rajapakse, R., Antoni, E., et al., 2004b. Role of gamma interferon and T cells in congenital Toxoplasma transmission. Parasite Immunol. 26, 315
318. Adams, F.H., Cooney, M., Adams, J.M., Kabler, P., 1949. Experimental toxoplasmosis. Proc. Soc. Exp. Biol. Med. 70, 258
260. Aguirre-Cruz, L., Sotelo, J., 1998. Lack of therapeutic effect of colchicine on murine toxoplasmosis. J. Parasitol. 84, 163
164. Aguirre-Cruz, L., Velasco, O., Sotelo, J., 1998. Nifurtimox plus pyrimethamine for treatment of murine toxoplasmosis. J. Parasitol. 84, 1032
1033. Alder, J., Hutch, T., Meulbroek, J.A., Clement, J.C., 1994. Treatment of experimental Toxoplasma gondii infection by clarithromycin-based combination therapy with minocycline or pyrimethamine. J. Acquir. Immune Defic. Syndr. 7, 1141
1148. Ali, S.M., Allam, S.R., Negm, A.Y., El Zawawy, L.A., 2003. Vaccination against congenital toxoplasmosis. J. Egypt. Soc. Parasitol. 33, 863
874. Anderson, M.L., Barr, B.C., Conrad, P.A., 1994. Protozoal causes of reproductive failure in domestic ruminants. Vet. Clin. North Am. Food Anim. Pract. 10, 439
461. Araujo, F.G., Remington, J.S., 1974. Effect of clindamycin on acute and chronic toxoplasmosis in mice. Antimicrob. Agents Chemother. 5, 647
651. Araujo, F.G., Remington, J.S., 1975. IgG antibody suppression of the IgM antibody response to Toxoplasma gondii in newborn rabbits. J. Immunol. 115, 335
338. Araujo, F.G., Williams, D.M., Grumet, F.C., Remington, J.S., 1976. Strain-dependent differences in murine susceptibility to Toxoplasma. Infect. Immun. 13, 1528
1530. Araujo, F.G., Huskinson, J., Remington, J.S., 1991a. Remarkable in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts of Toxoplasma gondii. Antimicrob. Agents Chemother. 35, 293
299. Araujo, F.G., Shepard, R.M., Remington, J.S., 1991b. In vivo activity of the macrolide antibiotics azithromycin, roxithromycin and spiramycin against Toxoplasma gondii. Eur. J. Clin. Microbiol. Infect. Dis. 10, 519
524. Araujo, F.G., Lin, T., Remington, J.S., 1992a. Synergistic combination of azithromycin and sulfadiazine for treatment of toxoplasmosis in mice. Eur. J. Clin. Microbiol. Infect. Dis. 11, 71
73. Araujo, F.G., Prokocimer, P., Remington, J.S., 1992b. Clarithromycin-minocycline is synergistic in a murine model of toxoplasmosis. J. Infect. Dis. 165, 788.

Toxoplasma Gondii

References

Araujo, F.G., Suzuki, Y., Remington, J.S., 1996. Use of rifabutin in combination with atovaquone, clindamycin, pyrimethamine, or sulfadiazine for treatment of toxoplasmic encephalitis in mice. Eur. J. Clin. Microbiol. Infect. Dis. 15, 394
397. Araujo, F.G., Khan, A.A., Slifer, T.L., Bryskier, A., Remington, J.S., 1997. The ketolide antibiotics HMR 3647 and HMR 3004 are active against Toxoplasma gondii in vitro and in murine models of infection. Antimicrob. Agents Chemother. 41, 2137
2140. Araujo, F.G., Khan, A.A., Bryskier, A., Remington, J.S., 1998. Use of ketolides in combination with other drugs to treat experimental toxoplasmosis. J. Antimicrob. Chemother 42, 665
667. Arribas, J.R., de Diego, J.A., Gamallo, C., Vazquez, J.J., 1995. A new murine model of severe acute Toxoplasma encephalitis. J. Antimicrob. Chemother. 36, 503
512. Ashour, D.S., Saad, A.E., El Bakary, R.H., El Barody, M.A., 2018. Can the route of Toxoplasma gondii infection affect the ophthalmic outcomes? Pathog. Dis. 76, fty056. Azami, S.J., Teimouri, A., Keshavarz, H., Amani, A., Esmaeili, F., Hasanpour, H., et al., 2018. Curcumin nanoemulsion as a novel chemical for the treatment of acute and chronic toxoplasmosis in mice. Int. J. Nanomed. 13, 7363
7374. Bajohr, L.L., Ma, L., Platte, C., Liesenfeld, O., Tietze, L.F., Groß, U., et al., 2010. In vitro and in vivo activities of 1hydroxy-2-alkyl-4(1H)quinolone derivates against Toxoplasma gondii. Antimicrob. Agents Chemother. 54, 517
521. Barbosa, B.F., Silva, D.A.O., Costa, I.N., Pena, J.D.O., Mineo, J.R., Ferro, E.A.V., 2007. Susceptibility to vertical transmission of Toxoplasma gondii in temporally dependent on the preconceptional infection in Calomys callosus. Plancenta 28, 624
630. Beaman, M.H., Araujo, F.G., Remington, J.S., 1994. Protective reconstitution of the SCID mouse against reactivation of toxoplasmic encephalitis. J. Infect. Dis. 169, 375
383. Beghetto, E., Nielsen, H.V., Del Porto, P., Buffolano, W., Guglietta, S., Felici, F., et al., 2005. A combination of antigenic regions of Toxoplasma gondii microneme proteins induces protective immunity against oral infection with parasite cysts. J. Infect. Dis. 191, 637
645. Behnke, M.S., Khan, A., Wootton, J.C., Dubey, J.P., Tang, K., Sibley, L.D., 2011. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc. Natl. Acad. Sci. U.S. A. 108, 9631
9636. Belal, U.S., Norose, K., Aosai, F., Mun, H.S., Ahmed, A.K., Chen, M., et al., 2004. Evaluation of the effects of sulfamethoxazole on Toxoplasma gondii loads and stage

355

conversion in IFN-gamma knockout mice using QCPCR. Microbiol. Immunol. 48, 185
193. Benmerzouga, I., Checkley, L.A., Ferdig, M.T., Arrizabalaga, G., Wek, R.C., Sullivan Jr., W.J., 2015. Guanabenz repurposed as an antiparasitic with activity against acute and chronic toxoplasmosis. Antimicrob. Agents Chemother. 59, 6939
6945. Berard-Badier, M., Laugier, M., Louchet, E., Payan, H., 1968. The placenta in experimental acute congenital toxoplasmosis in guinea pigs. Pathol. Biol. 16, 829
835. Beverley, J.K.A., 1958. A rational approach to the treatment of toxoplasmic uveitis. Trans. Opthal. Soc. U.K. 78, 109
121. Beverley, J.K.A., 1959. Congenital transmission of toxoplasmosis through successive generation of mice. Nature 183, 1348
1349. Beverley, J.K.A., 1961. Experimental ocular toxoplasmosis. Surv. Ophthalmol. 6, 897
923. Beverley, J.K.A., Henry, L., 1970. Congenital toxoplasmosis in mice. J. Pathol. 101, xxi. Beverley, J.K.A., Beattie, C.P., Fry, B.A., 1954. Experimental toxoplasmosis of the uveal tract. Br. J. Ophthalmol. 38, 489
496. Brown, C.R., McLeod, R., 1990. Class I MHC genes and CD8 1 T cells determine cyst number in Toxoplasma gondii infection. J. Immunol. 145, 3438
3441. Brown, C.R., Hunter, C.A., Estes, R.G., Beckmann, E., Forman, J., David, C., et al., 1995. Definitive identification of a gene that confers resistance against Toxoplasma cyst burden and encephalitis. Immunology 85, 419
428. Cavailles, P., Sergent, V., Bisanz, C., Papapietro, O., Colacios, C., Mas, M., et al., 2006. The rat Toxo1 locus directs toxoplasmosis outcome and controls parasite proliferation and spreading by macrophage-dependent mechanisms. Proc. Natl. Acad. Sci. U.S.A. 103, 744
749. Cavailles, P., Flori, P., Papapietro, O., Bisanz, C., Lagrange, D., Pilloux, L., et al., 2014. A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage. PLoS Pathog. 10, e1004005. Chang, H.R., Pechere, J.C., 1987. Effect of roxithromycin on acute toxoplasmosis in mice. Antimicrob. Agents Chemother. 31, 1147
1149. Chang, H.R., Rudareanu, F.C., Pechere, J.C., 1988. Activity of A-56268 (TE-031), a new macrolide, against Toxoplasma gondii in mice. J. Antimicrob. Chemother. 22, 359
361. Chang, H.R., Comte, R., Piguet, P.F., Pechere, J.C., 1991. Activity of minocycline against Toxoplasma gondii infection in mice. J. Antimicrob. Chemother. 27, 639
645.

Toxoplasma Gondii

356

7. Toxoplasma animal models and therapeutics

Chang, H.R., Arsenijevic, D., Comte, R., Polak, A., Then, R. L., Pechere, J.C., 1994. Activity of epiroprim (Ro 118958), a dihydrofolate reductase inhibitor, alone and in combination with dapsone against Toxoplasma gondii. Antimicrob. Agents Chemother. 38, 1803
1807. Charles, E., Callegan, M.C., Blader, I.J., 2007. The SAG1 Toxoplasma gondii surface protein is not required for acute ocular toxoplasmosis in mice. Infect. Immun. 75, 2079
2083. Cornelissen, J.B., van der Giessen, J.W., Takumi, K., Teunis, P.F., Wisselink, H.J., 2014. An experimental Toxoplasma gondii dose response challenge model to study therapeutic or vaccine efficacy in cats. PLoS One 9, e104740. Costa, I.N., Angeloni, M.B., Santana, L.A., Barbosa, B.F., Silva, M.C.P., Rodrigues, A.A., et al., 2009. Azithromycin inhibits vertical transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae). Placenta 30, 884
890. Couper, K.N., Nielsen, H.V., Petersen, E., Roberts, F., Roberts, C.W., Alexander, J., 2003. DNA vaccination with the immunodominant tachyzoite surface antigen (SAG-1) protects against adult acquired Toxoplasma gondii infection but does not prevent maternofoetal transmission. Vaccine 21, 2813
2820. Culbertson, W.W., Tabbara, K.F., O’Connor, R., 1982. Experimental ocular toxoplasmosis in primates. Arch. Ophthalmol. 100, 321
323. Darcy, F., Zenner, L., 1993. Experimental models of toxoplasmosis. Res. Immunol. 144, 16
23. Davidson, M.G., Lappin, M.R., English, R.V., Tompkins, M. B., 1993. A feline model of ocular toxoplasmosis. Invest. Ophthalmol. Vis. Sci. 34, 3653
3660. Davidson, M.G., Lappin, M.R., Rottman, J.R., Tompkins, M. B., English, R.V., Bruce, A.T., et al., 1996. Paradoxical effect of clindamycin in experimental, acute toxoplasmosis in cats. Antimicrob. Agents Chemother. 40, 1352
1359. De Champs, C., Imbert-Bernard, C., Belmeguenai, A., Ricard, J., Pelloux, H., Brambilla, E., et al., 1997. Toxoplasma gondii: in vivo and in vitro cystogenesis of the virulent RH strain. J. Parasitol. 83, 152
155. Deckert-Schlu¨ter, M., Schlu¨ter, D., Schmidt, D., Schwendemann, G., Wiestler, O.D., Hof, H., 1994. Toxoplasma encephalitis in congenic B10 and BALB mice: impact of genetic factors on the immune response. Infect. Immun. 62, 221
228. Degerli, K., Kilimcioglu, A.A., Kurt, O., Tamay, A.T., Ozbilgin, A., 2003. Efficacy of azithromycin in a murine toxoplasmosis model, employing a Toxoplasma gondii strain from Turkey. Acta. Trop. 88, 45
50. Denkers, E.Y., Gazzinelli, R.T., 1998. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin. Microbiol. Rev. 11, 569
588.

De Roever-Bonnet, H., 1960. Congenital Toxoplasma infections in mice and hamsters with avirulent strain. Trop. Geogr. Med. 21, 443
450. Derouin, F., Piketty, C., Chastang, C., Chau, F., Rouveix, B., Pocidalo, J.J., 1991. Anti-Toxoplasma effects of dapsone alone and combined with pyrimethamine. Antimicrob. Agents Chemother. 35, 252
255. Derouin, F., Almadany, R., Chau, F., Rouveix, B., Pocidalo, J. J., 1992. Synergistic activity of azithromycin and pyrimethamine or sulfadiazine in acute experimental toxoplasmosis. Antimicrob. Agents Chemother. 36, 997
1001. Derouin, F., Lacroix, C., Sumyuen, M.H., Romand, S., Garin, Y.J., 1995. Experimental models of toxoplasmosis. Pharmacological applications. Parasite 2, 243
256. Desmonts, G., 1966. Definitive serological diagnosis of ocular toxoplasmosis. Arch. Ophthalmol. 76, 839
851. Desmonts, G., Couvreur, J., 1984. Congenital toxoplasmosis. Prospective study of the outcome of pregnancy in 542 women with toxoplasmosis acquired during pregnancy. Ann. Pediatr. 31, 805
809. Djurkovic-Djakovic, O., Milenkovic, V., 2001. Murine model of drug-induced reactivation of Toxoplasma gondii. Acta Protozoologica 40, 99
106. Djurkovic-Djakovic, O., Nikolic, T., Robert-Gangneux, F., Bobic, B., Nikolic, A., 1999. Synergistic effect of clindamycin and atovaquone in acute murine toxoplasmosis. Antimicrob. Agents Chemother. 43, 2240
2244. Djurkovic-Djakovic, O., Milenkovic, V., Nikolic, A., Bobic, B., Grujic, J., 2002. Efficacy of atovaquone combined with clindamycin against murine infection with a cystogenic (Me49) strain of Toxoplasma gondii. J. Antimicrob. Chemother. 50, 981
987. Djurkovic-Djakovic, O., Nikolic, A., Bobic, B., Klun, I., Aleksic, A., 2005. Stage conversion of Toxoplasma gondii RH parasites in mice by treatment with atovaquone and pyrrolidine dithiocarbamate. Microbes. Infect. 7, 49
54. Doggett, J.S., Nilsen, A., Forquer, I., Wegmann, K.W., Jones-Brando, L., Yolken, R.H., et al., 2012. Endochinlike quinolones are highly efficacious against acute and latent experimental toxoplasmosis. Proc. Natl. Acad. Sci. U.S.A. 109, 15936
15941. Doggett, J.S., Ojo, K.K., Fan, E., Maly, D.J., Van Voorhis, W. C., 2014. Bumped kinase inhibitor 1294 treats established Toxoplasma gondii infection. Antimicrob. Agents Chemother. 58, 3547
3549. Dro¨gemu¨ller, K., Helmuth, U., Brunn, A., SakowiczBurkiewicz, M., Gutmann, D.H., Mueller, W., et al., 2008. Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis. J. Immunol. 181, 2683
2693. Dubey, J.P., 1996. Pathogenicity and infectivity of Toxoplasma gondii oocysts for rats. J. Parasitol. 82, 951
956.

Toxoplasma Gondii

References

Dubey, J.P., 1998. Re-examination of resistance of Toxoplasma gondii tachyzoites and bradyzoites to pepsin and trypsin digestion. Parasitology 116 (Pt 1), 43
50. Dubey, J.P., Frenkel, J.K., 1998. Toxoplasmosis of rats: a review, with considerations of their value as an animal model and their possible role in epidemiology. Vet. Parasitol. 77, 1
32. Dubey, J.P., Rommel, M., 1992. Abortions caused by protozoa in agricultural animals. Dtsch. Tierarztl. Wochenschr. 99, 355
362. Dubey, J.P., Shen, S.K., 1991. Rat model of congenital toxoplasmosis. Infect. Immun. 59, 3301
3302. Dubey, J.P., Shen, S.K., Kwok, O.C., Thulliez, P., 1997. Toxoplasmosis in rats (Rattus norvegicus): congenital transmission to first and second generation offspring and isolation of Toxoplasma gondii from seronegative rats. Parasitology 115 (Pt 1), 9
14. Dubey, J.P., Ferreira, L.R., Alsaad, M., Verma, S.K., Alves, D.A., Holland, G.N., et al., 2016. Experimental toxoplasmosis in rats induced orally with eleven strains of Toxoplasma gondii of seven genotypes: tissue tropism, tissue cyst size, neural lesions, tissue cyst rupture without reactivation and ocular lesions. PLoS One 11, e0156255. Dukaczewska, A., Tedesco, R., Liesenfeld, O., 2015. Experimental models of ocular infection with Toxoplasma gondii. Eur. J. Microbiol. Immunol. (Bp) 5, 292
305. Dumas, J.L., Chang, R., Mermillod, B., Piguet, P.F., Comte, R., Pechere, J.C., 1994. Evaluation of the efficacy of prolonged administration of azithromycin in a murine model of chronic toxoplasmosis. J. Antimicrob. Chemother. 34, 111
118. Dumas, J.L., Pizzolato, G., Pechere, J.C., 1999. Evaluation of trimethoprim and sulphamethoxazole as monotherapy or in combination in the management of toxoplasmosis in murine models. Int. J. Antimicrob. Agents 13, 35
39. Dunay, I.R., Heimesaat, M.M., Bushrab, F.N., Muller, R.H., Stocker, H., Arasteh, K., et al., 2004. Atovaquone maintenance therapy prevents reactivation of toxoplasmic encephalitis in a murine model of reactivated toxoplasmosis. Antimicrob. Agents Chemother. 48, 4848
4854. Dunay, I.R., Chan, W.C., Haynes, R.K., Sibley, L.D., 2009. Artemisone and artemiside control acute and reactivated toxoplasmosis in a murine model. Antimicrob. Agents Chemother. 53, 4450
4456. Dunay, I.R., Gajurel, K., Dhakal, R., Liesenfeld, O., Montoya, J.G., 2018. Treatment of toxoplasmosis: historical perspective, animal models, and current clinical practice. Clin. Microbiol. Rev. 31, e00057-17. Duncanson, P., Terry, R.S., Smith, J.E., Hide, G., 2001. High levels of congenital transmission of Toxoplasma gondii in

357

a commercial sheep flock. Int. J. Parasitol. 31, 1699
1703. Dutton, G.N., Hay, J., Hair, D.M., Ralston, J., 1986. Clinicopathological features of a congenital murine model of ocular toxoplasmosis. Graefes Arch. Clin. Exp. Ophthalmol. 224, 256
264. Elbez-Rubinstein, A., Ajzenberg, D., Darde´, M.L., Cohen, R., Dume`tre, A., Yera, H., et al., 2009. Congentital toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J. Infect. Dis. 199, 280
285. Elsaid, M.M., Martins, M.S., Frezard, F., Braga, E.M., Vitor, R.W., 2001. Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes. Mem. Inst. Oswaldo Cruz. 96, 99
104. Escoffier, P., Jeanny, J.C., Marinach-Patrice, C., Jonet, L., Raoul, W., Behar-Cohen, F., et al., 2010. Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice. Exp. Parasitol. 126, 259
262. Ferguson, D.J., Hutchison, W.M., Pettersen, E., 1989. Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study. Parasitol. Res. 75, 599
603. Ferreira, R.A., Oliveira, A.B., Gualberto, S.A., Vitor, R.W., 2002. Activity of natural and synthetic naphthoquinones against Toxoplasma gondii, in vitro and in murine models of infection. Parasite 9, 261
269. Ferro, E.A.V., Silva, D.A.O., Bevilacqua, E., Mineo, J.R., 2002. Effect of Toxoplasma gondii infection kinetics on trophoblast cell population in Calomys callosus, a model of congenital toxoplasmosis. Infect. Immun. 70, 7089
7094. Flori, P., Hafid, J., Bourlet, T., Raberin, H., Genin, C., Sung, R.T., 2002. Experimental model of congenital toxoplasmosis in guinea-pigs: use of quantitative and qualitative PCR for the study of maternofoetal transmission. J. Med. Microbiol. 51, 871
878. Flori, P., Hafid, J., Thonier, V., Bellete, B., Raberin, H., Tran Manh Sung, R., 2003. Parasite load in guinea pig foetus with real time PCR after maternofoetal transmission of Toxoplasma gondii. Parasite 10, 133
140. Foulet, A., Zenner, L., Darcy, F., Cesbron-Delauw, M.F., Capron, A., Gosselin, B., 1994. Pathology of Toxoplasma gondii infection in the nude rat. An experimental model of toxoplasmosis in the immunocompromised host? Pathol. Res. Pract. 190, 775
781. Franco, P.S., Silva, D.A.O., Costa, I.N., Gomes, A.O., Silva, A.L.N., Pena, J.D.O., et al., 2011. Evaluation of vertical transmission of Toxoplasma gondii in Calomys callosus model after reinfection with heterologous and virulent strain. Placenta 32, 116
120.

Toxoplasma Gondii

358

7. Toxoplasma animal models and therapeutics

Frenkel, J.K., 1953. Host, strain and treatment variation as factors in the pathogenesis of toxoplasmosis. Am. J. Trop. Med. Hyg. 2, 390
415. Frenkel, J.K., 1955. Ocular lesions in hamsters; with chronic Toxoplasma and Besnoitia infection. Am. J. Ophthalmol. 39, 203
225. Frenkel, J.K., Escajadillo, A., 1987. Cyst rupture as a pathogenic mechanism of toxoplasmic encephalitis. Am. J. Trop. Med. Hyg. 36, 517
522. Frenkel, J.K., Nelson, B.M., Arias-Stella, J., 1975. Immunosuppression and toxoplasmic encephalitis: clinical and experimental aspects. Hum. Pathol. 6, 97
111. Freyre, A., Correa, O., Falcon, J., Mendez, J., Gonzalez, M., Venzal, J.M., 2001a. Some factors influencing transmission of Toxoplasma in pregnant rats fed cysts. Parasitol. Res. 87, 941
944. Freyre, A., Falcon, J., Correa, O., Mendez, J., Gonzalez, M., Venzal, J.M., 2001b. Residual infection of 15 Toxoplasma strains in the brain of rats fed cysts. Parasitol. Res. 87, 915
918. Freyre, A., Falcon, J., Correa, O., Mendez, J., Gonzalez, M., Venzal, J.M., et al., 2003a. Cyst burden in the brains of Wistar rats fed Toxoplasma oocysts. Parasitol. Res. 89, 342
344. Freyre, A., Falcon, J., Mendez, J., Gonzalez, M., Venzal, J. M., Morgades, D., 2003b. Foetal Toxoplasma infection after oocyst inoculation of pregnant rats. Parasitol. Res. 89, 352
353. Freyre, A., Falcon, J., Mendez, J., Correa, O., Morgades, D., Rodriguez, A., 2004. An investigation of sterile immunity against toxoplasmosis in rats. Exp. Parasitol. 107, 14
19. Freyre, A., Falcon, J., Mendez, J., Rodreguez, A., Correa, L., Gonzalez, M., 2006a. Refinement of the mouse model of congenital toxoplasmosis. Exp. Parasitol. 113, 154
160. Freyre, A., Falcon, J., Mendez, J., Rodriguez, A., Correa, L., Gonzalez, M., 2006b. Toxoplasma gondii: partial crossprotection among several strains of the parasite against congenital transmission in a rat model. Exp. Parasitol. 112, 8
12. Freyre, A., Falcon, J., Mendez, J., Gonzalez, M., 2008. Toxoplasma gondii: an improved rat model of congenital infection. Exp. Parasitol. 120, 142
146. Freyre, A., Fialho, C.G., Bigatti, L.E., Araujo, F.A., Falcon, J. D., Mendez, J., et al., 2009. Toxoplasma gondii: congenital transmission in a hamster model. Exp. Parasitol. 122, 140
144. Freyre, A., Araujo, F.A.P., Fialho, C.G., Bigatti, L.E., Falcon, J.D., 2012. Protection in a hamster model of congenital toxoplasmosis. Vet. Parasitol. 183, 359
363. Friedrich, R., Mu¨ller, W.A., 1989. The effect of a subretinal injection of Toxoplasma gondii on the serum antibody titer in a rabbit model of ocular toxoplasmosis. Angew. Parasitol. 30, 15
17.

Fujii, H., Kamiyama, T., Hagiwara, T., 1983. Species and strain differences in sensitivity to Toxoplasma infection among laboratory rodents. Jpn. J. Med. Sci. Biol. 36, 343
346. Fux, B., Ferreira, A., Cassali, G., Tafuri, W.L., Vitor, R.W., 2000. Experimental toxoplasmosis in Balb/c mice. Prevention of vertical disease transmission by treatment and reproductive failure in chronic infection. Mem. Inst. Oswaldo Cruz. 95, 121
126. Gao, J.M., Yi, S.Q., Wu, M.S., Geng, G.Q., Shen, J.L., Lu, F. L., et al., 2015. Investigation of infectivity of neonates and adults from different rat strains to Toxoplasma gondii Prugniaud shows both variation which correlates with iNOS and arginase-1 activity and increased susceptibility of neonates to infection. Exp. Parasitol. 149, 47
53. Garcia, J.L., Burrells, A., Bartley, P.M., Bartley, K., Innes, E. A., Katzer, F., 2017. The use of ELISA, nPCR and qPCR for diagnosis of ocular toxoplasmosis in experimentally infected pigs. Res. Vet. Sci. 115, 490
495. Garweg, J.G., Boehnke, M., 2006. The antibody response in experimental ocular toxoplasmosis. Graefes Arch. Clin. Exp. Ophthalmol. 244, 1668
1679. Garweg, J.G., Kuenzli, H., Boehnke, M., 1998. Experimental ocular toxoplasmosis in naive and primed rabbits. Ophthalmologica 212, 136
141. Gazzinelli, R.T., Hartley, J.W., Fredrickson, T.N., Chattopadhyay, S.K., Sher, A., Morse III, H.C., 1992. Opportunistic infections and retrovirus-induced immunodeficiency: studies of acute and chronic infections with Toxoplasma gondii in mice infected with LP-BM5 murine leukemia viruses. Infect. Immun. 60, 4394
4401. Gazzinelli, R.T., Brezin, A., Li, Q., Nussenblatt, R.B., Chan, C.C., 1994. Toxoplasma gondii: acquired ocular toxoplasmosis in the murine model, protective role of TNFalpha and IFN-gamma. Exp. Parasitol. 78, 217
229. Goldmann, H., Witmer, R., 1954. Antiko¨rper im Kammerwasser. Ophthalmologica 127, 323
330. Gormley, P.D., Pavesio, C.E., Minnasian, D., Lightman, S., 1998. Effects of drug therapy on Toxoplasma cysts in an animal model of acute and chronic disease. Invest. Ophthalmol. Vis. Sci. 39, 1171
1175. Gormley, P.D., Pavesio, C.E., Luthert, P., Lightman, S., 1999. Retinochoroiditis is induced by oral administration of Toxoplasma gondii cysts in the hamster model. Exp. Eye Res. 68, 657
661. Grossman, P.L., Remington, J.S., 1979. The effect of trimethoprim and sulfamethoxazole on Toxoplasma gondii in vitro and in vivo. Am. J. Trop. Med. Hyg. 28, 445
455. Grujic, J., Djurkovic-Djakovic, O., Nikolic, A., Klun, I., Bobic, B., 2005. Effectiveness of spiramycin in murine models of acute and chronic toxoplasmosis. Int. J. Antimicrob. Agents 25, 226
230. Hakimi, M.A., Olias, P., Sibley, L.D., 2017. Toxoplasma effectors targeting host signaling and transcription. Clin. Microbiol. Rev. 30, 615
645.

Toxoplasma Gondii

References

Ha¨ndel, U., Brunn, A., Dro¨gemu¨ller, K., Mu¨ller, W., Deckert, M., Schlu¨ter, D., 2012. Neuronal gp130 expression is crucial to prevent neuronal loss, hyperinflammation, and lethal course of murine Toxoplasma encephalitis. Am. J. Pathol. 181, 163
173. Haumont, M., Delhaye, L., Garcia, L., Jurado, M., Mazzu, P., Daminet, V., et al., 2000. Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in a guinea pig model. Infect. Immun. 68, 4948
4953. Hay, J., Dutton, G.N., 1996. Fundal with dots: observations from a murine model of congenital ocular toxoplasmosis. Br. J. Ophthalmol. 80, 189. Hay, J., Kerrigan, P., 1982. Photography of cataract in mice congenitally infected with Toxoplasma gondii. Ann. Trop. Med. Parasitol. 76, 363
365. Hay, J., Hutchison, W.M., Lee, W.R., Siim, J.C., 1981. Cataract in mice congenitally infected with Toxoplasma gondii. Ann. Trop. Med. Parasitol. 75, 455
457. Hay, J., Lee, W.R., Dutton, G.N., Hutchison, W.M., Siim, J. C., 1984. Congenital toxoplasmic retinochoroiditis in a mouse model. Ann. Trop. Med. Parasitol. 78, 109
116. Hellbru¨gge, T.F., 1955. Foetal infection during the acute and chronic phase of latent toxoplasmosis in rats. Arch. Gynakol. 186, 384
388. Hellbru¨gge, T.F., Dahme, E., 1953. Experimental toxoplasmosis. Klin. Wochenschr. 31, 789
791. Hellbru¨gge, T.F., Dahme, E., Hellbru¨gge, F.K., 1953. Experimental animal observations on transplacental infection with Toxoplasma. Z. Tropenmed. Parasitol. 4, 312
322. Hofflin, J.M., Remington, J.S., 1987a. Clindamycin in a murine model of toxoplasmic encephalitis. Antimicrob. Agents Chemother. 31, 492
496. Hofflin, J.M., Remington, J.S., 1987b. In vivo synergism of roxithromycin (RU 965) and interferon against Toxoplasma gondii. Antimicrob. Agents Chemother. 31, 346
348. Hofflin, J.M., Conley, F.K., Remington, J.S., 1987. Murine model of intracerebral toxoplasmosis. J. Infect. Dis. 155, 550
557. Hogan, M.J., Lewis, A., Zweigart, P.A., 1956. Persistence of Toxoplasma gondii in ocular tissues. I. Am. J. Ophthalmol. 42, 84
89. Holland, G.N., 1999. Reconsidering the pathogenesis of ocular toxoplasmosis. Am. J. Ophthalmol. 128, 502
505. Holland, G.N., 2003. Ocular toxoplasmosis: a global reassessment. Part I: Epidemiology and course of disease. Am. J. Ophthalmol. 136, 973
988. Holland, G.N., Engstrom Jr., R.E., Glasgow, B.J., Berger, B. B., Daniels, S.A., Sidikaro, Y., et al., 1988a. Ocular toxoplasmosis in patients with the acquired immunodeficiency syndrome. Am. J. Ophthalmol. 106, 653
667. Holland, G.N., O’Connor, G.R., Diaz, R.F., Minasi, P., Wara, W.M., 1988b. Ocular toxoplasmosis in

359

immunosuppressed nonhuman primates. Invest. Ophthalmol. Vis. Sci. 29, 835
842. Holtschke, T., Lohler, J., Kanno, Y., Fehr, T., Giese, N., Rosenbauer, F., et al., 1996. Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell 87, 307
317. Howe, D.K., Sibley, L.D., 1995. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J. Infect. Dis. 172, 1561
1566. Howe, D.K., Summers, B.C., Sibley, L.D., 1996. Acute virulence in mice is associated with markers on chromosome VIII in Toxoplasma gondii. Infect. Immun. 64, 5193
5198. Hu, M.S., Schwartzman, J.D., Lepage, A.C., Khan, I.A., Kasper, L.H., 1999a. Experimental ocular toxoplasmosis induced in naive and preinfected mice by intracameral inoculation. Ocul. Immunol. Inflamm. 7, 17
26. Hu, M.S., Schwartzman, J.D., Yeaman, G.R., Collins, J., Seguin, R., Khan, I.A., et al., 1999b. Fas-FasL interaction involved in pathogenesis of ocular toxoplasmosis in mice. Infect. Immun. 67, 928
935. Hu, M.S., Schwartzman, J.D., Kasper, L.H., 2001. Apoptosis within mouse eye induced by Toxoplasma gondii. Chin. Med. J. (Engl.) 114, 640
644. Huldt, G., 1960. Experimental toxoplasmosis transplacental transmission in guinea pigs. Acta Pathol. Microbiol. Scand 49, 176
188. Hunn, J.P., Feng, C.G., Sher, A., Howard, J.C., 2011. The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens. Mamm. Genome 22, 43
54. Hutchison, W.M., Hay, J., Lee, W.R., Siim, J.C., 1982. A study of cataract in murine congenital toxoplasmosis. Ann. Trop. Med. Parasitol. 76, 53
70. Ismael, A.B., Dimier-Poisson, I., Lebrun, M., Dubremetz, J. F., Bout, D., Mevelec, M.N., 2006. Mic1-3 knockout of Toxoplasma gondii is a successful vaccine against chronic and congenital toxoplasmosis in mice. J. Infect. Dis. 194, 1176
1183. Israelski, D., Remington, J., 1990. Activity of gamma interferon in combination with pyrimethamine or clindamycin in treatment of murine toxoplasmosis. Eur. J. Clin. Microbiol. Infect. Dis. 9, 358
360. Jacobs, L., Melton, M.L., Kaufman, H.E., 1964. Treatment of experimental ocular toxoplasmosis. Arch. Ophthalmol. 71, 111
118. Johnson, A.M., 1984. Strain dependent, route of challengedependent susceptibility to toxoplasmosis. Z. Parasitenk. 70, 303
309. Johnson, L.L., 1992. SCID mouse models of acute and relapsing chronic Toxoplasma gondii infections. Infect. Immun. 60, 3719
3724. Johnson, J., Suzuki, Y., Mack, D., Mui, E., Estes, R., David, C., et al., 2002. Genetic analysis of influences on survival following Toxoplasma gondii infection. Int. J. Parasitol. 32, 179
185.

Toxoplasma Gondii

360

7. Toxoplasma animal models and therapeutics

Jost, C., Reiter-Owona, I., Liesenfeld, O., 2007. The timing of sulfadiazine therapy impacts the reactivation of latent Toxoplasma infection in IRF-8 2 / 2 mice. Parasitol. Res. 101, 1603
1609. Jungersen, G., Bille-Hansen, V., Jensen, L., Lind, P., 2001. Transplacental transmission of Toxoplasma gondii in minipigs infected with strains of different virulence. J. Parasitol. 87, 108
113. Kannan, G., Pletnikov, M.V., 2012. Toxoplasma gondii and cognitive deficits in schizophrenia: an animal model perspective. Schizophr. Bull. 38, 1155
1161. Kaufman, H.E., 1960. The effect of corticosteroids on experimental ocular toxoplasmosis. Am. J. Ophthalmol. 50, 919
926. Kaufman, H.E., Remington, J.S., Jacobs, L., 1958. Toxoplasmosis: the nature of virulence. Am. J. Ophthalmol. 46, 255
261. Kempf, M.C., Cesbron-Delauw, M.F., Deslee, D., Gross, U., Herrmann, T., Sutton, P., 1999. Different manifestations of Toxoplasma gondii infection in F344 and LEW rats. Med. Microbiol. Immunol. (Berl.) 187, 137
142. Khan, A.A., Slifer, T., Araujo, F.G., Remington, J.S., 1996. Trovafloxacin is active against Toxoplasma gondii. Antimicrob. Agents Chemother. 40, 1855
1859. Khan, A.A., Nasr, M., Araujo, F.G., 1998. Two 2-hydroxy-3alkyl-1,4-naphthoquinones with in vitro and in vivo activities against Toxoplasma gondii. Antimicrob. Agents Chemother. 42, 2284
2289. Khan, I.A., Green, W.R., Kasper, L.H., Green, K.A., Schwartzman, J.D., 1999. Immune CD8(1) T cells prevent reactivation of Toxoplasma gondii infection in the immunocompromised host. Infect. Immun. 67, 5869
5876. Khan, A.A., Araujo, F.G., Craft, J.C., Remington, J.S., 2000. Ketolide ABT-773 is active against Toxoplasma gondii. J. Antimicrob. Chemother 46, 489
492. Kobayashi, M., Aosai, F., Hata, H., Mun, H.S., Tagawa, Y., Iwakura, Y., et al., 1999. Toxoplasma gondii: difference of invasion into tissue of digestive organs between susceptible and resistant strain and influence of IFN-gamma in mice inoculated with the cysts perorally. J. Parasitol. 85, 973
975. Lacroix, C., Levacher-Clergeot, M., Chau, F., Sumuyen, M.H., Sinet, M., Pocidalo, J.J., et al., 1994. Interactions between murine AIDS (MAIDS) and toxoplasmosis in co-infected mice. Clin. Exp. Immunol 98, 190
195. Lahmar, I., Guinard, M., Sauer, A., Marcellin, L., Abdelrahman, T., Roux, M., et al., 2010. Murine neonatal infection provides an efficient model for congenital ocular toxoplasmosis. Exp. Parasitol. 124, 190
196. Lahmar, I., Pfaff, A.W., Marcellin, L., Sauer, A., Moussa, A., Babba, H., et al., 2014. Mu¨ller cell activation and

photoreceptor depletion in a mice model of congenital ocular toxoplasmosis. Exp. Parasitol. 144, 22
26. Lee, W.R., Hay, J., Hutchison, W.M., Dutton, G.N., Siim, J. C., 1983. A murine model of congenital toxoplasmic retinochoroiditis. Acta. Ophthalmol. (Copenh.) 61, 818
830. Lescano, S.A., Amato Neto, V., Chieffi, P.P., Bezerra, R.C., Gakiya, E., Ferreira, C.S., et al., 2004. [Evaluation of the efficacy of azithromycin and pyrimethamine, for treatment of experimental infection of mice with Toxoplasma gondii cystogenic strain]. Rev. Soc. Bras. Med. Trop. 37, 460
462. Letscher-Bru, V., Pfaff, A.W., Abou-Bacar, A., Filisetti, D., Antoni, E., Villard, O., et al., 2003. Vaccination with Toxoplasma gondii SAG-1 protein is protective against congenital toxoplasmosis in BALB/c mice but not in CBA/J mice. Infect. Immun. 71, 6615
6619. Lilue, J., Mu¨ller, U.B., Steinfeldt, T., Howard, J.C., 2013. Reciprocal virulence and resistance polymorphism in the relationship between Toxoplasma gondii and the house mouse. Elife 2, e01298. Ling, Y., Sahota, G., Odeh, S., Chan, J.M., Araujo, F.G., Moreno, S.N., et al., 2005. Bisphosphonate inhibitors of Toxoplasma gondi growth: in vitro, QSAR, and in vivo investigations. J. Med. Chem. 48, 3130
3140. Loke, Y.W., 1982. Transmission of parasites across the placenta. Adv. Parasitol 21, 155
228. Lopes, C.D., Silva, M.N., Ferro, E.A., Sousa, R.A., Firminot, M.L., Bernardes, E.S., et al., 2009. Azithromycin reduces ocular infection during congenital transmission of toxoplasmosis in the Calomys callosus model. J. Parasitol. 95, 1005
1010. Lu, F., Huang, S., Kasper, L.H., 2004. CD4 1 T cells in the pathogenesis of murine ocular toxoplasmosis. Infect. Immun. 72, 4966
4972. Lu, F., Huang, S., Hu, M.S., Kasper, L.H., 2005. Experimental ocular toxoplasmosis in genetically susceptible and resistant mice. Infect. Immun. 73, 5160
5165. Lu, F., Huang, S., Kasper, L.H., 2009. The temperaturesensitive mutants of Toxoplasma gondii and ocular toxoplasmosis. Vaccine 27, 573
580. Lu¨der, C.G.K., Reichard, U., Gross, U., 2014. Toxoplasma animal models and therapeutics. In: Weiss, L., Kim, K. (Eds.), Toxoplasma gondii. The Model Apicomplexan
Perspectives and Methods. Elsevier Academic Press, San Diego, CA; London, UK, pp. 217
255. Lyons, R.E., Anthony, J.P., Ferguson, D.J., Byrne, N., Alexander, J., Roberts, F., et al., 2001. Immunological studies of chronic ocular toxoplasmosis: up-regulation of major histocompatibility complex class I and transforming growth factor beta and a protective role for interleukin-6. Infect. Immun. 69, 2589
2595.

Toxoplasma Gondii

References

Martinez, A., Allegra, C.J., Kovacs, J.A., 1996. Efficacy of epiroprim (Ro11-8958), a new dihydrofolate reductase inhibitor, in the treatment of acute Toxoplasma infection in mice. Am. J. Trop. Med. Hyg. 54, 249
252. Martins-Duarte, E.S., Lemgruber, L., de Souza, W., Vommaro, R.C., 2010. Toxoplasma gondii: fluconazole and itraconalzole activity against toxoplasmosis in a murine model. Exp. Parasitol. 124, 466
469. Martins-Duarte, E.S., Dubar, F., Lawton, P., da Silva, C.F., Soeiro Mde, N., de Souza, W., et al., 2015. Ciprofloxacin derivatives affect parasite cell division and increase the survival of mice infected with Toxoplasma gondii. PLoS One 10, e0125705. McConnell, E.V., Bruzual, I., Pou, S., Winter, R., Dodean, R. A., Smilkstein, M.J., et al., 2018. Targeted structureactivity analysis of endochin-like quinolones reveals potent Qi and Qo site inhibitors of Toxoplasma gondii and Plasmodium falciparum cytochrome bc1 and identifies ELQ-400 as a remarkable effective compound against acute experimental toxoplasmosis. ACS Infect. Dis. 4, 1574
1584. McLeod, R., Estes, R.G., Mack, D.G., Cohen, H., 1984. Immune response of mice to ingested Toxoplasma gondii: a model of Toxoplasma infection acquired by ingestion. J. Infect. Dis. 149, 234
244. McLeod, R., Frenkel, J.K., Estes, R.G., Mack, D.G., Eisenhauer, P.B., Gibori, G., 1988. Subcutaneous and intestinal vaccination with tachyzoites of Toxoplasma gondii and acquisition of immunity to peroral and congenital Toxoplasma challenge. J. Immunol. 140, 1632
1637. McLeod, R., Eisenhauer, P., Mack, D., Brown, C., Filice, G., Spitalny, G., 1989. Immune responses associated with early survival after peroral infection with Toxoplasma gondii. J. Immunol. 142, 3247
3255. Mevelec, M.N., Bout, D., Desolme, B., Marchand, H., Magne, R., Bruneel, O., et al., 2005. Evaluation of protective effect of DNA vaccination with genes encoding antigens GRA4 and SAG1 associated with GM-CSF plasmid, against acute, chronical and congenital toxoplasmosis in mice. Vaccine 23, 4489
4499. Miedouge, M., Bessieres, M.H., Cassaing, S., Swierczynski, B., Seguela, J.P., 1997. Parasitemia and parasitic loads in acute infection and after anti-gamma-interferon treatment in a toxoplasmic mouse model. Parasitol. Res. 83, 339
344. Mitchell, S.M., Zajac, A.M., Kennedy, T., Davis, W., Dubey, J.P., Lindsay, D.S., 2006. Prevention of recrudescent toxoplasmic encephalitis using ponazuril in an immunodeficient mouse model. J. Eucaryot. Microbiol. 53, S164
S165. Mokua Mose, J., Muchina Kamau, D., Kagira, J.M., Maina, N., Ngotho, M., Njuguna, A., et al., 2017. Development

361

of neurological mouse model for toxoplasmosis using Toxoplasma gondii isolated from chicken in Kenya. Patholog. Res. Int. 2017, 4302459. Montoya, J.G., Kovacs, J.A., Remington, J.S., 2004. Toxoplasma gondii. In: Mandell, G.L., Bennett, J.E., Dolin, R. (Eds.), Principles and Practice of Infectious Diseases, vol. 2. Churchill Livingston, Philadelphia, PA; London, pp. 3170
3198. Moshkani, S.K., Dalimi, A., 2000. Evaluation of the efficacy of atovaquone alone or in combination with azithromycin against acute murine toxoplasmosis. Vet. Res. Commun. 24, 169
177. Mui, E.J., Jacobus, D., Milhous, W.K., Schiehser, G., Hsu, H., Roberts, C.W., et al., 2005. Triazine inhibits Toxoplasma gondii tachyzoites in vitro and in vivo. Antimicrob. Agents Chemother. 49, 3463
3467. Mu¨ller, J., Aguado-Martı´nez, A., Ortega-Mora, L.M., Moreno-Gonzalo, J., Ferre, I., Hulverson, M.A., et al., 2017. Development of a murine vertical transmission model for Toxoplasma gondii oocyst infection and studies on the efficacy of bumped kinase inhibitor (BKI)-1294 and the naphthoquinone buparvaquone against congenital toxoplasmosis. J. Antimicrob. Chemother 72, 2334
2341. Newman, P.E., Ghosheh, R., Tabbara, K.F., O’Connor, G.R., Stern, W., 1982. The role of hypersensitivity reactions to Toxoplasma antigens in experimental ocular toxoplasmosis in nonhuman primates. Am. J. Ophthalmol. 94, 159
164. Nguyen, B.T., Stadtsbaeder, S., 1985. Comparative effects of cotrimoxazole (trimethoprim-sulphamethoxazole) and spiramycin in pregnant mice infected with Toxoplasma gondii (Beverley strain). Br. J. Pharmacol 85, 713
716. Nicoll, S., Wright, S., Maley, S.W., Burns, S., Buxton, D., 1997. A mouse model of recrudescence of Toxoplasma gondii infection. J. Med. Microbiol. 46, 263
266. Norose, K., Mun, H.S., Aosai, F., Chen, M., Piao, L.X., Kobayashi, M., et al., 2003. IFN-gamma-regulated Toxoplasma gondii distribution and load in the murine eye. Invest. Ophthalmol. Vis. Sci. 44, 4375
4381. Norose, K., Aosai, F., Mizota, A., Yamamoto, S., Mun, H.S., Yano, A., 2005. Deterioration of visual function as examined by electroretinograms in Toxoplasma gondii-infected IFN-gamma-knockout mice. Invest. Ophthalmol. Vis. Sci. 46, 317
321. Norose, K., Aosai, F., Mun, H.S., Yano, A., 2006. Effects of sulfamethoxazole on murine ocular toxoplasmosis in interferon-gamma knockout mice. Invest. Ophthalmol. Vis. Sci. 47, 265
271. Nozik, R.A., O’Connor, G.R., 1968. Experimental toxoplasmic retinochoroiditis. Arch. Ophthalmol. 79, 485
489.

Toxoplasma Gondii

362

7. Toxoplasma animal models and therapeutics

O’Connor, G.R., 1984. Experimental models of toxoplasmosis. In: Tabbara, K.F., Cello, R.M. (Eds.), Animal Models of Ocular Disease. William Thomas Publishing, Springfield, MA, pp. 97
104. Oliveira, C.B., Meurer, Y.S., Medeiros, T.L., Pohlit, A.M., Silva, M.V., Mineo, T.W., et al., 2016. Anti-Toxoplasma activity of estragole and thymol in murine models of congenital and non-congenital toxoplasmosis. J. Parasitol. 102, 369
376. Olle, P., Bessieres, M.H., Cassaing, S., Esteve, T., Cazabonne, P., Seguela, J.P., 1994. Experimental murine toxoplasmic retinochoroiditis. J. Eukaryot. Microbiol. 41, 16S. Olle, P., Bessieres, M.H., Malecaze, F., Seguela, J.P., 1996. The evolution of ocular toxoplasmosis in anti-interferon gamma treated mice. Curr. Eye Res. 15, 701
707. Olliaro, P., Gorini, G., Jabes, D., Regazzetti, A., Rossi, R., Marchetti, A., et al., 1994. In vitro and in vivo activity of rifabutin against Toxoplasma gondii. J. Antimicrob. Chemother. 34, 649
657. Oz, H.S., Tobin, T., 2012. Atovaquone ameliorate gastrointestinal toxoplasmosis complications in a pregnancy model. Med. Sci. Monit. 18, BR337
BR345. Oz, H.S., Tobin, T., 2014. Diclazuril protects against maternal gastrointestinal syndrome and congenital toxoplasmosis. Int. J. Clin. Med. 5, 93
101. Pavesio, C.E., Chiappino, M.L., Gormley, P., Setzer, P.Y., Nichols, B.A., 1995. Acquired retinochoroiditis in hamsters inoculated with ME 49 strain Toxoplasma. Invest. Ophthalmol. Vis. Sci. 36, 2166
2175. Perea, E.J., Daza, R.M., 1976. The effect of minocycline, doxycycline and oxytetracycline on experimental mouse toxoplasmosis. Bull. Soc. Pathol. Exot. Filiales 69, 367
372. Pereira Mde, F., Silva, D.A., Ferro, E.A., Mineo, J.R., 1999. Acquired and congenital ocular toxoplasmosis experimentally induced in Calomys callosus (Rodentia, Cricetidae). Mem. Inst. Oswaldo Cruz. 94, 103
114. Petersen, E., Kijlstra, A., Stanford, M., 2012. Epidemiology of ocular toxoplasmosis. Ocul. Immunol. Inflamm. 20, 68
75. Pezerico, S.B., Langoni, H., Da Silva, A.V., Da Silva, R.C., 2009. Evaluation of Toxoplasma gondii placental transmission in BALB/c mice model. Exp. Parasitol. 123, 168
172. Pomeroy, C., Kline, S., Jordan, M.C., Filice, G.A., 1989. Reactivation of Toxoplasma gondii by cytomegalovirus disease in mice: antimicrobial activities of macrophages. J. Infect. Dis. 160, 305
311. Que, X., Wunderlich, A., Joiner, K.A., Reed, S.L., 2004. Toxopain-1 is critical for infection in a novel chicken embryo model of congenital toxoplasmosis. Infect. Immun. 72, 2915
2921.

Ramsey, E.M., Harris, J.W.S., 1966. Comparison of Uteroplacental Vasculature and Circulation in the Rhesus Monkey and Man, vol. 38. Contributions to Embryology/ Carnegie Institution of Washington, pp. 59
70. Remington, J.S., Jacobs, L., Kaufman, H.E., 1958. Studies on chronic toxoplasmosis; the relation of infective dose to residual infection and to the possibility of congenital transmission. Am. J. Ophthalmol. 46, 261
267. discussion 268. Remington, J.S., Jacobs, L., Melton, M.L., 1961. Congenital transmission of toxoplasmosis from mother animals with acute and chronic infections. J. Infect. Dis. 108, 163
173. Roberts, C.W., Alexander, J., 1992. Studies on a murine model of congenital toxoplasmosis: vertical disease transmission only occurs in BALB/c mice infected for the first time during pregnancy. Parasitology 104 (Pt 1), 19
23. Roberts, C.W., Brewer, J.M., Alexander, J., 1994. Congenital toxoplasmosis in the Balb/c mouse: prevention of vertical disease transmission and foetal death by vaccination. Vaccine 12, 1389
1394. Rodriguez-Diaz, J.C., Martinez-Grueiro, M.M., MartinezFernandez, A.R., 1993. Comparative activity of several antibiotics against Toxoplasma gondii in a mouse model. Enferm. Infecc. Microbiol. Clin. 11, 543
546. Rollins, D.F., Tabbara, K.F., Ghosheh, R., Nozik, R.A., 1982. Minocycline in experimental ocular toxoplasmosis in the rabbit. Am. J. Ophthalmol. 93, 361
365. Romand, S., Pudney, M., Derouin, F., 1993. In vitro and in vivo activities of the hydroxynaphthoquinone atovaquone alone or combined with pyrimethamine, sulfadiazine, clarithromycin, or minocycline against Toxoplasma gondii. Antimicrob. Agents Chemother. 37, 2371
2378. Romand, S., Bryskier, A., Moutot, M., Derouin, F., 1995. In vitro and in vivo activities of roxithromycin in combination with pyrimethamine or sulphadiazine against Toxoplasma gondii. J. Antimicrob. Chemother. 35, 821
832. Romand, S., Della Bruna, C., Farinotti, R., Derouin, F., 1996. In vitro and in vivo effects of rifabutin alone or combined with atovaquone against Toxoplasma gondii. Antimicrob. Agents Chemother. 40, 2015
2020. Rosowski, E.E., Lu, D., Julien, L., Rodda, L., Gaiser, R.A., Jensen, K.D.C., et al., 2011. Strain-specific activation of the NF-kB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208, 195
212. Sabin, A.B., 1941. Toxoplasmic encephalitis in children. JAMA 116, 801
807. Saeij, J.P.J., Boyle, J.P., Grigg, M., Arrizabalaga, G., Boothroyd, J.C., 2005. Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals

Toxoplasma Gondii

References

dramatic differences between strains. Infect. Immun. 73, 695
702. Saeij, J.P.J., Boyle, J.P., Coller, S., Taylor, S., Sibley, L.D., Brooke-Powell, E.T., et al., 2006. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314, 1780
1783. Samuel, B.U., Hearn, B., Mack, D., Wender, P., Rothbard, J., Kirisits, M.J., et al., 2003. Delivery of antimicrobials into parasites. Proc. Natl. Acad. Sci. U.S.A. 100, 14281
14286. Sarciron, M.E., Lawton, P., Saccharin, C., Petavy, A.F., Peyron, F., 1997. Effects of 20 ,30 -dideoxyinosine on Toxoplasma gondii cysts in mice. Antimicrob. Agents Chemother. 41, 1531
1536. Scharton-Kersten, T., Contursi, C., Masumi, A., Sher, A., Ozato, K., 1997. Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction. J. Exp. Med. 186, 1523
1534. Schlu¨ter, D., Kwok, L.Y., Lu¨tjen, S., Soltek, S., Hoffmann, S., Ko¨rner, H., et al., 2003. Both lymphotoxin-alpha and TNF are crucial for control of Toxoplasma gondii in the central nervous system. J. Immunol. 170, 6172
6182. Scho¨ler, N., Krause, K., Kayser, O., Muller, R.H., Borner, K., Hahn, H., et al., 2001. Atovaquone nanosuspensions show excellent therapeutic effect in a new murine model of reactivated toxoplasmosis. Antimicrob. Agents Chemother. 45, 1771
1779. Schoondermark-van de Ven, E., Melchers, W., Galama, J., Camps, W., Eskes, T., Meuwissen, J., 1993. Congenital toxoplasmosis: an experimental study in rhesus monkeys for transmission and prenatal diagnosis. Exp. Parasitol. 77, 200
211. Schoondermark-van de Ven, E., Galama, J., Camps, W., Vree, T., Russel, F., Meuwissen, J., et al., 1994a. Pharmacokinetics of spiramycin in the rhesus monkey: transplacental passage and distribution in tissue in the foetus. Antimicrob. Agents Chemother. 38, 1922
1929. Schoondermark-van de Ven, E., Melchers, W., Camps, W., Eskes, T., Meuwissen, J., Galama, J., 1994b. Effectiveness of spiramycin for treatment of congenital Toxoplasma gondii infection in rhesus monkeys. Antimicrob. Agents Chemother. 38, 1930
1936. Schoondermark-van de Ven, E., Galama, J., Vree, T., Camps, W., Baars, I., Eskes, T., et al., 1995. Study of treatment of congenital Toxoplasma gondii infection in rhesus monkeys with pyrimethamine and sulfadiazine. Antimicrob. Agents Chemother. 39, 137
144. Schultz, W., Bauer, H., 1952. Experimentelle Toxoplasmose. Klin. Wochenschr. 30, 850
851. Schultz, T.L., Hencken, C.P., Woodard, L.E., Posner, G.H., Yolken, R.H., Jones-Brando, L., et al., 2014. A thiazole derivative of artemisinin moderately reduces

363

Toxoplasma gondii cyst burden in infected mice. J. Parasitol. 100, 516
521. Sergent, V., Cautain, B., Khalife, J., Deslee, D., Bastien, P., Dao, A., et al., 2005. Innate refractoriness of the Lewis rat to toxoplasmosis is a dominant trait that is intrinsic to bone marrow-derived cells. Infect. Immun. 73, 6990
6997. Shen, D.F., Matteson, D.M., Tuaillon, N., Suedekum, B.K., Buggage, R.R., Chan, C.C., 2001. Involvement of apoptosis and interferon-gamma in murine toxoplasmosis. Invest. Ophthalmol. Vis. Sci. 42, 2031
2036. Shubar, H.M., Mayer, J.P., Hopfenmu¨ller, W., Liesenfeld, O., 2008. A new combined flow-cytometry-based assay reveals excellent activity against Toxoplasma gondii and low toxicity of new bisphosphonates in vitro and in vivo. J. Antimicrob. Chemother. 61, 1110
1119. Shubar, H.M., Dunay, I.R., Lachenmaier, S., Dathe, M., Bushrab, F.N., Mauludin, R., et al., 2009. The role of apolipoprotein E in uptake of atovaquone into the brain in murine acute and reactivated toxoplasmosis. J. Drug Target 17, 257
267. Shubar, H.M., Lachenmaier, S., Heimesaat, M.M., Lohmann, U., Maudulin, R., Mu¨ller, R.H., et al., 2011. SDS-coated atovaquone nanosuspensions show improved therapeutic efficacy against experimental acquired and reactivated toxoplasmosis by improving passage of gastrointestinal and blood-brain barriers. J. Drug Target. 19, 114
124. Sibley, L.D., Ajioka, J.W., 2008. Population structure of Toxoplasma gondii: clonal expansion driven by infrequent recombination and selective sweeps. Ann. Rev. Microbiol. 62, 329
351. Sibley, L.D., Boothroyd, J.C., 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 359, 82
85. Sordet, F., Aumjaud, Y., Fessi, H., Derouin, F., 1998. Assessment of the activity of atovaquone-loaded nanocapsules in the treatment of acute and chronic murine toxoplasmosis. Parasite 5, 223
229. Stahl, W., Matsubayashi, H., Akao, S., 1966. Modification of subclinical toxoplasmosis in mice by cortisone, 6mercaptopurine and splenectomy. Am. J. Trop. Med. Hyg 15, 869
874. Sumyuen, M.H., Garin, Y.J., Derouin, F., 1996. Effect of immunosuppressive drug regimens on acute and chronic murine toxoplasmosis. Parasitol. Res. 82, 681
686. Suzuki, Y., Joh, K., Orellana, M.A., Conley, F.K., Remington, J.S., 1991. A gene(s) within the H-2D region determines the development of toxoplasmic encephalitis in mice. Immunology 74, 732
739. Suzuki, Y., Joh, K., Kwon, O.C., Yang, Q., Conley, F.K., Remington, J.S., 1994. MHC class I gene(s) in the D/L region but not the TNF-alpha gene determines

Toxoplasma Gondii

364

7. Toxoplasma animal models and therapeutics

development of toxoplasmic encephalitis in mice. J. Immunol. 153, 4649
4654. Suzuki, Y., Yang, Q., Remington, J.S., 1995. Genetic resistance against acute toxoplasmosis depends on the strain of Toxoplasma gondii. J. Parasitol. 81, 1032
1034. Suzuki, Y., Kang, H., Parmley, S., Lim, S., Park, D., 2000. Induction of tumor necrosis factor-alpha and inducible nitric oxide synthase fails to prevent toxoplasmic encephalitis in the absence of interferon-gamma in genetically resistant BALB/c mice. Microbes Infect. 2, 455
462. Tabbara, K.F., Nozik, R.A., O’Connor, G.R., 1974. Clindamycin effects on experimental ocular toxoplasmosis in the rabbit. Arch. Ophthalmol. 92, 244
247. Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., et al., 2006. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314, 1776
1780. Tedesco, R.C., Smith, R.L., Corte-Real, S., Calabrese, K.S., 2005. Ocular toxoplasmosis in mice: comparison of two routes of infection. Parasitology 131, 303
307. Tenter, A.M., Heckeroth, A.R., Weiss, L.M., 2000. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30, 1217
1258. Thouvenin, M., Candolfi, E., Villard, O., Klein, J.P., Kien, T., 1997. Immune response in a murine model of congenital toxoplasmosis: increased susceptibility of pregnant mice and transplacental passage of Toxoplasma gondii are type 2-dependent. Parasitologia 39, 279
283. Uhlikova, M., Hubner, J., 1973. Congenital transmission of toxoplasmosis in domestic rabbits. Folia Parasitol. (Praha) 20, 285
291. Usmanova, A.F., 1965. Apropos of the effect of chloridine and sulfadimezin on the course of pregnancy. (Experimental study). Sov. Zdravookhr. Kirg. 2, 7
10. Vargas-Villavicencio, J.A., Besne´-Me´rida, A., Correa, D., 2016a. Vertical transmission and fetal damage in animal models of congenital toxoplasmosis: a systematic review. Vet. Parasitol. 223, 195
204. Vargas-Villavicencio, J.A., Cedillo-Pela´ez, C., Rico-Torres, C.P., Besne´-Me´rida, A., Garcia-Va´zquez, F., Saldana, J.I., et al., 2016b. Mouse model of congenital infection with a non-virulent Toxoplasma gondii strain: vertical transmission, “sterile” fetal damage, or both? Exp. Parasitol. 166, 116
123. Vidadala, R.S., Rivas, K.L., Ojo, K.K., Hulverson, M.A., Zambriski, J.A., Bruzual, I., et al., 2016. Development of an orally available and central nervous system (CNS) penetrant Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) inhibitor with minimal human ether-a-go-go-related gene (hERG) activity for the treatment of toxoplasmosis. J. Med. Chem. 59, 6531
6546.

Wang, T., Liu, M., Gao, X.J., Zhao, Z.J., Chen, X.G., Lun, Z. R., 2011. Toxoplasma gondii: the effects of infection at different stages of pregnancy on the offspring of mice. Exp. Parasitol. 127, 107
112. Watanabe, H., Suzuki, Y., Makino, M., Fujiwara, M., 1993. Toxoplasma gondii: induction of toxoplasmic encephalitis in mice with chronic infection by inoculation of a murine leukemia virus inducing immunodeficiency. Exp. Parasitol. 76, 39
45. Watson, G.F., Davis, P.H., 2019. Systematic review and meta-analysis of variation in Toxoplasma gondii cyst burden in the murine model. Exp. Parasitol. 196, 55
62. Watts, E., Zhao, Y., Dhara, A., Eller, B., Patwardhan, A., Sinai, A.P., 2015. Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. mBio 6, e0115515. Webb, R.M., Tabbara, K.F., O’Connor, G.R., 1984. Retinal vasculitis in ocular toxoplasmosis in nonhuman primates. Retina 4, 182
188. Weiss, L.M., Udem, S.A., Salgo, M., Tanowitz, H.B., Wittner, M., 1991. Sensitive and specific detection of Toxoplasma DNA in an experimental murine model: use of Toxoplasma gondii-specific cDNA and the polymerase chain reaction. J. Infect. Dis. 163, 180
186. Weiss, L.M., Luft, B.J., Tanowitz, H.B., Wittner, M., 1992. Pyrimethamine concentrations in serum during treatment of acute murine experimental toxoplasmosis. Am. J. Trop. Med. Hyg 46, 288
291. Werner, H., Janitschke, K., Masihi, M., Adusu, E., 1977. The effect of Toxoplasma antibodies after reinfection with T. gondii. III. Communication: investigations on the question of placental transmission of Toxoplasma in immunised pregnant animals. Zentralbl. Bakteriol. [Orig A] 238, 128
142. Williams, D.M., Grumet, F.C., Remington, J.S., 1978. Genetic control of murine resistance to Toxoplasma gondii. Infect. Immun. 19, 416
420. Williams, R.H., Morley, E.K., Hughes, J.M., Duncanson, P., Terry, R.S., Smith, J.E., et al., 2005. High levels of congenital transmission of Toxoplasma gondii in longitudinal and cross-sectional studies on sheep farms provides evidence of vertical transmission in ovine hosts. Parasitology 130, 301
307. Wong, M.M., Kozek, W.J., Karr Jr., S.L., Brayton, M.A., Theis, J.H., Hendrickx, A.G., 1979. Experimental congenital infection of Toxoplasma gondii in Macaca arctoides. Asian J. Infect. Dis. 3, 61
67. Wright, I., 1972. Transmission of Toxoplasma gondii across the guinea-pig placenta. Lab. Anim. 6, 169
180. Yardley, V., Khan, A.A., Martin, M.B., Slifer, T.R., Araujo, F.G., Moreno, S.N.J., et al., 2002. In vivo activities of farnesyl pyrophosphate synthase inhibitors against

Toxoplasma Gondii

References

Leishmania donovani and Toxoplasma gondii. Antimicrob. Agents Chemother. 46, 929
931. Youssef, M.Y., el-Ridi, A.M., Arafa, M.S., el-Sawy, M.F., elSayed, W.M., 1985. Effect of levamisole on toxoplasmosis during pregnancy in guinea-pigs. J. Egypt. Soc. Parasitol. 15, 41
48. Zenner, L., Darcy, F., Cesbron-Delauw, M.F., Capron, A., 1993. Rat model of congenital toxoplasmosis: rate of transmission of three Toxoplasma gondii strains to foetuses and protective effect of a chronic infection. Infect. Immun. 61, 360
363. Zenner, L., Darcy, F., Capron, A., Cesbron-Delauw, M.F., 1998. Toxoplasma gondii: kinetics of the dissemination in

365

the host tissues during acute phase of infection of mice and rats. Exp. Parasitol. 90, 86
94. Zenner, L., Estaquier, J., Darcy, F., Maes, P., Capron, A., Cesbron-Delauw, M.F., 1999a. Protective immunity in the rat model of congenital toxoplasmosis and the potential of excreted-secreted antigens as vaccine components. Parasite Immunol. 21, 261
272. Zenner, L., Foulet, A., Caudrelier, Y., Darcy, F., Gosselin, B., Capron, A., et al., 1999b. Infection with Toxoplasma gondii RH and Prugniaud strains in mice, rats and nude rats: kinetics of infection in blood and tissues related to pathology in acute and chronic infection. Pathol. Res. Pract. 195, 475
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8 Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake Isabelle Coppens1 and Cyrille Botte´2 1

Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, United States 2Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, Universite´ Grenoble Alpes, Grenoble, France

8.1 Introduction Lipid inventory in Toxoplasma gondii reveals the presence of neutral and polar lipids, as found in any eukaryotic organism. Lipid metabolism in the parasite is essential for the production of infectious progeny, signal transduction for proper interactions with mammalian cells, and the long-term persistence in the host. Among lipids, glycero(phospho)lipids, sterols, and sphingolipids are major building blocks of biological membranes, whose composition and homeostasis define the physical properties and functions of the compartment where they reside. Some lipids, in the form of triacylglycerols, cholesteryl esters, and other neutral lipids usually found in lipid droplets (LDs), are also important storage molecules for T. gondii and used for energetic purposes or lipotoxicity prevention. Other lipids, especially

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00008-6

fatty acids and phosphatidic acid, are important signal transducers involved in several cellular and metabolic pathways. Bioinformatic tools, for example, Kyoto Encyclopedia of Genes and Genomes framework have delineated the metabolic maps for lipid synthesis in T. gondii and have concomitantly revealed auxotrophies for several lipid species. In addition, recent lipidomic and fluxomic approaches have unveiled that some phospholipid species have a mixed composition, containing one fatty acyl chain synthesized by the parasite and the other one coming from the host cell as a result of scavenging activities. Toxoplasma has also uncommon lipid molecular species that can serve as signatures, indicating the presence of unique biosynthetic enzymes and translocators/transporters for lipid acquisition. The discovery of the apicoplast and the unique fatty acid synthetic pathways operational in this

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organelle has highlighted that the parasite synthesizes prokaryotic-like lipids that are essential for its survival. This raises the prospect that lipid homeostatic, trafficking, and remodeling pathways in the parasite may abound in potential drug targets. As a prime example, fatty acid biosynthetic pathways are being successfully exploited as antimicrobial targets against Toxoplasma infections. This chapter summarizes the different classes of lipids present in Toxoplasma, as a result of biosynthesis in the parasite and/or salvage from the host cell.

8.2 Fatty acids 8.2.1 Fatty acid biosynthetic pathways— generalities Fatty acid synthesis (FAS) is a critical anabolic pathway in most organisms. In addition to being the hydrophobic building blocks of membrane lipids, fatty acids are important energy storage molecules, and fatty acyl derivatives possess a variety of physiological functions, including posttranslational modification of numerous proteins. The fundamental process of fatty acid biosynthesis is highly conserved among species and involves major metabolites in the central carbon metabolism as substrates, including acetyl-CoA. The central feature of the pathway is the sequential extension of an alkanoic chain, two carbons at a time, by a series of decarboxylative condensation reactions. This process is generally initiated with the carboxylation of acetyl-CoA to yield malonyl-CoA (Smith et al., 2003). The malonate group of malonyl-CoA is transferred to the phosphopantetheine prosthetic group of a small, acidic protein or protein domain, called the acyl carrier protein (ACP). MalonylACP is then condensed with acetyl-CoA, reduced, dehydrated, and reduced once again yielding an acyl-ACP. The elongation of the

chain occurs by condensing another malonylACP with the acyl-ACP and repeating the reaction cycle. In nature, there are two basic types of FAS architectures based on their origin. The prototypical FASI, found in vertebrates and fungi, is an associated system consisting of a mega, single gene that encodes a multifunctional protein that contains all of the enzymatic reaction centers capable of producing a fatty acid molecule (Smith et al., 2003). In contrast, bacteria, algae, plants, organisms bearing chloroplast-like organelles including Apicomplexa, most mitochondria, and some lower eukaryotes express multiple enzymes that act as one complex from a prokaryotic origin (White et al., 2005). This dissociated system named FASII encodes each enzymatic component that catalyzes a single step in the pathway. FASI is thought to have evolved by the fusion of the prokaryotic type II complex into a single protein. The multifunctional protein of FASI is usually localized in the cytosol of mammalian cells. In plants, photosynthetic and plastidbearing organisms, FASII, is located in the plastid (chloroplast) that is derived from a cyanobacterial endosymbiont, for example, the apicoplast in Toxoplasma. The genes for these enzymes are all encoded in the nuclear genome, and the proteins are posttranslationally exported to the plastid, as it is common with plastid enzymes due to a massive lateral plastid genome transfer during evolution (McFadden, 1999). Some FASII intermediates are used in the synthesis of key cellular constituents and cofactors, such as lipoic acid that is essential for the proper function of most dehydrogenase complexes (such as pyruvate dehydrogenases, a central component of the central carbon metabolism). This enormous diversity of FASII products is possible because the acyl-ACP intermediates (activated forms of fatty acids) are diffusible entities that are trafficked and diverted into many biosynthetic pathways.

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In addition, some fatty acids can further be elongated into (very) long-chain fatty acids chains by individual membrane-bound enzymes, named elongases (ELO), usually located in the endoplasmic reticulum (ER). The synthesis of very long-chain fatty acids is a ubiquitous system is found in different organisms and cell types, and specific “elongated” fatty acids serve commonly as building blocks of sphingolipids, but they can be also constituents of glycerophospholipids, triacylglycerols (TAG), and steryl- and wax-esters (Jakobsson et al., 2006; Uttaro, 2006).

8.2.2 Fatty acid synthesis in Toxoplasma Toxoplasma has the great ability to infect virtually any warm-blooded animals and to multiply in any type of nucleated cells as their hosts. In these different environments the

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parasite must encounter different nutritional and metabolic challenges. It is no surprise that T. gondii has evolved to express a broad set of fatty acid-related genes for de novo synthesis (Mazumdar and Striepen, 2007). Bioinformatic, genetic, and biochemical studies document that T. gondii expresses three fatty acid synthetic pathways localized to different cellular compartments (Waller et al., 1998; Seeber, 2003; Ramakrishnan et al., 2012): a cytosolic FASI-like pathway; a FASII present in the apicoplast producing myristic (C14:0) and palmitic (C14:0) acids, in addition to lipoic acid; and an elaborate fatty acid elongation pathway compartmentalized in the ER and responsible for the production of very long-chain monounsaturated fatty acids (Fig. 8.1). The FASI pathway consists of a single large polypeptide that harbors the ACP, FabD, FabH, FabG, FabZ, and FabI activities.

FIGURE 8.1 Fatty acid salvage and biosynthetic pathways in Toxoplasma. The parasite harbors three fatty acid synthetic pathways: (A) Cytosolic-located FASI. (B) Apicoplast-localized FASII producing myristic and palmitic acid in addition to lipoic acid. (C) The parasite is also able to scavenge several fatty acids from the environment. (D) ER-associated elongase system that synthesizes very long-chain monounsaturated fatty acids using the activity of ELO. Major products are highlighted in red. The parasite is also able to scavenge several fatty acids from the environment. Ac, Acetate; ELO, elongases; ER, endoplasmic reticulum; hcell, host cell; Mal, malonate; PV, parasitophorous vacuole. Source: Adapted from Ramakrishnan, S., Docampo, M.D., Macrae, J.I., Pujol, F.M., Brooks, C.F., van Dooren, G.G., et al., 2012. Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 287, 4957 4971.

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In Toxoplasma the apicoplast possesses a complete prokaryotic FASII. This pathway is essential during the tachyzoite (proliferative) life stage because it provides the bulk of fatty acids used for the synthesis of major membrane lipid classes (Mazumdar et al., 2006; Ramakrishnan et al., 2012; Amiar et al., 2016; Sidik et al., 2016). T. gondii uses glucose as a major source for energetic purposes. The apicoplastic FASII is dependent on the import of host glucose and the parasite’s glycolytic pathway to generate the proper substrate for FAS, that is, acetyl-ACP (Ramakrishnan et al., 2012; Amiar et al., 2016). Glucose and acetyl-CoA do not enter the apicoplast to fuel FASII (as initially thought), but instead two triose phosphate glycolytic products, that is, dihydroxyacetone phosphate and phsophoenolpyruvate (PEP), are imported into the apicoplast by the apicoplast phosphate transporter, named TgAPT (Lim et al., 2009; Ramakrishnan et al., 2012; Amiar et al., 2016). Recent stable isotope labeling assays combined to lipidomics confirm that the apicoplast metabolizes glycolytic products (Ramakrishnan et al., 2012; Amiar et al., 2016). Imported PEP can then be converted into pyruvate and finally to acetyl-CoA by the apicoplastic pyruvate kinase, named TgPKII, and the apicoplast pyruvate dehydrogenase complex, named TgPDH (Oppenheim et al., 2014; Tymoshenko et al., 2015b; Amiar et al., 2016; Dubois et al., 2018). One of the subunits of the TgPDH complex, E2 PDH, must be lipoylated de novo in the apicoplast to be functional (Mazumdar et al., 2006). In the next major step of FASII in T. gondii, acetyl-CoA is carboxylated to form malonylCoA by an acetyl coenzyme A carboxylase (ACC) using bicarbonate as a source of the carboxyl group, biotin as a cofactor, and ATP as a source of energy (Jelenska et al., 2001). Indeed, ACC consists of three major functional domains: the biotin carboxylase domain, the carboxyltransferase domain, and the biotin carboxyl carrier domain containing covalently

attached biotin. The first step of the ACCcatalyzed reaction is an ATP-dependent transfer of the carboxyl group from bicarbonate to the biotin residue (first half-reaction). The carboxyl group is then transferred to acetyl-CoA producing malonyl-CoA (second half-reaction). Malonyl-CoA is used for de novo fatty acid biosynthesis as well as in fatty acid elongation. Incubation of radiolabeled malonyl-CoA with T. gondii extracts results in the production of C16:0. Based on recent lipidomics studies, C16:0 is likely produced through the elongase pathway (Ramakrishnan et al., 2012; Dubois et al., 2018). Intriguingly, T. gondii expresses a second ACC, most likely cytosolic although having the multidomain type as the ACC prototype found in the cytoplasm of eukaryotes and in plastids of some plants. This cytosolic ACC may generate substrates for the cytosolic FASI but experimental evidence is still missing. Subsequently to the ACC activity, acetylCoA, and malonyl-CoA are transferred to an ACP, which transfers the nascent fatty acid chain to the different enzymes of the pathway, through the actions of acetyl-CoA ACP transacylase and malonyl-CoA:ACP transacylase (FabD), respectively. β-Ketoacyl:ACP is then synthesized from acetyl-ACP and malonyl-ACP by a β-ketoacyl:ACP synthase (FabH). β-KetoacylACP is reduced by β-ketoacyl:ACP reductase (FabG) to form β-hydroxyacyl-ACP that is dehydrated by β-hydroxyacyl-ACP dehydrase (FabZ) to form α,β-trans enoyl-ACP. This is further reduced to butyryl-ACP by the action of enoylACP reductase (FabI). This cycle occurs up to 6 8 times in T. gondii (Ramakrishnan et al., 2012; Amiar et al., 2016; Dubois et al., 2018). ACP plays a central role in fatty acid biosynthesis by holding the forming acyl chain, whereas FabH and FabZ are involved in the condensation and dehydration steps, respectively, of acetyl addition during acyl chain elongation. Loss of FASII severely compromises the replication and the virulence of Toxoplasma

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(Mazumdar et al., 2006; Ramakrishnan et al., 2012; Martins-Duarte et al., 2016; Amiar et al., 2016). In particular, genetic disruption of ACP leads to defects in apicoplast biogenesis, with loss of the organelle. Stable isotope labeling using 13C-U-glucose as a precursor for FASII reveals that the pathway relies on glycolytic intermediates and not on import of acetate to generate fatty acid products (Ramakrishnan et al., 2012; Amiar et al., 2016), and that the main products of FASII are medium to long fatty acid chains, mainly C12:0 (dodecanoate), C14:0 (myristate), C16:0, and C18:0 (stearate). Comprehensive fluxomics using 13C-U-glucose and 13C-acetate in combination with mass spectrometry-based lipidomics, performed to assess whether C12:0, C14:0, C16:0, and C18:0 produced by FASII are important for parasite growth, shows that these fatty acids are used to generate the bulk of major phospholipid classes, especially phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) for membrane biosynthesis and organelle biogenesis (Amiar et al., 2016; see below for detailed mechanisms in phospholipid synthesis by FASII products). In addition to providing medium-long fatty acids, FASII is required for de novo synthesis of lipoic acid, a short fatty acid chain, C8:0. Lipoic acid is an essential cofactor for oxidative decarboxylases and is usually involved in the response to oxidative stress in eukaryotic systems. ACP-knockdown parasites are impaired in protein lipoylation by the apicoplast PDH (pyruvate dehydrogenase complex), the sole source of the metabolic precursor acetyl-CoA. In particular, the apicoplast produces lipoic acid that is required for the functional maintenance of FASII and is thus important for membrane biogenesis and progeny formation. Fundamentally different from the cytosolic type I pathway of the mammalian host, FASII in T. gondii has a tremendous potential for the development of parasite-specific inhibitors. Many components of this pathway are already

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the target for existing antibiotics and herbicides (Roberts et al., 2003; Sonda and Hehl, 2006; Goodman and McFadden, 2007; MartinsDuarte et al., 2009), although some promising molecules against FASII in Apicomplexa have been shown to have off-target effects (Botte´ et al., 2012). In vitro and in vivo tests with selected aryloxyphenoxypropionate herbicides show that the carboxyltransferase domain of the apicoplast T. gondii ACC is the binding target for this class of inhibitors (Jelenska et al., 2002). Expectedly, the cytosolic form of T. gondii ACC and human ACC are resistant to aryloxyphenoxypropionates. Triclosan is also a potent inhibitor of type II FabI (Baldock et al., 1996). This compound restricts the growth of T. gondii in vitro (McLeod et al., 2001). Triclosan blocks the incorporation of radioactive acetate into the fatty acids of Toxoplasma and specifically inhibits FASII. Morphological analyses on triclosan-treated parasites reveal that this compound affects apicoplast inheritance and parasite division by preventing cytokinesis completion, resulting in incomplete daughter cell budding (Martins-Duarte et al., 2016). Long-chain fatty acid supplementation in the medium rescues the cytokinesis and proliferation defects of FASII inhibition, which confirms that FASII is essential to generate lipid substrates. Thiolactomycin, a fungal secondary metabolite (Oishi et al., 1982) selectively inhibits type II FabH of T. gondii (Waller et al., 1998; Martins-Duarte et al., 2009). Thiolactomycin decreases rapidly the growth of this parasite. Cerulenin, a metabolite of Cephalosporium caerulens, is an inhibitor of both types I and II FabH (Waller et al., 1998; Heath et al., 2001). Cerulenin is found to act synergistically with triclosan in inhibiting FAS II in the related malaria parasite (Waller et al., 1998; Botte´ et al., 2012). Thiolactomycin and cerulenin represent potential drugs that may also affect the two FAS in T. gondii, and therefore growth. If the apicoplast represents a significant source of fatty acids, the latter products can

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further be modified in the ER of Toxoplasma (Ramakrishnan et al., 2012; Dubois et al., 2018). The existence of membrane contact sites between the apicoplast outermost membrane and the ER as shown at the ultrastructural level suggests a nonvesicular trafficking of lipid between the two organelles (Tomavo et al., 2009), although the two organelles share phosphatidylinositol phosphate vesicular-based trafficking pathways sustaining apicoplast biogenesis (Tawk et al., 2011). The synthesis of very long-chain fatty acids is primarily dependent on the fatty acid elongation system comprising three ELO, two reductases, and a dehydratase expressed by T. gondii (Ramakrishnan et al., 2012). Metabolic labeling studies with radioactive glucose show that intracellular parasites synthesize a range of long to very long-chain fatty acids (C14:0 26:1). The enzymatic steps involved in the fatty acid elongation process are similar to those in FASII, but the growing chain is held by CoA instead of ACP. Genetic ablation of each individual fatty acid elongase (ELO-A, ELO-B, and ELO-C) does not result in global growth defect despite observations of decreased amounts of very long fatty acids for most single elongase-deficient parasites. This suggests functional redundancy between these complexes and/or that other fatty acid biosynthetic or salvage mechanisms compensate for the loss of individual ELO complexes. Nevertheless, conditional knockdown of the nonredundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth (Ramakrishnan et al., 2015). 13 C-U-glucose and 13C-acetate labeling and comprehensive lipidomics analyses of these mutants show selective defect in synthesis of unsaturated long to very long-chain fatty acids and depletion of PI and PE species containing unsaturated long to very long-chain fatty acids. Supplementing the media with these fatty acids is insufficient to compensate the loss of

hydroxyacyl-CoA dehydratase and enoyl-CoA reductase. In T. gondii the initial substrate for the ER-based elongation pathway is acetyl-CoA produced by two enzymes: a mitochondrial citrate lyase, named TgACL, and a single cytosolic acetyl-CoA synthetase, named TgACS, that uses acetate as substrate (Tymoshenko et al., 2015; Sidik et al., 2016; Dubois et al., 2018). Neither single knockout of TgACL nor TgACS affects the parasite growth, but parasites in which TgACL has been silenced and TgACS ablated are not viable, suggesting that these enzymes have redundant functions for the parasite. Based on 13C-stable isotope labeling combined with mass spectrometry-based lipidomics analyses, TgACS is involved in providing acetyl-CoA for the fatty elongation pathway in the ER to generate very long fatty acids (C18:0, C18:1, C20:0, C20:1, C22:0, and C24:0). Disruption of TgACS has a minor effect on the global fatty acid composition, likely due to the metabolic flux changes induced to compensate for its loss. The precise role of TgACL in fatty acid elongation is currently unknown.

8.2.3 Fatty acid salvage by Toxoplasma In addition to producing fatty acids through three biosynthetic pathways, Toxoplasma imports selected fatty acids from the host cell (Tomavo et al., 1989; Quittnat et al., 2004; Polonais and Soldati-Favre, 2010; Charron and Sibley, 2002; Ramakrishnan et al., 2012; Hu et al., 2017; Nolan et al., 2017, 2018; Pernas et al., 2018). Exogenous fatty acids are used for incorporation into complex lipids. Intracellular T. gondii scavenges the fluorescent fatty acid analog 5-butyl-4,4-difluoro-4-bora-3a,4a-diazas-indacene-3-nonanoic acid (C4-BODIPY-C9) from the medium and delivers this lipid to the parasitophorous vacuole (PV) membrane, Golgi/ER (Charron and Sibley, 2002) and thin tubules filling the lumen forming the

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intravacuolar network (IVN) (Sibley et al., 1995). In infected cells pulsed with BODIPY-FL-C 12 (FL C12), a 12-carbon saturated fatty acid was covalently bound to the BODIPY fluorophore at its hydrophobic end (hence equivalent to a long-chain fatty acid), FL C12 is rerouted to the PV (Pernas et al., 2018). Another study showed that upon incubation of mammalian cells for 24 hours with the nonmetabolized fatty acid 4,4difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diazas-indacene (BODIPY493/503) that accumulates in LDs, followed by infection with Toxoplasma, the signal for BODIPY493/503 is detected within parasite LD, indicating that Toxoplasma is able to retrieve fatty acids from host LD for storage (Nolan et al., 2017). This selective compartmentalization of diverted lipids reflects sorting activities mediated by the parasite to adeptly distribute exogenous lipids into proper organelles. Intracellular and extracellular T. gondii are competent to accrue various free fatty acids from their environment, including C16:0, oleic (C18:1), stearic (C18:0), linoleic (C18:1), arachidonic (C20:4) acids, with a preferential internalization of C16:0 (Quittnat et al., 2004), and also the polyunsaturated fatty acids C18:2, C20:4, and C20:5 that the parasite is unable to synthesize (Amiar et al., 2016). Within the parasite, exogenous fatty acids are manufactured into TAG (Quittnat et al., 2004), cholesteryl esters (Nishikawa et al., 2005; Lige et al., 2013), and phospholipids (Amiar et al., 2016). Unsaturated fatty acids [C18:1, palmitoleate (C16:1), linoleate (C18:2)] added at physiological concentrations accumulate in large LD in Toxoplasma and impair parasite replication, whereas saturated fatty acids (C16:0, C18:0) neither stimulate LD formation nor impact growth (Nolan et al., 2018). Examination of parasite growth defects with 0.4 mM oleate shows massive lipid deposits outside LD, indicating enzymatic inadequacies for storing neutral lipids in LD in response to the copious salvage of oleate. Toxoplasma exposure to 0.5 mM oleate leads to irreversible

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growth arrest and lipid-induced damage, confirming a major disconnect between fatty acid uptake and the parasite’s cellular lipid requirements. However, exogenous fatty acids are required to generate parasite membrane phospholipids and sustain survival (Amiar et al., 2016). Therefore the parasite needs to maintain a balance in fatty acid fluxes between the two sources, salvage, and synthesis. Exogenous fatty acids can also be acquired from scavenged phospholipids that are then recycled by the parasite (Charron and Sibley 2002; Amiar et al., 2016). Extracellular parasites incorporate C16:0 and C14:0 into GPI anchors, such as that of SAG1 (Tomavo et al., 1989). Based on a chemical proteomic approach, a comprehensive analysis of palmitoylated proteins in T. gondii, identifies 282 cytosolic, membrane-associated or transmembrane proteins, involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host-cell invasion (apical membrane antigen 1, AMA1) (Foe et al., 2015). Treating tachyzoites with the palmitoyl acyltransferase (PAT) inhibitor, 2-bromopalmitate, inhibits motility and host-cell invasion and disrupts parasite morphology (Alonso et al., 2012). A rhoptry-localized PAT, named TgDHHC7, has been characterized and functions to properly affix the rhoptries at the apical end of the parasite (Beck et al., 2013). Conditional disruption of TgDHHC7 results in defects in rhoptry localization and function. This indicates that palmitoylation is ubiquitous throughout the T. gondii proteome and that palmitate is a critical fatty acid for the parasite’s survival. After the uptake of butyric acid (C4:0) by the parasite, this short fatty acid chain is anabolized to generate PC (Charron and Sibley, 2002). Similarly, most phospholipid classes (PC, PI, and PE) require fatty acids both scavenged and produced through FASII (Amiar et al., 2016). Interestingly, the reduced

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growth of T. gondii consecutive to ablation of type II fatty acid synthase activities is partially restored by addition of long-chain fatty acids in the medium (Ramakrishnan et al., 2012), confirming that both salvaged and de novo synthesized fatty acids have compensatory activities, although neither sources can be fully disrupted. Conserved among bacteria, archaea, and eukaryotes, the sterol-carrier protein-2 (SCP-2) is a nonspecific lipid transfer protein for phospholipids, fatty acids, and fatty acyl-CoA, (Gallegos et al., 2001). A D-bifunctional protein consisting of one N-terminal D-3-hydroxyacylCoA dehydrogenase domain fused to two tandem SCP-2 domains, TgHAD-2SCP-2, has been identified in Toxoplasma and promotes the circulation of cholesterol, phospholipids, and fatty acids between parasite organelles and the plasma membrane (Lige et al., 2009). The parasite expresses TgACBP1, a cytosolic acyl-CoA binding protein believed to be a fatty acid transporter, whose disruption causes dysfunction of fatty acid uptake from host cells (Fu et al., 2018). 13C-labeling assays combined with gas chromatography-mass spectrometry (GC MS) show that double disruption of TgACBP1 and TgHAD-2SCP-2 leads to reduced synthesis rates of C18:0, C22:1, and C24:1. Another acyl-CoA transporter, TgACBP2, localized to the parasite mitochondrion may provide acyl chains for phosphatidic acid and phosphatidylglycerol (PG) and also be involved in the synthesis of cardiolipins (phospholipid species with a diphosphatidylglycerol (DPG) structure combined with four acyl chains, which is the typical signature lipid class of mammalian mitochondria), based on reduction in the amounts of PA, PG, and cardiolipins in TgACBP2-deficient Toxoplasma (Fu et al., 2019). These mutants from cystogenic type II strains have attenuated virulence in mice. Type I but not type II strains of Toxoplasma recruit host mitochondria at their PV by secreting a mitochondrial association factor-1 (MAF-1) localized at the PV membrane. The

role of host mitochondria-PV association is still unknown but when TgACBP2-deficient type II parasites are transfected with MAF-1, they partially regain virulence, suggesting a metabolic network and lipid exchange between host mitochondria and the PV. Obviously, the diversity and redundancy of the fatty acid pathways might be taken as an indication that the availability of the correct fatty acids is an essential determinant for successful adaptation of the parasite to various host cells. If fatty acid uptake is as essential as FAS for the parasite growth and pathogenesis, fatty acid homeostatic pathways hold promise for much more Toxoplasma-specific drug targets.

8.2.4 Fatty acid fluxes in Toxoplasma T. gondii synthesizes some phospholipids, made on a “patchwork” of fatty acids that are synthesized by FASII and/or salvaged from the host (Amiar et al., 2016). Mechanisms allowing the parasite to strike a balance between the two different sources of fatty acids remain largely unknown. The advent of 13C-U-glucose based stable isotope labeling, allowing the determination of apicoplast-derived (labeled) versus scavenged host fatty acids, has surpassed major lacunae. MS/MS analysis reveals that some major phospholipid species in the parasite contain one 13C-labeled fatty acid moiety and one that is unlabeled, thus originating from the host environment, raising the issue how the parasite could achieve/generate such patchwork lipids (Amiar et al., 2016). In the apicoplast, TgATS1 generates lysophosphatidic acid (LPA) using fatty acids synthesized by FASII, which in turn fuels bulk phospholipid biosynthesis reactions. Disruption of ATS1 is lethal to Toxoplasma, resulting in significant decrease of C14:0 into bulk synthesis of major phospholipid classes (Amiar et al., 2016). This implicates the

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importance of apicoplast-derived fatty acid flux within the parasite, which cannot always be bypassed by fatty acid salvage. The apicoplast possesses a second enzyme for PA synthesis, named ATS2, which construes the fatty acid scavenging capacities of the parasite. Disruption of TgATS2 results in an increase of polyunsaturated fatty acids that are readily scavenged, such as C20:4, eicosapentaenoic acid (C20:5), and docosadienoic acid (C22:6) along with C18:0 as a compensatory mechanism for the loss of FASII-derived fatty acids (mostly C14:0). TgATS2-deficient parasites import significantly more exogenous lipids, such as PA and PC, which compensates for the inability of the parasite to synthesize these lipids adequately (Katris, Botte, and Dass, unpublished). Fatty acids can also be enzymatically recycled for membrane homeostasis. Phospholipases participate in lipid turnover by generating lysophospholipids and free fatty acid moieties for recycling/reshuffling to various membranous compartments within the parasite. Toxoplasma has an apicoplastic patatin-like phospholipase, named TgPL2, which is involved in recycling of fatty acids by generating lysophosphatidylcholine from PC (Le´veˆque et al., 2017). Phospholipases (host and parasite derived) are also involved in recruiting fatty acids and/or lipids from host LD that localize near and within the PV (Nolan et al., 2017). Another group of enzymes facilitating the remodeling of host versus apicoplast-derived lipids are fatty acid transporters and acyl-CoA synthetases. 13C-U-labeling assays combined with GC MS show that double disruption of the acyl-CoA transporter TgACBP1 and of TgSCP2 reduces the abundance of major scavenged fatty acids C18:0, C22:1, and nervonic acid (C24:1) (Fu et al., 2018). Understanding this complex, yet essential, fatty acid metabolism will help in the identification of key weak points in the parasite “biological armor” for developing novel therapeutics.

8.3 Glycerophospholipids 8.3.1 Phospholipid biosynthetic pathways—generalities Glycerophospholipids, known as phospholipids, are key molecules that contribute to the structural definition of cells and that participate in the regulation of many cellular processes. Phospholipid metabolism is a major activity that engages cells throughout their growth (Carman and Zeimetz, 1996). These amphiphilic lipids insert in cell membranes and form into a sheet two molecules thick with the fat-soluble portions inside, shielded on both sides by the water-soluble portions. This stable structure provides the cell membrane with its integrity. In mammalian cells the most abundant glycerophospholipids are PC, PE, PI, phosphatidylserine (PS), PG, and cardiolipin. All phospholipids are synthesized de novo from the unique and central precursor PA and its two direct downstream products, diacylglycerol (DAG) and CDP-DAG. PA is synthesized de novo by the sequential esterification of acyl-CoA/ACP onto a glycerol-3-phosphate backbone by acylglycerol-3-phosphate acyltransferase (AGPAT/ATS1), forming LPA and an acylglycerol-3-phosphate acyltransferase (AGPAT/ATS2) forming PA from LPA. PS, PI, PG, and cardiolipins are synthesized from the so-called cytidine diphosphate-DAG (CDPDAG) pathway, while PC and PE are synthesized from DAG by the Kennedy pathway where polar heads precursors (CDP-choline and CDP-ethanolamine) are transferred onto a DAG, made from PA by a phosphatidic acid phosphatase (PAP).

8.3.2 Phospholipid composition and physiological relevance in Toxoplasma Quantification of the phospholipid profiles of Toxoplasma reveals that PC is the most prevalent lipid, accounting for about 75% of total

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phospholipids (Gupta et al., 2005). The next most abundant lipids are PE (10%), PI (7.5%), PS (6%), and PA (1%). Compared to mammalian cells, T. gondii has higher levels of PC but lower levels of sphingomyelin and PS (Welti et al., 2007). However, a recent highperformance liquid chromatography analysis reveals the presence of a novel lipid eluting next to PS and corresponding to phosphatidylthreonine (PThr) (Arroyo-Olarte et al., 2015). In contrast to mammalian in which PThr is a trace component, this lipid is significantly more abundant in parasite membranes, and considerably more preponderant than PS. The role of PThr in T. gondii is still unknown, but its presence may be due to high amounts of threonine scavenged from the host, directly used by a PS synthase 2 named TgPSS2. In other systems, PSS2 enzymes usually catalyze PS synthesis from serine but TgPSS2 in the parasite may have more affinity for threonine than for PS. Being one of the most abundant anionic lipids, PThr may regulate Ca21 homeostatic events. Calcium signaling is central for Toxoplasma infection as it governs motility, egression, and invasion (Lourido and Moreno, 2015), potentially making PThr an important contributor to these events. Compared to eukaryotic cells, T. gondii has a wider range of polar lipids with unique lipid composition and unusual abundance, such as a relatively high level of ceramide phosphoethanolamine (B2% of the total polar lipids), which has a fatty acid profile solely constituted of 16 and 18 carbon species (Welti et al., 2007). The parasite has also a greater amount of short to medium chain fatty acid incorporated into polar lipids. For example, PC containing two saturated acyl chains with 12, 14, or 16 carbons makes up over 11% of PC but less than 3% of the mammalian PC molecular species. This specificity is directly linked to the metabolic capacities of the apicoplastic FASII, in which C12:0, C14:0, and C16:0 are the major products. Furthermore, fluxomics reveals patchwork

lipid classes where most phospholipid classes (especially PC, PE, and PI) are composed of one fatty acid coming from the FASII and one scavenged from the host. PA plays central in Toxoplasma cell cycle and infectivity, as it controls the secretion of micronemal proteins involved in parasite invasion (Bullen et al., 2016). In addition, PA is found in the PV lumen where it triggers the naturel egress of the parasite when the host cell becomes a hostile environment (Bisio et al., 2019). A dynamin-related protein, named TgDrpC participates in the parasite’s endodyogeny, which in turn relies on the local PA and its precursor LPA to generating appropriate membrane curvature, thereby assisting in cytokinesis of daughter cells (Katris, Botte´, unpublished). The distinctive T. gondii phospholipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host’s immune system. Some PS molecules are secreted by Toxoplasma in the PV (Gupta et al., 2012). Present at outer leaflet of the plasma membrane of eukaryotic cells, PS is a major ligand involved in the uptake of apoptotic cells (Fadok et al., 2001). Phagocytosis of apoptotic cells by macrophages induces a noninflammatory response based on the exposure of PS that leads to TGFβ1 secretion (Fadok et al., 1998). PS exposure on the cell surface has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. T. gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-β1 by infected activated macrophages that leads to the degradation of inducible nitric oxide synthase and inhibition of nitric oxide production, and consequently parasite persistence in macrophages (Santos et al., 2011). A PS-negative subpopulation of Toxoplasma enters macrophages by phagocytosis and is unable to inhibit nitric oxide synthesis, whereas a PS-positive subpopulation invade macrophages by active penetration, and

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no sign of inflammation is detected in mouse. This indicates that the escape mechanism of T. gondii is dependent on the exposure of PS, making this lipid essential for a successful infection and survival. Interfering with the PS biosynthetic pathways would dramatically reduce the parasite burden in their hosts. Among PI species, T. gondii contains PI 3monophosphate that is involved in a vesicular trafficking process involved in the biogenesis of the apicoplast, allowing the fusion of vesicles containing nuclear-encoded apicoplast proteins with this organelle (Tawk et al., 2011). Pharmacological studies have revealed that PC is also a central phospholipid for Toxoplasma physiology. The choline analog N, N-dimethylethanolamine is taken up by intracellular parasites as efficiency as choline (Gupta et al., 2005). As a result, T. gondii growth is progressively arrested, probably due to dramatic PC depletion and/or toxic phosphatidyldimethylethanolamine amassing in parasite membranes. In mammalian cells, phosphatidyldimethylethanolamine is normally produced as a short-live intermediate in the conversion of PE to PC (Vance and Vance, 2004). Clearly, dimethylethanolamine interferes with choline uptake and metabolism to PC, resulting in selective alteration in parasite membrane morphology at concentrations nontoxic for the host cell. This indicates that the dominance of PC as a major lipid in T. gondii membranes offers great potentialities to disrupt the membrane biogenesis of the parasite.

8.3.3 Phospholipid synthesis in Toxoplasma As mentioned above, glycerophospholipid synthesis is initiated by the synthesis of the central phospholipid precursor, PA. Toxoplasma possesses four genes encoding two separate sets of the acyltransferases required for PA synthesis de novo (Amiar et al., 2016). One

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gene product is predicted to be a glycerol-3phosphate acyltransferase (GPAT) from a eukaryotic origin and another one, a prokaryotic acylglycerol-phosphate-acyltransferase (AGPAT), both located in the ER. In addition, T. gondii expresses TgATS1 in the apicoplast (see Section 8.2). The apicoplast-located FASII and TgATS1, which are primarily used to generate plastid galactolipids in plants and algae, have been repurposed to generate bulk phospholipids for membrane biogenesis in T. gondii. In mammalian systems, DAG is interconverted to PA via DAG kinases (DGK) and PAPs. In the T. gondii genome, three genes code for putative PAP: two cytosolic TgPAP1 and TgPAP2 (Bullen et al., 2016), and a TgPAP2-like located to the inner membrane complex sutures (Chen et al., 2017), but the biochemical activities of these enzymes remain to be characterized. Furthermore, three genes encode three putative DGK: TgDGK1 localized to the parasite plasma membrane, TgDGK2 to dense granules and to the PV, and TgDGK3 to micronemes (Bullen et al., 2016). Depletion of TgDGK1 impairs egress and causes parasite death. This enzyme contains an acylated pleckstrin-homology domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. TgDGK2 is responsible for the production of PA into the PV lumen (Bisio et al., 2019). PA acts as an intrinsic signal that elicits natural egress upstream of an atypical guanylate cyclase, which is composed of a P4-ATPase and two guanylate cyclase catalytic domains. The P4-ATPase domain is predicted to be responsible for sensing of vacuolar levels of PA and pH. This suggests the existence of a signaling platform that responds to an intrinsic lipid mediator and extrinsic signals to control programmed and induced egress. CDP-DAG synthesis is particularly relevant in T. gondii as the parasite has two CDP-DAG pools (Kong et al., 2017). It expresses two

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phylogenetically divergent CDS enzymes: the eukaryotic-type TgCDS1 located in the ER and the prokaryotic-type TgCDS2 residing in the apicoplast. Conditional knockdown of TgCDS1 severely attenuates parasite growth and results in severe loss of virulence in mice. The residual growth of TgCDS1 mutant is abolished by consecutive deletion of TgCDS2. Lipidomics

analyses on the TgCDS1-deficient parasites show declines in PI levels in the Golgi apparatus while TgCDS2-deficient parasites have less PG for the mitochondrion. Toxoplasma is also enzymatically equipped to synthesize de novo glycerophospholipids via the Kennedy pathway (Fig. 8.2). T. gondii incorporates choline, ethanolamine, and serine Choline

Ethanolamine

DME

CK

EK

Phosphoethanolamine

Phosphocholine

PhosphoDME

PCT

PET

CDP-choline

CDP-ethanolamine

EPT

CDP-DME

CPT PEMT

PtdEtn

PSS2

PtdCho

PtdDME

PSS1

PSD

PtdSer Serine

FIGURE 8.2 Biosynthetic pathways of three major phospholipids in human and Toxoplasma. The Homo sapiens pathways are adapted from literature and that of Toxoplasma gondii are constructed based on the reported enzyme activities and annotations in the parasite database (www.ToxoDB.org). The common pathways are shown in black; those specific to human are depicted in green. Initial precursors are shown in blue; the intermediates of lipid synthesis are in black; phospholipids are in red, and the enzymes are in brown color. DME is metabolized via the CDP-choline route and produces PE, which is not methylated to PC in T. gondii causing the disruption of membrane biogenesis. CDP, Cytidine diphosphate; CK, choline kinase (forming clusters in the cytosol in green); CPT, CDP-choline phosphotransferase (localized to the ER in green); DME, dimethylethanolamine; EK, ethanolamine kinase; EPT, CDP-ethanolamine phosphotransferase; PC, phosphatidylcholine; PCT, phosphocholine cytidylyltransferase (localized to nucleus in green); PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine methyltransferase; PET, phosphoethanolamine cytidylyltransferase; PSD, phosphatidylserine decarboxylase; PSS, phosphatidylserine synthase; SD, serine decarboxylase. Source: Adapted from Sampels, V., Hartmann, A., Dietrich, I., Coppens, I., Sheiner, L., Striepen, B., et al., 2012. Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii. J. Biol. Chem. 287, 16289 16299.

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into PC and PS, respectively. Unlike its mammalian host (Vance and Vance, 2004), Toxoplasma does not show any activities from a serine decarboxylase, a PE decarboxylase, and a PE methyltransferase and thus appears incompetent in making PC from serine and/or ethanolamine (Gupta et al., 2005; Sampels et al., 2012). Conversely, a parasite choline kinase has potential to counteract the loss of ethanolamine kinase and to sustain de novo synthesis of PE in T. gondii. The parasite also harbors a PS decarboxylase route to produce PE from PS. The parasite resilience to a perturbation of choline kinase, compositional flexibility of its membranes, and likely redundant routes of PE synthesis confer an ability for T. gondii to adjust membrane biogenesis in response to dissimilar nutrient environments in host cells. This observed metabolic plasticity allows T. gondii to fine-tune membrane biogenesis according to intracellular niche and may have contributed to its evolution as a promiscuous pathogen. The parasite produces PE in the mitochondrion and in the PV by decarboxylation of PS, and in the ER by the fusion of CDPethanolamine and DAG (Hartmann et al., 2014). PE in the mitochondrion is synthesized by the PS decarboxylase TgPSD1mt a type I class PSD that harbors a targeting peptide required for mitochondrial localization. Ablation of TgPSD1mt expression leads to growth impairment in the parasite, however, the PE content remains unchanged, suggesting the presence of compensatory mechanisms. Toxoplasma secretes a soluble form of PS decarboxylase, TgPSD1, from dense granules releasing PE in the PV lumen (Gupta et al., 2012). It remains possible that secreted TgPSD1 could reduce externalized PS on host cells, enabling the evasion of phagocytosis. The parasite expresses TgPTS (see Section 8.2) that produces PThr in the ER (Arroyo-Olarte et al., 2015). Genetic disruption of TgPTS abrogates de novo synthesis of PThr and results in a threefold

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gain in PS. However, TgPTS-deficient parasites have impaired lytic cycle and virulence in mice. DPG, a mitochondrial cardiolipin representative, and PG are produced by Toxoplasma as monitored by metabolic experiments using radioactive acetate (Bisanz et al., 2006). Mammalian lecithin: cholesterol acyltransferase (LCAT) is characterized by dual activity, PLA2, and acyltransferase (Glomset, 1968). This enzyme catalyzes the transacylation of the sn-2 fatty acid liberated from various phospholipids (e.g., PC and PE) to the 3-β-hydroxyl group on the A-ring of cholesterol, thereby forming cholesteryl esters. Toxoplasma expresses a LCAT homolog TgLCAT. Unlike other LCAT enzymes, TgLCAT is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity (Pszenny et al., 2016). TgLCAT uses PC as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT localizes to the plasma membrane on extracellular parasites, contributing to Toxoplasma egress, likely by disrupting the hostcell membrane during exit (Pszenny et al., 2016; Schultz and Carruthers, 2018). During the intracellular stage of the parasite, TgLCAT is secreted into the PV and distributes to the membranous tubules forming the IVN. The role of intravacuolar TgLCAT would be to process host vesicles and organelles trapped in the PV (Coppens et al., 2006; Romano et al., 2013) by disrupting their membranes, thus making their nutrient content available to the parasite (Romano et al., 2017). In support to this hypothesis, less intact host organelles are detected inside the PV of parasite-overexpressing TgLCAT, probably as a result of host organelle processing and degradation (Romano et al., 2017), and these overexpressors are more virulent than wild-type parasites (Pszenny et al., 2016).

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In many organisms, patatin-like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. T. gondii codes for six proteins predicted to bear a patatin-like domain. TgPL1 is secreted in the PV upon immune stresses but seems lacking lipase activities (Mordue et al., 2007; Tobin and Knoll, 2012). TgPL2 is on the apicoplast and is involved in lipid metabolism. TgPL2 contributes to the maintenance of this organelle as TgPL2-depleted parasites have degenerated apicoplast concomitant to decrease in the amounts of PC, PE, and PI consistent to reduced FAS from FASII (Le´veˆque et al., 2017). PC is a probable substrate for TgPL2, which could use it to generate lysophosphatidylcholine and liberate fatty acids available for phospholipid synthesis in the apicoplast.

8.3.4 Phospholipid salvage by Toxoplasma Quantitative data on the rates of phospholipid syntheses reveal that T. gondii has an adequate synthetic capacity to produce all of the PE species, but only 50% of PS and B5% 10% of PC as required for a parasite doubling. This indicates that T. gondii must be auxotrophic for PS and PC—or their precursors to acquire sufficient amounts of all phospholipids (Gupta et al., 2005; Charron and Sibley, 2002). Activities of phospholipid uptake by T. gondii have been exemplified by using fluorescent glycerophospholipid analogs 2-(4,4-difluoro5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diazas-indacene-3-pentanoyl)-1-hexadecanoyl-snglycero-3-phosphocholine (BODIPY-PC) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-pentanoyl)-1-hexadecanoyl-snglycero-3 phosphate (BODIPY-PA) that label parasite compartments after internalization in host cells (Charron and Sibley, 2002). BODIPY-PC is mobilized to plasma membrane and dispersed

small vesicles, while BODIPY-PA moves to the parasite compartments similar to those containing fluorescent C4-BODIPY-C9, corresponding presumably to the Golgi/ER and the PV membrane. Interestingly, diversion of BODIPY-PA loaded in host cells prior to infection shows a bright fluorescent labeling in the PV membrane. By contrast, BODIPY-PC is completely excluded from parasites that have invaded prelabeled hosts. Intracellular and extracellular T. gondii are able to take up host-derived PA, but only the parasites inside host cells further metabolize scavenged PA into PC (Charron and Sibley, 2002). T. gondii acquires the phospholipid head group precursors from its environment and use them for the synthesis of major lipids (Gupta et al., 2005). Labeled serine internalized by free parasites is metabolized into PS and PE after PS decarboxylation, as well as in minor sphingolipids. PE is the main polar lipid generated after the uptake of ethanolamine. Like serine, the metabolism of ethanolamine shows a time-dependent increase in lipid synthesis that progressively slows over a 6 hours period. No significant radioactive PC is detected in Toxoplasma membranes after incubation in the presence of either tritiated serine or ethanolamine, suggesting that the parasite has a negligible PE methyltransferase activity (Gupta et al., 2005). However, another study conducted on parasites grown in host cells fed with labeled serine or ethanolamine showed that T. gondii synthesizes PC as a major resultant end product using these precursors (Charron and Sibley, 2002). This discrepancy is probably ascribed to differences in metabolic requirements between parasites released from cells or growing inside cells. When host cells are loaded with labeled serine or ethanolamine prior to infection, no subsequent metabolization of radioactive serine or ethanolamine is observed (Charron and Sibley, 2002). This may be linked to the rapid conversion of serine and ethanolamine into PC by mammalian cells and

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8.5 Acylglycerol synthesis and storage in Toxoplasma

the inability of the parasite to scavenge PC intact from host cells, as corroborated in fluorescence studies (as stated previously). Exposure of parasites to labeled choline or methylcholine results in the uptake and metabolization of these compounds into PC (Charron and Sibley, 2002; Gupta et al., 2005). Choline preaccumulated into host cells before infection is also further metabolized into PC by the parasites, in accordance with the competence of the intravacuolar T. gondii to readily take up choline. Various forms of radioactive choline-containing lipids are only observed in intravacuolar parasites. This parallels the observation showing that the metabolism of choline is increased by about twofold in hostfree parasites incubated in an intracellular-type medium compared to parasites maintained in an extracellular-type medium. This leads to the assumption that choline metabolism and PC synthesis are stimulated in response to parasitic invasion and replication within host cells.

8.4 Acylglycerols 8.4.1 Acylglycerol biosynthetic pathways—generalities Bacteria, yeast, plants, and animals all have the ability to synthesize acylglycerols, mainly TAG and DAGs, a critical function during periods of nutritional excess and/or nutritional stress (Coleman and Lee, 2004). In higher eukaryotes, TAG are packaged in circulating lipoproteins for distribution to peripheral tissues where they can be used immediately or stored in cytosolic LD. Such energy-dense TAG stores can free organisms temporally and spatially from the need for an immediate energy supply and provide a reserve depot that can be used when local resources fail or when specific kinds of fatty acids or lipid precursors are required. TAG stores can also be partially hydrolyzed to form DAG, which performs two

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distinct roles: supporting the biosynthesis/degradation of phospholipids and regulating the protein kinase C activity that controls cell growth. In animals, TAG are thus energy stores, repositories of fatty acids and precursors for phospholipid biosynthesis, and depots of signaling molecules (van Blitterswijk and Houssa, 1999). By contrast, in bacteria and lower eukaryotes, TAG are solely synthesized during times of stress or resource depletion, and they are used primarily for phospholipid synthesis. It is not surprising that higher organisms have developed several pathways for TAG synthesis and regulation, as compared to unicellular organisms. Commonly, the formation of TAG is catalyzed by the activity of microsomal acyl-CoA:DAG acyltransferases (DGAT).

8.5 Acylglycerol synthesis and storage in Toxoplasma In T. gondii, TAG synthesis occurs via the glycerol-3-phosphate pathway and involves DGAT homolog TgDGAT1 (Quittnat et al., 2004). Fatty acids can be incorporated into Toxoplasma DAG, revealing that this latter lipid is the acyl acceptor. TgDGAT1 contains signature motifs characteristic of the DGAT1 family. TgDGAT1 is an integral membrane protein localized to the ER. When a Saccharomyces cerevisiae mutant strain lacking neutral lipid production is transformed with TgDGAT1, significant DGAT activity is reconstituted, resulting in the biogenesis of cytosolic lipid inclusions. In contrast to human DGAT1 that lacks fatty acid specificity, TgDGAT1 preferentially incorporates palmitate into TAG. TAG are stored in parasite cytosolic LD. Stored TAG may be a reservoir of fatty acids utilizable for phospholipid biosynthesis and/or exploitable as respiratory substrates in Toxoplasma although no evidence for fatty acid β-oxidation machinery exists in the parasite.

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The apicoplast lacks typical plastidsignature galactolipids as evidenced by mass spectrometry-based lipidomic analysis, but instead the parasite contains galactosyl- and digalactosyl-ceramides (Marechal et al., 2002; Welti et al., 2007, Botte´ et al., 2008). The absence of galactolipids is likely explained by the evolution of apicomplexa from autotrophic photosynthetic organisms to obligate intracellular parasites, which led to the loss of galactosyltransferase and eventually to the repurposing of apicoplast acyltransferases (Botte´ et al., 2008; Amiar et al., 2016) Toxoplasma infection increases host TAG production in infected cells (Hu et al., 2017) and the number of host LD at the beginning of infection of fibroblasts (Nolan et al., 2017) and up to 2 days in skeletal muscle cells (Gomes et al., 2014) and macrophages (Mota et al., 2014). Increase in host LD would benefit Toxoplasma for the copious lipid content of these organelles. The parasite is able to salvage lipids stored in host LD (Nolan et al., 2017). The LD are also sites of storage and synthesis of cytokines. Stimulation of host LD biogenesis may increase the levels of prostaglandin E2, contributing to the control of the synthesis of IL-12 and IFN-γ during infection and decrease in nitric oxide (NO) synthesis (Mota et al., 2014; Gomes et al., 2014). It has been proposed that increase of IL-12 and IFN-γ, which are involved in the repair and homeostasis of muscle cells after injury, might contribute to the maintenance of the chronic phase of T. gondii infection in skeleton muscle tissues. Parasiteinduced LD accumulation seems linked to the inhibition of host mTOR and JNK signaling pathways throughout the secretion of a parasite component in the host cell (Hu et al., 2017). This process requires the involvement of the parasite effector MYR1 that mediates the passage of protein throughout the PV membrane. Indeed, no change in neutral lipid storage has been observed in fibroblats infected with Myr1-lacking parasites.

Upon supplementation of 0.2 and 0.4 mM oleate in the culture medium, Toxoplasma activates TgDGAT transcription by 1.5-fold, resulting in increased acylgycerol synthesis to control the influx of oleate in the parasite (Nolan et al., 2017). However, such an increase in TgDGAT transcripts seems insufficient, as lipids also accumulate outside LD (Nolan et al., 2018). At 0.5 mM oleate, Toxoplasma is puffed with lipids and unable to response adequality to excess oleate by activating DGAT and stops replicating. TAG synthesis is essential for buffering excess fatty acids taken up by Toxoplasma and preventing lipotoxicity in Toxoplasma. TgDGAT shares 31% identity and 49% similarity with human DGAT1 (HsDGAT1) (Quittnat et al., 2004). T863 is a selective inhibitor of HsDGAT1, blocking the acyl-CoA binding site of the enzyme, resulting in a blockade of DGAT1mediated TAG formation (Cao et al., 2011). Treatment of Toxoplasma tachyzoites with T863 at subtoxic concentrations for mammalian cells results in rapid parasite growth arrest (Fig. 8.3) and impaired TAG storage (Nolan et al., 2018). T863-treated tachyzoites show abnormal ERderived membranous structures that are stockpiled in the cytoplasm, likely impeding normal endodyogeny. It seems that, in the face of TgDGAT inhibition, DAG and fatty acids may be diverted by the parasite to the synthesis of PC and PE through the Kennedy pathway. Dual addition of OA (0.5 mM) and T863 is synergically detrimental for the parasite. Toxoplasma bradyzoites also contain LD for TAG and cholesteryl ester storage (Nolan et al., 2018). T863 treatment leads to misshapen cysts and reduced LD in bradyzoites (Fig. 8.3), although sensitivity occurs at concentrations higher than those observed for tachyzoites, perhaps due to the lower growth rate and more quiescent metabolism of bradyzoites. Inhibiting TgDGAT1 enzymatic activity to interfere with energy storage in Toxoplasma may be a reasonable therapeutic intervention against toxoplasmosis, including the control of the chronic stage of infection.

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8.6 Sterols and steryl esters

FIGURE 8.3 The treatment of tachyzoites and bradyzoites of Toxoplasma with T863. Immunofluorescence images of tachyzoites (RFP-parasites) and bradyzoites (TRITC-lectin for cyst wall) showing misshapen parasites upon incubation with T863.

8.6 Sterols and steryl esters 8.6.1 Sterol lipid biosynthetic pathways—generalities Cholesterol is the major sterol molecule ubiquitously present in mammalian cells. This lipid has been selected in the long natural evolution process for its ability to maintain a delicate balance between membrane rigidity (e.g., to allow large cell volumes) and membrane fluidity (e.g., to allow membrane-embedded proteins to function properly; Bretscher and Munro, 1993). Mammalian cells obtain cholesterol both by internalization of plasma low density lipoprotein (LDL) particles or by de novo synthesis via the mevalonate pathway in

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the ER (Goldstein and Brown, 1990). The cholesterol molecule is formed from acetate units. The acetate units are joined in a series of reactions to form farnesyl pyrophosphate, a branch point for the biosynthesis of other isoprenoid compounds such as ubiquinone, dolichol, and farnesylated proteins. Hydroxy-3methylglutaryl-CoA reductase from the mevalonate pathway is the rate determining enzyme for the entire pathway from acetate to cholesterol. Deposition of excess cellular cholesterol in the form of cholesteryl esters is catalyzed by acyl-CoA:cholesterol acyltransferases (ACAT) and ER resident enzymes. Native and exogenous cholesterol has several possible fates: incorporation into membranes, efflux to extracellular acceptors, conversion into cholesteryl esters, or depending on the cell type, metabolism into bile acids, or steroid hormones. Rates of cholesterol biosynthesis, LDL internalization, and cholesterol esterification are exquisitely sensitive to cellular levels of free cholesterol. Three possible mechanisms of cholesterol movement include aqueous diffusion, vesicle-mediated transport, and soluble carriers, which may work together or separately to mobilize cholesterol within the cell (Liscum and Underwood, 1995). Evidence has accrued that biological membranes are made of a mosaic of lipids domains. Maintenance of domain structure is critical for cell function. Cholesterol plays a key role in organizing signaling lipids and proteins within these membrane domains (Anderson and Jacobson, 2002).

8.6.2 Sterol salvage and transport in Toxoplasma Toxoplasma membranes contain ß-hydroxysterols, as probed using the polyene antibiotic filipin routinely used to reveal the steady-state distribution of sterols by fluorescence microscopy (Coppens et al., 2000). A predominantly

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parasite staining is located to the plasma membrane and the rhoptries, apical secretory organelles whose content is discharged upon parasite invasion (Coppens and Joiner, 2003). T. gondii diverts LDL-derived cholesterol that has transited through host lysosomes (Coppens et al., 2000) as demonstrated after incubation of infected cells with labeled cholesterol incorporated into LDL. Cholesterol movement from lysosomes to the PV requires temperatures permissive for vesicular transport, metabolic energy, and host microtubules, but no fusion (Sehgal et al., 2005) Toxoplasma sequesters cholesterol-filled host lysosomes within invaginations of the PV membrane (Coppens et al., 2006). Cholesterol delivery into the PV requires functional host Niemann-Pick type C proteins (Sehgal et al., 2005) that mediate cholesterol egress across the endo-lysosomal membranes (Sleat et al., 2004). In addition, a host cell P-glycoprotein transporter, a membrane-bound efflux pump, is required for cholesterol transport to the PV (Bottova et al., 2009). Cholesterol incorporation into the parasite is abolished after protease treatment on parasite plasma membrane (Sehgal et al., 2005). A lipid-translocating importer of the ATP-binding cassette (ABC) transporter G subfamily (ABCG family) located to the PV and parasite plasma membrane delivers cholesterol to the parasite interior (Ehrenman et al., 2010). A D-bifunctional protein containing two sterol-carrier protein-2 domains promotes the circulation of cholesterol within the parasite, in addition to phospholipids and fatty acids (Lige et al., 2009). Other parasite ABCG transporters are involved in cholesterol and phospholipid movement from the parasite to the PV and may contribute to the expansion of PV membrane size (Ehrenman et al., 2010). The availability of host cholesterol has a direct impact on Toxoplasma development inside its PV. LDL deprivation impairs parasite growth, whereas the overabundance of these

lipoproteins stimulates parasite replication (Coppens et al., 2000; Nishikawa et al., 2005). LDL-derived cholesterol levels also play a crucial role in bradyzoite conversion as LDL starvation induces expression of bradyzoite proteins (Ihara and Nishikawa, 2014). A study based on gene data mining shows that acute infection with T. gondii is associated with a decrease in cholesterol content in the liver and brain, as a part of the host defense response to deprive the parasite of cholesterol necessary for tachyzoite proliferation and development (Milovanovi´c et al., 2017). Other studies reveal a strong link between toxoplasmosis and several lipid disorders, including hypercholesterolemia and obesity in humans (Portugal et al., 2009; Reeves et al., 2013), a correlation supported by data in mice (Portugal et al., 2004). Increased blood lipoprotein/cholesterol deposition in the arterial wall in atherosclerotic patients is a consequence of a Th1-mediated pro-inflammatory reaction. Toxoplasma induces a dysregulation in host lipid metabolism throughout its scavenging activities. In the brain a reduction in cholesterol levels is observed during acute infection, perhaps as part of the host defensive reaction to the parasite, potentially serving to decrease parasite replication through cholesterol deprivation. On the other hand, T. gondii infection elicits a systemic Th1 inflammatory response, possibly imposing a proatherogenic environment in the host. As a result, the association between atherosclerosis and toxoplasmosis in one individual could change the outcome of each disease, depending on the availability of intracellular cholesterol and the intensity of the inflammatory reaction triggered by the parasite. Studying host cholesterol manipulation by the Toxoplasma cysts could then be relevant for a clinical point of view. Delivery of sterol analogs incorporated into LDL could be an efficient strategy to substitute the indispensable cholesterol by structural-related compound with growth-

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8.6 Sterols and steryl esters

reducing activity. The sterol analogs 22,26azasterol and 24,25-(R,S)-epiminolanosterol, inhibitors of sterol-24-methyl transferase producing 24-alkyl sterols, have potent and selective antiproliferative activity against T. gondii (Dantas-Leite et al., 2004; Martins-Duarte et al., 2009). The molecular mechanism of these lipids is unclear since 24-alkyl sterols are not detected in this parasite. It is observed, however, that the rapid accumulation of these lipid analogs in diverse membranes alters maintenance, fusogenicity, and function of the parasite organelles. It is reported that selected sterol analogs (e.g., cholesteryl chloride, cholestanone, or thiocholesterol) can affect the growth of various cholesterol-auxotroph organisms (Clayton, 1964). Their antiproliferative properties should promisingly be extended to Toxoplasma.

8.6.3 Sterol storage in Toxoplasma Nile red that strongly fluoresces in the presence of steryl esters detects the presence of cytosolic LD in Toxoplasma (Sonda et al., 2001; Charron and Sibley, 2002; Quittnat et al., 2004), indicating the ability of the parasites for cholesterol storage. Of the 21 molecular species detected in Toxoplasma, cholesteryl oleate (42%) and palmitate (26%) are the main esters as for mammalian cells but the parasite also has uniquely large amounts of cholesteryl eicosanoate (7%). In addition, the parasite contains to a lesser extent, cholesteryl palmitoleate, stearate, linoleate, arachidonate, and some polyunsaturated fatty acids. Other cholesteryl ester fatty acid species represented 3.5% of the total species (Lige et al., 2011). Toxoplasma is competent to synthesize cholesteryl esters by two ACAT-related enzymes located in the ER, TgACAT1, and TgACAT2 (Nishikawa et al., 2005; Lige et al., 2013). Parasite ACATs present a broad sterol substrate affinity but preferentially use palmitate to form cholesteryl esters.

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The loss of individual ACAT can be tolerated by the parasite though slower growth compared to wild-type parasites has been observed, due to increase in toxic-free cholesterol within membrane. This suggests that the parasites can endure the resulting slight differences in their cholesteryl ester pools and that the ACAT enzymes partially complement each other. Loss of both ACAT results in synthetic lethality, indicating that cholesterol storage is an important function for T. gondii. Another source of cholesteryl esters for the parasite is provided by TgLCAT using the fatty acid chains liberated from phospholipids to esterify cholesterol (Pszenny et al., 2016). In addition, Toxoplasma is able to retrieve neutral lipids stored in LD and sequester host LD in the PV lumen, suggesting that it may have access to host cholesteryl esters, as a source of cholesterol (Nolan et al., 2017). Host LDL and fatty acids scavenged by Toxoplasma serve as ACAT activators by stimulating cholesteryl ester synthesis and LD biogenesis in the parasite. Upon supplementation in the medium with 0.2 and 0.4 mM oleate, Toxoplasma activates TgACAT2 transcription by B1.3-fold, resulting in increased cholesteryl ester synthesis to control the influx of oleate in the parasite (Nolan et al., 2017). Lipoprotein depletion causes a progressive consumption of material stored in parasite’s LD. Under the conditions of excess LDL the activity of cholesterol esterification is significantly increased, entailing that the parasites adeptly control the massive supply of cholesterol by producing the storage form of cholesterol. A Niemann-Pick, type C1-related protein in Toxoplasma controls the intracellular levels of several lipids (Lige et al., 2009). Parasites lacking the NiemannPick I-related protein accumulate LD enriched in cholesteryl esters. The replication rate of intracellular T. gondii correlates with the LDL concentration in the medium. Excess cholesterol diverted by the parasite is rapidly neutralized and stored in

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LD. Blockade of cholesteryl ester synthesis is deleterious for the parasite, leading to a rapid induction of free cholesterol crystallization in parasite membranes and rupture of the plasma membrane (Nishikawa et al., 2005). ACATdeficient parasites are particularly sensitive to ACAT inhibitors (Lige et al., 2013). The higher vulnerability of T. gondii toward ACAT inhibitors compared with mammalian cells is probably linked to the absence of cholesterol acceptors (mainly lipoproteins) in the PV, which are known to desorb excess cholesterol from membranes.

8.7 Sphingolipids 8.7.1 Sphingolipid biosynthetic pathways—generalities Glycosylphosphatidylinositols (GPI) are a class of glycolipids that are used by a wide variety of eukaryotic cells to anchor proteins, polysaccharides, and small oligosaccharides to the plasma membrane through covalent linkages (Ferguson and Williams, 1988). Comparison of the chemical structures of GPI membrane anchors of different organisms indicates that the anchors contain a remarkably conserved core glycan structure, suggesting that a common biosynthetic pathway may have been conserved throughout eukaryotic evolution. The transfer of GPI anchors to proteins occurs in the ER with concurrent displacement of a C-terminal hydrophobic peptide, followed by the rapid substitution of the peptide tail by the GPI anchor. Ceramides are the principal lipid components present in sphingomyelin, complex glycolipids, cerebrosides, and gangliosides (Sharma and Shi, 1999). Ceramides are broadly recognized as vital second messengers in the signal transduction process mediated by receptors of many cytokines and growth factors.

8.7.2 Sphingolipid synthesis in Toxoplasma Our lipidomic analysis of T. gondii revealed that the parasite contains .20 species of sphingolipids consisting of both saturated and unsaturated fatty acids (Lige et al., 2011). The parasite is capable of the de novo synthesis of sphingolipids, based on inhibitor studies (Sonda et al., 2005), as well as on metabolic labeling studies of extracellular or released intracellular parasites (Azzouz et al., 2002; Bisanz et al., 2006). Toxoplasma can readily incorporate sugars and amino acids as precursors of sphingolipids. De novo synthesis of ceramide, glycosylated ceramide, and sphingomyelin in T. gondii has been demonstrated by metabolic labeling studies using tritiated serine and galactose. After internalization, labeled galactose is metabolized in various glycosphingolipids (e.g., di- and triglycosylated ceramide; Azzouz et al., 2002). Incubation of the parasite with labeled serine leads to the production of ceramide. After uptake, labeled glucosamine serves as a GPI glycolipid precursor and is associated with the dominant surface protein, SAG1 (Striepen et al., 1997; Zinecker et al., 2001). Toxoplasma has a sphingolipid synthase homolog, named TgSLS, and recombinant TgSLS exhibits an inositol phosphorylceramide synthase activity in vitro (Pratt et al., 2013). The parasite also expresses on the ER a serine palmitoyltransferase, named TgSPT1, that catalyzes the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA (Mina et al., 2017). Unlike in eukaryotes that express a serine palmitoyltransferase forming a conserved heterodimeric enzyme complex, TgSPT1 is homodimeric and evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. TgSPT1 could have been acquired by Toxoplasma via lateral gene transfer from a prokaryote.

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8.7 Sphingolipids

GPI in T. gondii serve as membrane anchors for a large number of plasma membrane proteins (Tomavo et al., 1992; Striepen et al., 1997; Zinecker et al., 2001). Their biosynthetic pathway is initiated on the parasite ER with the transfer of N-acetylglucosamine to PI involving a PI-glycan class A (PIGA)-like protein (Wichroski and Ward, 2003). The GPI core glycan is then assembled via sequential glycosylation of PI (Tomavo et al., 1992). Toxoplasma PIGA sequence contains a potential transmembrane domain followed by a stretch of mostly hydrophilic residues extending to the Cterminus. A functional copy of PIGA is required for viability, demonstrating that GPI biosynthesis is an essential process in T. gondii. Glycosphingolipids, for example, inositol phosphorylceramide are synthesized de novo via the 3-ketosphinganine pathway from serine and palmitoyl-CoA (Azzouz et al., 2002; Sonda et al., 2005) with ceramide as an intermediate. Metabolic studies show that T. gondii readily incorporates radioactive acetate into glycosylcerebroside, lactosylcerebroside, and globotriaosylceramide, while only intracellular parasites produce globoside (Bisanz et al., 2006). GPI-anchored proteins dominate the surface of T. gondii and are implicated in both host-cell attachment and modulation of the host immune response (Lekutis et al., 2001). Although the GPI core glycan is conserved in all organisms, some differences in additional modifications to GPI structures and biosynthetic pathways have been reported for T. gondii (de Macedo et al., 2003). This indicates that the GPI biosynthetic pathway is a potential target for the development of new chemotherapeutics against this parasite. Indeed the lethal consequences of PIGA disruption in T. gondii may result from a deficiency in GPI-anchored proteins, free GPI, or both (Wichroski and Ward, 2003). In vitro and in vivo studies reveal that sugars and amino acid analogs, synthetic mannoside acceptor substrates and natural

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compounds specifically interfere with GPI biosynthesis in many different pathogenic organisms (de Macedo et al., 2003). Synthesis of parasite ceramide is dramatically decreased after incubation of intracellular Toxoplasma with either threo-phenyl-2-palmitoylamino-3morpholino-1-propanol, a specific inhibitor of glucosylceramide synthesis, or L-cycloserine that blocks the serine palmitoyltransferase activity (Azzouz et al., 2002). The antibiotic aureobasidin A, a potent inhibitor of inositol phosphorylceramide that is absent from mammalian cells, abrogates T. gondii replication by the severe reduction of total complex sphingolipids’ synthesis without noticeable host-cell alterations (Sonda et al., 2005).

8.7.3 Sphingolipid salvage by Toxoplasma Intracellular T. gondii is also able to retrieve sphingolipids intact from the culture and accumulate the scavenged lipids into the Golgi apparatus. In mammalian cells, exogenous ceramides concentrate the Golgi complex to be further metabolized into major sphingolipids and glucosylceramides. After incubation with NBD-C6-ceramide the Golgi of intravacuolar T. gondii is stained, suggesting that the parasite can intercept the ceramide pathway of the host cell to acquire exogenous ceramides or other sphingolipids manufactured in the host Golgi (de Melo and de Souza, 1996). A morphological study shows that the PV of Toxoplasma preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection (Romano et al., 2013). The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi derived vesicles within the PV. The vesicles, marked with Rab14, Rab30, or Rab43 colocalize with host-derived sphingolipids in

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8. Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake

the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite’s growth. Host Rab vesicles enter the PV via invaginations of the vacuolar membrane extending into the PV lumen (Fig. 8.4). Some invaginations are formed following the fusion of an IVN tubule with the vacuolar membrane or are generated by host microtubules poking the PV membrane (Coppens et al., 2006; Romano et al., 2017). Host vesicles use either type of invagination of the PV membrane as conduits, resulting in their sequestration into the vacuole, based on EM observations. TgLCAT secreted by dense granules (Pszenny et al., 2016), localizes to the IVN and seems involved in the degradation of host intravacuolar vesicles (Romano et al., 2017). Parasites overexpressing TgLCAT contain fewer host Rab vesicles in the PV,

suggesting that host organelles in the PV are processed to liberate and make available to the parasite their content.

8.8 Isoprenoid derivatives 8.8.1 Isoprenoid biosynthetic pathways—generalities The posttranslational modification of proteins by isoprenoid residues such as farnesyl and geranylgeranyl, is a major mechanism by which cytosolic proteins interact with cellular membranes (Swiezewskaa and Danikiewiczb 2005). Isoprenylation is also required for the proper membrane localization and the biological activity of several cellular proteins implicated in the regulation of DNA replication and cell cycling, therefore having important roles in the regulation of cell proliferation. Two coexisting isoprenoid pathways exist in organisms, the mevalonate pathway present in the cytosol of mammalian cells (as stated previously) and the recently described 1-deoxy-D-xylulose-5-phosphate pathway. This latter pathway seems to be restricted so far to bacteria, plastids in plants and apicoplast in Apicomplexa (Jomaa et al., 1999).

8.8.2 Isoprenoid synthesis in Toxoplasma

FIGURE 8.4 Proposed model of host Rab vesicle trapping inside the PV for sphingolipid acquisition. (1) Transport of sphingolipid-loaded Rab14, Rab30, or Rab43 vesicles to the PV using host microtubules (green); (2) docking via parasite proteins; (3) internalization via tubular invaginations; (4) detachment from the PV membrane and encircling by IVN tubules; (5) disruption of Rab vesicle membranes by TgLCAT and liberation of sphingolipids in the PV lumen; and (6) uptake of sphingolipids by the parasite. IVN, Intravacuolar network; PV, parasitophorous vacuole.

T. gondii membranes contains both farnesylated and geranylgeranylated proteins (Ibrahim et al., 2001; Ling et al., 2005). The parasite is able to make its own isoprenoids (Fig. 8.5). Enzymes of the 1-deoxy-D-xylulose-5-phosphate pathway seem to have an apicoplast origin and may contribute to the production of farnesyl and geranylgeranyl molecules for the parasite (Seeber, 2003). Two farnesyl-diphosphate synthase homologs, named TgFPPS, have been identified in Toxoplasma and localized to the mitochondria; this enzyme is bifunctional, catalyzing the formation of both farnesyl diphosphate and geranylgeranyl diphosphate

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FIGURE 8.5

Isoprenoid synthesis and salvage by Toxoplasma gondii. The DOXP pathway localizes to the parasite apicoplast (green), and the mevalonate pathway is only present in the mammalian host. Green arrows show metabolites imported from the host cell. TgFPPS synthesizes FPP and GGPP while the host uses two enzymes (FPPS and GGPPS) to make the same metabolites. Enzymes and their known inhibitors are indicated. Alkyl-BP, alkyl bisphosphonates; DMAPP, dimethyl allyl diphosphate; Fos, fosmidomycin; FPP, farnesyl diphosphate; G3P, glyceraldehyde 3-phosphate; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; N-BP, nitrogen bisphosphonates. Source: From Li, Z.H., Ramakrishnan, S., Striepen, B., Moreno, S.N.J., 2013. Toxoplasma gondii relies on both host and parasite isoprenoids and can be rendered sensitive to atorvastatin. PLoS Pathog. 9, e1003665.

(Ling et al., 2007). Intracellular TgFPPS-deficient parasites survive without endogenous production of farnesyl diphosphate and/or geranylgeranyl diphosphate because they are able to scavenge these lipids from the host cells (Li et al., 2017). Prenylated proteins are ubiquitously important for cell proliferation regulation. Two categories of protein farnesyltransferase inhibitors have been described so far (Qian et al., 1997): isoprene analogues and peptidomimetics based on the consensus CAAX motif that is required

for isoprenylation. Peptidomimetics act as alternative substrates in vitro, thereby competitively block protein farnesylation. Clearly, specific inhibition of T. gondii protein farnesyltransferase activity is observed using selected modified heptapeptides (Ibrahim et al., 2001). Bisphosphonates, diphosphate analogs in which a carbon atom replaces the oxygen atom bridge between the two phosphorus atoms of the diphosphate, are potent inhibitors of farnesyl-diphosphate synthase and inhibit the growth of Toxoplasma (Martin et al., 2001) by

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8. Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake

inhibiting the parasite farnesyl diphosphate/ geranylgeranyl-diphosphate synthase. Moreover, in vivo testing of bisphosphonates against T. gondii in mice has shown that one of the nitrogen-containing bisphosphonates, risedronate, significantly increases the survival of mice infected by T. gondii (Yardley et al., 2002). All these results indicate that bisphosphonates are promising candidate drugs to treat infections caused by T. gondii as well as some other protozoan parasites.

8.8.3 Isoprenoid salvage by Toxoplasma Alternatively, the parasite can salvage isoprenoids such as labeled trans, trans-farnesol and labeled trans,trans,cis geranylgeraniol, from the medium to produce its prenylated proteins (Ibrahim et al., 2001) and farnesyl diphosphate and geranylgeranyl diphosphate from the host cell (Li et al., 2013) (Fig. 8.5). This implies the presence of functional protein farnesyltransferase and geranylgeranyl transferase in T. gondii. Indeed, a protein farnesyltransferase activity has been detected in the parasite, responsible for the catalysis of isoprene lipid modifications. The survival of parasites lacking TgFPPS depends on isoprenoids salvaged from the host cells. Inhibition of the host mevalonate pathway with statin enhances the requirement for parasite isoprenoid synthesis (Li et al., 2013). Dual blockade of the parasite isoprenoid pathway with fosmidomycin/bisphosphonates and the host mevalonate pathway with atorvastatin results in parasite lethality in vitro and mice survival, emphasizing this double-hit strategy combining inhibitors as a promising therapeutic approach (Li et al., 2017).

genetic capacity to express unique and redundant lipid biosynthetic pathways, and it has developed efficient mechanisms to scavenge several host lipids or lipidic precursors (Sonda and Hehl, 2006; Goodman and McFadden, 2007; Zhang et al., 2010; Ramakrishnan et al., 2013; Coppens, 2013,2014; Amiar et al., 2016). The parasite shows amazingly diverse features in lipid metabolic pathways, with some of sharing close similarities to mammalian pathways, whereas others are more evolutionary related to bacteria and plant pathways. From a cell biological viewpoint, no doubt exists that the lipid metabolism and great plasticity of T. gondii will likely reveal many more metabolic surprises in the future. One of the most striking feature of the parasite lipid synthetic pathways is the parasite’s ability to mix fatty acids that are scavenged and de novo synthesized to generate most major phospholipid classes, forming membrane made of “patchwork lipids.” T. gondii uses the two sources of fatty acids concomitantly resulting in complex trafficking and lipid remodeling pathways. Characterization of more lipid-based target molecules and knowledge about mechanisms promoting host lipid delivery to the PV, lipid processing and trafficking pathways hold considerable potential for toxoplasmosis chemotherapy. To this end, rationally designed lipid synthesis/uptake inhibitors would represent exciting prospects for the next generation of anti-Toxoplasma agents.

Acknowledgments The authors are grateful to the members of their laboratories for helpful discussions on lipid metabolism in Toxoplasma, in particular, Sheena Dassa for critical reading of the manuscript and thoughtful comments.

References

8.9 Concluding remarks In this Section covering lipid metabolism, we illustrate that T. gondii has sophisticated lipid homeostatic pathways. It has retained the

Alonso, A.M., Coceres, V.M., De Napoli, M.G., Nieto, Guil, A.F., Angel, S.O., et al., 2012. Protein palmitoylation inhibition by 2-bromopalmitate alters gliding, host cell invasion and parasite morphology in Toxoplasma gondii. Mol. Biochem. Parasitol. 184, 39 43.

Toxoplasma Gondii

References

Amiar, S., MacRae, J.I., Callahan, D.L., Dubois, D., van Dooren, G.G., Shears, M.J., et al., 2016. Apicoplastlocalized lysophosphatidic acid precursor assembly is required for bulk phospholipid synthesis in Toxoplasma gondii and relies on an algal/plant-like glycerol 3phosphate acyltransferase. PLoS Pathog. 12, e1005765. Anderson, R.G., Jacobson, K., 2002. A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296, 1821 1825. Arroyo-Olarte, R.D., Brouwers, J.F., Kuchipudi, A., Helms, J.B., Biswas, A., Dunay, I.R., et al., 2015. Phosphatidylthreonine and lipid-mediated control of parasite virulence. PLoS Biol. 13, e1002288. Azzouz, N., Rauscher, B., Gerold, P., Cesbron-Delauw, M. F., Dubremetz, J.F., Schwarz, R.T., 2002. Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii. Int. J. Parasitol. 32, 677 6784. Baldock, C., Rafferty, J.B., Sedelnikova, S.E., Baker, P.J., Stuitje, A.R., Slabas, A.R., et al., 1996. A mechanism of drug action revealed by structural studies of enoyl reductase. Science 274, 2107 2110. Beck, J.R., Fung, C., Straub, K.W., Coppens, I., Vashisht, A. A., Wohlschlegel, J.A., et al., 2013. A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress. PLoS Pathog. 9, e1003162. Bisanz, C., Bastien, O., Grando, D., Jouhet, J., Marechal, E., Cesbron-Delauw, M.F., 2006. Toxoplasma gondii acyllipid metabolism: de novo synthesis from apicoplast generated fatty acids versus scavenging of host cell precursors. Biochem. J. 394, 197 205. Bisio, H., Lunghi, M., Brochet, M., Soldati-Favre, D., 2019. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. Nat. Microbiol. 4, 420 428. van Blitterswijk, W.J., Houssa, B., 1999. Diacylglycerol kinases in signal transduction. Chem. Phys. Lipids 98, 95 108. Botte´, C., Saı¨dani, N., Mondragon, R., Mondrago´n, M., Isaac, G., Mui, E., et al., 2008. Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii. J. Lipid Res. 49, 746 762. Botte´, C.Y., Dubar, F., McFadden, G.I., Mare´chal, E., Biot, C., 2012. Plasmodium falciparum apicoplast drugs: targets or off-targets? Chem. Rev. 112, 1269 1283. Bottova, I., Hehl, A.B., Stefani´c, S., Fabria`s, G., Casas, J., Schraner, E., et al., 2009. Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii. J. Biol. Chem. 284, 17438 17448. Bretscher, M.S., Munro, S., 1993. Cholesterol and the Golgi apparatus. Science 261, 1280 1281.

391

Bullen, H.E., Jia, Y., Yamaryo-Botte´, Y., Bisio, H., Zhang, O., Jemelin, N.K., et al., 2016. Phosphatidic acidmediated signaling regulates microneme secretion in toxoplasma. Cell Host Microbe 19, 349 360. Cao, J., Zhou, Y., Peng, H., Huang, X., Stahler, S., Suri, V., et al., 2011. Targeting acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases. J. Biol. Chem. 286, 41838 41851. Carman, G.M., Zeimetz, G.M., 1996. Regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 271, 13293 13296. Charron, A.J., Sibley, L.D., 2002. Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii. J. Cell Sci. 115, 3049 3059. Chen, A.L., Moon, A.S., Bell, H.N., Huang, A.S., Vashisht, A.A., Toh, J.Y., et al., 2017. Novel insights into the composition and function of the Toxoplasma IMC sutures. Cell. Microbiol. 19. Available from: https://doi.org/ 10.1111. Clayton, R.B., 1964. The utilization of sterols by insects. J. Lipid Res. 15, 3 19. Coleman, R.A., Lee, D.P., 2004. Enzymes of triacylglycerol synthesis and their regulation. Prog. Lipid Res. 43, 134 176. Coppens, I., 2013. Targeting lipid biosynthesis and salvage in apicomplexan parasites for improved chemotherapies. Nat. Rev. Microbiol. 11, 823 835. Coppens, I., 2014. Exploitation of auxotrophies and metabolic defects in Toxoplasma as therapeutic approaches. Int. J. Parasitol. 44, 109 120. Coppens, I., Joiner, K.A., 2003. Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge. Mol. Biol. Cell 14, 3804 3820. Coppens, I., Sinai, A.P., Joiner, K.A., 2000. Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J. Cell Biol. 149, 167 180. Coppens, I., Dunn, J.D., Romano, J.D., Pypaert, M., Zhang, H., Boothroyd, J.C., et al., 2006. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space. Cell 125, 261 274. Dantas-Leite, L., Urbina, J.A., de Souza, W., Vommaro, R. C., 2004. Selective anti-Toxoplasma gondii activities of azasterols. Int. J. Antimicrob. Agents 23, 620 626. Dubois, D., Fernandes, S., Amiar, S., Dass, S., Katris, N.J., Botte´, C.Y., et al., 2018. Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages. J. Lipid Res. 59, 994 1004. Ehrenman, K., Sehgal, A., Lige, B., Stedman, T.T., Joiner, K. A., Coppens, I., 2010. Novel roles for ATP-binding

Toxoplasma Gondii

392

8. Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake

cassette G transporters in lipid redistribution in Toxoplasma. Mol. Microbiol. 76, 1232 1249. Fadok, V.A., Bratton, D.L., Henson, P.M., 2001. Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences. J. Clin. Invest. 108, 957 962. Fadok, V.A., Bratton, D.L., Frasch, S.C., Warner, M.L., Henson, P.M., 1998. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ 5, 551 562. Ferguson, M.A., Williams, A.F., 1988. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu. Rev. Biochem. 57, 285 320. Foe, I.T., Child, M.A., Majmudar, J.D., Krishnamurthy, S., van der Linden, W.A., Ward, G.E., et al., 2015. Global analysis of palmitoylated proteins in Toxoplasma gondii. Cell Host Microbe 18, 501 511. Fu, Y., Cui, X., Fan, S., Liu, J., Zhang, X., Wu, Y., et al., 2018. Comprehensive characterization of Toxoplasma acyl coenzyme A-binding protein TgACBP2 and its critical role in parasite cardiolipin metabolism. mBio 9, e01597. Fu, Y., Cui, X., Liu, J., Zhang, X., Zhang, H., Yang, C., et al., 2019. Synergistic roles of acyl-CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism. Cell. Microbiol. 21, e12970. Gallegos, A.M., Atshaves, B.P., Storey, S.M., Starodub, O., Petrescu, A.D., Huang, H., et al., 2001. Gene structure, intracellular localization, and functional roles of sterol carrier protein-2. Prog. Lipid Res. 40, 498 563. Glomset, J.A., 1968. The plasma lecithins:cholesterol acyltransferase reaction. J. Lipid Res. 9, 155 167. Goldstein, J.L., Brown, M.S., 1990. Regulation of the mevalonate pathway. Nature 343, 425 430. Gomes, A.F., Magalha˜es, K.G., Rodrigues, R.M., de Carvalho, L., Molinaro, R., Bozza, P.T., et al., 2014. Toxoplasma gondii-skeletal muscle cells interaction increases lipid droplet biogenesis and positively modulates the production of IL-12, IFN-γ and PGE2. Parasit. Vectors 7, 47. Goodman, C.D., McFadden, G.I., 2007. Fatty acid biosynthesis as a drug target in apicomplexan parasites. Curr. Drug Targets 8, 15 30. Gupta, N., Zahn, M.M., Coppens, I., Joiner, K.A., Voelker, D.R., 2005. Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth. J. Biol. Chem. 280, 16345 16353. Gupta, N., Hartmann, A., Lucius, R., Voelker, D.R., 2012. The obligate intracellular parasite Toxoplasma gondii secretes a soluble phosphatidylserine decarboxylase. J. Biol. Chem. 287, 22938 22947. Hartmann, A., Hellmund, M., Lucius, R., Voelker, D.R., Gupta, N., 2014. Phosphatidylethanolamine synthesis in

the parasite mitochondrion is required for efficient growth but dispensable for survival of Toxoplasma gondii. J. Biol. Chem. 289, 6809 6824. Heath, R.J., White, S.W., Rock, C.O., 2001. Lipid biosynthesis as a target for antibacterial agents. Prog. Lipid Res. 40, 467 497. Hu, X., Binns, D., Reese, M.L., 2017. The coccidian parasites Toxoplasma and Neospora dysregulate mammalian lipid droplet biogenesis. J. Biol. Chem. 292, 11009 11020. Ibrahim, M., Azzouz, N., Gerold, P., Schwarz, R.T., 2001. Identification and characterisation of Toxoplasma gondii protein farnesyltransferase. Int. J. Parasitol. 31, 1489 1497. Ihara, F., Nishikawa, Y., 2014. Starvation of low-density lipoprotein-derived cholesterol induces bradyzoite conversion in Toxoplasma gondii. Parasites Vectors 7, 248. Jakobsson, A., Westerberg, R., Jacobsson, A., 2006. Fatty acid elongases in mammals: their regulation and roles in metabolism. Prog. Lipid Res. 45, 237 249. Jelenska, J., Crawford, M.J., Harb, O.S., Zuther, E., Haselkorn, R., Roos, D.S., et al., 2001. Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 98, 2723 2728. Jelenska, J., Sirikhachornkit, A., Haselkorn, R., Gornicki, P., 2002. The carboxyltransferase activity of the apicoplast acetyl-CoA carboxylase of Toxoplasma gondii is the target of aryloxyphenoxypropionate inhibitors. J. Biol. Chem. 277, 23208 23215. Jomaa, H., Wiesner, J., Sanderbrand, S., Altincicek, B., Weidemeyer, C., Hintz, M., et al., 1999. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285, 1573 1576. Kong, P., Ufermann, C.M., Zimmermann, D.L.M., Yin, Q., Suo, X., Helms, J.B., et al., 2017. Two phylogenetically and compartmentally distinct CDP-diacylglycerol synthases cooperate for lipid biogenesis in Toxoplasma gondii. J. Biol. Chem. 292, 7145 7159. Lekutis, C., Ferguson, D.J., Grigg, M.E., Camps, M., Boothroyd, J.C., 2001. Surface antigens of Toxoplasma gondii: variations on a theme. Int. J. Parasitol. 31, 1285 1292. Le´veˆque, M.F., Berry, L., Yamaryo-Botte´, Y., Nguyen, H.M., Galera, M., Botte´, C.Y., et al., 2017. TgPL2, a patatin-like phospholipase domain-containing protein, is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma. Mol. Microbiol. 105, 158 174. Li, Z.H., Ramakrishnan, S., Striepen, B., Moreno, S.N.J., 2013. Toxoplasma gondii relies on both host and parasite isoprenoids and can be rendered sensitive to atorvastatin. PLoS Pathog. 9, e1003665. Li, Z.H., Li, C., Szajnman, S.H., Rodriguez, J.B., Moreno, S. N.J., 2017. Synergistic activity between statins and

Toxoplasma Gondii

References

bisphosphonates against acute experimental toxoplasmosis. Antimicrob. Agents Chemother. 61, e02628. Lige, B., Romano, J.D., Bandaru, V.V., Ehrenman, K., Levitskaya, J., Sampels, V., et al., 2011. Deficiency of a Niemann-Pick, type C1-related protein in Toxoplasma is associated with multiple lipidoses and increased pathogenicity. PLoS Pathog. 7, e1002410. Lige, B., Sampels, V., Coppens, I., 2013. Characterization of a second sterol-esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite. Mol. Microbiol. 87, 951 967. Lim, L., Kalanon, M., McFadden, G.I., 2009. New proteins in the apicoplast membranes: time to rethink apicoplast protein targeting. Trends Parasitol. 25, 97 200. Ling, Y., Sahota, G., Odeh, S., Chan, J.M., Araujo, F.G., Moreno, S.N., et al., 2005. Bisphosphonate inhibitors of Toxoplasma gondii growth: In vitro, QSAR, and in vivo investigations. J. Med. Chem. 48, 3130 3140. Ling, Y., Li, Z.H., Miranda, K., Oldfield, E., Moreno, S.N., 2007. The farnesyl-diphosphate/geranylgeranyl-diphosphate synthase of Toxoplasma gondii is a bifunctional enzyme and a molecular target of bisphosphonates. J. Biol. Chem. 282, 30804 30816. Liscum, L., Underwood, K.W., 1995. Intracellular cholesterol transport and compartmentation. J. Biol. Chem. 270, 15443 15446. Lourido, S., Moreno, S.N.J., 2015. The calcium signaling toolkit of the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell Calcium 57, 186 193. de Macedo, C.S., Shams-Eldin, H., Smith, T.K., Schwarz, R. T., Azzouz, N., 2003. Inhibitors of glycosylphosphatidylinositol anchor biosynthesis. Biochimie 85, 465 472. Marechal, E., Azzouz, N., de Macedo, C.S., Block, M.A., Feagin, J.E., Schwarz, R.T., et al., 2002. Synthesis of chloroplast galactolipids in apicomplexan parasites. Eukaryot. Cell 1, 653 656. Martin, M.B., Grimley, J.S., Lewis, J.C., Heath 3rd, H.T., Bailey, B.N., et al., 2001. Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy. J. Med. Chem. 44, 909 916. Martins-Duarte, E.S., Jones, S.M., Gilbert, I.H., Atella, G.C., de Souza, W., Vommaro, R.C., 2009. Thiolactomycin analogues as potential anti-Toxoplasma gondii agents. Parasitol. Int. 58, 411 415. Martins-Duarte, E´.S., Carias, M., Vommaro, R., Surolia, N., de Souza, W., 2016. Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii. J. Cell Sci. 129, 3320 3331. Mazumdar, J., Striepen, B., 2007. Make it or take it: fatty acid metabolism of apicomplexan parasites. Eukaryot. Cell 6, 1727 1735.

393

Mazumdar, J., Wilson, E.H., Masek, K., Hunter, C.A., Striepen, B., 2006. Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103, 13192 13197. McFadden, G.I., 1999. Plastids and protein targeting. J. Eukaryot. Microbiol. 46, 339 346. McLeod, R., Muench, S.P., Rafferty, J.B., Kyle, D.E., Mui, E.J., Kirisits, M.J., et al., 2001. Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I. Int. J. Parasitol. 31, 109 113. de Melo, E.J., de Souza, W., 1996. Pathway of C6-NBDCeramide on the host cell infected with Toxoplasma gondii. Cell Struct. Funct. 21, 47 52. Milovanovi´c, I., Busarˇcevi´c, M., Trbovich, A., Ivovi´c, V., Uzelac, A., Djurkovi´c-Djakovi´c, O., 2017. Evidence for host genetic regulation of altered lipid metabolism in experimental toxoplasmosis supported with gene data mining results. PLoS One 12, e0176700. Mina, J.G., Thye, J.K., Alqaisi, A.Q.I., Bird, L.E., Dods, R.H., Grøftehauge, M.K., et al., 2017. Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites. J. Biol. Chem. 292, 12208 12219. Mordue, D.G., Scott-Weathers, C.F., Tobin, C.M., Knoll, L. J., 2007. A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages. Mol. Microbiol. 63, 482 496. Mota, L.A., Roberto, Neto, J., Monteiro, V.G., Lobato, C.S., Oliveira, M.A., et al., 2014. Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii. Mem. Inst, Oswaldo Cruz 109, 767 774. Nishikawa, Y., Quittnat, F., Stedman, T.T., Voelker, D.R., Choi, J.Y., Zahn, M., et al., 2005. Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma. Cell. Microbiol. 7, 849 867. Nolan, S.J., Romano, J.D., Coppens, I., 2017. Host lipid droplets: an important source of lipids salvaged by the intracellular parasite Toxoplasma gondii. PLoS Pathog. 13, e1006362. Nolan, S.J., Romano, J.D., Kline, J.T., Coppens, I., 2018. Novel approaches to kill Toxoplasma gondii by exploiting the uncontrolled uptake of unsaturated fatty acids and vulnerability to lipid storage inhibition of the parasite. Antimicrob. Agents Chemother. 62, e00347. Oishi, H., Noto, T., Sasaki, H., Suzuki, K., Hayashi, T., Okazaki, H., et al., 1982. Thiolactomycin, a new antibiotic. I. Taxonomy of the producing organism, fermentation and biological properties. J. Antibiot. 35, 391 395. Oppenheim, R.D., Creek, D.J., Macrae, J.I., Modrzynska, K. K., Pino, P., Limenitakis, J., et al., 2014. BCKDH: the

Toxoplasma Gondii

394

8. Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake

missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei. PLoS Pathog. 1, e1004263. Pernas, L., Bean, C., Boothroyd, J.C., Scorrano, L., 2018. Mitochondria restrict growth of the intracellular parasite Toxoplasma gondii by limiting its uptake of fatty acids. Cell Metab. 27, 886 897. Polonais, V., Soldati-Favre, D., 2010. Versatility in the acquisition of energy and carbon sources by the Apicomplexa. Biol. Cell 102, 435 445. Portugal, L.R., Fernandes, L.R., Cesar, G.C., Santiago, H.C., Oliveira, D.R., Silva, N.M., et al., 2004. Infection with Toxoplasma gondii increases atherosclerotic lesion in ApoE-deficient mice. Infect. Immun. 72, 3571 3576. Portugal, L.R., Fernandes, L.R., Alvarez-Leite, J.I., 2009. Host cholesterol and inflammation as common key regulators of toxoplasmosis and atherosclerosis development. Expert. Rev. Anti Infect. Ther. 7, 807 819. Pratt, S., Wansadhipathi-Kannangara, N.K., Bruce, C.R., Mina, J.G., Shams-Eldin, H., Casas, J., et al., 2013. Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii. Mol. Biochem. Parasitol. 187, 43 51. Pszenny, V., Ehrenman, K., Romano, J.D., Kennard, A., Schultz, A., Roos, D.S., et al., 2016. A lipolytic lecithin: cholesterol acyltransferase secreted by toxoplasma facilitates parasite replication and egress. J. Biol. Chem. 291, 3725 3746. Qian, Y., Sebti, S.M., Hamilton, A.D., 1997. Farnesyltransferase as a target for anticancer drug design. Biopolymers 43, 25 41. Quittnat, F., Nishikawa, Y., Stedman, T.T., Voelker, D.R., Choi, J.Y., Zahn, M.M., et al., 2004. On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme. Mol. Biochem. Parasitol. 138, 107 122. Ramakrishnan, S., Docampo, M.D., Macrae, J.I., Pujol, F.M., Brooks, C.F., van Dooren, G.G., et al., 2012. Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 287, 4957 4971. Ramakrishnan, S., Serricchio, M., Striepen, B., Bu¨tikofer, P., 2013. Lipid synthesis in protozoan parasites: a comparison between kinetoplastids and apicomplexans. Prog. Lipid. Res. 52, 488 512. Ramakrishnan, S., Docampo, M.D., MacRae, J.I., Ralton, J.E., Rupasinghe, T., McConville, M.J., et al., 2015. The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell. Mol. Microbiol. 97, 64 76. Reeves, G.M., Mazaheri, S., Snitker, S., Langenberg, P., Giegling, I., Hartmann, A.M., et al., 2013. A positive

association between T. gondii seropositivity and obesity. Front. Public Health 1, 73. Roberts, C.W., McLeod, R., Rice, D.W., Ginger, M., Chance, M.L., Goad, L.J., 2003. Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Mol. Biochem. Parasitol. 126, 129 142. Romano, J.D., Sonda, S., Bergbower, E., Smith, M.E., Coppens, I., 2013. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole. Mol. Biol. Cell 24, 1974 1995. Romano, J.D., Nolan, S.J., Porter, C., Ehrenman, K., Hartman, E.J., Hsia, R.C., et al., 2017. The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network. J. Cell Biol. 216, 4235 4254. Sampels, V., Hartmann, A., Dietrich, I., Coppens, I., Sheiner, L., Striepen, B., et al., 2012. Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii. J. Biol. Chem. 287, 16289 16299. Santos, T.A., Portes Jde, A., Damasceno-Sa´, J.C., Caldas, L. A., de Souza, W., Damatta, R.A., et al., 2011. Phosphatidylserine exposure by Toxoplasma gondii is fundamental to balance the immune response granting survival of the parasite and of the host. PLoS One 6, e27867. Schultz, A.J., Carruthers, V.B., 2018. Toxoplasma gondii LCAT primarily contributes to tachyzoite egress. mSphere 3, e00073. Seeber, F., 2003. Biosynthetic pathways of plastid-derived organelles as potential drug targets against parasitic apicomplexa. Curr. Drug Targets Immune Endocr. Metabol. Disord. 3, 99 109. Sehgal, A., Bettiol, S., Wenk, M.R., Pypaert, M., Kaasch, A., Blader, I., et al., 2005. Peculiarities of host cholesterol transport to the unique intracellular compartment containing Toxoplasma gondii. Traffic 6, 1 17. Sharma, K., Shi, Y., 1999. The yins and yangs of ceramide. Cell Res. 9, 1 10. Sibley, L.D., Niesman, I.R., Parmley, S.F., Cesbron-Delauw, M.F., 1995. Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J. Cell Sci. 108, 1669 1677. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166, 1423 1435. Sleat, D.E., Wiseman, J.A., El-Banna, M., Price, S.M., Verot, L., Shen, M.M., et al., 2004. Genetic evidence for nonredundant functional cooperativity between NPC1 and

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Further reading

NPC2 in lipid transport. Proc. Natl. Acad. Sci. U.S.A. 101, 5886 5891. Smith, S., Witkowski, A., Joshi, A.K., 2003. Structural and functional organization of the animal fatty acid synthase. Prog. Lipid Res. 42, 289 317. Sonda, S., Hehl, A.B., 2006. Lipid biology of Apicomplexa: perspectives for new drug targets, particularly for Toxoplasma gondii. Trends Parasitol. 22, 41 47. Sonda, S., Ting, L.M., Novak, S., Kim, K., Maher, J.J., Farese, R.V., et al., 2001. Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii. J. Biol. Chem. 276, 34434 34440. Sonda, S., Sala, G., Ghidoni, R., Hemphill, A., Pieters, J., 2005. Inhibitory effect of aureobasidin A on Toxoplasma gondii. Antimicrob. Agents Chemother 49, 1794 1801. Striepen, B., Zinecker, C.F., Damm, J.B., Melgers, P.A., Gerwig, G.J., Koolen, M., et al., 1997. Molecular structure of the “low molecular weight antigen” of Toxoplasma gondii: a glucose alpha 1-4 N-acetylgalactosamine makes free glycosyl-phosphatidylinositols highly immunogenic. J. Mol. Biol. 266, 797 813. Swiezewskaa, E., Danikiewiczb, W., 2005. Polyisoprenoids: structure, biosynthesis and function. Prog. Lipid Res. 44, 235 258. Tawk, L., Dubremetz, J.-F., Montcourrier, P., Chicanne, G., Merezegue, F., Richard, V., et al., 2011. Phosphatidylinositol 3-Monophosphate Is Involved in Toxoplasma Apicoplast Biogenesis. PLoS Pathog. 7, e1001286. Tobin, C.M., Knoll, L.J., 2012. A patatin-like protein protects Toxoplasma gondii from degradation in a nitric oxide-dependent manner. Infect. Immun. 80, 55 61. Tomavo, S., Schwarz, R.T., Dubremetz, J.F., 1989. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens. Mol. Cell Biol. 9, 4576 4580. Tomavo, S., Dubremetz, J.F., Schwarz, R.T., 1992. Biosynthesis of glycolipid precursors for glycosylphosphatidylinositol membrane anchors in a Toxoplasma gondii cell-free system. J. Biol. Chem. 267, 21446 21458. Tomavo, C., Humbel, B.M., Geerts, W.J., Entzeroth, R., Holthuis, J.C., Verkleij, A.J., 2009. Membrane contact sites between apicoplast and ER in Toxoplasma gondii revealed by electron tomography. Traffic 10, 1471 1480. Tymoshenko, S., Oppenheim, R.D., Agren, R., Nielsen, J., Soldati-Favre, D., Hatzimanikatis, V., 2015. Metabolic Needs and Capabilities of Toxoplasma gondii through Combined Computational and Experimental Analysis. PLoS Comput Biol. 11 (5), e1004261. Available from:

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https://doi.org/10.1371/journal.pcbi.1004261. eCollection 2015 May. PubMed PMID: 26001086; PubMed Central PMCID: PMC4441489. Uttaro, A.D., 2006. Biosynthesis of polyunsaturated fatty acids in lower eukaryotes. IUBMB Life 58, 563 5671. Vance, J.E., Vance, D.E., 2004. Phospholipid biosynthesis in mammalian cells. Biochem. Cell Biol. 82, 113 128. Waller, R.F., Keeling, P.J., Donald, R.G., Striepen, B., Handman, E., Lang-Unnasch, N., et al., 1998. Nuclearencoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S. A. 95, 12352 12357. Welti, R., Mui, E., Sparks, A., Wernimont, S., Isaac, G., Kirisits, M., et al., 2007. Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids. Biochemistry 46, 13882 13890. White, S.W., Zheng, J., Zhang, Y.M., Rock, C.O., 2005. The structural biology of type II fatty acid biosynthesis. Annu. Rev. Biochem. 74, 791 831. Wichroski, M.J., Ward, G.E., 2003. Biosynthesis of glycosylphosphatidylinositol is essential to the survival of the protozoan parasite Toxoplasma gondii. Eukaryot. Cell 2, 1132 1136. Yardley, V., Khan, A.A., Martin, M.B., Slifer, T.R., Araujo, F.G., Moreno, S.N., et al., 2002. In vivo activities of farnesyl pyrophosphate synthase inhibitors against Leishmania donovani and Toxoplasma gondii. Antimicrob. Agents Chemother. 46, 929 931. Zhang, K., Bangs, J.D., Beverley, S.M.I., 2010. Sphingolipids in parasitic protozoa. Adv. Exp. Med. Biol. 688, 238 248. Zinecker, C.F., Striepen, B., Geyer, H., Geyer, R., Dubremetz, J.F., Schwarz, R.T., 2001. Two glycoforms are present in the GPI-membrane anchor of the surface antigen 1 (P30) of Toxoplasma gondii. Mol. Biochem. Parasitol. 116, 127 135.

Further reading Caffaro, C.E., Boothroyd, J.C., 2011. Evidence for host cells as the major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells. Eukaryot. Cell 10, 1095 1099. Hoffmann, P.R., de Cathelineau, A.M., Ogden, C.A., Leverrier, Y., Bratton, D.L., Daleke, D.L., et al., 2001. Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells. J. Cell Biol. 155, 649 659.

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9 Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa Barbara A. Fox and David J. Bzik Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States

9.1 Introduction Purines and pyrimidines and their nucleotides are of fundamental importance in the replication and development of Toxoplasma gondii and other apicomplexan parasites. Nucleotides are essential for replication of DNA and transcription of RNA in rapidly dividing stages. DNA synthesis relies on an ample supply of pyrimidine (deoxycytidine triphosphate and deoxythymidine triphosphate) and purine (deoxyadenosine triphosphate and deoxyguanosine triphosphate) deoxynucleotide 50 -triphosphates, while RNA synthesis utilizes the ribonucleotides adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine 50 -triphosphate (GTP), as well as uridine 50 -triphosphate (UTP). Uridine 50 -monophosphate is a key nucleotide in the generation of UTP and

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00009-8

CTP. Nucleotides are also essential in providing the cellular energy sources (ATP and GTP) and are involved in numerous other metabolic roles. Nucleotides provide precursors of more complex molecules such as folate, serve as nucleotide-based enzyme cofactors such as nicotinamide adenine dinucleotide or flavin adenine dinucleotide, serve in regulatory roles as intracellular messengers such as cyclic adenosine monophosphate, and also play roles in controlling metabolic and gene regulation. Nucleotides are either synthesized from small molecules and amino acids, or they are acquired via salvage pathways from preformed host-derived nucleobases and nucleosides. The apicomplexan parasites considered in this chapter, T. gondii, Plasmodium falciparum, and Cryptosporidium parvum, are important pathogens of humans that cause significant

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© 2020 Elsevier Ltd. All rights reserved.

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morbidity and mortality. Notably, current treatment strategies in human infections caused by T. gondii or P. falciparum are based on blocking the accumulation of nucleotides. This validated approach to chemotherapy highlights the significance of further research to dissect details of nucleotide metabolism in the Apicomplexa as well as the potential for this research to lead to the development of improved treatments. Apicomplexa are obligate intracellular lower eukaryotic single-cell organisms that are only capable of replication when they properly associate with their parasitized host cell. They may exist in a viable form extracellularly for short periods of time but are incapable of even a single-cell division. A comparative approach examining three selected apicomplexan pathogens that differ greatly in their interactions with the human host was undertaken to begin to address the possible relationships between the host cell and environment occupied by each parasite and the strategy that each parasite has adopted to ensure the delivery of a sufficient supply of purines, pyrimidines, and related metabolites that are necessary to support rapid parasite replication. The complete genome sequences for T. gondii (http://toxodb.org), P. falciparum (http://plasmodb.org), C. parvum (http://cryptodb.org, which also has genome sequences for Cryptosporidium hominis associated with human infections) permits new predictions about parasite biology, permits new opportunities for development of chemotherapy directed at selected parasite targets, and permits a retrospective comparison of newly identified orthologs of genes involved in purine and pyrimidine metabolism with previous biochemical, cell biological, and genetic studies. The study of purine and pyrimidine metabolism in Apicomplexa is extraordinarily complicated by the obligatory presence of the host cell, which is necessary for proliferation of the parasite. Mammalian host cells are much larger than their apicomplexan invaders, and the host cell itself has a rich supply of purines,

pyrimidines, and nucleotides. Mammalian host cells also possess their own extensive metabolic capacities to transport, synthesize, interconvert, and catabolize purines and pryimidines and related metabolites. Our understanding of purine and pyrimidine metabolism in the Apicomplexans is still incomplete due to difficulty of deciphering the complex interactions between the parasite and its host cell. While studies have examined the extracellular form of the parasite in regard to the transport, synthesis, and interconversion of nucleotides, the biochemical isolation of free parasites without contaminating host material is uncertain. Consequently, genetic studies have been a particularly informative approach to examine the phenotype of engineered parasites lacking or gaining a gene involved in purine or pyrimidine metabolism or, alternatively, by examining the interaction of normal or mutant parasites in a mutant host cell. Most of the genetic studies have been performed in T. gondii due to the more rapid and robust genetic models available for manipulating this parasite (Kim and Weiss, 2004; Suarez et al., 2017). Several important genetic selection models developed for T. gondii are also based on parasite purine or pyrimidine metabolism. Genetic selection models based on purine or pyrimidine metabolism are also available in P. falciparum. While genetic selection based on nucleotide metabolism is not yet available, targeted genetic manipulation of C. parvum using a CRISPR/cas9 system has been recently developed (Vinayak et al., 2015). The topics of purine and pyrimidine metabolism span a large swath of metabolomics. This chapter reviews the primary comparative aspects of purine and pyrimidine acquisition pathways in three Apicomplexans (T. gondii, C. parvum, and P. falciparum) and touches on certain aspects of amino acid metabolism, such as polyamine and arginine acquisition, that are linked to purine or pyrimidine pathways. Other biological intersections of T. gondii purine and pyrimidine metabolism can be

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found in Chapter 8, Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake, in Chapter 10, Metabolic networks and metabolomics, in Chapter 20, Genetic manipulation of Toxoplasma gondii, and in Chapter 26, Adaptive immunity.

9.2 Purines Purines are crucial to all cells as required components of nucleic acids, a cellular energy source, and cofactors or substrates for specific aspects of cellular metabolism. All aspects of the apicomplexan lifestyle including motilitydependent invasion of host cells is powered by nucleotides (Kimata and Tanabe, 1982). Remarkably, parasitic protozoa, with the exception of Acanthamoeba (Hassan and Coombs, 1986), lack the ability to synthesize the purine ring de novo (Berens et al., 1981; Fish et al., 1982; Marr et al., 1978; Miller and Linstead, 1983; Wang, 1984; Wang and Aldritt, 1983; Wang and Simashkevich, 1981). Since T. gondii and other apicomplexan parasites lack the machinery to synthesize the purine ring de novo, they rely on essential capture, transport, and salvage machinery to steal purines from their hosts for incorporation into the parasite nucleotide pools (Chaudhary et al., 2004; Krug et al., 1989; Perotto and Keister, 1971; Schwartzman and Pfefferkorn, 1982; Ting et al., 2005). The integration of these pathways in Apicomplexa reflects the specific needs of each parasite, as well as the differing purine resources potentially available in various host cells, tissues, and environments inhabited by each parasite. Although incapable of replication extracellularly, even in the richest medium, early investigations in T. gondii demonstrated that this parasite could obtain purines and meet all other demands for normal intracellular replication in host cells that were blocked in protein synthesis (Pfefferkorn and Pfefferkorn, 1981; Triglia and Cowman, 1994), in enucleated host cells that were blocked in host RNA and

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DNA synthesis (Jones, 1973; Sethi et al., 1973), as well as in host cells that were operating glycolytically due to deficient mitochondrial function (Schwartzman and Pfefferkorn, 1981). The capture, transport, and salvage pathways necessary for purine acquisition have long been viewed as a significant Achille’s heel of the parasite that may be targeted for chemotherapy (Avila and Avila, 1981; Avila et al., 1987; Berens et al., 1984; Fish et al., 1985; Gero et al., 1989, 2003; Marr and Berens, 1988; Pfefferkorn et al., 2001). Parasite purine transporters, located on the parasite plasma membrane, are required to transport purine nucleobases and nucleosides into the parasite cytosol (Carter et al., 2003; de Koning et al., 2005). For apicomplexan parasites such as T. gondii or P. falciparum, this model suggests that host cellderived purines either passively or actively accumulate in the parasitophorous vacuole space. Therefore the purines acquired from the host by the intracellular parasite could be derived from some combination of potential resources including de novo purine synthesis in the parasitized host cell, existing purine pools within the host cell, host-cell purine catabolism, or purine transport flux into the parasitized host cell. Few studies have experimentally addressed whether there is any requirement for host-cell purine transporters. Significant progress has been made in understanding the purine transport capability of the apicomplexan parasites, as well as the parasite enzymes that facilitate interconversion, salvage, and incorporation of host purines into the parasite purine nucleotide pools. The host-cell menu of purine compounds, the ability to transport and capture purines, and the specific activity levels of purine salvage and interconversion enzymes determine the metabolic capabilities and the potential flux of purines from the host to the parasite.

9.2.1 Capture and transport Purine transport capacity and purine transporters have been described from several

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protozoans with the majority of work being reported on transporter functions in Leishmania spp. and Trypanosoma spp. (de Koning et al., 2005; Landfear, 2011; Landfear et al., 2004). Nucleobase and nucleoside transporters fall into two general groups: sodium-dependent concentrative nucleoside transporters (CNT) and nonconcentrative equilibrative nucleoside transporters (ENT). Mammals and bacteria possess CNT and ENT transporters while the protozoa only have ENT-type transporters (Boswell-Casteel and Hays, 2017; Chaudhary, 2005; de Koning et al., 2005; Landfear et al., 2004; Valdes et al., 2014). Other genes for nucleobase transporters in bacteria, fungi, and plants are classified as either the plant purinerelated transporter family, the microbial purine-related transporter family, or the nucleobase/ascorbate transporter family (de Koning and Diallinas, 2000). Genes corresponding to these latter three groups of purine transporters have not been identified among the protozoa. 9.2.1.1 Genome analysis of purine transporters in Apicomplexa The complete genome sequences of T. gondii, P. falciparum, and C. parvum have identified putative ENT-type transporter genes, putative channels, and other transporters that may play a role in purine transport. The genome of C. parvum revealed a remarkable repertoire of transport capacities and several putative purine transporters that still await functional characterization (Abrahamsen et al., 2004; Xu et al., 2004). The P. falciparum genome encodes a previously characterized purine transporter, PfNT1 [PfENT1], as well as three additional putative ENT orthologs (Bahl et al., 2002, 2003; Chaudhary, 2005; Kirk et al., 2005; Martin et al., 2005). Similarly, the T. gondii genome revealed the TgAT1 transporter and three additional ENT orthologs designated as TgNT1, TgNT2, and TgNT3 (Chaudhary, 2005;

Kissinger et al., 2003; Li et al., 2003). Recent work has demonstrated that TgAT1, TgNT1, and TgNT3 are expressed in tachyzoites, bradyzoites, and sporulated oocysts, whereas TgNT2 is not (Chaudhary, 2005). TgAT1 and TgNT3 proteins localized to the plasma membrane of intracellular tachyzoites, while TgNT1 was localized as a punctate labeling of the parasite cytosol within a compartment that did not colocalize with any other marker of a known T. gondii organelle (Chaudhary, 2005). Other than T. gondii TgAT1 and P. falciparum PfNT1, the functional role of each of these recently identified ENT orthologs is currently unknown. 9.2.1.2 Model of purine acquisition in Toxoplasma gondii Purine acquisition by T. gondii was initially proposed to rely on host ATP, the most abundant host purine present at greater than 4 mM in the mammalian host-cell cytosol (Plagemann, 1986; Plagemann et al., 1988; Traut, 1994). The hypothesis that T. gondii steals host-cell ATP as its purine source was based on data from dual-label experiments showing that extracellular T. gondii readily incorporated the nucleoside component of ATP or AMP into nucleic acids but did not incorporate the phosphate moiety (Schwartzman and Pfefferkorn, 1982). In addition, the parasitophorous vacuole surrounding the intracellular tachyzoite is covered in a layer of host-cell mitochondria, an organelle rich in ATP (Jones et al., 1972). The identification of the parasitophorous vacuole surrounding intracellular tachyzoites as a passive permeation barrier suggested that the high concentration of cytosolic or mitochondrial-derived host ATP potentially could permeate into the parasitophorous vacuole space and equilibrate at mM concentrations (Schwab et al., 1994). The discovery of a remarkably abundant nucleoside triphosphate hydrolase (NTPase) activity secreted into

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the parasitophorous vacuole space suggested host ATP may be hydrolyzed to AMP within the vacuolar space (Asai et al., 1995; Bermudes et al., 1994; Sibley et al., 1994). Consequently, it was postulated that T. gondii might obtain its purine requirement from the flux created through permeation of ATP into the vacuolar space, conversion of ATP to AMP by vacuolar NTPase activity, and the subsequent conversion of AMP to adenosine by a hypothesized parasite plasma membrane 50 -ectonucleotidase. Since the pool of host-cell adenosine is very small at B1 μM (Plagemann, 1986; Plagemann et al., 1988; Traut, 1994), utilization of the more abundant host ATP pools was an attractive model to explain how host adenosine may be concentrated within the parasitophorous vacuole space. The first adenosine transporter characterized for T. gondii, TgAT1, was described as a nonconcentrative low-affinity (KmB120 μM) adenosine transport system that likely would require a higher concentration of adenosine than the 1 μM present in host-cell cytosol for physiological significance (Schwab et al., 1995). Subsequent work has demonstrated that intracellular tachyzoites as well as the parasitophorous vacuole space, and membrane, have no detectable 50 -ectonucleotidase activity (Ngo et al., 2000). Based on these studies, T. gondii may have no significant direct access to intracellular pools of host-cell nucleotides. Intriguingly, CD73 knockout mice are protected from chronic cyst formation, because the parasites are unable to differentiate into bradyzoites (Mahamed et al., 2012). This study proposed that host-cell CD73 expression, which is required to convert AMP to adenosine, promotes bradyzoite differentiation in a process that is dependent upon adenosine generation. Furthermore, based on kinetic evidence, a nonspecific high-affinity transporter for adenosine was identified and designated as TgAT2 (de Koning et al., 2003). Thus the most likely pathway for acquisition of adenosine is reliance on

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the host-cell 50 -ectonucleotidase activity specific for AMP (CD73), and then transport of this adenosine from the parasitophorous vacuole space by parasite plasma membrane localized TgAT2. 9.2.1.3 Properties of purine transporters in Apicomplexa Functional studies of purine transporters in C. parvum are not yet available. By contrast, several purine transporters have been functionally characterized in T. gondii. TgAT1 is a lowaffinity transporter for adenosine, guanosine and inosine (Chiang et al., 1999; Schwab et al., 1995). Low-affinity transport of adenosine by TgAT1 is blocked by dipyridamole and inhibited by excess inosine, formycin B, or hypoxanthine, but not pyrimidines, suggesting a role in transport that is purine selective and broad spectrum. The subsequent selection and characterization of adenine arabinoside (ara-A)resistant mutants identified mutations at a genetic locus corresponding to a gene, designated as TgAT, and characterized as a putative 11 membrane-spanning region protein of the ENT family (Chiang et al., 1999). Expression of TgAT in Xenopus laevis oocytes reconstituted low-affinity adenosine transport function similar to the previously characterized TgAT1 suggesting TgAT is likely to represent the cloned gene of TgAT1 (Chiang et al., 1999; Schwab et al., 1995). Disruption of the TgAT gene caused a complete loss of all adenosine transport function in extracellular tachyzoites (Chiang et al., 1999). Additional high-affinity nucleoside and nucleobase transporters were identified in extracellular tachyzoites of T. gondii (de Koning et al., 2003). This study characterized a low-affinity adenosine (KmB105 μM) and inosine (KmB134 μM) transporter equivalent to the previously characterized TgAT1 (TgAT). Kinetic evidence revealed the presence of a high-affinity adenosine (KmB0.49 μM) and

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inosine (KmB0.77 μM) transport system that was also a high-affinity and broad-spectrum transporter capable of transporting a large number of different purine and pyrimidine nucleosides. This second adenosine transport system was designated as TgAT2 (de Koning et al., 2003). The high affinity and broad specificity of TgAT2 suggested it could be an efficient route for uptake of various therapeutic nucleosides (de Koning et al., 2003). The same study also revealed a third transport system, TgNT1, which is the first purine transporter that has been demonstrated to selectively transport purine nucleobases. Based on competition kinetics, TgNT1 has a high affinity for hypoxanthine (KmB0.91 μM) as well as guanine and xanthine. The presence of TgAT2 and TgNT1 on the tachyzoite plasma membrane of extracellular tachyzoites suggests an extraordinary capacity of T. gondii to salvage exceedingly low concentrations of purine nucleobases and nucleosides from within the parasitophorous vacuole space in the intracellular environment. This extraordinary capacity to scavenge purines may in part explain the remarkable ability of T. gondii to replicate in virtually any mammalian cell type. Yet while the T. gondii parasitophorous vacuole membrane is proposed to be a molecular sieve that permits passive permeation of neutral or charged molecules up to 13001900 Da between the host-cell cytoplasm and the parasitophorous vacuole space (Joiner et al., 1994, 1996; Schwab et al., 1994), experimental studies specifically addressing permeation or concentration of nucleobases, nucleosides, or nucleotides inside the T. gondii parasitophorous vacuole space have not been reported. P. falciparum possesses genes encoding four potential ENT transporters (PfNT1, PFNT2, PfNT3, and PfNT4) (Landfear, 2011). Transport properties of PfNT3 are unknown. PfNT2 is proposed to be a transporter for uridine (Downie et al., 2010), although the significance

of uridine transport is unclear, because it is not currently known whether P. falciparum inside the erythrocyte has any significant pyrimidine salvage capacity. PfNT4 (aka PfENT4) transports adenine, adenosine, and 20 deoxyadenosine with millimolar range affinity but has no ability to transport hypoxanthine or AMP (Frame et al., 2012). The significance of these transport activities remains unknown, as PfENT4 is not thought to be the major purine transporter. P. falciparum expresses an 11-membranespanning ENT family nucleoside transporter designated PfNT1 (or PfENT1), which is localized to the parasite plasma membrane (Carter et al., 2000; Parker et al., 2000; Rager et al., 2001). PfNT1 is expressed in all life stages, and expression of this gene is markedly up regulated in young trophozoites, corresponding with an increased need for purines in P. falciparum replication. PfNT1 expression in X. laevis oocytes confers high-affinity adenosine transport (KmB13 μM) and low-affinity inosine transport (KmB253 μM). Previous inhibition studies revealed PfNT1 is broad spectrum for a number of purine and pyrimidine nucleosides, and unlike its mammalian host can transport both Dand L-nucleosides. More recent genetic studies suggest PfNT1 is a transporter for purines. Genetic deletion of the gene for PfNT1 established a null strain that could grow if medium was supplemented with 50 μM, or greater concentrations, of hypoxanthine, adenosine, or inosine (El Bissati et al., 2006). Hypoxanthine, adenosine, or inosine concentrations below 50 μM could not support parasite replication. PfNT1 transported hypoxanthine, xanthine, guanine, and inosine (El Bissati et al., 2008). Thus, either PfNT1 directly transports adenosine and inosine or alternatively, adenosine in the host red cell cytosol is converted to inosine by host adenosine deaminase and inosine in the host-cell cytosol is converted to hypoxanthine by host purine nucleoside phosphorylase (PNP) (El Bissati et al., 2006, 2008). Inhibitors have

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been identified for this PfNT1 [PfENT1] transporter, and these compounds are being evaluated as potential antimalarial drugs (Arora et al., 2016; Frame et al., 2015).

9.2.2 Purine transport in the parasitized host cell The P. falciparum parasitized erythrocyte exhibits remarkable alterations in transport functions compared with the normal erythrocyte (Gero et al., 2003; Kirk et al., 1994). Nucleosides enter parasitized erythrocytes through at least three parasite-induced permeation pathways, including a saturable highaffinity adenosine transport system (Upston and Gero, 1995), through the tubulovesicular membrane (TVM) network connecting the parasitophorous vacuole to the erythrocyte periphery (Lauer et al., 1997), and through a nonsaturable, anion selective-type channel that also has capacity to transport L-nucleosides, amino acids, sugars, and cations (Kirk et al., 1994). P. falciparum has a TVM network extending from the parasitophorous vacuole as well as the requisite machinery to enable regulated protein secretion from the parasite cytoplasm to the vacuolar space, the erythrocyte cytoplasm, and to the erythrocyte periphery (Kyes et al., 2001; Lauer et al., 1997). The ability of parasitized erythrocytes to selectively transport L-nucleosides was used to elegantly deliver therapeutic agents to intracellular P. falciparum parasites, selectively killing them (Gero et al., 2003). Conjugating 5fluorouridine to L-adenosine or L-thymidine selectively delivered cytotoxic 5-fluorouridine to intracellular P. falciparum, whereas these hybrid molecules showed no transport or cytotoxicity in mammalian cells. It is unknown whether the new transport functions of the parasitized erythrocyte are due to modification or incorporation of new transporters, channels, ducts, or TVM near the erythrocyte periphery.

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While mammalian cells lack any ability to transport the mammalian adenosine transporter inhibitor 6-thiobenzylthioinosine (NBMPR), T. gondiiparasitized host cells selectively transports NBMPR (Al safarjalani et al., 2003). NBMPR is selectively cytotoxic to intracellular T. gondii (El kouni et al., 1999). The activation of NBMPR cytotoxicity is associated with the ability of T. gondiiderived enzyme extracts, but not host cellderived enzyme extracts, to phosphorylate NBMPR to its nucleoside 50 -monophosphate. Adenosine kinase (AK)-deficient T. gondii fails to phosphorylate NBMPR to its nucleoside 50 -monophosphate, showing AK is the major pathway to selective incorporation and cytotoxicity of NBMPR (Al safarjalani et al., 2003; Rais et al., 2005). Similar to the P. falciparum parasitized erythrocyte, T. gondii parasitized host cells also selectively transport nonphysiological β-Lenantiomers of purine nucleosides, β-L-adenosine, β-L-deoxyadenosine, and β-L-guanosine. Uninfected host cells do not transport NBMPR or the β-L-nucleosides. NBMPR also inhibits the transport function of the host-cell nucleoside transporter ENT1 (es) (Gupte et al., 2005). Dipyridamole, another inhibitor of nucleoside transport, inhibited transport of NBMPR and β-L-nucleosides into parasitized host cells. Transport of NBMPR and β-L-nucleosides in the parasitized host cell required a functional TgAT1 transporter (Al safarjalani et al., 2003; Chiang et al., 1999). While these observations explain a requirement for transport into the intracellular tachyzoite from the parasitophorous vacuole space, these studies do not specifically address why the T. gondiiparasitized host cell selectively transports these compounds. Therefore infection with T. gondii confers a broad repertoire of parasite-specific transport mechanisms to the host cell. The novel transport capacity of parasitized host cells opens a new avenue toward developing chemotherapeutic approaches, as well as addressing other biological modifications of

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the parasitized host. The novel transport mechanisms specific to the T. gondii parasitized host cell may entail numerous possibilities including the recently reported equilibratory high-affinity TgAT2 adenosine transporter (de Koning et al., 2003), a concentrative iondependent channel, a TVM system interconnecting the parasitophorous vacuole to the host-cell periphery and a duct for transport of macromolecules that bypasses the host-cell membrane (Gero et al., 2003). The elegant electrophysiological description of the mechanism of parasitophorous vacuole formation in T. gondii suggested that after T. gondii invasion and vacuole formation, a fission pore remnant is left on the host-cell surface (Suss-Toby et al., 1996). Shortly after invasion of the host cell, a protein- and membrane-rich intravacuolar network derived from electron-dense granules is formed in the parasitophorous vacuole space (Mercier et al., 1998, 2002; Sibley et al., 1995). Three-dimensional imaging of T. gondii within recently formed vacuoles revealed fibrous and tubular material that connects the parasite plasma membrane on intracellular tachyzoites within the parasitophorous vacuole to the remnant of the fission pore at the host-cell plasma membrane (Schatten and Ris, 2004). Collectively, these observations suggest that the transport of nutrients such as purines to the tachyzoite within the parasitophorous vacuole may be facilitated by additional mechanisms beyond simple diffusion of nutrients within host-cell cytosol through proposed pores in the parasitophorous vacuole membrane. This concept is supported by recent evidence indicating that the T. gondii parasitophorous vacuole actively internalizes host cytosol, host vesicles, and host organelles (Coppens et al., 2006; Dou et al., 2014; Nolan et al., 2017; Romano et al., 2013), suggesting that bulk cargo is delivered into the vacuole through heterophagy. These mechanisms will further concentrate host cellderived nutrients in the parasitophorous vacuole space

surrounding the replicating parasites. Further studies are necessary to assign functional roles to the ENT gene orthologs identified in T. gondii, the requirement for host-cell transporters, the potential roles of the tubulovesicular network in the parasitophorous vacuole space, and the parasitophorus vacuole membrane in ingesting host-cell cargo, vesicles, and organelles, and to elucidate additional mechanisms that may promote the availability and permeation of host purines to the intracellular parasite.

9.2.3 Purine interconversion and salvage pathways in Apicomplexa Early studies in nonreplicating extracellular tachyzoites demonstrated that the purine ring precursor, glycine, was poorly incorporated into T. gondii nucleic acids (Perotto and Keister, 1971). By utilizing a mutant host cell deficient in de novo purine synthesis, intracellular T. gondii was also shown to poorly incorporate glucose and formate precursors of the guanine and adenine ring (Schwartzman and Pfefferkorn, 1982). T. gondii could not synthesize the purine nucleotides de novo from formate, glycine, or serine (Krug et al., 1989). Collectively these pioneering studies clearly demonstrated that T. gondii cannot synthesize purines de novo, and therefore the parasite strictly relies on stealing a supply of purines from the host. These findings illustrated the potential of targeting the purine auxotrophy of apicomplexan parasites to inhibit parasite replication. Replication of T. gondii is efficiently inhibited by ara-A, and the establishment of parasite mutants resistant to ara-A demonstrated that a parasite AK activity is required for activation and incorporation of ara-A (Pfefferkorn and Pfefferkorn, 1976, 1978). Most investigations of purine auxotrophy in apicomplexan parasites focused on the machinery responsible for interconverting purines and

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incorporating host purine nucleobases and nucleosides into the parasite nucleotide pools. These studies have included (1) studies of enzyme activities, (2) gene cloning, expression, kinetic analysis, structure determination, (3) studies on regulation and cellular localization, (4) gene knockouts, (5) studies in mutant host cells, and (6) genome and evolutionary analysis. In the apicomplexans, T. gondii has presented the most amenable model for applying varied approaches to decipher purine acquisition in Apicomplexa. T. gondii possesses significant machinery for purine salvage showing the importance of this nutrient to parasite metabolism and replication. By contrast, C. parvum possesses the most diminished purine salvage machinery of the apicomplexans, and this diminished capacity is likely to be related to the nutrient rich, but highly restricted niche of this parasite in the gut. The P. falciparum purine pathways are only slightly less robust than pathways present in T. gondii. Plasmodium spp. is atypical in infecting and replicating within erythrocytes, a cell type that does transcribe RNA or replicate DNA, suggesting a diminished host-cell requirement for pyrimidines and purines. This niche specialization is likely to be associated with novel adaptations in the Plasmodium purine pathways that are not observed in other apicomplexan parasites. 9.2.3.1 Purine salvage pathways in Toxoplasma gondii Genetic studies in T. gondii have complemented biochemical approaches to more clearly define the transport and purine salvage capacity of this parasite. In early studies, [3H] hypoxanthine labeling of T. gondii infected LeschNyhan mutant human host cells, deficient in hypoxanthineguanine phosphoribosyltransferase activity, demonstrated that only intracellular parasites were labeled with no detectable incorporation into host-cell nucleic acids (Pfefferkorn and Pfefferkorn, 1977c).

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Therefore the flux of purines is unidirectional from the host cell to the replicating T. gondii parasite. A comprehensive biochemical investigation of parasite enzyme activities involved in salvage, interconversion, and incorporation of host purines in T. gondii was performed in viable extracellular tachyzoites (Krug et al., 1989). However, interpretation of results from this type of investigation is complicated by contamination of parasite preparations with host-cell membranes and host purine metabolism enzymes (Ngo et al., 2000). Interpretation of results from this study is also complicated by the use of high, nonphysiological, concentrations of radiolabeled purines to maximize the detection of transport and incorporation of purines in extracellular tachyzoites. Such high concentrations of purines are unlikely to be actually available to the intracellular tachyzoite. Extracellular tachyzoites also are not replicating organisms and could have a reduced ability to acquire or use purines; thus this model may not accurately reflect the complexity of the purine interactions between intracellular parasites and the host cell. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated and indicated the presence of a hypoxanthinexanthineguanine phosphoribosyltransferase (HXGPRT) activity as well as an adenine phosphoribosyltransferase (APRT) activity (Krug et al., 1989). Adenine was incorporated one-half as efficiently as hypoxanthine. Guanine was incorporated at 55% and xanthine at 67% of the rate at which hypoxanthine was incorporated (Krug et al., 1989). Subsequent studies have demonstrated T. gondii has no APRT gene or activity (Chaudhary et al., 2004). The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated into nucleic acids. Adenosine was incorporated more than 12-fold as well as any other purine nucleoside or nucleobase and suggested a parasite AK to be the major route to AMP. By contrast, hypoxanthine was

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incorporated at 8.3%, inosine at 8.2%, xanthine at 5.6%, guanine at 4.6%, adenine at 3.9%, guanosine at 2.5% and xanthosine at 0.3% of the rate at which adenosine was incorporated (Krug et al., 1989). Correspondingly, in parasite protein extracts prepared from extracellular tachyzoites, AK activity was greater than 15-fold more active than the next most active enzyme. Guanine, guanosine, xanthine, and xanthosine labeled only guanylate nucleotides. Therefore T. gondii has no pathway from guanylate to adenylate nucleotides (Krug et al., 1989). Adenosine, inosine, and hypoxanthine labeled adenylate and guanylate nucleotides pool at approximately equal ratios (Krug et al., 1989; Pfefferkorn et al., 2001). PNP activities were detected only for inosine and guanosine. Deaminase activities were detected for guanine (GUAD), adenine (ADE), adenosine (ADA), and AMP (AMPD) (Krug et al., 1989). While the reported GUAD may be present in T. gondii, this activity is not shown on the current model of purine pathways, because this activity was low, there is highly abundant host GUAD that may contaminate tachyzoite preparations, and no gene ortholog has been identified for a T. gondii GUAD (Chaudhary et al., 2004) (Fig. 9.1). While a T. gondii ADA activity was demonstrated in tachyzoites grown in mutant host cell deficient in host adenosine deaminase (Krug et al., 1989), the AK pathway is by far the most significant pathway for incorporation of adenosine due to the high specific activity of AK. While no putative gene ortholog for ADA has been yet identified this pathway is present and may be significant, particularly to parasites lacking AK activity (Chaudhary et al., 2004). Adenine was variably but generally poorly incorporated into T. gondii nucleic acids during infection of normal host cells, but in host cells that are deficient in APRT activity, adenine incorporation was low (Chaudhary et al., 2004). While a putative gene ortholog for T. gondii ADE has been identified, the ADE

pathway to hypoxanthine appears to be a minor pathway for this parasite (Chaudhary et al., 2004; Krug et al., 1989). Hypoxanthine is converted to inosine 50 -monophosphate (IMP) by HXGPRT. Once IMP is available, AMP can be made in two steps by adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ADSL) and GMP can be made in two steps by IMP dehydrogenase (IMPDH) and GMP synthetase (GMPS). Therefore interconversion of nucleotides occurs only in the direction of adenylate to guanylate nucleotides via AMPD (Krug et al., 1989; Pfefferkorn et al., 2001) (Fig. 9.1). There is no GMP reductase, and other than AK no other nucleoside kinase or phosphotransferase activities are present (Krug et al., 1989). Therefore T. gondii possesses a minimum of 10 enzymes involved in interconversion and salvage of host purines. Gene orthologs have been reported in nine of these enzymes in T. gondii (ADE, PNP, AK, HXGPRT, ADSS, ADSL, AMPD, IMPDH and GMPS) (Chaudhary et al., 2004). T. gondii can transport and salvage the host nucleosides adenosine, inosine, and guanosine, as well as the host nucleobases adenine, hypoxanthine, xanthine, and guanine (Fig. 9.1). The parasite can incorporate host purines into the parasite nucleotide pool by two major routes, via AK and HXGPRT. Adenosine kinase and HXGPRT Incorporation of host adenosine into the AMP pool by AK appears to be the most significant purine salvage pathway (Chaudhary et al., 2004; Krug et al., 1989; Ngo et al., 2000). Yet resistance to ara-A due to mutation and disruption of parasite AK was described even in early studies (Pfefferkorn and Pfefferkorn, 1976, 1978), suggesting the AK gene may not be essential. Subsequently, a genome-wide insertional mutagenesis screen was used to select ara-Aresistant mutants. One class of isolated mutants was disrupted in the AK gene and activity, demonstrating again that a parasite with disrupted AK function still replicates

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FIGURE 9.1

Model of purine transport and salvage pathways in Toxoplasma gondii. The host-cell cytosol, parasitophorous vacuole membrane, parasitophorous vacuole space, and the parasite plasma membrane are indicated. Purine transporters are shown as cylinders resting in the parasite plasma membrane. Relevant purines and the enzymic machinery managing purine interconversion and salvage of host purine nucleobases and nucleosides into the adenylate (AMP) and guanylate (GMP) nucleotide pools are shown. Solid lines and arrows depict active pathways. Where information on purine flux is available, the weighting of the pathway is emphasized by the weight of the lines and arrows. The weighting of pathways described in this figure reflects the most likely predictions from available data; however, the weightings shown in this figure are only hypothetical and the purine flux of host purine to the parasite, as well as interconversion and incorporation within the intracellular parasite, remains to be experimentally tested. Substrates of each enzyme activity are shown on the side of the solid line and the product(s) of each enzyme activity are shown on the arrowhead side. The enzyme activity responsible for each interconversion step is shown in capital italicized text beside the arrowhead line. Adenosine kinase (AK) and HXGPRT represent the major pathways for salvage and incorporation into the nucleotide pool. ADA, Adenosine deaminase; ADE, adenine deaminase; ADSL, adenylosucccinate lyase; ADSS, adenylosuccinate synthetase; AMPD, AMP deaminase; GMPS, GMP synthetase; HXGPRT, hypoxanthinexanthineguanine phosphoribosyltransferase; IMPDH, inosine 50 -monophosphate dehydrogenase; PNP, purine nucleoside phosphorylase; PRPP, 5phosphoribosyl-1-pyrophosphate.

normally HXGPRT has been chemical,

most likely by salvage through the (Sullivan et al., 1999). T. gondii AK expressed in Escherichia coli for biokinetic, and structural studies that

have revealed significant differences between the parasite AK and the mammalian AK which may be exploited for drug design (Darling et al., 1999; Schumacher et al., 2000a,b).

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Subversive substrates of T. gondii AK have been shown to be selectively toxic to the parasite (El kouni et al., 1999; Iltzsch et al., 1995; Pfefferkorn and Pfefferkorn, 1976). Interestingly, the AK gene from C. parvum complements AK-deficient T. gondii. This complementation system may present a useful high-throughput model to screen for potential inhibitors of C. parvum AK (Striepen and Kissinger, 2004; Striepen et al., 2004). HXGPRT represents the second major route of incorporation of host purines. Yet, as observed for AK, disruption of T. gondii HXGPRT has no highly significant effect on replication or viability of tachyzoites (Donald et al., 1996; Pfefferkorn and Borotz, 1994). The unique xanthine phosphoribosyltransferase activity of HXGPRT, which is absent in the human host, may be exploited for drug design using toxic analogs of xanthine. This approach has been validated in studies using 6thioxanthine, a compound with selective toxicity to T. gondii (Pfefferkorn and Borotz, 1994; Pfefferkorn et al., 2001). The crystal structure and enzyme mechanisms of T. gondii HXGPRT have been determined and the parasite HXGPRT is a potential drug target (Heroux et al., 1999a,b; Schumacher et al., 1996). Parasites completely deficient in HXGPRT or AK are viable, suggesting that either pathway can suffice for purine incorporation and parasite replication (Donald et al., 1996; Sullivan et al., 1999). In the T. gondii HXGPRT knockout parasite, host adenosine would be required for parasite replication and would be incorporated into adenylate nucleotides by AK and then into guanylate nucleotides by AK, AMP deaminase, IMPDH and GMPS (Fig. 9.1). Conversely, in a T. gondii AK knockout mutant, host guanine, guanosine, and xanthine would provide guanylate nucleotides, but no adenylate nucleotides could be formed from these purines. In this mutant host adenine, adenosine, inosine, or hypoxanthine could potentially satisfy the parasite’s demand for both

adenylate and guanylate nucleotides (Fig. 9.1). All of the potential host purine precursors would funnel through hypoxanthine into the IMP pool, which can go to guanylate nucleotides through IMPDH and GMPS and to adenylate nucleotides through ADSS and ADSL. Therefore unlike P. falciparum and C. parvum (discussed next), T. gondii possesses a functionally redundant purine salvage pathway (AK and HXGPRT) with the capacity to meet the purine requirement by using an assortment of potential host-cell purine nucleobases and nucleosides. This feature of T. gondii may help to explain how the parasite is capable of replicating in such a wide variety of host cells and tissues that are likely to present quite varied potential purine resources. The complex purine salvage pathway of T. gondii also suggests that this pathway is likely to be subjected to complex regulatory mechanisms (Fig. 9.1). For instance, parasites that are growth inhibited by treatment with 6-thioxanthine incorporate fourfold more hypoxanthine and xanthine into nucleic acids that untreated control parasites. This increase in salvage of hypoxanthine and xanthine was not due to any increase in specific activity of HXGPRT but involves some other compensatory aspect of the salvage pathways (Pfefferkorn et al., 2001). Genetic studies have been performed on T. gondii AK and HXGPRT that help to clarify previous observations. HXGPRT and AK activities cannot be simultaneously disrupted in T. gondii, suggesting that these are the only functional routes to purine nucleotides in the parasite (Chaudhary et al., 2004). Consequently, it is feasible to knockout the parasite AK and HXGPRT activities as long as at least one functional pathway to purine nucleotides is provided in trans. As predicted, a parasite possessing no functional endogenous T. gondii genes for AK and HXGPRT is viable when complemented by a functional APRT gene from Leishmania donovani (Chaudhary et al., 2004). This genetic study also demonstrated that a single gene knockout of

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parasite AK or HXGPRT has small but detectable defects in fitness as determined by growth rate of tachyzoites. AK-deficient parasites exhibit a fitness defect in growth rate of 7.6% per generation, while HXGPRT deficient parasites exhibit a fitness defect in growth rate of 3.7% per generation (Chaudhary et al., 2004). This significant finding suggests that purine acquisition may be rate limiting to parasite growth rate. In the case of the AK knockout parasite the same host supply of adenosine would be available as in the wild-type parent, thus the flux of adenosine, when diverted by lack of AK activity, to inosine to hypoxanthine to IMP, then to guanylate and adenylate nucleotides, is not sufficient to fully support the normal parasite growth rate. Therefore parasite transport and incorporation of host adenine, adenosine, inosine, hypoxanthine, guanosine, guanine, and xanthine through parasite HXGPRT are insufficient to fully support normal parasite replication (Fig. 9.1). Similarly, in an HXGPRT knockout, transport and incorporation of host adenosine through the high activity AK pathway are insufficient to fully support parasite replication, suggesting the host supply of adenosine itself is not quite sufficient to fully support the maximum parasite replication rate. Collectively, these observations suggest multiple host purine nucleobases and/or nucleosides and both pathways of incorporation of host purines into the nucleotide pools of T. gondii are likely to be required for supporting a maximum replication rate. Considering the very high specific activity of parasite AK (Krug et al., 1989), the bottleneck for purine flux to the replicating intracellular tachyzoite is most likely due to a limited availability of host purines, or a limited transport capacity of the parasitophorous vacuole and intracellular tachyzoite. Multiple isoforms and localization of HXGPRT A novel feature of the T. gondii purine salvage pathway is the expression of two forms of HXGPRT from a single gene locus by alternatively spliced mRNA (Donald et al., 1996; White

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et al., 2000). The two isoforms differ by a 49 amino acid segment comprising an extra exon in isoform-II. Both isoforms behave in a kinetically similar manner, although isoform-II is slightly less efficient in recognizing guanine as a substrate (White et al., 2000). The cellular compartmentalization of the two HXGPRT isoforms is different. Isoform-I is cytosolic, while the longer version isoform-II is localized to the inner membrane complex (IMC) of the tachyzoite (Chaudhary et al., 2005). The 49 amino acid insert at the N-terminus of isoform-II is required for the localization to the IMC. The mechanism of IMC localization was identified to be palmitoylation that occurred at three adjacent cysteine residues within the 49 amino acid insert. Mutation of these three cysteines blocked palmitoylation and localization of HXGPRT to the IMC (Chaudhary et al., 2005). The biological basis of functional redundancy of HXGPRT in T. gondii is not obvious because both isoforms are functionally competent HXGPRT activities that can support parasite replication (Donald et al., 1996; Donald and Roos, 1998). It is possible that this functional redundancy in HXGPRT enables T. gondii to grow in a wider variety of cell types where purines may be limiting and additional mechanisms may be required for purine transport and salvage. Alternatively, this novel dual localization of HXGPRT may reflect an economy of purine metabolism within the parasite itself, or perhaps some unknown aspect of purine regulation. T. gondii PNP. The T. gondii PNP gene has been cloned and expressed in E. coli (Chaudhary et al., 2006). The structure and inhibitors of the T. gondii PNP have been reported (Donaldson et al., 2014a). Similar to human and P. falciparum PNP, the recombinant T. gondii PNP enzyme recognizes inosine and guanosine as good substrates, and adenosine and xanthosine as poor substrates. T. gondii PNP, however, is unusual in recognizing deoxynucleosides as poor substrates (Chaudhary et al., 2006). This unusual substrate property of

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the parasite PNP suggests that the intracellular parasite may have access to the host pool of guanosine and inosine but would poorly incorporate host deoxyguanosine or deoxyinosine transported by the parasite. T. gondii PNP demonstrated no activity against 50 0 methylthioadenosine (MTA) or 5 -methylthioinosine (MTI) and was insensitive to inhibition by methylthio (MT)-immucillin-H (Chaudhary et al., 2006). Immucillin-H is a strong nM inhibitor of the parasite enzyme in vitro (Chaudhary et al., 2006). While the replication of wild-type T. gondii parasites is completely unaffected by immucillin-H in vitro, an AK knockout mutant of T. gondii is inhibited by immucillin-H (Chaudhary et al., 2006). The growth inhibition of immucillin-H in the AK knockout background is reversed in the presence of excess hypoxanthine in the in vitro culture medium (Chaudhary et al., 2006). If host adenine and hypoxanthine were being supplied in a sufficient amount to the replicating parasite, this purine supply should confer resistance to immucillin-H even in an AK knockout mutant (Fig. 9.1). Therefore based on the growth inhibition of an AK knockout mutant by immucillin-H, it is possible that the supply of host adenosine and inosine in the intracellular environment exceeds the potential supply of host adenine and hypoxanthine, suggesting that host nucleosides rather than host nucleobases are the more important purine pool (Fig. 9.1). A caveat with this interpretation of the inhibition of parasite PNP by immucillin-H is the possibility that hypoxanthine may, partly, antagonize inhibition of PNP, or that host PNP is also inhibited by immucillin-H thus reducing host purine pools (Kicska et al., 2002a). All together, these studies further validate the proposed pathways for purine incorporation and salvage in T. gondii (Fig. 9.1). Genetic selection based on T. gondii HXGPRT Early studies established the parasite HXGPRT as both a potential drug target and a gene that would be amenable for both positive and

negative genetic selection in T. gondii (Pfefferkorn and Borotz, 1994). The mechanism of 6-thioxanthine inhibition of parasite replication is based on activation of 6-thioxanthine to 6-thioxanthine 50 -monophosphate by the parasite HXGPRT (Pfefferkorn et al., 2001). Unlike mercaptopurine in mammals (Elion, 1989a,b), 6-thioxanthine and its nucleotide product 6-thioxanthine 50 -monophosphate is not a substrate for T. gondii GMPS and is not incorporated into nucleic acids. The mechanism of inhibition is parasitostatic and has been suggested to inhibit parasite IMPDH by accumulation of 6-thioxanthine 50 -monophosphate (Pfefferkorn et al., 2001). T. gondii mutants deficient in HXGPRT are completely resistant to the toxic effects of 6thioxanthine (Pfefferkorn and Borotz, 1994). Once resistance to 6-thioxanthine is selected by knockout of HXGPRT, parasites with a functional HXGPRT can be positively selected by growth in mycophenolic acid (MPA) with supplements of xanthine or guanine (Pfefferkorn and Borotz, 1994). This selection scheme is based on the ability of MPA to specifically inhibit IMPDH, selectively blocking the conversion of IMP to xanthine monophosphate (XMP) (Fig. 9.1). Thus parasites with a nonfunctional HXGPRT cannot be rescued with xanthine or guanine when IMPDH is inhibited, whereas parasites with a functional HXGPRT will be rescued by xanthine or guanine supplementation of growth medium and this pathway will bypass the inhibition at IMPDH. The biochemical description of this selection strategy also proved that T. gondii is perfectly capable of obtaining adenylate nucleotides by the AK pathway, and guanylate nucleotides by HXGPRT salvage of xanthine to XMP, or salvage of guanine to GMP (Pfefferkorn and Borotz, 1994). The identification of the HXGPRT gene enabled a test of this biochemical prediction and resulted in the establishment of a robust genetic selection scheme for positive and negative selection using the selection

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principles described above (Donald et al., 1996). The HXGPRT selection scheme established the first genetic system for hit and run mutagenesis in T. gondii where a stable pseudodiploid could be established during positive selection, then negative selection can be used to force out the HXGPRT gene to create a subtle or major mutation within the gene locus of interest (Donald and Roos, 1998). The HXGPRT genetic model has been extensively used to generate knockout mutants in nonessential parasite genes as well as in genetic studies of parasite development. This genetic system was also adapted for functional cloning studies and has resulted in the initial identification of the ε-proteobacterium-type IMPDH gene from C. parvum (Striepen et al., 2002). 9.2.3.2 Purine salvage pathways in Cryptosporidium parvum Experimental work on purine pathways in C. parvum has been limited by the difficulty of culturing this parasite, the previous difficulty of developing targeted gene knockouts, and the difficulty of obtaining purified parasites for biochemical analysis. The Cryptosporidium genomes have given the first detailed insights into the strategy this parasite has adopted to satisfy its appetite for host-cell purines (Abrahamsen et al., 2004; Striepen and Kissinger, 2004; Xu et al., 2004). In addition to its limited ability to synthesize amino acids and pyrimidines (discussed next), C. parvum also possesses a very limited repertoire of purine salvage and interconversion enzymes compared to other apicomplexans (Chaudhary et al., 2004; Striepen et al., 2004). While an early study using crude parasite extracts suggested C. parvum expressed HXGPRT and APRT activities (Doyle et al., 1998), C. parvum lacks any gene ortholog for APRT or HXGPRT, and the parasite is insensitive to the HXGPRT inhibitor 6thioxanthine (Chaudhary et al., 2004; Striepen and Kissinger, 2004; Striepen et al., 2004).

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Remarkably, adenosine is the only host purine that was previously predicted to be of any physiological significance to C. parvum. Adenosine is readily supplied in the gut environment (Fig. 9.2A). Similar to T. gondii and P. falciparum, C. parvum possesses an adenosine transporter gene ortholog that is likely to be responsible for transport of host adenosine into the parasite cytosol (Abrahamsen et al., 2004). When host adenosine is available in the parasite cytosol, the parasite can incorporate adenosine into the nucleotide pool by the AK pathway to AMP. Once the parasite has a nucleotide pool of AMP, this pool meets the entire demand for all adenylate and guanylate nucleotides (Abrahamsen et al., 2004). AMP is first deaminated to IMP by AMPD. IMP is then converted to XMP by IMPDH and XMP is converted to GMP by GMPS (Fig. 9.2A). Since there is no reverse pathway from IMP to AMP, the nucleotide flux extends only from AMP to GMP. The C. parvum IMPDH gene was acquired by horizontal gene transfer from an ε-proteobacterium (Striepen and Kissinger, 2004; Striepen et al., 2002, 2004). The parasite genome was previously predicted to express only these four enzymes to direct the unidirectional flow of transported host adenosine to parasite adenylate and guanylate nucleotides (Fig. 9.2A). Potential drug targets in C. parvum purine salvage pathways: Unlike T. gondii, which has redundant purine salvage pathways, each of the C. parvum purine pathway enzymes (AK, AMPD, IMPDH or GMPS) was previously predicted to be a potential drug target. The C. parvum AK activity was previously evaluated as a drug target in genetic experiments using an AK deficient mutant of T. gondii that was complemented with the C. parvum AK gene activity (Striepen et al., 2004). In addition, IMPDH inhibition using drugs such as ribavirin and MPA was shown to inhibit C. parvum development (Woods and Upton, 1998) in a dose-dependent manner by these

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FIGURE 9.2 Model of purine transport and salvage pathways in Cryptosporidium parvum. (A) Previous model of purine acquisition. (B) Current model of purine acquisition. The host-cell cytosol, parasitophorous vacuole membrane, parasitophorous vacuole space, and the parasite plasma membrane are indicated. Purine transporters are shown as cylinders resting in the parasite plasma membrane. Relevant purines and the enzymic machinery managing purine interconversion and salvage of host purine nucleobases and nucleosides into the adenylate (AMP) and guanylate (GMP) nucleotide pools are shown. Solid lines and arrows depict active pathways. Where information on purine flux is available, the weighting of the pathway is emphasized by the weight of the lines and arrows. Substrates of each enzyme activity are shown on the side of the solid line and the product(s) of each enzyme activity are shown on the arrowhead side. The enzyme activity responsible for each interconversion step is shown in capital italicized text beside the arrowhead line. AK is a major pathway for salvage and incorporation into the nucleotide pool. A second major pathway of purine acquisition is via import of host cell ATP and GTP. AK, Adenosine kinase; AMPD, AMP deaminase; GMPS, GMP synthetase; IMPDH, inosine 50 monophosphate dehydrogenase.

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IMPDH inhibitors (Striepen et al., 2004). A screening pipeline for drugs that inhibit C. parvum IMPDH was previously developed (Sharling et al., 2010), the structure of C. parvum IMPDH has been determined (Kim et al., 2015; Sun et al., 2014), and various inhibitors of this enzyme have been identified (Gorla et al., 2012; Sarwono et al., 2019; Teotia et al., 2016). However, recent gene knockout experiments refute the previous evidence suggesting that the C. parvum adenosine transporter, AK, IMPDH, and GMPS activities are potential drug targets. C. parvum mutants lacking the adenosine transporter, AK, IMPDH, or GMPS are viable and maintain the ability to replicate (Pawlowic et al., 2019). In addition, the growth of purine enzyme deficient C. parvum mutants was perturbed in the presence of pharmacological inhibitors that also targeted host cell purine metabolism, suggesting that high levels of host purines were required for parasite replication to compensate for the loss of a parasite purine pathway enzyme (Pawlowic et al., 2019). This combination of genetic and pharmacological results supports a revised model for purine acquisition based on evidence that C. parvum directly imports host cell GTP and ATP (Fig. 9.2B) (Pawlowic et al., 2019). The import of host cell ATP energy by C. parvum is consistent with the parasite’s lack of oxidative phosphorylation and inconsistent patterns of expression of glycolytic exzymes during the parasite life cycle (Pawlowic et al., 2019). Additional studies are still necessary to identify the ATP and GTP import mechanism and to elucidate whether C. parvum possesses additional novel purine pathway activities that are not currently predicted by the genome sequence. 9.2.3.3 Purine salvage pathways in Plasmodium falciparum The purine salvage and interconversion pathways are more diverse in P. falciparum than C. parvum and may be slightly less robust

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than the pathways present in T. gondii. Yet, P. falciparum (and Plasmodium spp.) uniquely possess enzymes of purine metabolism that are highly adapted to the intracellular challenges faced by this parasite. Within the Apicomplexa, Plasmodium spp. is unique in surviving and replicating within erythrocytes. The erythrocyte is a highly differentiated cell type that has lost capabilities normally present in other mammalian cell types and therefore has very limited nucleotide requirements. The human erythrocyte also has no nucleus, does not synthesize DNA, RNA, or protein, and also lacks both purine and pyrimidine de novo synthetic pathways. To fulfill its nucleotide requirements, P. falciparum appears to be capable of transporting the purine nucleobases hypoxanthine, xanthine and guanine, as well as the purine nucleosides, adenosine, inosine and guanosine. P. falciparum possesses appropriate enzymic machinery for the interconversion and incorporation of any of these six potential purine sources (Chaudhary et al., 2004) (Fig. 9.3). P. falciparum has no reverse pathway from guanylate to adenylate nucleotides, and while the host-supplied xanthine, guanine, or guanosine, collectively, could meet the parasite’s demand for guanylate nucleotides, these precursors could not supply any adenylate nucleotides. Host adenosine, inosine, and hypoxanthine, individually or collectively, are therefore likely to be necessary for meeting the parasite’s demands for adenylate and guanylate nucleotides. While the parasite can transport additional purine compounds, their physiological relevance, if any, is not clear at this time. For example, adenine is transported by P. falciparum, yet this purine is not incorporated into nucleotides because the parasite lacks APRT and ADE genes and activities (Chaudhary et al., 2004). Similar to T. gondii, P. falciparum can convert adenosine to inosine via ADA. Inosine is then

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FIGURE 9.3 Model of purine transport and salvage pathways in Plasmodium falciparum. The host-cell cytosol, parasitophorous vacuole membrane, parasitophorous vacuole space, and the parasite plasma membrane are indicated. Purine transporters are shown as cylinders resting in the parasite plasma membrane. Relevant purines and the enzymic machinery managing purine interconversion and salvage of host purine nucleobases and nucleosides into the adenylate (AMP) and guanylate (GMP) nucleotide pools are shown. Solid lines and arrows depict active pathways. Where information on purine flux is available, the weighting of the pathway is emphasized by the weight of the lines and arrows. The weighting of pathways described in this figure reflects the most likely predictions from available data; however, the weightings shown in this figure are only hypothetical and the purine flux of host purine to the parasite, as well as interconversion and incorporation within the intracellular parasite, remains to be experimentally tested. Substrates of each enzyme activity are shown on the side of the solid line and the product(s) of each enzyme activity are shown on the arrowhead side. The enzyme activity responsible for each interconversion step is shown in capital italicized text beside the arrowhead line. HXGPRT represents the major pathways for salvage and incorporation into the nucleotide pool. ADA, Adenosine deaminase; ADSL, adenylosucccinate lyase; ADSS, adenylosuccinate synthetase; AMPD, AMP deaminase; GMPS, GMP synthetase; HXGPRT, hypoxanthinexanthineguanine phosphoribosyltransferase; IMPDH, inosine 50 -monophosphate dehydrogenase; MTA, 50 -methylthioadenosine; MTI, 50 methylthioinosine; PNP, purine nucleoside phosphorylase; PRPP, 5-phosphoribosyl-1-pyrophosphate. SS, spermidine synthetase. Enclosed triangles indicate: PS, polyamine synthesis; S, spermidine.

converted to hypoxanthine by parasite PNP (Fig. 9.3). Similar to human and T. gondii PNP, P. falciparum PNP does not recognize either xanthosine or adenosine as a good substrate

(Kicska et al., 2002a). Hypoxanthine is incorporated into the nucleotide pool as IMP by HXGPRT. Once IMP is available the parasite can meet its entire demand for adenylate and

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guanylate nucleotides. Notably, P. falciparum lacks any detectable AK gene or activity. IMP is converted to AMP in two steps by sequential reactions of ADSS and ADSL. This is the only pathway to AMP. The parasite can balance IMP and AMP pools in the reverse reaction of AMP deamination to IMP (Fig. 9.3). Since P. falciparum has no direct route to AMP from adenosine the most important purine for incorporation into purine nucleotides becomes hypoxanthine. Hypoxanthine is the only purine compound that can completely satisfy the parasite’s demand for both adenylate and guanylate nucleotide pools. As also seen in both T. gondii and C. parvum, IMP is converted to XMP by parasite IMPDH and subsequently to GMP by parasite GMPS (Figs. 9.1 and 9.2). XMP and GMP are also incorporated in P. falciparum through the phosphoribosylation of xanthine and guanine, respectively. Thus the parasite ultimately incorporates all purine nucleotides via parasite HXGPRT activity and possesses a minimum of seven distinct purine interconversion or incorporation activities (Fig. 9.3). Potential drug targets in the P. falciparum purine salvage pathway: The machinery used by P. falciparum to satisfy its purine auxotrophy suggests that purine transporters, parasite HXGPRT, certain purine interconversion enzymes, or a recently discovered novel purine recycling or salvage pathway may all be amenable targets for drug development. Due to the absence of AK activity in P. falciparum, ADA and PNP activities were recognized as potential drug targets in early studies (Daddona et al., 1984, 1986; Webster et al., 1984). Hypoxanthine could be directly transported by the parasite from the parasitized host cell, or alternatively, hypoxanthine could be supplied from transported inosine and adenosine via ADA and PNP activities. Host erythrocyte hypoxanthine pools may increase during infection from host catabolism of ATP in deteriorating parasitized erythrocytes. Host erythrocyte

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ATP is catabolized sequentially, via ADP, AMP, adenosine, and inosine to produce hypoxanthine via host PNP (Ting et al., 2005). It is known that depletion of hypoxanthine effectively inhibits P. falciparum replication in vitro (Berman and Human, 1991; Berman et al., 1991). Metabolic analysis under conditions of a limiting hypoxanthine supply has shown that P. falciparum invokes specific purine recycling pathways to compensate for hypoxanthine deprivation. However, these compensatory mechanisms are not sufficient to maintain the viability of the parasite (Tewari et al., 2019). Thus the physiological source of hypoxanthine is a critical question to be answered to optimize strategies that can inhibit purine acquisition in Plasmodium ssp. infections. If the bulk of incorporated hypoxanthine is derived strictly in the parasite cytosol from transported host purines, the parasite ADA and PNP activities may be the optimal drug targets. Complicating this analysis, however, are studies that demonstrate P. falciparum to be extremely unusual in possessing a metabolic pathway from 50 methylthiopurines to hypoxanthine. If P. falciparum can transport 50 -methylthiopurines from the erythrocyte environment the parasite can potentially salvage additional hypoxanthine by this novel pathway (discussed next). Genetic disruption of P. falciparum PNP produced a null strain that replicated normally with excess hypoxanthine supplementation, but this strain exhibited a significant growth defect at low physiological concentrations of hypoxanthine (Madrid et al., 2008). The P. falciparum PNP null strain exhibited no PNP activity on inosine or MTI (Madrid et al., 2008). Inhibition of PNP in P. falciparum: P. falciparum possesses a structurally novel PNP enzyme that is inhibited by immucillin compounds (Kicska et al., 2002a). Several immucillin compounds bind P. falciparum PNP with low nanomolar inhibition constants and inhibit enzyme activity. Human PNP is also the target of immucillins, with subnanomolar inhibition

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constants, and currently, these compounds are being investigated for therapy in cancer and immunosuppression chemotherapy. ImmucillinH is an immucillin that inhibits both human and P. falciparum PNP. Immucillin-H induced purine-less death in P. falciparum infection (Kicska et al., 2002b). The parasite growth inhibition induced by immucillin-H is reversed by supplementing culture medium with high doses of hypoxanthine, suggesting that the effect of immucillin-H is primarily to block acquisition of essential purines resulting in purine-less death (Kicska et al., 2002b). Crystal structure and energetic mapping studies of P. falciparum PNP and immucillin interactions are under investigation to identify more selective inhibitors of the parasite PNP (Lewandowicz et al., 2005; Shi et al., 2004). A highly potent immucillin, DADMe-immucillin-G, which inhibits both Plasmodium ssp. and human PNP, is able to control P. falciparum infection in the Aotus primate model (Cassera et al., 2011). A drug screen has identified several new and highly selective inhibitors of P. falciparum PNP (Kagami et al., 2017). Inhibition of HXGPRT in P. falciparum: Early studies on inhibitors of P. falciparum growth supported the concept that the parasite HXGPRT activity was a candidate target for drug development (Queen et al., 1990). P. falciparum growth in vitro is inhibited by purine analogs 6-mercaptopurine, 6-thioguanine, and 8-azaguanine. Studies have not been performed to establish whether the mechanism of 6-mercaptopurine inhibition in P. falciparum is similar to the mechanism in mammals, which requires activation by phosphoribosylation via host HGPRT, followed by recognition of GMPS to incorporate the toxic analog into the guanylate nucleotide pool for its toxic incorporation into nucleic acids (Elion, 1989a,b). Early studies characterized the enzymic properties and the gene encoding the P. falciparum HXGPRT (Queen et al., 1988; Vasanthakumar et al., 1989,

1990). The crystal structure of the P. falciparum HXGPRT revealed unique structural features of the parasite enzyme compared to the structure of the human enzyme (Shi et al., 1999a,b). Additional studies have investigated enzyme mechanisms and features directing substrate specificity, and lead transition state inhibitors have been reported (Li et al., 1999; Sarkar et al., 2004; Shi et al., 1999a; Thomas and Field, 2002). Based on the purine incorporation pathways elucidated for P. falciparum, selectively blocking incorporation of hypoxanthine by inhibition of HXGPRT has been generally predicted to induce purine-less death in P. falciparum (Fig. 9.3). Screens have been established to identify selective substrates of the P. falciparum HXGPRT (Shivashankar et al., 2001). It is also possible that the xanthine phosphoribosyltransferase activity of P. falciparum HXGPRT, which is absent in the human host, may also support a mechanism for incorporating toxic xanthine analogs to block parasite replication, analogous to inhibition of T. gondii replication by 6-thioxanthine (Pfefferkorn et al., 2001). Acyclic immucillin phosphonates (AIPs) and cell permeable AIP prodrugs have been developed as inhibitors of P. falciparum HXGPRT (Hazleton et al., 2012). The early immucillin transition state inhibitors of HGXPRT were potent but were unable to cross biological membranes and, consequently, were ineffective against cultured parasites. The AIP prodrugs are biologically stable inhibitors that block proliferation of cultured parasites and may represent a new strategy to target purine pathways of malaria. Inhibition of guanylate nucleotide pools in P. falciparum: Guanylate nucleotides could arise from guanine supplied by the host, guanine derived from host guanosine by the parasite PNP, and xanthine via HXGPRT. The erythrocyte host of P. falciparum lacks a guanosine kinase activity, and biochemical evidence suggests that the host guanine and guanosine

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pools available to P. falciparum are limited (Reyes et al., 1982). P. falciparum growth in vitro is inhibited by psicofuranine, an inhibitor of bacterial GMPS (Mcconkey, 2000). Psicofuranine inhibition of parasite growth is antagonized in the presence of guanine supplemented parasite growth medium (Mcconkey, 2000). These observations provide strong evidence that the primary route to GMP is through GMPS and demonstrates that during in vitro cultivation, the potential supply of host guanine and guanosine is limited (Fig. 9.3). Unfortunately, the possible importance of host xanthine in GMP formation is not clarified by these studies. Xanthine can be incorporated into the GMP pool by the sequential reactions of HXGPRT and GMPS. Human erythrocytes are reported to have a concentration of xanthine of B3.6 μM, which may be sufficient for physiological transport and incorporation by HXGPRT and GMPS (Traut, 1994). MPA, a highly specific inhibitor of IMPDH, blocks P. falciparum replication at micromolar doses in vitro (Queen et al., 1990). While these observations suggest that host xanthine pools are not sufficient to bypass the inhibition at parasite IMPDH, these results cannot be conclusively interpreted because MPA also inhibits host IMPDH and depletes host guanylate nucleotide pools. Inhibition of adenylate nucleotide pools in P. falciparum: The only recognized pathway to AMP from the IMP nucleotide pool is through sequential reactions catalyzed by ADSS and ADSL. P. falciparum ADSL has been cloned, expressed in E. coli for kinetic analysis, and a crystal structure was determined (Eaazhisai et al., 2004; Jayalakshmi et al., 2002). A unique reaction mechanism has been described for this essential parasite enzyme (Raman et al., 2004). Targeting either ADSS and ADSL, or both, is predicted to completely deplete P. falciparum of essential adenylate nucleotides and inhibit parasite replication (Fig. 9.3).

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9.2.3.4 Alternative purine pathways in Apicomplexa Of the apicomplexans considered in this chapter, only P. falciparum possesses a unique alternative pathway to purines via recycling of 50 -methylthiopurines generated during polyamine metabolism (Ting et al., 2005) (Fig. 9.3). While earlier studies hinted at the existence of this novel pathway (Sufrin et al., 1995), other studies have helped to illustrate an important physiological role for the pathway and in vitro studies have validated components of the pathway to be drug targets. The growth inhibition achieved by treating parasite-infected erythrocytes with immucillins (Kicska et al., 2002a,b) may be multifaceted, because the P. falciparum PNP plays a dual role in both conversion of inosine to hypoxanthine, as well as in recycling MTI to hypoxanthine (Ting et al., 2005). Therefore P. falciparum has a novel pathway to hypoxanthine. Current evidence suggests this novel pathway plays an important role in parasite metabolism during replication in erythrocytes (Ting et al., 2005). The intersection of polyamine and purine metabolism in P. falciparum: Erythrocytes do not synthesize polyamines, and P. falciparum, unlike T. gondii, must synthesize its own polyamines. Therefore P. falciparum replication can be blocked by difluoromethylornithine (DFMO), a mechanism-based inhibitor of ornithine decarboxylase (ODC) involved in conversion of ornithine to putrescine in polyamine synthesis (Muller et al., 2001). The polyamine biosynthesis pathway forms a molecule of 5’methylthioadenosine (MTA) for each molecule of spermidine, or spermine, that is synthesized. In humans and other organisms the MTA is typically recycled to regenerate both adenine and methionine pools. Surprisingly, the genes associated with purine salvage (APRT) and recycling of MTA into the methionine pool are absent in the P. falciparum genome (Chaudhary et al., 2004; Gardner et al., 2002a,b). However,

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P. falciparum ADA was demonstrated to have the novel ability to recognize MTA as a substrate and convert MTA to MTI (Ting et al., 2005). In addition, P. falciparum PNP was found to have the unique ability to recognize MTI as a substrate and convert MTI to hypoxanthine. The structural basis for specificity of P. falciparum PNP for MTI has been reported (Donaldson et al., 2014b). This novel pathway was proven to be functional when exogenously supplied MTI was incorporated into P. falciparum nucleic acids (Ting et al., 2005). Therefore the unique substrate properties of P. falciparum ADA and PNP comprise a novel metabolic pathway from MTA to MTI to hypoxanthine for selective recycling of purines from polyamine biosynthesis (Fig. 9.3). Early studies suggested that P. falciparum expressed very abundant levels of ADA, PNP, and HXGPRT when compared to corresponding activities present in the host erythrocyte, therefore indicating a potentially important secondary role beyond their role in normal purine pathways (Reyes et al., 1982). Although P. falciparum grows normally in PNP and ADA deficient host erythrocytes (Daddona et al., 1984, 1986), inhibitors of parasite and host ADA such as coformycin, deoxycoformycin, and L-ribosyl analogs of corformycins block P. falciparum replication (Daddona et al., 1984; Ting et al., 2005). Selective inhibition of P. falciparum PNP, but not host PNP, with MTimmucillin-H blocked parasite replication nearly as efficiently as immucillin-H (Ting et al., 2005). This apparent selective inhibition indicates P. falciparum PNP is an essential parasite activity. This conclusion is further supported by the significantly decreased ability of MTimmucillin-H to inhibit the PNP knockout strain of P. falciparum (Madrid et al., 2008), mechanistically supporting parasite PNP as a viable drug target for selective inhibition. It is unlikely that MT-immucillin-H inhibits the polyamine pathway by product inhibition at

MTA since P. falciparum ADA converts MTA to MTI, and significantly, exogenously supplied hypoxanthine reverses the in vitro inhibition achieved by treatment with MT-immucillin-H or immucillin-H (Kicska et al., 2002b; Ting et al., 2005) (Fig. 9.3). These observations suggest that the host pool (or flux) of hypoxanthine alone is likely to be insufficient to fully support parasite growth during in vitro cultivation. Thus the host inosine pool, the host adenosine pool, or hypoxanthine recovered in the novel purine recycling pathway is likely to be required for normal replication of P. falciparum (Fig. 9.3). These in vitro observations validate the P. falciparum enzymes ADA and PNP for further drug discovery, as well as further validating the importance of enzymes involved in polyamine biosynthesis (Kicska et al., 2002b; Ting et al., 2005). Highly selective inhibitors of P. falciparum ADA have been identified (Tyler et al., 2007). However, the selection of drug resistance in these targets could be a problem for drug design. For example, while DADMeimmucillin-G causes purine starvation and parasite death in vitro and in primate infection models, the exposure of P. falciparum to this drug over many generations led to increased copy number and point mutations in the parasite PNP that were associated with drug resistance (Ducati et al., 2018). In addition, emerging evidence suggests that PNP may be the target of some “old” antimalarial drugs with unknown mechanism of action. Quinine and mefloquine, with previously unknown mechanisms of action, were recently shown to target PNP by binding to its active site (Dziekan et al., 2019). It still remains to be determined in confirmatory studies whether parasite resistance to quinine or mefloquine is associated with genetic adaptations in PNP. Recycling or salvage? Another key question still to be answered is whether the novel purine recycling pathway in P. falciparum plays any direct role in purine salvage. Exogenously supplied MTI is transported by P. falciparum

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and incorporated into parasite nucleic acids (Ting et al., 2005) (Fig. 9.3). Any supply of host MTA transported by P. falciparum could be salvaged to hypoxanthine and incorporated into the IMP nucleotide pool (Fig. 9.3). MTA may represent a significant source of purines for P. falciparum if MTA is abundant in erythrocytes and the parasite has a transporter capable of stealing this molecule from the host cytosol/ parasitophorous vacuole space. Alternatively, it is also plausible that P. falciparum may either secrete ADA into the erythrocyte cytosol or parasitophorous vacuole space to convert host MTA to MTI or secrete PNP into the erythrocyte cytosol or parasitophorous vacuole space to convert host MTI to hypoxanthine. Both of these strategies could increase the supply of purines to P. falciparum because both MTI and hypoxanthine are transported and incorporated into parasite nucleic acids (Ting et al., 2005) (Fig. 9.3). Therefore studies of the localization of P. falciparum ADA and PNP in parasitized erythrocytes, as well as determining the capabilities of MTI and MTA transport in parasitized erythrocytes are necessary to fully resolve the major physiological function of this novel pathway. 9.2.3.5 Polyamines in Apicomplexa Host erythrocytes do not synthesize polyamines. However, growth of P. falciparum is blocked by DFMO, a selective ODC inhibitor (Muller et al., 2001). ODC catalyzes the conversion of ornithine to putrescine in the first step in the synthesis of spermidine and spermine (Tabor and Tabor, 1985). Incorporation of radiolabeled glutamine into the ornithine pool in P. falciparum was minor, suggesting that the parasite possesses another major route to acquire or synthesize ornithine (Gafan et al., 2001). In organisms that synthesize polyamines, the other major routes are through enzymes for arginase and ODC, or arginine decarboxylase (conversion of arginine to agmatine) and agmatinase. Arginase directly converts arginine to

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ornithine, while agmatinase bypasses ornithine entirely by converting agmatine to putrescine (Wu and Morris, 1998). Sensitivity of P. falciparum to DFMO suggests that the ODC activity is essential. A P. falciparum arginase gene has been cloned and the recombinant enzyme when expressed possessed arginase activity with no detectable agmatinase activity (Muller et al., 2005). As discussed in the pyrimidine sections of this chapter, all of the apicomplexan parasites are incapable of de novo arginine synthesis and therefore must acquire arginine from the host. Arginine taken from the host by P. falciparum is utilized in protein synthesis as well as polyamine biosynthesis. The protozoan parasite Leishmania spp. also utilizes an arginase activity that is essential for polyamine biosynthesis (Roberts et al., 2004; Satriano, 2003). P. falciparum has a novel ODC activity that exists as a bifunctional enzyme with Sadenosylmethionine decarboxylase. This bifunctional enzyme enables a balanced synthesis of putrescine from ornithine without involving domaindomain interactions (Birkholtz et al., 2004; Krause et al., 2000; Muller et al., 2000; Wrenger et al., 2001). P. falciparum can transport putrescine and spermidine (Niemand et al., 2012). Putrescine and deoxyadenosylmethionine are combined by spermidine synthase generating a single molecule of spermidine and a molecule of MTA, which is recycled to MTI by ADA and then to hypoxanthine by PNP (Chaudhary, 2005; Muller et al., 2000; Ting et al., 2005) (Fig. 9.3). P. falciparum also transports host spermidine (Muller et al., 2001). No P. falciparum gene ortholog for a spermine synthase has been identified (Chaudhary, 2005; Chaudhary and Roos, 2005). By contrast to P. falciparum, C. parvum appears to biosynthesize polyamines by the pathway predominantly present in bacteria and plants involving arginine decarboxylase and agmatinase (Yarlett et al., 1996). Based on the extraordinary transport capacities of

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C. parvum, it is also likely that this parasite may also transport putrescine or other polyamines from the rich environment of the human gut. C. parvum ADC activity is inhibited by difluoromethylarginine and is unaffected by DFMO (Keithly et al., 1997). Since C. parvum lacks any gene ortholog or activity for PNP (Fig. 9.2), this parasite is incapable of recycling 50 -methylthiopurines back into the parasite purine pool. The origin of polyamines in T. gondii is not clearly experimentally resolved. The weight of the current evidence suggests that this parasite is auxotrophic for polyamines and must inhabit host cells that can supply polyamines. The parasite appears to possess transporters capable of transporting ornithine, putrescine, and other polyamines (Chaudhary, 2005; Chaudhary et al., 2006). T. gondii appears to lack ODC activity (Sarkar et al., 2004), and growth of tachyzoites is unaffected by DFMO (Chaudhary, 2005; Chaudhary and Roos, 2005). However, the T. gondii genome does reveal the presence of a gene ortholog with homology to the ADC/ODC gene family (Kissinger et al., 2003). If functional, this gene ortholog would represent a potential ADC based on lack of sensitivity to DFMO. Currently, no gene ortholog can be identified for a member of the related arginase/agmatinase gene family or any other gene member of the polyamine biosynthetic pathway (Chaudhary, 2005; Chaudhary and Roos, 2005). Collectively, these data suggest that T. gondii is incapable of polyamine biosynthesis and most likely relies upon direct transport and salvage of preformed polyamines supplied by the host cell. The absence of any gene ortholog for spermidine synthase or spermine synthase suggests the parasite does not produce MTA. 50 -methylthiopurines could not be recycled into the parasite purine pool since T. gondii PNP cannot utilize MTI as a valid substrate (Chaudhary et al., 2006). Adaptation of the polyamine and purine pathways in Apicomplexa: The selective adaptation of P. falciparum ADA and PNP enzymes to recycle 50 -methylthiopurines to hypoxanthine

may have arisen in response to the loss of AK activity (Ting et al., 2005). The potential purine flux is reflected by both the demands of the parasite and the purine limitations of the host erythrocyte. Replication of P. falciparum occurs by schizogony, where DNA replication and nuclear division are coordinated in a narrow window of time. Schizogony, where many daughter nuclei are rapidly formed, may demand an increased requirement for purine flux into P. falciparum. By contrast, T. gondii replicates by endodyogeny and creation of daughter parasites every B7 hours. Endodyogeny, where one daughter nucleus is formed from one mother nucleus, may inherently require a lower flux of purines to sustain replication. The only feature of the polyamine pathway shared in all apicomplexans and protozoan parasites is the use of spermidine as a substrate for deoxyhypusine synthase (DHS). DHS with deoxyhypusine hydroxylase (DOHH) modify the translation elongation factor eIF5A with covalent attachment the amino acid hypusine. DHS and DOHH are present in a wide range of parasitic protozoa (Phillips, 2018). C. parvum can synthesize hypusine (Mittal et al., 2014), and DHS and DOHH are essential activities in P. falciparum (Zhang et al., 2018). The identification of attractive drug targets in polyamine acquisition (Phillips, 2018; Roberts and Ullman, 2017) as well as the recent identification of new drugs that can reduce polyamine acquisition (El Bissati et al., 2019; Panozzo-Zenere et al., 2018) is revealing the important roles of this pathway in parasite biology in Apicomplexa.

9.3 Pyrimidines Pyrimidines are essential components of nucleic acids and molecules in pyrimidine metabolism intersect with many aspects of cellular metabolism in apicomplexans. Most apicomplexan parasites have retained the ability

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for de novo synthesis of the parental pyrimidine molecule, uridine 50 -monophosphate (UMP). The apicomplexan parasites that rely on the de novo synthetic pathway are therefore dependent on an appropriate supply of precursor molecules for the biosynthesis of pyrimidines. These precursor molecules include small molecules such as bicarbonate, amino acids, and purine nucleotides. Consequently, acquisition of amino acids and satisfying the purine auxotrophy of apicomplexan parasites are necessary for pyrimidine biosynthesis. Most apicomplexans, including T. gondii and P. falciparum, have functional pathways for de novo pyrimidine synthesis. T. gondii is unusual in also possessing significant pyrimidine salvage activities. However, the pyrimidine biosynthetic pathway is essential for replication of both T. gondii and P. falciparum. Some apicomplexans, such as C. parvum, have lost the ability to synthesize UMP by the de novo pathway and completely rely on salvage pathways to acquire required pyrimidines from the host. In these apicomplexans, acquisition of pyrimidines is completely dependent on transport and salvage of preformed pyrimidine nucleobases and nucleosides from the host. C. parvum has uniquely acquired several pyrimidine salvage activities not observed in any other apicomplexan parasite. Relatively little experimental work has been performed on the associated transport pathways essential for pyrimidine biosynthesis or the relationships and likely cross-regulatory talk likely to occur between the pyrimidine and purine pathways. Interestingly, pyrimidine starvation is one of the triggers used to experimentally induce in vitro stage differentiation in T. gondii from the tachyzoite to the bradyzoite stages (Bohne and Roos, 1997; Roos et al., 1997). Recent studies have clarified the functional organization of pyrimidine metabolism in the Apicomplexa and have revealed new strategies to target essential pyrimidine acquisition pathways in apicomplexan parasites.

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9.3.1 De novo pyrimidine synthesis in Apicomplexa The human host of T. gondii, P. falciparum, and C. parvum is capable of significant salvage and biosynthesis of pyrimidines such that either pathway can supply the pyrimidine requirements. Early investigation of nucleotide metabolism determined that the Apicomplexa are generally capable of synthesizing pyrimidines from amino acid precursors glutamine and aspartic acid by the same six-step pathway present in their hosts. While C. parvum cannot synthesize pyrimidines de novo, T. gondii and P. falciparum possess the intact pyrimidine biosynthetic pathway (Asai et al., 1983; Hill et al., 1981a,b; O’sullivan et al., 1981; Reyes et al., 1982; Schwartzman and Pfefferkorn, 1981) (Fig. 9.4). The pyrimidine biosynthetic pathway starts with carbamoyl phosphate synthetase (CPS) II (CPSII). CPSII combines two molecules of ATP, L-glutamine, and bicarbonate in a sequence of elegant chemical reactions at multiple active sites to produce a molecule of carbamoyl phosphate (Holden et al., 1999; Kothe et al., 2005). Carbamoyl phosphate is then fused with L-aspartate in the second step by aspartate carbamoyltransferase (ATC) to produce carbamoyl aspartate. Dihydroorotase (DHO) then converts carbamoyl aspartate to dihydroorotate in the third step. In the fourth step of the pathway, dihydroorotate dehydrogenase (DHODH) creates orotate from dihydrooratate and in doing so also creates electrons to coenzyme Q (CoQ) in the mitochondrion. Orotate is combined with 5phosphoribosyl-1-pyrophosphate (PRPP) in the fifth step by orotate phosphoribosyltransferase (OPRT) to produce orotidine-50 -monophosphate (OMP). UMP, the parent pyrimidine mononucleotide and the precursor of all other pyrimidine nucleotides, is finally produced in the sixth step via the decarboxylation of OMP by OMP decarboxylase (OMPDC). In all Apicomplexa, UMP is phosphorylated to UTP

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FIGURE 9.4 Pyrimidine biosynthesis and salvage pathways in Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. Pathways are shown for pyrimidine biosynthesis in T. gondii and P. falciparum (top), for pyrimidine salvage in T. gondii (bottom left), and for pyrimidine salvage in C. parvum (bottom right). Solid lines and arrows depict active pathways. Substrates of each enzyme are shown on the side of the solid line and the product(s) of each enzyme activity are shown on the arrowhead side. The enzyme activity responsible for each conversion step is shown in capital italicized text beside the arrowhead line. ATC, Aspartate carbamoyltransferase; CMP, cytidine 50 -monophosphate; CoQ, mitochondrial coenzyme Q; CPSII, carbamoyl phosphate synthetase II; CYTD, cytidine deaminase; dCYTD, deoxycytidine deaminase; DHO, dihydroorotase; DHODH, dihydroorotate dehydrogenase; dTMP, thymidine 50 -monophosphate; dUP, deoxyuridine phosphorylase; OMPDC, orotidine 50 -monophosphate decarboxylase; OPRT, orotate phosphoribosyltransferase; PRPP, 50 -phosphoribosyl-1-pyrophosphate; TK, thymidine kinase; UMP, uridine 50 -monophosphate; UP, uridine phosphorylase; UPRT, uracil phosphoribosyltransferase; UPRTUK, uracil phosphoribosyltransferaseuridine kinase.

in two sequential steps by UMP kinase and nucleoside diphosphate kinase. UTP is converted to CTP by CTP synthase (CTPS) in a rate-limiting step in all organisms. CTPS is the only known route for de novo synthesis of cytidine nucleotides. T. gondii, P. falciparum and C.

parvum all express a CTPS gene (Abrahamsen et al., 2004; Hendriks et al., 1998; Kissinger et al., 2003; Xu et al., 2004). CTPS was found to be essential for T. gondii, and the localization of the enzyme is spatially regulated (NarvaezOrtiz et al., 2018). Conversion of ribonucleotides

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to deoxyribonucleosides occurs at the level of nucleoside diphosphate kinase and is catalyzed by a ubiquitous ribonucleotide reductase present in all apicomplexans. The structure of the T. gondii nucleoside diphosphate kinase was recently determined (Lykins et al., 2018). 9.3.1.1 Organization and regulation of carbamoyl phosphate synthetase II in Apicomplexa A key regulatory enzyme in the apicomplexan pyrimidine biosynthetic pathway is the first step encoded by CPSII. In mammalian cells, CPSII catalyzes the rate-limiting step and controls the flux through the pyrimidine biosynthetic pathway (Evans and Guy, 2004; Jones, 1980). The architecture of CPSII enzymes from T. gondii and P. falciparum is unique to the Apicomplexa and other protozoan parasites. Leishmania spp., Trypanosoma spp., Babesia bovis, Plasmodium spp., and T. gondii encode a novel glutamine-dependent CPSII activity fused with an N-terminal glutamine amidotransferase (GAT) activity (Aoki et al., 1994; Chansiri and Bagnara, 1995; Flores et al., 1994; Fox and Bzik, 2003; Gao et al., 1998, 1999; Nara et al., 1998). By contrast, mammalian pyrimidine-specific CPSII is contained on the CAD gene encoding a multifunctional protein possessing the GAT domain fused via linkers of various lengths in order with CPSII, DHO and ATC (Davidson et al., 1993; Mori and Tatibani, 1978). In Saccharomyces cerevisiae a multifunctional protein contains, in order, GAT, CPSII, and ATC domains, but is missing the DHO activity found on mammalian CAD (Davidson et al., 1993). A different strategy was taken by plants, eubacteria, and archebacteria, which express a gene containing the monofunctional CPS and a separate gene encoding GAT (Jones, 1980; Zhou et al., 2000). While the P. falciparum CPSII gene possesses no introns, the T. gondii CPSII is encoded by a gene with a complex organization of 37 exons

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and 36 introns specifying a polypeptide of 1687 amino acids (Fox and Bzik, 2003). CPSII from T. gondii, P. falciparum (2391 amino acids), and B. bovis (1645 amino acids) are also much larger polypeptides than is CPSII from other species (Chansiri and Bagnara, 1995; Flores et al., 1994; Fox and Bzik, 2003). Relative to other CPS and GAT domains, the apicomplexan CPSII enzymes have unique sites of insertion within GAT domains, within CPS domains, in the linker region fusing the two CPS halves, as well as within the C-terminal allosteric regulatory domain (Fox and Bzik, 2003). Surprisingly, the architecture of CPSII activity in T. gondii and P. falciparum revealed the presence of a single glutamine-dependent CPSII gene and activity (Flores et al., 1994; Fox and Bzik, 2002, 2003). The existence of a single CPSII in T. gondii is highly unusual for a eukaryotic organism. In many prokaryotes a single CPS polypeptide is typically found in this CPS activity and is responsible for producing carbamoyl phosphate, the precursor molecule for both pyrimidines and arginine. Consequently, disruption of E. coli CPS produces a dual pyrimidine and arginine auxotrophy (Beckwith et al., 1962). Consequently, in many eukaryotes, two distinct CPS genes and activities are found, a glutamine-dependent CPSII linked with pyrimidine biosynthesis and a mitochondria-associated CPSI dedicated to arginine biosynthesis (Davis, 1986; Makoff and Radford, 1978). The carbamoyl phosphate produced by CPSI in many eukaryotes is sequestered in the mitochondria for immediate conversion to citrulline via ornithine carbamoyltransferase. Arginine is produced from citrulline in two steps by the sequential actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) (Davis, 1986; Makoff and Radford, 1978). The availability of a CPSII knockout mutant in T. gondii (discussed next) enabled a functional determination of whether the sole CPSII in Apicomplexa is responsible for pyrimidine

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and arginine biosynthesis (Fox et al., 2004). This study conclusively demonstrated that the T. gondii CPSII and the carbamoyl phosphate product are dedicated only toward pyrimidine biosynthesis. T. gondii is a natural arginine auxotroph. The arginine auxotrophy of T. gondii is rescued by supplementing growth media with either arginine or citrulline. Using mutant host cells, it was demonstrated that rescue with citrulline was dependent on the presence of host-cell AS and AL activities. These experiments demonstrated the functional absence of any arginine biosynthetic enzyme activity in T. gondii, and this conclusion has been verified by the absence of corresponding gene orthologs in the T. gondii genome. Other apicomplexans such as C. parvum and P. falciparum also are natural arginine auxotrophs. This natural arginine auxotrophy and arginine depletion in T. gondii infection have been linked with the differentiation of tachyzoites to bradyzoite containing cysts in vitro (Fox et al., 2004). These observations suggested that local depletion of arginine by inducible nitric oxide synthase during the host immune response to T. gondii infection is likely to trigger cyst development (Fox et al., 2004; Wu and Morris, 1998). 9.3.1.2 Pyrimidine biosynthetic pathways in Apicomplexa Conceptually, inhibition of UMP synthesis by the pyrimidine biosynthetic pathway is likely to be a more potent strategy than blocking only thymine nucleotide synthesis and DNA synthesis indirectly through inhibition of folate metabolism (discussed next), because inhibition of pyrimidine biosynthesis will cause starvation for UTP, CTP, and dTTP nucleotides essential for both RNA and DNA synthesis (Fig. 9.4). The apicomplexan CPSII enzymes exhibit several differences from the mammalian CPSII that provides a basis for chemotherapy. The novel and large amino acids insertions in T. gondii and P. falciparum CPSII may provide parasite-specific targets for

inhibiting CPSII and de novo pyrimidine synthesis in chemotherapy. Targeting ribozymes to a site corresponding to a novel P. falciparum CPSII insertion blocked replication of P. falciparum in vitro (Flores et al., 1997). A rapid microassay has been developed for P. falciparum CPSII activity that may be amenable for high-throughput assays (Flores and Stewart, 1998). The GAT activity of T. gondii CPSII has also been shown to be a possible target of acivicin (Fox and Bzik, 2003). Mammalian CPSII is an allosterically regulated enzyme with activity activated by 5phosphoribosyl-1-pyrophosphate (PRPP) and activity suppressed by UTP (Jones, 1980). P. falciparum CPSII is activated by PRPP, and high UTP concentrations cause UTP inhibition (Gero et al., 1984). T. gondii CPSII is insensitive to activation by PRPP but is inhibited by UTP (Asai et al., 1983). The regulatory domain controlling allosteric regulation of CPS activity is contained within the C-terminal B150 amino acids of the polypeptide (Evans and Guy, 2004; Fresquet et al., 2000; Mora et al., 1999). Both the P. falciparum and the T. gondii CPSII enzymes possess significant amino acid insertions in the allosteric regulatory domain, as well as divergent amino acid composition (Flores et al., 1994; Fox and Bzik, 2003). The allosteric regulatory domain is the most divergent domain within the entire CPSII, suggesting that regulation of CPSII is unique in apicomplexans (Fox and Bzik, 2003). Genetic studies have identified several of the novel CPSII domains to be essential functional domains for enzyme activity or regulation of CPSII (Fox et al., 2009a). Other enzymes in the de novo pathway of T. gondii and P. falciparum are also under investigation as potential drug targets. T. gondii aspartate transcarbamoylase (ATC) catalyzes the second step and is a cytosolic monofunctional enzyme (Asai et al., 1983). Recombinant T. gondii ATC product has been produced and characterized (Mejias-Torres and Zimmermann,

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2002). The lack of any observed regulation on T. gondii ATC further supports the key regulatory role of CPSII in the de novo pathway of pyrimidine biosynthesis (Asai et al., 1983; Mejias-Torres and Zimmermann, 2002). The crystal structure of P. falciparum ATC has been determined (Lunev et al., 2016), and noncompetitive inhibitors of P. falciparum ATC have been discovered (Lunev et al., 2018). T. gondii DHO catalyzes the third step and is a cytosolic monofunctional enzyme, which is unresponsive to any nucleotide (Asai et al., 1983). DHO from T. gondii has been expressed and enzymatically characterized (Robles lopez et al., 2006). DHO has also been expressed and characterized in Plasmodium (Christopherson et al., 2004). The structure of the DHO enzyme from Plasmodium ssp. has been determined (Rashmi et al., 2017). The DHODH from T. gondii has been cloned and expressed (Satriano, 2003). A large number of inhibitors were tested on T. gondii DHODH, and several micromolar inhibitors have been identified (Hortua Triana et al., 2012). The T. gondii DHODH localizes to the parasite mitochondrion (Hortua Triana et al., 2016). The T. gondii DHODH catalytic activity is essential for synthesis of pyrimidines (Hortua Triana et al., 2016). Surprisingly, a second function, independent of catalytic activity, has been identified for the T. gondii DHODH. Mitochondrial localization of a catalytically deficient T. gondii DHODH was essential for parasite viability in the presence of uracil auxotrophy (discussed next) (Hortua Triana et al., 2016). This second noncatalytic function of DHODH may be essential for the maintenance of mitochondrial functions and membrane potential, possibly through the association and complexing of mitochondrial membrane proteins into functional respiratory chain complexes. The catalytic function of DHODH is essential for pyrimidine synthesis in T. gondii and P. falciparum. Understanding of structure, catalytic activity, and developing DHODH

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inhibitors has advanced rapidly for the P. falciparum DHODH (Phillips et al., 2008; Phillips and Rathod, 2010). Several compounds are demonstrating exquisite selectivity and low toxicity, and some of these compounds are rapidly approaching potential clinical use in drug therapy for malaria infection (Hoelz et al., 2018; Kokkonda et al., 2018; Llanos-Cuentas et al., 2018; Pavadai et al., 2016; Phillips et al., 2016; White et al., 2018; Xu et al., 2018). In T. gondii and P. falciparum, OPRT and OMPDC are encoded by separate genes (Krungkrai et al., 2004b) but appear to function in a multienzyme complex (Krungkrai and Krungkrai, 2016). The functional and kinetic properties of these activities have been reported (Krungkrai et al., 2004a,b, 2005). The structure of P. falciparum OPRT has been determined (Kumar et al., 2015; Takashima et al., 2012b) as well as the structure of the OMPDC (Novak et al., 2018; Takashima et al., 2012a). The OMPDC activity has great potential as a drug target due to the structure and mechanism of the enzyme (Novak et al., 2018) and due to the fact that the OMPDC gene and its activity are essential for synthesis of UMP in Plasmodium and T. gondii (Fox and Bzik, 2015). Inhibitors of parasite OMPDC have been investigated (Crandall et al., 2013; Krungkrai and Krungkrai, 2016; Novak et al., 2018; Takashima et al., 2012a). The properties of an artificially (genetically) fused recombinant OPRT-OMPDC enzyme were studied. Remarkably, the fused enzymes exhibit a nearly perfect catalytic activity. Compared to the natural enzymes that are expressed as independent gene products that complex together, the fused enzymes exhibited up to a 1000-fold enhancement in the Kcat (Paojinda et al., 2018). 9.3.1.3 Indirect inhibition of pyrimidine biosynthesis The pyrimidine biosynthetic pathway of apicomplexans is indirectly inhibited by atovaquone. Atovaquone, a naphthoquinone derivative, in

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combination with proguanil (PG) (Malarone) is clinically used to treat human malaria infections. Atovaquone is a structural analog of CoQ (ubiquinone) in the mitochondrial electron transport chain. Atovaquone collapses the membrane potential of Plasmodium spp. by inhibition of cytochrome b in the bc1 complex (complex III) of the parasite electron transport chain (Korsinczky et al., 2000; Srivastava et al., 1999). Blocking electron flow inhibits mitochondrial membraneassociated enzymes such as DHODH that requires electron transfer to CoQ when it oxidizes dihydroorotate to orotate in the fourth step of the pyrimidine biosynthetic pathway (Fig. 9.4). Interestingly, the sole function of mitochondrial electron transport in P. falciparum appears to be its role in regeneration of ubiquinone required as the electron acceptor for DHODH to sustain the essential de novo pyrimidine synthesis pathway (Painter et al., 2007). Resistance of Plasmodium spp. to atovaquone is associated with specific mutations within the parasite cytochrome b (Korsinczky et al., 2000; Srivastava et al., 1999). Atovaquone treatment of P. falciparum in vitro causes major accumulations of carbamoyl aspartate and dihydroorotate demonstrating the breakdown in electron flow disrupts DHODH and causes significant substrate accumulation leading to starvation of UMP (Seymour et al., 1994). Atovaquone is also an approved drug for treatment of acute toxoplasmosis. As with P. falciparum, the DHODH of T. gondii is most likely associated with mitochondrial membranes. DHODH purifies from T. gondii in the particulate fraction of tachyzoites and is inhibited by respiratory chain inhibitors (Asai et al., 1983). The DHODH gene of T. gondii is highly similar to the family of DHODH enzymes linked to the respiratory chain in mitochondria for their catalytic redox force (Satriano, 2003). T. gondii mutants resistant to atovaquone can be selected in vitro (Pfefferkorn et al., 1993), and these mutants possess mutations within

the T. gondii cytochrome b gene suggesting this function to be the target of atovaquone (McFadden et al., 2000).

9.3.2 Pyrimidine salvage in Apicomplexa While de novo pyrimidine synthesis is essential for T. gondii and P. falciparum, C. parvum lacks all six enzymes required for synthesis of UMP (Fig. 9.4). Therefore pyrimidine salvage in C. parvum presents a key target for drug development. 9.3.2.1 Salvage of pyrimidines in Cryptosporidium parvum The genome sequences of C. parvum and C. hominis demonstrated that Cryptosporidium spp. do not retain any of the six enzymes comprising the pyrimidine biosynthetic pathway (Abrahamsen et al., 2004; Puiu et al., 2004; Striepen and Kissinger, 2004; Xu et al., 2004) (Fig. 9.4). Thus C. parvum is completely dependent on the host for supplying pyrimidines that must be salvaged to meet the pyrimidine demand of the parasite. Remarkably, C. parvum expresses only three distinct pyrimidine salvage activities. C. parvum encodes a thymidine kinase (TK), a monofunctional uracil phosphoribosyltransferase (UPRT), and a bifunctional polypeptide having UPRT and uridine kinase (UK) (UPRTUK) activities (Striepen and Kissinger, 2004; Striepen et al., 2004). With this repertoire of salvage activities, C. parvum can potentially convert thymidine (TK), cytidine and uridine (UK), and uracil (UPRT) to their respective pyrimidine-50 -monophosphate (Fig. 9.4). Biochemical evidence supports the expression of active TK and UK enzymes in C. parvum infection based on incorporation of bromodeoxyuridine and cytosinearabinoside, respectively (Striepen and Kissinger, 2004; Striepen et al., 2004; Woods and Upton, 1998). However, recent expression of active TK indicates that AraT or AraC are not substrates for

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C. parvum TK (Sun et al., 2010). The proposed UPRT gene activity has not yet been experimentally validated by either enzyme assay or demonstrating the incorporation of uracil during C. parvum infection. The presence of TK and UK activities in C. parvum is highly unusual when compared with their absence in other apicomplexan parasites such as T. gondii and P. falciparum. Phylogenetic analysis indicates genes involved in nucleotide metabolism in C. parvum were incorporated by horizontal gene transfer from bacterial (TK), algal (UKUPRT), or protozoan sources (Abrahamsen et al., 2004; Huang et al., 2004; Striepen and Kissinger, 2004; Striepen et al., 2004). C. parvum may use the parasitized host cell, the gut environment itself, or both as the source of the pyrimidine nucleobase and nucleoside precursors. C. parvum growth in vitro is inhibited by cytosinearabinoside, a prodrug that is activated by UK (Pfefferkorn and Pfefferkorn, 1976; Woods and Upton, 1998). The UPRT as well as the bacterial-type TK activity of C. parvum potentially could be similarly exploited for selectively targeting incorporation of toxic analogs. C. parvum TK has been shown to activate the prodrugs 5-fluorodeoxyuridine (FUDR) and trifluorothymidine, and these compounds inhibit in vitro replication (Sun et al., 2010). Thus the genome sequence of C. parvum has revealed the lack of biosynthetic capability and several newly identified essential salvage activities that may be amenable for drug development. 9.3.2.2 Salvage of pyrimidines in Plasmodium falciparum P. falciparum is highly restricted in its ability to salvage pyrimidines marking the de novo pyrimidine synthetic pathway a key drug target. Even though salvage is highly restricted, P. falciparum is capable of significant transport and accumulation of pyrimidine nucleobases and nucleosides (Lauer et al., 1997).

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For example, P. falciparum is capable of accumulating thymidine and can incorporate exogenously supplied orotic acid and cytotoxic 5fluoroorotic acid (Lauer et al., 1997; Rathod et al., 1989). In parasite extracts, P. falciparum pyrimidine salvage activities for deaminase, nucleoside phosphorylase, nucleoside kinase, phosphoribosyltransferase, and nucleoside-50 kinase have been reported (Naguib et al., 2018). However, whether the increased activities observed in the infected erythrocyte in comparison to the uninfected erythrocyte is attributed to a parasite encoded gene activity or to an altered host enzyme activity is unknown. P. falciparum was reported to lack certain gene such as UPRT, TK, and UK that are required for the incorporation of pyrimidine nucleobases or nucleosides into the parasite nucleotide pool (Gardner et al., 2002a,b). It is also possible that the increased pyrimidine salvage enzyme activities observed in the infected erythrocyte (Naguib et al., 2018) are involved in the balancing or rebalancing of pyrimidine pools, rather than for significant incorporation into nucleotide pools. Consequently, conclusive evidence is still lacking as to whether P. falciparum has pyrimidine salvage capabilities that could support parasite growth in the erythrocyte. 9.3.2.3 Salvage of pyrimidines in Toxoplasma gondii The pyrimidine salvage capabilities of T. gondii, P. falciparum, and C. parvum are summarized in Fig. 9.4. Unlike P. falciparum or C. parvum, T. gondii has the six-step de novo synthetic pathway yet has also retained potentially significant salvage activities. Early labeling studies demonstrated that uracil was well incorporated into intracellular or extracellular tachyzoites but did not label host-cell nucleic acids (Pfefferkorn and Pfefferkorn, 1977a). Importantly, these initial studies demonstrated efficient and selective labeling of T. gondii nucleic acids by uracil, a method still in use

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today to assess parasite replication. The authors of this work also made the important biological observation that the host cell has no access to the parasite pyrimidine nucleotide pools. A parasite mutant was first selected to be resistant to FUDR and both intracellular (or extracellular) tachyzoites of this mutant were found to be deficient in their ability to incorporate uridine, deoxyuridine, and uracil (Pfefferkorn and Pfefferkorn, 1977a,b). Correspondingly, this mutant was also determined to be coresistant to 5-fluorouracil and 5fluoruridine. In pioneering work, the basis of the FUDR-resistant mutant was identified as a biochemical defect in the parasite UPRT activity (Pfefferkorn, 1978), thus demonstrating that the UPRT activity was nonessential to the tachyzoite stage. Genetic evidence also reveals that the UPRT activity is not essential for development or maintenance of latent T. gondii infection (Fox et al., 2011). These observations suggest that the de novo pyrimidine synthesis pathway is fully capable of supporting parasite replication during the acute and chronic stages of infection. Carbon dioxide starvation was used to suppress T. gondii de novo pyrimidine synthesis and growth of wild-type parasites could be rescued with uracil supplementation, whereas growth of the FUDR-resistant mutant was not rescued. These observations suggested that T. gondii is fully capable of growth in vitro by salvage of uracil in the absence of a functioning pyrimidine biosynthetic pathway to UMP (Pfefferkorn, 1978). T. gondii salvage capacities were comprehensively investigated using biochemical measurements of enzyme activities present in protein extracts prepared from isolated tachyzoites. T. gondii possesses salvage activities enabling the parasite to salvage a variety of pyrimidines including deoxycytidine, deoxyuridine, cytidine, uridine, and uracil (Iltzsch, 1993) (Fig. 9.4). T. gondii can also salvage pyrimidines arising from degradation of parasite nucleic

acids through degradation of dUMP, dCMP, and CMP to their corresponding nucleosides by nucleoside 50 -monophosphate phosphorylase (Iltzsch, 1993). The parasite cannot directly obtain phosphorylated nucleotides from the host cell but can transport and incorporate pyrimidine nucleobases and nucleosides (Iltzsch, 1993; Pfefferkorn and Pfefferkorn, 1977a,b). Uracil is the only pyrimidine compound that can be directly incorporated into the pyrimidine pool by conversion to UMP by the major UPRT activity. All other pyrimidine compounds are first catabolized to uracil mediated by parasite activities for uridine phosphorylase (UP), deoxyuridine phosphorylase, cytidine deaminase, and deoxycytidine deaminase (Fig. 9.4). This unique salvage strategy has been described as a “salvage funnel” to uracil (Pfefferkorn, 1978). The T. gondii genome sequence suggests that the uridine/deoxyuridine/thymidine phosphorylase activities and the cytidine/deoxycytidine deaminase activities are likely to be present on single polypeptides (Iltzsch, 1993; Kissinger et al., 2003). Although T. gondii has retained the ability to interconvert thymine and thymidine, the parasite lacks any TK activity and is incapable of salvaging thymidine. However, these compounds can be incorporated into T. gondii if the parasite is genetically modified with a TK gene derived from herpes simplex virus (Fox et al., 2001). Similarly, T. gondii cannot salvage cytosine unless the parasite is genetically modified with a bacterial cytosine deaminase (CD) gene (Fox et al., 1999). Potential inhibitors of pyrimidine salvage in T. gondii: Genetic studies on the pyrimidine salvage pathway have been performed in T. gondii. In early mutagenesis and biochemical studies, UPRT was demonstrated to be dispensable, and therefore UPRT could be used as a target for genetic inactivation (Pfefferkorn, 1978). Integration of plasmid DNA into the UPRT locus was selected using 5-fluorouracil (Donald and Roos, 1995). Mutants selected to

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be resistant to 5-fluorouracil compounds were defective in UPRT activity. Isolation of plasmid transgenes and surrounding chromosomal sequences demonstrated integration of plasmid in these mutants occurred at the UPRT locus, which was cloned and characterized in the same study. The T. gondii UP activity is not essential (Fox and Bzik, 2010; Fox et al., 2011). Therefore direct inhibition of salvage enzymes in T. gondii is unlikely to perturb parasite replication during acute or chronic infection. Nonetheless, parasite enzymes may have unique substrate properties and potential inhibitors of T. gondii UP have been assessed (El kouni et al., 1996; Iltzsch and Klenk, 1993). UPRT activity expressed by T. gondii is largely absent in the mammalian host, and this suggests another pathway of drug development. In addition to its natural substrate uracil, T. gondii UPRT also recognizes 2,4-dithiouracil and incorporates this analog into parasite nucleic acids. Interestingly, this property of UPRT has been adapted to enable cell-specific microarray analysis of mRNA synthesis and decay (Cleary et al., 2005; Zeiner et al., 2008). UPRT also recognizes 50 -fluorouracil compounds as well as other uracil analogs that suggest a pathway of drug development based on selective incorporation of toxic analogs by parasite UPRT. This approach has been validated in studies using 50 -fluorouracil, 50 -fluorouridine, FUDR, and emimycin (Pfefferkorn, 1978; Pfefferkorn et al., 1989; Pfefferkorn and Pfefferkorn, 1977a,b). 50 Fluorouracil compounds are incorporated by UPRT then ultimately become an inhibitor of thymidylate synthase (TS) that prevents synthesis of thymine nucleotides. The crystal structure of T. gondii UPRT has been determined, and efforts are under way to identify potential analogs that may be selectively incorporated into parasite nucleic acids to block replication of tachyzoites (Carter et al., 1997; Iltzsch, 1993; Iltzsch and Tankersley, 1994; Schumacher et al., 1998).

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9.3.3 Pyrimidine synthesis and salvage pathways related to parasite niche 9.3.3.1 Plasmodium falciparum and Cryptosporidium parvum The different strategies used by P. falciparum and C. parvum to acquire their pyrimidines are likely to be related to their particular niche within the human host. C. parvum resides in the gut, an environment rich in a nutrient supply, whereas P. falciparum resides initially in hepatocytes, and subsequently during within the erythrocyte, a host cell that loses its ability to synthesize pyrimidines and is not engaged in the business of transcription or replication. The erythrocyte niche of P. falciparum has a low abundance of pyrimidines. P. falciparum has compensated over time by abandoning the enzymes, if initially present, that can salvage pyrimidines. P. falciparum has retained a robust de novo pyrimidine synthetic pathway fully capable of supporting rapid replication. By contrast, the C. parvum genome encodes no component of the pyrimidine biosynthetic pathway (Abrahamsen et al., 2004). C. parvum as a gut pathogen discovered free food and acquired enzymes via horizontal transfer from other organism to take advantage of pyrimidine nucleobases and nucleosides present in the environment. The C. parvum genome reveals a plethora of transporters including putative transporters of nucleobases and nucleosides (Abrahamsen et al., 2004; Puiu et al., 2004). While recent evidence supports the hypothesis that C. parvum imports the purine nucleotides GTP and ATP, this parasite does not have the ability to import pyrimidine nucleotides (Pawlowic et al., 2019). 9.3.3.2 Toxoplasma gondii By contrast to P. falciparum and C. parvum, T. gondii (in vitro) can invade and replicate in any nucleated mammalian cell type and correspondingly has more significant capabilities in possessing both pyrimidine biosynthesis and

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salvage pathways. T. gondii can rely strictly on the de novo pathway, and this pathway is required for virulence in mammals (Fox and Bzik, 2002). These observations demonstrate T. gondii can readily obtain the amino acid precursors and the necessary ATP from purine salvage to synthesize pyrimidines de novo in the wide variety of cells and tissues that this parasite can infect. Surprisingly, T. gondii tachyzoites blocked in the pyrimidine biosynthetic pathway can grow strictly on the salvage pathway in vitro with uracil supplements (Fox and Bzik, 2002; Pfefferkorn, 1978). Yet complete disruption of the salvage pathway through mutation and disruption of UPRT has no detectable effect on tachyzoite growth or virulence (Donald and Roos, 1995; Fox and Bzik, 2010; Pfefferkorn, 1978). These observations raise the puzzling question as to why T. gondii has retained the capacity to grow on either pathway, whereas C. parvum and P. falciparum have not. Potential roles of T. gondii UPRT: It remains a mystery why uracil is so well incorporated into nucleic acids in tachyzoites and why the UPRT activity of the parasite is such a major activity (Pfefferkorn, 1978). The retention of a nonessential gene for the parasite UPRT and other salvage activities suggests that some advantage may be conferred by its expression. T. gondii UPRT recognizes uracil as its only natural substrate for pyrimidine compounds that are normally available in the mammalian host (Carter et al., 1997; Pfefferkorn, 1978). It is possible that the expression of UPRT confers some minor advantage to intracellular parasites by enabling the recovery and reincorporation of pyrimidines into the UMP pool that are catabolized from T. gondii or host nucleic acids and nucleotides. The ability of T. gondii to grow or survive on uracil alone in the absence of the de novo pyrimidine pathway may be essential during another life stage of the parasite other than the tachyzoite stage. Because UPRT is an enzyme

that is absent in mammals but commonly found in bacteria and plants, it is plausible that uracil may be a nutrient that is present in certain nonmammalian environmental niches. Within these niches the environmentally stable oocyst stage may have some access to uracil. Indirect biochemical evidence suggests T. gondii UPRT may interact with the soil environment. The naturally occurring antibiotic emimycin produced by Streptomyces spp. (primarily an inhabitant of soil) is an equivalent substrate to uracil for T. gondii UPRT (Pfefferkorn et al., 1989; Terao, 1963; Terao et al., 1960). Emimycin is a selective inhibitor of T. gondii growth and nucleic acid synthesis and parasite mutants with disrupted UPRT activity are resistant to emimycin. Emymycin is incorporated to emimycin 50 -monophosphate by T. gondii UPRT and further to emimycin riboside diphosphate and emimycin riboside triphosphate (ETP). However, ETP is not well incorporated into nucleic acids, and the inhibition of T. gondii RNA and DNA synthesis may occur at the level of parasite RNA and DNA polymerases (Pfefferkorn et al., 1989). It is also possible that the T. gondii UPRT activity was retained, because it plays an important role in another aspect of cell biology or metabolism. The structure and biochemical properties of UPRT demonstrated that in the absence of substrates or its activator GTP, the enzyme behaves as a homodimer composed of two identical subunits. In the presence of GTP, GTP binding stabilizes an active tetrameric structure of UPRT exhibiting high enzyme activity compared to the homodimer (Schumacher et al., 2002). Based on these observations, it was suggested that T. gondii UPRT is likely to play some role in balancing purine and pyrimidine pools in T. gondii (Schumacher et al., 2002). If UPRT is involved in balancing pyrimidine and purine pools, this balance may be best achieved in the nonmammalian environment, because UPRT cannot substantially

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contribute to parasite pyrimidine nucleotide pools in infected mammalian cells or animals (Fox and Bzik, 2002).

9.3.4 Folate pathways and synthesis of thymine nucleotides For decades, the folate pathway has been a key target for antimicrobial agents directed against T. gondii and P. falciparum and is under investigation for targeting C. parvum (Anderson, 2005). Thymine nucleotides are formed during the thymidylate cycle by the methylation of dUMP to produce dTMP in a reaction mediated by TS in folate metabolism (Fig. 9.5). During conversion of dUMP to dTMP a molecule of tetrahydrofolate is oxidized to dihydrofolate. During the thymidylate cycle, T. gondii, P. falciparum, and C. parvum rely on TS to produce dTMP from dUMP, on dihydrofolate reductase (DHFR) for recycling of dihydrofolate to tetrahydrofolate and on serine hydroxymethyltransferase to produce 5,10methylene-tetrahydrofolate from serine (Fig. 9.5). Therefore thymidine starvation can be induced in T. gondii and P. falciparum, as well as humans, by reducing the pool of tetrahydrofolate via inhibition of DHFR and recycling of dihydrofolate to tetrahydrofolate. C. parvum is unusual in that it also expresses a TK activity that grants this parasite a potentially significant salvage pathway to dTMP (Abrahamsen et al., 2004) (Figs. 9.4 and 9.5). C. parvum can replicate normally when the parasite TK activity is the only available pathway to make TMP (Pawlowic et al., 2019). 9.3.4.1 Biosynthesis of folates in Apicomplexa The primary precursors for the de novo synthesis of folates are para-aminobenzoic acid and GTP, a purine nucleotide that protozoan parasites cannot synthesize de novo. Humans lack

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the ability to synthesize folates de novo and rely on folate transport from dietary sources. By contrast, several of the apicomplexans possess an endogenous folate biosynthetic pathway that is susceptible to antifolate inhibitors. T. gondii and P. falciparum possess the full complement of genes encoding all seven enzymic steps in the de novo biosynthesis of 7,8-dihydrofolate (Fig. 9.5). By contrast, genome analysis indicates that C. parvum lacks any identifiable gene ortholog for folate biosynthetic enzymes and is apparently dependent on folate salvage to obtain 7,8-dihydrofolate for the thymidylate cycle (Abrahamsen et al., 2004; Striepen and Kissinger, 2004; Striepen et al., 2004) (Fig. 9.5). P. falciparum, C. parvum, and T. gondii possess gene orthologs for putative folate transporters indicating these parasites have the potential to salvage folates from the host (Chaudhary and Roos, 2005; Striepen and Kissinger, 2004; Striepen et al., 2004). The salvage pathway to para-aminobenzoic acid as well as the de novo synthesis of 7,8-dihydrofolate from GTP and para-aminobenzoic acid are functional in P. falciparum, because labeled precursors are incorporated into end products (Wang et al., 2004b). Recent evidence suggests that Plasmodium can acquire sufficient para-aminobenzoic acid either by de novo synthesis or by salvage from the host cell (Mather and Ke, 2019; Matz et al., 2019). In Apicomplexa, serine is also required for synthesis of dTMP during the thymidylate cycle (Fig. 9.5), and inhibitors of serine hydroxylmethyltransferase have been identified (Schwertz et al., 2018). Functional data on folate salvage in T. gondii and C. parvum are not yet available. In addition to salvage from the host, a second pathway to para-aminobenzoic acid exists as a product of the shikimate pathway via chorismate. While C. parvum possesses none of the genes involved in this pathway, T. gondii and P. falciparum encode several of the enzymes of this pathway (Fig. 9.5) (Mcconkey et al., 2004; Roberts et al., 1998).

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FIGURE 9.5 Thymine nucleotide synthesis and pathways to folate in Apicomplexa. Pathways present in Tg, Pf, and Cp are indicated in rectangles. The thymidylate cycle is enclosed by a large triangle. Potential synthetic or salvage sources of folate entering the thymidylate cycle are shown. Solid lines and arrows depict active pathways present. Substrates of several enzyme activities are shown at the start of the solid line and the product(s) are shown on the arrowhead side. Several key enzymes are indicated in capital italicized text beside the arrowhead line. All pathways shown appear to be present as indicated; however, the significance of the folate salvage and pABA salvage pathways is unclear in Tg (indicated with a question mark). Polyglutamated forms of folate are not shown. dTMP synthesis is shown at the bottom of the thymidylate cycle. Salvage of thymidine in Cp is shown. Cp, Cryptosporidium parvum; DHFR, dihydrofolate reductase; DHFS, dihydrofolate synthetase; DHPS, dihydropteroate synthase; Pf, Plasmodium falciparum; SHMT, serine hydromethyltransferase; Tg, Toxoplasma gondii; TK, thymidine kinase; TS, thymidylate synthetase.

9.3.4.2 Antifolate chemotherapy and antifolate resistance For several decades, antifolates such as pyrimethamine (PYR) and PG that target DHFR and folate recycling, and sulfa drugs that target dihydropteroate synthase (DHPS), have been

in clinical use for the treatment of P. falciparum and T. gondii infections (Brooks et al., 1987; Gregson and Plowe, 2005) (Fig. 9.5). While humans depend upon a monofunctional DHFR for DNA replication, in Apicomplexa the DHFR activity is present on a bifunctional

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polypeptide with TS activity (DHFR-TS or TSDHFR) (Bzik et al., 1987). Sulfa drugs target DHPS, which is a monofunctional enzyme in humans, but is also a bifunctional enzyme in T. gondii and P. falciparum with hydroxymethylpterin pyrophosphokinase activity (Pashley et al., 1997; Triglia and Cowman, 1994). Mutations in P. falciparum DHPS have been correlated with resistance to sulfa drugs (Triglia et al., 1998; Wang et al., 2004a). Genetic studies have documented that various mutations in DHPS correlate with resistance to sulfa drugs in vivo (Triglia et al., 1998). Resistance to antifolates that target DHFR arose rapidly in P. falciparum. Current antifolate therapy is based on synergism achieved in combination therapy of a DHFR inhibitor combined with sulfa drugs (Gregson and Plowe, 2005). The current treatment for T. gondii infection employs a similar strategy using PYR and sulfadiazine. While T. gondii has not developed resistance to antifolates, long-term use of this therapy to treat toxoplasmosis in AIDS patients has proven to be difficult due to significant adverse clinical reactions (Haverkos, 1987; Leport et al., 1988). The T. gondii DHFR-TS has unique features that present potentially selective targets for drug therapy (Anderson, 2017). Consistent with the presence of these unique features, highly selective and potents inhibitors of T. gondii DHFR-TS were recently reported (Hopper et al., 2019). Antifolates have been investigated as potential inhibitors of C. parvum (Anderson, 2005), and new inhibitors were designed and tested (Kumar et al., 2014; Ruiz et al., 2019). However, genetic ablation of the parasite DHFR-TS activity has recently demonstrated that C. parvum can replicate normally via the TK pathway, showing that the DHFRTS activity is not essential (Pawlowic et al., 2019). A nonessential DHFR-TS and the lack of a DHPS gene suggest that drug targeting of C. parvum folate metabolism may not be feasible. Antifolate resistance in P. falciparum and C. parvum: In early experiments, mutation of

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DHFR in the bifunctional DHFR-TS enzyme was identified as the basis of resistance to PYR in P. falciparum (Inselburg et al., 1987; Inselburg et al., 1988). In laboratory isolates, gene duplication and chromosomal changes have also been reported as a mechanism of resistance to PYR (Inselburg et al., 1987; Tanaka et al., 1990a,b). Early studies of PYR resistance in isolates of P. falciparum identified mutations in key amino acid residues (codons 51, 59, and 108) that were associated with highlevel resistance to PYR (Cowman et al., 1988; Inselburg et al., 1988; Peterson et al., 1988). The crystal structure of wild-type and mutant P. falciparum DHFR-TS suggested that these key resistance mutations caused steric interactions with inhibitors or weakened the binding of inhibitors in the active site (Yuvaniyama et al., 2003). Resistance to antifolates continues to spread in parasite populations globally (Okell et al., 2017). The crystal structure of C. hominis DHFR-TS was determined (O’neil et al., 2003a,b) and revealed some new insights into the natural resistance of C. parvum DHFR to antifolates (O’neil et al., 2003a,b; Vasquez et al., 1996). However, recent genetic experiments have shown that DHFR-TS is nonessential for C. parvum (Pawlowic et al., 2019). The earlier hints that DHFR-TS may be a drug target is explained by the recent finding that perturbing the thymidine pools of the host cell limits the ability of C. parvum to replicate via its TK activity when DHFR-TS is genetically deleted (Pawlowic et al., 2019). Antifolate resistance in T. gondii: Resistance to PYR is uncommon in clinical treatment of toxoplasmosis. Even low-level resistance to PYR has been difficult to select under drug pressure in vitro but has been demonstrated in a genetic model of direct mutagenesis of a plasmid borne T. gondii DHFR-TS followed by transfection of PYR-sensitive T. gondii and growth selection under PYR in vitro (Reynolds et al., 2001). By modeling P. falciparum resistance

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mutations at codons equivalent to 59 and 108 (Bzik et al., 1987), high-level PYR resistance was obtained in a T. gondii DHFR-TS mutant (Donald and Roos, 1993; Roos, 1993). Plasmids conferring high-level PYR resistance in T. gondii have been useful in genetic dissection of evolution and mechanisms associated with PYR and cycloguanil resistance (Reynolds and Roos, 1998). This approach was adapted to investigate evolutionary fitness of DHFR-TS mutations associated with PYR resistance. This study revealed subtle, but potentially significant, effects on fitness of mutant DHFR-TS enzymes in vitro that appear to be associated with the natural appearance, or nonappearance, of these mutants in vivo (Fohl and Roos, 2003). Modeling studies of T. gondii DHFR-TS have suggested that the long linker domain connecting DHFR to TS domains in Apicomplexa donates a helix that crosses to the second DHFR domain of the homodimer complex and contacts the outer shell of the DHFR active site (Belperron et al., 2004; O’neil et al., 2003a). Genetic studies of T. gondii DHFR-TS revealed that various mutations within the linker domain inactivated PYR resistance and enzyme activity in vitro and in vivo (Belperron et al., 2001; Belperron et al., 2004).

9.3.5 Toxoplasma gondii pyrimidine genetic selection strategies High-level PYR resistance based on mutant DHFR-TS was used to establish an early model of positive genetic selection in T. gondii (Donald and Roos, 1993; Roos, 1993). The high efficiency of obtaining PYR-resistant parasites allowed the refinement of methods for efficiently incorporating plasmid DNA into the parasite (Roos et al., 1994). The isolation of both a genomic DNA version as well as a cDNA version of the PYR-resistant DHFR-TS enabled an assessment of general homology requirements for recombination in T. gondii.

The cDNA version of T. gondii DHFR-TS lacks numerous introns contained in the genomic DNA version and randomly incorporated into the genome of the parasite (Donald and Roos, 1993). By contrast, the genomic DNA version was demonstrated to primarily integrate into the homologous DHFR-TS locus (Donald and Roos, 1994, 1995). These studies established methods and tools for random insertional mutagenesis, as well as gene replacement strategies in T. gondii. Subsequently, the high-level PYR-resistant bifunctional DHFR-TS was converted into a trifunctional enzyme by the incorporation of either herpes simplex virus TK or bacterial CD. The TK and CD enzymes were inserted as inframe genes into the linker domain of T. gondii DHFR-TS (Fox et al., 1999, 2001). The DHFRCD-TS plasmid conferred high-level resistance to PYR (positive selection), and stable PYRresistant parasite clones were killed (negative selection) by treatment of parasites with low doses of the normally nontoxic prodrug 5fluorocytosine (Fox et al., 1999). This study established DHFR-CD-TS as a trifunctional enzyme capable of positive and negative selection in T. gondii. Transgenic T. gondii parasites that express CD are killed by treatment with 5fluorocytosine because it is converted to 5fluorouracil, and then salvaged by T. gondii UPRT to 5-fluorouridine 50 -monophosphate, and converted to 5-fluoro-dUMP, which is a suicide inhibitor of TS activity that blocks the accumulation of dTMP and therefore parasite replication (Fox et al., 1999). Recently, a highly efficient forward genetic selection scheme based on the CD activity was reported in T. gondii (Fox et al., 2016). Similarly, construction of a plasmid encoding the trifunctional enzyme DHFR-TK-TS on a single polypeptide enabled positive selection by high-level resistance to PYR, and negative selection in submicromolar doses of ganciclovir (Fox et al., 2001). The DHFR-TK-TS plasmid was used in positive and negative selection experiments to obtain

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the avirulent uracil auxotroph mutant by targeted knockout of the CPSII gene (Fox and Bzik, 2002). The T. gondii CPSII is the most amenable among the apicomplexans for genetic dissection of CPSII activities and regulation. Cloning of the cDNA for T. gondii CPSII enables a genetic scheme for positive selection based on complementation of the uracil auxotroph CPSII knockout mutant. Complementation of CPSII has been demonstrated (Fox and Bzik, 2002; Fox et al., 2009a), as well as complementation of OMPDC (Fox and Bzik, 2010).

9.3.6 Uracil auxotrophy, vaccination, and immunity Genetic inactivation of the pyrimidine biosynthetic pathway by knockout of the CPSII gene produced a strain of T. gondii that is auxotrophic for uracil (Fox and Bzik, 2002) (Fig. 9.4). The T. gondii uracil auxotroph replicates at a normal growth rate in vitro in the presence of high concentrations of exogenously supplied uracil. By contrast, if uracil is omitted from the in vitro culture medium, this mutant will invade host cells normally but has no detectable growth rate. Uracil auxotrophy can also be developed by genetic deletion of the DHODH catalytic activity (Hortua Triana et al., 2016), or deletion of the genes encoding OPRT or OMPDC (Fox and Bzik, 2010, 2015; Fox et al., 2011), showing that deletion of any of the six enzymes in the pyrimidine biosynthetic pathway causes uracil auxotrophy in T. gondii. These genetic studies validate the entire pyrimidine biosynthetic pathway as a drug target in T. gondii. In addition, it is likely that the de novo pyrimidine synthesis pathway is also required for sustaining latent infection since disruption of the critical salvage activity mediated by UPRT has no effect on latent infection (Fox et al., 2011). The uracil auxotroph phenotype is specific to T. gondii because C. parvum

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lacks pyrimidine synthesis and P. falciparum does not have salvage capabilities that can rescue parasite growth in the absence of pyrimidine biosynthesis (Fig. 9.4). T. gondii uracil auxotrophs inactivated for CPSII (Fox and Bzik, 2002) or deleted for OMPDC and UP, OMPDC/UP (Fox and Bzik, 2010, 2015) do not replicate in animals, or in the absence of uracil in cell culture (Fox and Bzik, 2002; Fox et al., 2009b), and do not induce any detectable pathology or virulence even after high-dose vaccination. The host immune response completely eliminates T. gondii uracil auxotrophs within 57 days after vaccination (Dupont et al., 2014; Fox et al., 2016). Uracil auxotrophs have a potent ability to elicit immunity to T. gondii infection. Remarkably, a single low-dose vaccination of just 10,000 nonreplicating uracil auxotrophs was sufficient to elicit immunity to reinfection (Shaw et al., 2006). Vaccination with nonreplicating uracil auxotrophs activates T. gondii specific CD81 T cells and interferon gamma that elicits a lifelong protective CD81 T-cell immunity to reinfection (Dupont et al., 2014; Fox and Bzik, 2002, 2010, 2015; Fox et al., 2009b, 2011, 2016; Gigley et al., 2009a,b; Jordan et al., 2009; Shaw et al., 2006; Sukhumavasi et al., 2008). Remarkably, when mice with established lethal tumors for melanoma, ovarian cancer, or pancreatic cancer were vaccinated with T. gondii uracil auxotrophs, the host immune response to this vaccination also induced a highly effective coimmunity to the cancer, resulting in clearance of the tumor cells and tumors (Baird et al., 2013a,b; Fox et al., 2013a,b; Sanders et al., 2015, 2016). The mechanism for this tumor immunity was dependent on the ability of vaccination with T. gondii uracil auxotrophs to elicit CD81 T cells that specifically targeted each type of tumor that was present at the time of vaccination (Baird et al., 2013a,b; Sanders et al., 2015). Moreover, mice that were cleared of their tumors and survived after vaccination with T. gondii uracil auxotrophs exhibited

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strong memory responses to the tumor and were protected from future rechallenge with the same tumor (Baird et al., 2013a). The mechanisms that elicited these tumor specific responses were dependent upon T. gondii biology and the peculiar features of uracil auxotrophs, particularly their inability to replicate. T. gondii uracil auxotrophs reversed tumor immune suppression and reawakened the natural ability of the host to eliminate cancer. T. gondii uracil auxotrophs performed this feat at the cellular level. To generate any protective immune response to T. gondii (Dupont et al., 2014) or to cancer (Fox et al., 2016), the T. gondii uracil auxotrophs must invade myeloid cells, particularly dendritic cells. This cellular invasion by T. gondii uracil auxotrophs reprograms the host cell and activates antigen presentation to CD81 T cells. In addition, the reprograming of the myeloid/dendritic cell and the stimulation of CD81 T cells is dependent on the secretion of particular T. gondii effector proteins, such as GRA24, into the host cell (Fox et al., 2016, 2017). In sum, investigation of purine and pyrimidine metabolism in Apicomplexa has revealed metabolomic strategies used by the parasite to acquire these essential nutrients from their host cells. Various enzymes in purine and pyrimidine, and related pathways, are targets of current treatments for Apicomplexan parasites, and other targets in these pathways offer great promise for the development of improved treatments. The purine and pyrimidine pathways have also been fruitful in offering valuable strategies for selecting mutant parasites based on targeted gene insertion or targeted gene deletion. And mutants in these pathways, particularly in the pyrimidine pathways (uracil auxotrophs), have led to fundamental insights into how the host responds to infection, have helped to define the immunological requirements to generate a life-long immunity by vaccination, and have advanced new microbial-based immunotherapeutic

treatments for cancer for the first time based on the unique biology of parasites in the phylum Apicomplexa.

References Abrahamsen, M.S., Templeton, T.J., Enomoto, S., Abrahante, J.E., Zhu, G., Lancto, C.A., et al., 2004. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304, 441445. Al safarjalani, O.N., Naguib, F.N., El kouni, M.H., 2003. Uptake of nitrobenzylthioinosine and purine beta-Lnucleosides by intracellular Toxoplasma gondii. Antimicrob. Agents Chemother. 47, 32473251. Allison, A.C., Eugui, E.M., 2005. Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation 80, S181S190. Anderson, A.C., 2005. Targeting DHFR in parasitic protozoa. Drug Discov. Today 10, 121128. Anderson, K.S., 2017. Understanding the molecular mechanism of substrate channeling and domain communication in protozoal bifunctional TS-DHFR. Protein Eng. Des. Sel. 30, 253261. Aoki, T., Shimogawara, R., Ochiai, K., Yamasaki, H., Shimada, J., 1994. Molecular characterization of a carbamoyl-phosphate synthetase II (CPS II) gene from Trypanosoma cruzi. Adv. Exp. Med. Biol. 370, 513516. Arora, A., Deniskin, R., Sosa, Y., Nishtala, S.N., Henrich, P. P., Kumar, T.R., et al., 2016. Substrate and inhibitor specificity of the Plasmodium berghei equilibrative nucleoside transporter type 1. Mol. Pharmacol. 89, 678685. Asai, T., O’sullivan, W.J., Kobayashi, M., Gero, A.M., Yokogawa, M., Tatibana, M., 1983. Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii. Mol. Biochem. Parasitol. 7, 89100. Asai, T., Miura, S., Sibley, L.D., Okabayashi, H., Takeuchi, T., 1995. Biochemical and molecular characterization of nucleoside triphosphate hydrolase isozymes from the parasitic protozoan Toxoplasma gondii. J. Biol. Chem. 270, 1139111397. Avila, J.L., Avila, A., 1981. Trypanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease. Exp. Parasitol. 51, 204208. Avila, J.L., Rojas, T., Avila, A., Polegre, M.A., Robins, R.K., 1987. Biological activity of analogs of guanine and guanosine against American Trypanosoma and Leishmania spp. Antimicrob. Agents Chemother. 31, 447451. Bahl, A., Brunk, B., Coppel, R.L., Crabtree, J., Diskin, S.J., Fraunholz, M.J., et al., 2002. PlasmoDB: the Plasmodium genome resource. An integrated database providing tools for accessing, analyzing and mapping expression and sequence data (both finished and unfinished). Nucleic Acids Res. 30, 8790.

Toxoplasma Gondii

References

Bahl, A., Brunk, B., Crabtree, J., Fraunholz, M.J., Gajria, B., Grant, G.R., et al., 2003. PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data. Nucleic Acids Res. 31, 212215. Baird, J.R., Byrne, K.T., Lizotte, P.H., Toraya-Brown, S., Scarlett, U.K., Alexander, M.P., et al., 2013a. Immunemediated regression of established B16F10 melanoma by intratumoral injection of attenuated Toxoplasma gondii protects against rechallenge. J. Immunol. 190, 469478. Baird, J.R., Fox, B.A., Sanders, K.L., Lizotte, P.H., CubillosRuiz, J.R., Scarlett, U.K., et al., 2013b. Avirulent Toxoplasma gondii generates therapeutic antitumor immunity by reversing immunosuppression in the ovarian cancer microenvironment. Cancer Res. 73, 38423851. Beckwith, J.R., Pardee, A.B., Austrian, R., Jacob, F., 1962. Coordination of the synthesis of the enzymes in the pyrimidine pathway of Escherichia coli. J. Mol. Biol. 5, 618635. Belperron, A.A., Fox, B.A., Horii, T., Bzik, D.J., 2001. Toxoplasma gondii: genetic selection of tethered dihydrofolate reductase-thymidylate synthase fusion proteins. Exp. Parasitol. 98, 167170. Belperron, A.A., Fox, B.A., O’neil, R.H., Peaslee, K.A., Horii, T., Anderson, A.C., et al., 2004. Toxoplasma gondii: generation of novel truncation mutations in the linker domain of dihydrofolate reductase-thymidylate synthase. Exp. Parasitol. 106, 179182. Berens, R.L., Marr, J.J., Lafon, S.W., Nelson, D.J., 1981. Purine metabolism in Trypanosoma cruzi. Mol. Biochem. Parasitol. 3, 187196. Berens, R.L., Marr, J.J., Looker, D.L., Nelson, D.J., Lafon, S. W., 1984. Efficacy of pyrazolopyrimidine ribonucleosides against Trypanosoma cruzi: studies in vitro and in vivo with sensitive and resistant strains. J. Infect. Dis. 150, 602608. Berman, P.A., Human, L., 1991. Hypoxanthine depletion induced by xanthine oxidase inhibits malaria parasite growth in vitro. Adv. Exp. Med. Biol. 309A, 165168. Berman, P.A., Human, L., Freese, J.A., 1991. Xanthine oxidase inhibits growth of Plasmodium falciparum in human erythrocytes in vitro. J. Clin. Invest. 88, 18481855. Bermudes, D., Peck, K.R., Afifi, M.A., Beckers, C.J., Joiner, K.A., 1994. Tandemly repeated genes encode nucleoside triphosphate hydrolase isoforms secreted into the parasitophorous vacuole of Toxoplasma gondii. J. Biol. Chem. 269, 2925229260. Birkholtz, L.M., Wrenger, C., Joubert, F., Wells, G.A., Walter, R.D., Louw, A.I., 2004. Parasite-specific inserts in the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase of Plasmodium falciparum modulate catalytic activities and domain interactions. Biochem. J. 377, 439448.

437

Bohne, W., Roos, D.S., 1997. Stage-specific expression of a selectable marker in Toxoplasma gondii permits selective inhibition of either tachyzoites or bradyzoites. Mol. Biochem. Parasitol. 88, 115126. Boswell-Casteel, R.C., Hays, F.A., 2017. Equilibrative nucleoside transporters—a review. Nucleosides Nucleotides Nucleic Acids 36, 730. Brooks, R.G., Remington, J.S., Luft, B.J., 1987. Drugs used in the treatment of toxoplasmosis. Antimicrob. Agents Annu. 2, 297306. Bzik, D.J., Li, W.B., Horii, T., Inselburg, J., 1987. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc. Natl. Acad. Sci. U.S.A. 84, 83608364. Carter, D., Donald, R.G., Roos, D., Ullman, B., 1997. Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii. Mol. Biochem. Parasitol. 87, 137144. Carter, N.S., Ben mamoun, C., Liu, W., Silva, E.O., Landfear, S.M., Goldberg, D.E., et al., 2000. Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum. J. Biol. Chem. 275, 1068310691. Carter, N.S., Rager, N., Ullman, B. (Eds.), 2003. Purine and Pyrimidine Transport and Metabolism. Academic Press, London. Cassera, M.B., Hazleton, K.Z., Merino, E.F., Obaldia 3RD, N., Ho, M.C., Murkin, A.S., et al., 2011. Plasmodium falciparum parasites are killed by a transition state analogue of purine nucleoside phosphorylase in a primate animal model, PLoS One, 6. p. e26916. Chansiri, K., Bagnara, A.S., 1995. The structural gene for carbamoyl phosphate synthetase from the protozoan parasite Babesia bovis. Mol. Biochem. Parasitol. 74, 239243. Chaudhary, K., 2005. Purine Transport and Salvage in Apicomplexan Parasites. University of Pennsylvania. Chaudhary, K., Roos, D.S., 2005. Protozoan genomics for drug discovery. Nat. Biotechnol. 23, 10891091. Chaudhary, K., Darling, J.A., Fohl, L.M., Sullivan JR., W.J., Donald, R.G., Pfefferkorn, E.R., et al., 2004. Purine salvage pathways in the apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 279, 3122131227. Chaudhary, K., Donald, R.G., Nishi, M., Carter, D., Ullman, B., Roos, D.S., 2005. Differential localization of alternatively spliced hypoxanthine-xanthine-guanine phosphoribosyltransferase isoforms in Toxoplasma gondii. J. Biol. Chem. 280, 2205322059. Chaudhary, K., Ting, L.M., Kim, K., Roos, D.S., 2006. Toxoplasma gondii purine nucleoside phosphorylase biochemical characterization, inhibitor profiles, and comparison with the Plasmodium falciparum ortholog. J. Biol. Chem. 281, 2565225658.

Toxoplasma Gondii

438

9. Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa

Chiang, C.W., Carter, N., Sullivan JR., W.J., Donald, R.G., Roos, D.S., Naguib, F.N., et al., 1999. The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression. J. Biol. Chem. 274, 3525535261. Christopherson, R.I., Cinquin, O., Shojaei, M., Kuehn, D., Menz, R.I., 2004. Cloning and expression of malarial pyrimidine enzymes. Nucleosides Nucleotides Nucleic Acids 23, 14591465. Cleary, M.D., Meiering, C.D., Jan, E., Guymon, R., Boothroyd, J.C., 2005. Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cellspecific microarray analysis of mRNA synthesis and decay. Nat. Biotechnol. 23, 232237. Coppens, I., Dunn, J.D., Romano, J.D., Pypaert, M., Zhang, H., Boothroyd, J.C., et al., 2006. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space. Cell 125, 261274. Cowman, A.F., Morry, M.J., Biggs, B.A., Cross, G.A., Foote, S.J., 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S.A. 85, 91099113. Crandall, I.E., Wasilewski, E., Bello, A.M., Mohmmed, A., Malhotra, P., Pai, E.F., et al., 2013. Antimalarial activities of 6-iodouridine and its prodrugs and potential for combination therapy. J. Med. Chem. 56, 23482358. Daddona, P.E., Wiesmann, W.P., Lambros, C., Kelley, W. N., Webster, H.K., 1984. Human malaria parasite adenosine deaminase. Characterization in host enzymedeficient erythrocyte culture. J. Biol. Chem. 259, 14721475. Daddona, P.E., Wiesmann, W.P., Milhouse, W., Chern, J. W., Townsend, L.B., Hershfield, M.S., et al., 1986. Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors. J. Biol. Chem. 261, 1166711673. Darling, J.A., Sullivan JR., W.J., Carter, D., Ullman, B., Roos, D.S., 1999. Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase. Mol. Biochem. Parasitol. 103, 1523. Davidson, J.N., Chen, K.C., Jamison, R.S., Musmanno, L.A., Kern, C.B., 1993. The evolutionary history of the first three enzymes in pyrimidine biosynthesis. Bioessays 15, 157164. Davis, R.H., 1986. Compartmental and regulatory mechanisms in arginine pathways of Neurospora crassa and Saccharomyces cerevisiae. Microbiol. Rev. 50, 280313. de Koning, H., Diallinas, G., 2000. Nucleobase transporters (review). Mol. Membr. Biol. 17, 7594.

de Koning, H.P., Al-Salabi, M.I., Cohen, A.M., Coombs, G. H., Wastling, J.M., 2003. Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii. Int. J. Parasitol. 33, 821831. de Koning, H.P., Bridges, D.J., Burchmore, R.J., 2005. Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy. FEMS Microbiol. Rev. 29, 9871020. Donald, R.G., Roos, D.S., 1993. Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drugresistance mutations in malaria. Proc. Natl. Acad. Sci. U.S.A. 90, 1170311707. Donald, R.G., Roos, D.S., 1994. Homologous recombination and gene replacement at the dihydrofolate reductasethymidylate synthase locus in Toxoplasma gondii. Mol. Biochem. Parasitol. 63, 243253. Donald, R.G., Roos, D.S., 1995. Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 92, 57495753. Donald, R.G., Roos, D.S., 1998. Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis. Mol. Biochem. Parasitol. 91, 295305. Donald, R.G., Carter, D., Ullman, B., Roos, D.S., 1996. Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. J. Biol. Chem. 271, 1401014019. Donaldson, T.M., Cassera, M.B., Ho, M.C., Zhan, C., Merino, E.F., Evans, G.B., et al., 2014a. Inhibition and structure of Toxoplasma gondii purine nucleoside phosphorylase. Eukaryot. Cell 13, 572579. Donaldson, T.M., Ting, L.M., Zhan, C., Shi, W., Zheng, R., Almo, S.C., et al., 2014b. Structural determinants of the 50 -methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase. PLoS One 9, e84384. Dou, Z., Mcgovern, O.L., Di cristina, M., Carruthers, V.B., 2014. Toxoplasma gondii ingests and digests host cytosolic proteins. mBio 5, e01188-14. Downie, M.J., El Bissati, K., Bobenchik, A.M., Nic lochlainn, L., Amerik, A., Zufferey, R., et al., 2010. PfNT2, a permease of the equilibrative nucleoside transporter family in the endoplasmic reticulum of Plasmodium falciparum. J. Biol. Chem. 285, 2082720833. Doyle, P.S., Kanaani, J., Wang, C.C., 1998. Hypoxanthine, guanine, xanthine phosphoribosyltransferase activity in Cryptosporidium parvum. Exp. Parasitol. 89, 915.

Toxoplasma Gondii

References

Ducati, R.G., Namanja-Magliano, H.A., Harijan, R.K., Fajardo, J.E., Fiser, A., Daily, J.P., et al., 2018. Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S. A. 115, 21142119. Dupont, C.D., Christian, D.A., Selleck, E.M., Pepper, M., Leney-Greene, M., Harms pritchard, G., et al., 2014. Parasite fate and involvement of infected cells in the induction of CD4 1 and CD8 1 T cell responses to Toxoplasma gondii. PLoS Pathog. 10, e1004047. Dziekan, J.M., Yu, H., Chen, D., Dai, L., Wirjanata, G., Larsson, A., et al., 2019. Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay. Sci. Transl. Med. 11. Available from: https://doi.org/10.1126/scitranslmed.aau3174. Eaazhisai, K., Jayalakshmi, R., Gayathri, P., Anand, R.P., Sumathy, K., Balaram, H., et al., 2004. Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum. J. Mol. Biol. 335, 12511264. El Bissati, K., Zufferey, R., Witola, W.H., Carter, N.S., Ullman, B., Ben mamoun, C., 2006. The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S.A. 103, 92869291. El Bissati, K., Downie, M.J., Kim, S.K., Horowitz, M., Carter, N., Ullman, B., et al., 2008. Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum. Mol. Biochem. Parasitol. 161, 130139. El Bissati, K., Redel, H., Ting, L.M., Lykins, J.D., Mcphillie, M.J., Upadhya, R., et al., 2019. Novel synthetic polyamines have potent antimalarial activities in vitro and in vivo by decreasing intracellular spermidine and spermine concentrations. Front. Cell. Infect. Microbiol. 9, 9. Elion, G.B., 1989a. Nobel Lecture. The purine path to chemotherapy. Biosci. Rep. 9, 509529. Elion, G.B., 1989b. The purine path to chemotherapy. Science 244, 4147. El kouni, M.H., Naguib, F.N., Panzica, R.P., Otter, B.A., Chu, S.H., Gosselin, G., et al., 1996. Effects of modifications in the pentose moiety and conformational changes on the binding of nucleoside ligands to uridine phosphorylase from Toxoplasma gondii. Biochem. Pharmacol. 51, 16871700. El kouni, M.H., Guarcello, V., Al safarjalani, O.N., Naguib, F.N., 1999. Metabolism and selective toxicity of 6nitrobenzylthioinosine in Toxoplasma gondii. Antimicrob. Agents Chemother. 43, 24372443. Evans, D.R., Guy, H.I., 2004. Mammalian pyrimidine synthesis: fresh insights into an ancient pathway. J. Biol. Chem. 279, 3303533038.

439

Fish, W.R., Marr, J.J., Berens, R.L., 1982. Purine metabolism in Trypanosoma brucei gambiense. Biochim. Biophys. Acta 714, 422428. Fish, W.R., Marr, J.J., Berens, R.L., Looker, D.L., Nelson, D. J., Lafon, S.W., et al., 1985. Inosine analogs as chemotherapeutic agents for African trypanosomes: metabolism in trypanosomes and efficacy in tissue culture. Antimicrob. Agents Chemother. 27, 3336. Flores, M.V., Stewart, T.S., 1998. Plasmodium falciparum: a microassay for the malarial carbamoyl phosphate synthetase. Exp. Parasitol. 88, 243245. Flores, M.V., O’sullivan, W.J., Stewart, T.S., 1994. Characterisation of the carbamoyl phosphate synthetase gene from Plasmodium falciparum. Mol. Biochem. Parasitol. 68, 315318. Flores, M.V., Atkins, D., Wade, D., O’sullivan, W.J., Stewart, T.S., 1997. Inhibition of Plasmodium falciparum proliferation in vitro by ribozymes. J. Biol. Chem. 272, 1694016945. Fohl, L.M., Roos, D.S., 2003. Fitness effects of DHFR-TS mutations associated with pyrimethamine resistance in apicomplexan parasites. Mol. Microbiol. 50, 13191327. Fox, B.A., Bzik, D.J., 2002. De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii. Nature 415, 926929. Fox, B.A., Bzik, D.J., 2003. Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii. Int. J. Parasitol. 33, 8996. Fox, B.A., Bzik, D.J., 2010. Avirulent uracil auxotrophs based on disruption of orotidine-50 -monophosphate decarboxylase elicit protective immunity to Toxoplasma gondii. Infect. Immun. 78, 37443752. Fox, B.A., Bzik, D.J., 2015. Nonreplicating, cyst-defective type II Toxoplasma gondii vaccine strains stimulate protective immunity against acute and chronic infection. Infect. Immun. 83, 21482155. Fox, B.A., Belperron, A.A., Bzik, D.J., 1999. Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine. Mol. Biochem. Parasitol. 98, 93103. Fox, B.A., Belperron, A.A., Bzik, D.J., 2001. Negative selection of herpes simplex virus thymidine kinase in Toxoplasma gondii. Mol. Biochem. Parasitol. 116, 8588. Fox, B.A., Gigley, J.P., Bzik, D.J., 2004. Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation. Int. J. Parasitol. 34, 323331.

Toxoplasma Gondii

440

9. Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa

Fox, B.A., Ristuccia, J.G., Bzik, D.J., 2009a. Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii. Int. J. Parasitol. 39, 533539. Fox, B.A., Ristuccia, J.G., Gigley, J.P., Bzik, D.J., 2009b. Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining. Eukaryot. Cell 8, 520529. Fox, B.A., Falla, A., Rommereim, L.M., Tomita, T., Gigley, J. P., Mercier, C., et al., 2011. Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryot. Cell 10, 11931206. Fox, B.A., Sanders, K.L., Bzik, D.J., 2013a. Non-replicating Toxoplasma gondii reverses tumor-associated immunosuppression. Oncoimmunology 2, e26296. Fox, B.A., Sanders, K.L., Chen, S., Bzik, D.J., 2013b. Targeting tumors with nonreplicating Toxoplasma gondii uracil auxotroph vaccines. Trends Parasitol. 29, 431437. Fox, B.A., Sanders, K.L., Rommereim, L.M., Guevara, R.B., Bzik, D.J., 2016. Secretion of rhoptry and dense granule effector proteins by nonreplicating Toxoplasma gondii uracil auxotrophs controls the development of antitumor immunity. PLoS Genet. 12, e1006189. Fox, B.A., Butler, K.L., Guevara, R.B., Bzik, D.J., 2017. Cancer therapy in a microbial bottle: Uncorking the novel biology of the protozoan Toxoplasma gondii. PLoS Pathog. 13, e1006523. Frame, I.J., Merino, E.F., Schramm, V.L., Cassera, M.B., Akabas, M.H., 2012. Malaria parasite type 4 equilibrative nucleoside transporters (ENT4) are purine transporters with distinct substrate specificity. Biochem. J. 446, 179190. Frame, I.J., Deniskin, R., Rinderspacher, A., Katz, F., Deng, S.X., Moir, R.D., et al., 2015. Yeast-based high-throughput screen identifies Plasmodium falciparum equilibrative nucleoside transporter 1 inhibitors that kill malaria parasites. ACS Chem. Biol. 10, 775783. Fresquet, V., Mora, P., Rochera, L., Ramon-Maiques, S., Rubio, V., Cervera, J., 2000. Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals. J. Mol. Biol. 299, 979991. Gafan, C., Wilson, J., Berger, L.C., Berger, B.J., 2001. Characterization of the ornithine aminotransferase from Plasmodium falciparum. Mol. Biochem. Parasitol. 118, 110. Gao, G., Nara, T., Nakajima-Shimada, J., Aoki, T., 1998. Molecular characterization of a carbamoyl-phosphate

synthetase II (CPS II) gene from Leishmania mexicana. Adv. Exp. Med. Biol. 431, 237240. Gao, G., Nara, T., Nakajima-Shimada, J., Aoki, T., 1999. Novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in Trypanosoma cruzi. J. Mol. Biol. 285, 149161. Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R.W., et al., 2002a. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498511. Gardner, M.J., Shallom, S.J., Carlton, J.M., Salzberg, S.L., Nene, V., Shoaibi, A., et al., 2002b. Sequence of Plasmodium falciparum chromosomes 2, 10, 11 and 14. Nature 419, 531534. Gero, A.M., Brown, G.V., O’sullivan, W.J., 1984. Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum. J. Parasitol. 70, 536541. Gero, A.M., Scott, H.V., O’sullivan, W.J., Christopherson, R.I., 1989. Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites. Mol. Biochem. Parasitol. 34, 8797. Gero, A.M., Dunn, C.G., Brown, D.M., Pulenthiran, K., Gorovits, E.L., Bakos, T., et al., 2003. New malaria chemotherapy developed by utilization of a unique parasite transport system. Curr. Pharm. Des. 9, 867877. Gigley, J.P., Fox, B.A., Bzik, D.J., 2009a. Cell-mediated immunity to Toxoplasma gondii develops primarily by local Th1 host immune responses in the absence of parasite replication. J. Immunol. 182, 10691078. Gigley, J.P., Fox, B.A., Bzik, D.J., 2009b. Long-term immunity to lethal acute or chronic type II Toxoplasma gondii infection is effectively induced in genetically susceptible C57BL/6 mice by immunization with an attenuated type I vaccine strain. Infect. Immun. 77, 53805388. Gorla, S.K., Kavitha, M., Zhang, M., Liu, X., Sharling, L., Gollapalli, D.R., et al., 2012. Selective and potent urea inhibitors of Cryptosporidium parvum inosine 50 -monophosphate dehydrogenase. J. Med. Chem. 55, 77597771. Gregson, A., Plowe, C.V., 2005. Mechanisms of resistance of malaria parasites to antifolates. Pharmacol. Rev. 57, 117145. Gupte, A., Buolamwini, J.K., Yadav, V., Chu, C.K., Naguib, F.N., El kouni, M.H., 2005. 6-Benzylthioinosine analogues: Promising anti-toxoplasmic agents as inhibitors of the mammalian nucleoside transporter ENT1(es). Biochem. Pharmacol. 71, 6973. Hassan, H.F., Coombs, G.H., 1986. De novo synthesis of purines by Acanthamoeba castellani and A. astronyxis. IRCS Med. Sci. 14, 559560.

Toxoplasma Gondii

References

Haverkos, H.W., 1987. Assessment of therapy for toxoplasmic encephalitis. Am. J. Med. 82, 907914. Hazleton, K.Z., Ho, M.C., Cassera, M.B., Clinch, K., Crump, D.R., Rosario, I., et al., 2012. Acyclic immucillin phosphonates: second-generation inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase. Chem. Biol. 19, 721730. Hendriks, E.F., O’sullivan, W.J., Stewart, T.S., 1998. Molecular cloning and characterization of the Plasmodium falciparum cytidine triphosphate synthetase gene. Biochim. Biophys. Acta 1399, 213218. Heroux, A., White, E.L., Ross, L.J., Borhani, D.W., 1999a. Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex. Biochemistry 38, 1448514494. Heroux, A., White, E.L., Ross, L.J., Davis, R.L., Borhani, D. W., 1999b. Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg(2 1 ) ions bound: insights into the catalytic mechanism. Biochemistry 38, 1449514506. Hill, B., Kilsby, J., Mcintosh, R.T., Wrigglesworth, R., Ginger, C.D., 1981a. Pyrimidine biosynthesis in Plasmodium berghei. Int. J. Biochem. 13, 303310. Hill, B., Kilsby, J., Rogerson, G.W., Mcintosh, R.T., Ginger, C.D., 1981b. The enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and helminths. Mol. Biochem. Parasitol. 2, 123134. Hoelz, L.V., Calil, F.A., Nonato, M.C., Pinheiro, L.C., Boechat, N., 2018. Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria. Future Med. Chem. 10, 18531874. Holden, H.M., Thoden, J.B., Raushel, F.M., 1999. Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product. Cell. Mol. Life Sci. 56, 507522. Hopper, A.T., Brockman, A., Wise, A., Gould, J., Barks, J., Radke, J.B., et al., 2019. Discovery of selective Toxoplasma gondii dihydrofolate reductase inhibitors for the treatment of toxoplasmosis. J. Med. Chem. 62, 15621576. Hortua Triana, M.A., Cajiao herrera, D., Zimmermann, B. H., Fox, B.A., Bzik, D.J., 2016. Pyrimidine pathwaydependent and -independent functions of the Toxoplasma gondii mitochondrial dihydroorotate dehydrogenase. Infect. Immun. 84, 29742981. Hortua Triana, M.A., Huynh, M.H., Garavito, M.F., Fox, B. A., Bzik, D.J., Carruthers, V.B., et al., 2012. Biochemical and molecular characterization of the pyrimidine

441

biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii. Mol. Biochem. Parasitol. 184, 7181. Huang, J., Mullapudi, N., Lancto, C.A., Scott, M., Abrahamsen, M.S., Kissinger, J.C., 2004. Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum. Genome. Biol. 5, R88. Iltzsch, M.H., 1993. Pyrimidine salvage pathways in Toxoplasma gondii. J. Eukaryot. Microbiol. 40, 2428. Iltzsch, M.H., Klenk, E.E., 1993. Structure-activity relationship of nucleobase ligands of uridine phosphorylase from Toxoplasma gondii. Biochem. Pharmacol. 46, 18491858. Iltzsch, M.H., Tankersley, K.O., 1994. Structure-activity relationship of ligands of uracil phosphoribosyltransferase from Toxoplasma gondii. Biochem. Pharmacol. 48, 781792. Iltzsch, M.H., Uber, S.S., Tankersley, K.O., El kouni, M.H., 1995. Structure-activity relationship for the binding of nucleoside ligands to adenosine kinase from Toxoplasma gondii. Biochem. Pharmacol. 49, 15011512. Inselburg, J., Bzik, D.J., Horii, T., 1987. Pyrimethamine resistant Plasmodium falciparum: overproduction of dihydrofolate reductase by a gene duplication. Mol. Biochem. Parasitol. 26, 121134. Inselburg, J., Bzik, D.J., Li, W.B., 1988. Plasmodium falciparum: three amino acid changes in the dihydrofolate reductase of a pyrimethamine-resistant mutant. Exp. Parasitol. 67, 361363. Jayalakshmi, R., Sumathy, K., Balaram, H., 2002. Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli. Protein Expr. Purif. 25, 6572. Joiner, K.A., Beckers, C.J., Bermudes, D., Ossorio, P.N., Schwab, J.C., Dubremetz, J.F., 1994. Structure and function of the parasitophorous vacuole membrane surrounding Toxoplasma gondii. Ann. N.Y. Acad. Sci. 730, 16. Joiner, K.A., Bermudes, D., Sinai, A., Qi, H., Polotsky, V., Beckers, C.J., 1996. Structure and function of the Toxoplasma gondii vacuole. Ann. N.Y. Acad. Sci. 797, 17. Jones, M.E., 1980. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP synthesis. Ann. Rev. Biochem. 49, 253279. Jones, T.C., 1973. Multiplication of toxoplasmas in enucleate fibroblasts. Proc. Soc. Exp. Biol. Med. 142, 12681271. Jones, T.C., Yeh, S., Hirsch, J.G., 1972. The interaction between Toxoplasma gondii and mammalian cells. I.

Toxoplasma Gondii

442

9. Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa

Mechanism of entry and intracellular fate of the parasite. J. Exp. Med. 136, 11571172. Jordan, K.A., Wilson, E.H., Tait, E.D., Fox, B.A., Roos, D.S., Bzik, D.J., et al., 2009. Kinetics and phenotype of vaccine-induced CD8 1 T-cell responses to Toxoplasma gondii. Infect. Immun. 77, 38943901. Kagami, L.P., Das neves, G.M., Rodrigues, R.P., Da silva, V. B., Eifler-Lima, V.L., Kawano, D.F., 2017. Identification of a novel putative inhibitor of the Plasmodium falciparum purine nucleoside phosphorylase: exploring the purine salvage pathway to design new antimalarial drugs. Mol. Divers. 21, 677695. Keithly, J.S., Zhu, G., Upton, S.J., Woods, K.M., Martinez, M.P., Yarlett, N., 1997. Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy. Mol. Biochem. Parasitol. 88, 3542. Kicska, G.A., Tyler, P.C., Evans, G.B., Furneaux, R.H., Kim, K., Schramm, V.L., 2002a. Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum. J. Biol. Chem. 277, 32193225. Kicska, G.A., Tyler, P.C., Evans, G.B., Furneaux, R.H., Schramm, V.L., Kim, K., 2002b. Purine-less death in Plasmodium falciparum induced by immucillin-H, a transition state analogue of purine nucleoside phosphorylase. J. Biol. Chem. 277, 32263231. Kim, K., Weiss, L.M., 2004. Toxoplasma gondii: the model apicomplexan. Int. J. Parasitol. 34, 423432. Kim, Y., Makowska-Grzyska, M., Gorla, S.K., Gollapalli, D. R., Cuny, G.D., Joachimiak, A., et al., 2015. Structure of cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity. Acta Crystallogr. F: Struct. Biol. Commun. 71, 531538. Kimata, I., Tanabe, K., 1982. Invasion by Toxoplasma gondii of ATP-depleted and ATP-restored chick embryo erythrocytes. J. Gen. Microbiol. 128, 24992501. Kirk, K., Horner, H.A., Elford, B.C., Ellory, J.C., Newbold, C.I., 1994. Transport of diverse substrates into malariainfected erythrocytes via a pathway showing functional characteristics of a chloride channel. J. Biol. Chem. 269, 33393347. Kirk, K., Martin, R.E., Broer, S., Howitt, S.M., Saliba, K.J., 2005. Plasmodium permeomics: membrane transport proteins in the malaria parasite. Curr. Top. Microbiol. Immunol. 295, 325356. Kissinger, J.C., Gajria, B., Li, L., Paulsen, I.T., Roos, D.S., 2003. ToxoDB: accessing the Toxoplasma gondii genome. Nucleic Acids Res. 31, 234236. Kokkonda, S., El mazouni, F., White, K.L., White, J., Shackleford, D.M., Lafuente-Monasterio, M.J., et al., 2018. Isoxazolopyrimidine-based inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with antimalarial activity. ACS Omega 3, 92279240.

Korsinczky, M., Chen, N., Kotecka, B., Saul, A., Rieckmann, K., Cheng, Q., 2000. Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site. Antimicrob. Agents Chemother. 44, 21002108. Kothe, M., Purcarea, C., Guy, H.I., Evans, D.R., PowersLee, S.G., 2005. Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase. Protein Sci. 14, 3744. Krause, T., Luersen, K., Wrenger, C., Gilberger, T.W., Muller, S., Walter, R.D., 2000. The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs. Biochem. J. 352 (Pt 2), 287292. Krug, E.C., Marr, J.J., Berens, R.L., 1989. Purine metabolism in Toxoplasma gondii. J. Biol. Chem. 264, 1060110607. Krungkrai, S.R., Krungkrai, J., 2016. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target. Asian Pac. J. Trop. Med. 9, 525534. Krungkrai, S.R., Aoki, S., Palacpac, N.M., Sato, D., Mitamura, T., Krungkrai, J., et al., 2004a. Human malaria parasite orotate phosphoribosyltransferase: functional expression, characterization of kinetic reaction mechanism and inhibition profile. Mol. Biochem. Parasitol. 134, 245255. Krungkrai, S.R., Prapunwattana, P., Horii, T., Krungkrai, J., 2004b. Orotate phosphoribosyltransferase and orotidine 50 -monophosphate decarboxylase exist as multienzyme complex in human malaria parasite Plasmodium falciparum. Biochem. Biophys. Res. Commun. 318, 10121018. Krungkrai, S.R., Delfraino, B.J., Smiley, J.A., Prapunwattana, P., Mitamura, T., Horii, T., et al., 2005. A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 50 -monophosphate decarboxylase in human malaria parasite Plasmodium falciparum: physical association, kinetics, and inhibition characterization. Biochemistry 44, 16431652. Kumar, S., Krishnamoorthy, K., Mudeppa, D.G., Rathod, P. K., 2015. Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein-protein interactions. Acta Crystallogr. F: Struct. Biol. Commun. 71, 600608. Kumar, V.P., Cisneros, J.A., Frey, K.M., CastellanosGonzalez, A., Wang, Y., Gangjee, A., et al., 2014. Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase. Bioorg. Med. Chem. Lett. 24, 41584161.

Toxoplasma Gondii

References

Kyes, S., Horrocks, P., Newbold, C., 2001. Antigenic variation at the infected red cell surface in malaria. Annu. Rev. Microbiol. 55, 673707. Landfear, S.M., 2011. Nutrient transport and pathogenesis in selected parasitic protozoa. Eukaryot. Cell 10, 483493. Landfear, S.M., Ullman, B., Carter, N.S., Sanchez, M.A., 2004. Nucleoside and nucleobase transporters in parasitic protozoa. Eukaryot. Cell 3, 245254. Lauer, S.A., Rathod, P.K., Ghori, N., Haldar, K., 1997. A membrane network for nutrient import in red cells infected with the malaria parasite. Science 276, 11221125. Leport, C., Raffi, F., Katlama, C., Regneir, B., Saimot, A.G., Marche, C., et al., 1988. Treatment of central nervous system toxoplasmosis with pyrimethamine/sulfonamide combination in 35 patients with the acquired immunodeficiency syndrome. Am. J. Med. 84, 94100. Lewandowicz, A., Ringia, E.A., Ting, L.M., Kim, K., Tyler, P.C., Evans, G.B., et al., 2005. Energetic mapping of transition state analogue interactions with human and Plasmodium falciparum purine nucleoside phosphorylases. J. Biol. Chem. 280, 3032030328. Li, C.M., Tyler, P.C., Furneaux, R.H., Kicska, G., Xu, Y., Grubmeyer, C., et al., 1999. Transition-state analogs as inhibitors of human and malarial hypoxanthineguanine phosphoribosyltransferases. Nat. Struct. Biol. 6, 582587. Li, L., Brunk, B.P., Kissinger, J.C., Pape, D., Tang, K., Cole, R.H., et al., 2003. Gene discovery in the apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Res. 13, 443454. Llanos-Cuentas, A., Casapia, M., Chuquiyauri, R., Hinojosa, J.C., Kerr, N., Rosario, M., et al., 2018. Antimalarial activity of single-dose DSM265, a novel Plasmodium dihydroorotate dehydrogenase inhibitor, in patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria infection: a proof-of-concept, open-label, phase 2a study. Lancet. Infect. Dis. 18, 874883. Lunev, S., Bosch, S.S., Batista Fde, A., Wrenger, C., Groves, M.R., 2016. Crystal structure of truncated aspartate transcarbamoylase from Plasmodium falciparum. Acta Crystallogr. F: Struct. Biol. Commun. 72, 523533. Lunev, S., Bosch, S.S., Batista, F.A., Wang, C., Li, J., Linzke, M., et al., 2018. Identification of a non-competitive inhibitor of Plasmodium falciparum aspartate transcarbamoylase. Biochem. Biophys. Res. Commun. 497, 835842. Lykins, J.D., Filippova, E.V., Halavaty, A.S., Minasov, G., Zhou, Y., Dubrovska, I., et al., 2018. CSGID solves structures and identifies phenotypes for five enzymes in Toxoplasma gondii. Front. Cell. Infect. Microbiol. 8, 352.

443

Madrid, D.C., Ting, L.M., Waller, K.L., Schramm, V.L., Kim, K., 2008. Plasmodium falciparum purine nucleoside phosphorylase is critical for viability of malaria parasites. J. Biol. Chem. 283, 3589935907. Mahamed, D.A., Mills, J.H., Egan, C.E., Denkers, E.Y., Bynoe, M.S., 2012. CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system. Proc. Natl. Acad. Sci. U.S.A. 109, 1631216317. Makoff, A.J., Radford, A., 1978. Genetics and biochemistry of carbamoyl phosphate biosynthesis and its utilization in the pyrimidine biosynthetic pathway. Microbiol. Rev. 42, 307328. Marr, J.J., Berens, R.L., 1988. Hypoxanthine and inosine analogues as chemotherapeutic agents in Chagas’ disease. Mem. Inst. Oswaldo Cruz 83 (Suppl. 1), 301307. Marr, J.J., Berens, R.L., Nelson, D.J., 1978. Purine metabolism in Leishmania donovani and Leishmania braziliensis. Biochim. Biophys. Acta 544, 360371. Martin, R.E., Henry, R.I., Abbey, J.L., Clements, J.D., Kirk, K., 2005. The ‘permeome’ of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum. Genome. Biol. 6, R26. Mather, M.W., Ke, H., 2019. Para-aminobenzoate synthesis versus salvage in malaria parasites. Trends Parasitol. 35, 176178. Matz, J.M., Watanabe, M., Falade, M., Tohge, T., Hoefgen, R., Matuschewski, K., 2019. Plasmodium paraaminobenzoate synthesis and salvage resolve avoidance of folate competition and adaptation to host diet. Cell Rep. 26, 356363.e4. Mcconkey, G.A., 2000. Plasmodium falciparum: isolation and characterisation of a gene encoding protozoan GMP synthase. Exp. Parasitol. 94, 2332. Mcconkey, G.A., Pinney, J.W., Westhead, D.R., Plueckhahn, K., Fitzpatrick, T.B., Macheroux, P., et al., 2004. Annotating the Plasmodium genome and the enigma of the shikimate pathway. Trends Parasitol. 20, 6065. Mcfadden, D.C., Tomavo, S., Berry, E.A., Boothroyd, J.C., 2000. Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance. Mol. Biochem. Parasitol. 108, 112. Mejias-Torres, I.A., Zimmermann, B.H., 2002. Molecular cloning, recombinant expression and partial characterization of the aspartate transcarbamoylase from Toxoplasma gondii. Mol. Biochem. Parasitol. 119, 191201. Mercier, C., Cesbron-Delauw, M.F., Sibley, L.D., 1998. The amphipathic alpha helices of the toxoplasma protein GRA2 mediate post-secretory membrane association. J. Cell. Sci. 111 (Pt 15), 21712180. Mercier, C., Dubremetz, J.F., Rauscher, B., Lecordier, L., Sibley, L.D., Cesbron-Delauw, M.F., 2002. Biogenesis of nanotubular network in Toxoplasma parasitophorous

Toxoplasma Gondii

444

9. Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa

vacuole induced by parasite proteins. Mol. Biol. Cell. 13, 23972409. Miller, R.L., Linstead, D., 1983. Purine and pyrimidine metabolizing activities in Trichomonas vaginalis extracts. Mol. Biochem. Parasitol. 7, 4151. Mittal, D., Gubin, M.M., Schreiber, R.D., Smyth, M.J., 2014. New insights into cancer immunoediting and its three component phases—elimination, equilibrium and escape. Curr. Opin. Immunol. 27, 1625. Mora, P., Rubio, V., Fresquet, V., Cervera, J., 1999. Localization of the site for the nucleotide effectors of Escherichia coli carbamoyl phosphate synthetase using site-directed mutagenesis. FEBS Lett. 446, 133136. Mori, M., Tatibani, M., 1978. Multi-enzyme complex of glutamine-dependent carbamoyl phosphate synthetase with aspartate carbamoyltransferase and dihydroorotase from rat acites-hepatoma cells. Eur. J. Biochem. 86, 381388. Muller, S., Da’dara, A., Luersen, K., Wrenger, C., Das gupta, R., Madhubala, R., et al., 2000. In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase. J. Biol. Chem. 275, 80978102. Muller, S., Coombs, G.H., Walter, R.D., 2001. Targeting polyamines of parasitic protozoa in chemotherapy. Trends Parasitol. 17, 242249. Muller, I.B., Walter, R.D., Wrenger, C., 2005. Structural metal dependency of the arginase from the human malaria parasite Plasmodium falciparum. Biol. Chem. 386, 117126. Naguib, F.N.M., Wilson, C.M., El kouni, M.H., 2018. Enzymes of pyrimidine salvage pathways in intraerythrocytic Plasmodium falciparum. Int. J. Biochem. Cell. Biol. 105, 115122. Nara, T., Gao, G., Yamasaki, H., Nakajima-Shimada, J., Aoki, T., 1998. Carbamoyl-phosphate synthetase II in kinetoplastids. Biochim. Biophys. Acta 1387, 462468. Narvaez-Ortiz, H.Y., Lopez, A.J., Gupta, N., Zimmermann, B.H., 2018. A CTP synthase undergoing stage-specific spatial expression is essential for the survival of the intracellular parasite Toxoplasma gondii. Front. Cell. Infect. Microbiol. 8, 83. Ngo, H.M., Ngo, E.O., Bzik, D.J., Joiner, K.A., 2000. Toxoplasma gondii: are host cell adenosine nucleotides a direct source for purine salvage? Exp. Parasitol. 95, 148153. Niemand, J., Louw, A.I., Birkholtz, L., Kirk, K., 2012. Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum. Int. J. Parasitol. 42, 921929. Nolan, S.J., Romano, J.D., Coppens, I., 2017. Host lipid droplets: an important source of lipids salvaged by the

intracellular parasite Toxoplasma gondii. PLoS Pathog. 13, e1006362. Novak, W.R.P., West, K.H.J., Kirkman, L.M.D., Brandt, G. S., 2018. Re-refinement of Plasmodium falciparum orotidine 50 -monophosphate decarboxylase provides a clearer picture of an important malarial drug target. Acta Crystallogr. F: Struct. Biol. Commun. 74, 664668. Okell, L.C., Griffin, J.T., Roper, C., 2017. Mapping sulphadoxine-pyrimethamine-resistant Plasmodium falciparum malaria in infected humans and in parasite populations in Africa. Sci. Rep. 7, 7389. O’neil, R.H., Lilien, R.H., Donald, B.R., Stroud, R.M., Anderson, A.C., 2003a. The crystal structure of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis reveals a novel architecture for the bifunctional enzyme. J. Eukaryot. Microbiol. 50 (Suppl.), 555556. O’neil, R.H., Lilien, R.H., Donald, B.R., Stroud, R.M., Anderson, A.C., 2003b. Phylogenetic classification of protozoa based on the structure of the linker domain in the bifunctional enzyme, dihydrofolate reductasethymidylate synthase. J. Biol. Chem. 278, 5298052987. O’sullivan, W.J., Johnson, A.M., Finney, K.G., Gero, A.M., Hagon, E., Holland, J.W., et al., 1981. Pyrimidine and purine enzymes in Toxoplasma gondii. Aust. J. Exp. Biol. Med. Sci. 59, 763767. Painter, H.J., Morrisey, J.M., Mather, M.W., Vaidya, A.B., 2007. Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum. Nature 446, 8891. Panozzo-Zenere, E.A., Porta, E.O.J., Arrizabalaga, G., Fargnoli, L., Khan, S.I., Tekwani, B.L., et al., 2018. A minimalistic approach to develop new antiapicomplexa polyamines analogs. Eur. J. Med. Chem. 143, 866880. Paojinda, P., Imprasittichai, W., Krungkrai, S.R., Palacpac, N.M.Q., Horii, T., Krungkrai, J., 2018. Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 50 -monophosphate decarboxylase. Parasitol. Int. 67, 7984. Parker, M.D., Hyde, R.J., Yao, S.Y., Mcrobert, L., Cass, C.E., Young, J.D., et al., 2000. Identification of a nucleoside/ nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy. Biochem. J. 349, 6775. Pashley, T.V., Volpe, F., Pudney, M., Hyde, J.E., Sims, P.F., Delves, C.J., 1997. Isolation and molecular characterization of the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase gene from Toxoplasma gondii. Mol. Biochem. Parasitol. 86, 3747. Pavadai, E., El mazouni, F., Wittlin, S., De kock, C., Phillips, M.A., Chibale, K., 2016. Identification of new

Toxoplasma Gondii

References

human malaria parasite Plasmodium falciparum dihydroorotate dehydrogenase inhibitors by pharmacophore and structure-based virtual screening. J. Chem. Inf. Model. 56, 548562. Pawlowic, M.C., Somepalli, M., Sateriale, A., Herbert, G.T., Gibson, A.R., Cuny, G.D., et al., 2019. Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell. Proc. Natl. Acad. Sci. U.S.A. 116, 2116021165. Perotto, J., Keister, D.B., 1971. Incorporation of precursors into Toxoplasma DNA. J. Protozool. 18, 470473. Peterson, D.S., Walliker, D., Wellems, T.E., 1988. Evidence that a point mutation in dihydrofolate reductasethymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc. Natl. Acad. Sci. U.S. A. 85, 91149118. Pfefferkorn, E.R., 1978. Toxoplasma gondii: the enzymic defect of a mutant resistant to 5-fluorodeoxyuridine. Exp. Parasitol. 44, 2635. Pfefferkorn, E.R., Borotz, S.E., 1994. Toxoplasma gondii: characterization of a mutant resistant to 6-thioxanthine. Exp. Parasitol. 79, 374382. Pfefferkorn, E.R., Pfefferkorn, L.C., 1976. Arabinosyl nucleosides inhibit Toxoplasma gondii and allow the selection of resistant mutants. J. Parasitol. 62, 993999. Pfefferkorn, E.R., Pfefferkorn, L.C., 1977a. Specific labeling of intracellular Toxoplasma gondii with uracil. J. Protozool. 24, 449453. Pfefferkorn, E.R., Pfefferkorn, L.C., 1977b. Toxoplasma gondii: characterization of a mutant resistant to 5fluorodeoxyuridine. Exp. Parasitol. 42, 4455. Pfefferkorn, E.R., Pfefferkorn, L.C., 1977c. Toxoplasma gondii: specific labeling of nucleic acids of intracellular parasites in Lesch-Nyhan cells. Exp. Parasitol. 41, 95104. Pfefferkorn, E.R., Pfefferkorn, L.C., 1978. The biochemical basis for resistance to adenine arabinoside in a mutant of Toxoplasma gondii. J. Parasitol. 64, 486492. Pfefferkorn, E.R., Pfefferkorn, L.C., 1981. Toxoplasma gondii: growth in the absence of host cell protein synthesis. Exp. Parasitol. 52, 129136. Pfefferkorn, E.R., Eckel, M.E., Mcadams, E., 1989. Toxoplasma gondii: the biochemical basis of resistance to emimycin. Exp. Parasitol. 69, 129139. Pfefferkorn, E.R., Borotz, S.E., Nothnagel, R.F., 1993. Mutants of Toxoplasma gondii resistant to atovaquone (566C80) or decoquinate. J. Parasitol. 79, 559564. Pfefferkorn, E.R., Bzik, D.J., Honsinger, C.P., 2001. Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine. Exp. Parasitol. 99, 235243.

445

Phillips, M.A., 2018. Polyamines in protozoan pathogens. J. Biol. Chem. 293, 1874618756. Phillips, M.A., Rathod, P.K., 2010. Plasmodium dihydroorotate dehydrogenase: a promising target for novel antimalarial chemotherapy. Infect. Disord. Drug. Targets 10, 226239. Phillips, M.A., Gujjar, R., Malmquist, N.A., White, J., El mazouni, F., Baldwin, J., et al., 2008. Triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors with potent and selective activity against the malaria parasite Plasmodium falciparum. J. Med. Chem. 51, 36493653. Phillips, M.A., White, K.L., Kokkonda, S., Deng, X., White, J., El mazouni, F., et al., 2016. A triazolopyrimidinebased dihydroorotate dehydrogenase inhibitor with improved drug-like properties for treatment and prevention of malaria. ACS Infect. Dis. 2, 945957. Plagemann, P.G., 1986. Transport and metabolism of adenosine in human erythrocytes: effect of transport inhibitors and regulation by phosphate. J. Cell. Physiol. 128, 491500. Plagemann, P.G., Wohlhueter, R.M., Woffendin, C., 1988. Nucleoside and nucleobase transport in animal cells. Biochim. Biophys. Acta 947, 405443. Puiu, D., Enomoto, S., Buck, G.A., Abrahamsen, M.S., Kissinger, J.C., 2004. CryptoDB: the Cryptosporidium genome resource. Nucleic Acids Res. 32, D329D331. Queen, S.A., Vander jagt, D., Reyes, P., 1988. Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 30, 123133. Queen, S.A., Jagt, D.L., Reyes, P., 1990. In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism. Antimicrob. Agents Chemother. 34, 13931398. Rager, N., Mamoun, C.B., Carter, N.S., Goldberg, D.E., Ullman, B., 2001. Localization of the Plasmodium falciparum PfNT1 nucleoside transporter to the parasite plasma membrane. J. Biol. Chem. 276, 4109541099. Rais, R.H., Al safarjalani, O.N., Yadav, V., Guarcello, V., Kirk, M., Chu, C.K., et al., 2005. 6-Benzylthioinosine analogues as subversive substrate of Toxoplasma gondii adenosine kinase: activities and selective toxicities. Biochem. Pharmacol. 69, 14091419. Raman, J., Mehrotra, S., Anand, R.P., Balaram, H., 2004. Unique kinetic mechanism of Plasmodium falciparum adenylosuccinate synthetase. Mol. Biochem. Parasitol. 138, 18. Rashmi, M., Yadav, M.K., Swati, D., 2017. Targeting pyrimidine pathway of Plasmodium knowlesi: new strategies towards identification of novel antimalarial chemotherapeutic agents. Comb. Chem. High Throughput Screen. 20, 547558.

Toxoplasma Gondii

446

9. Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa

Rathod, P.K., Khatri, A., Hubbert, T., Milhous, W.K., 1989. Selective activity of 5-fluoroorotic acid against Plasmodium falciparum in vitro. Antimicrob. Agents Chemother. 33, 10901094. Reyes, P., Rathod, P.K., Sanchez, D.J., Mrema, J.E., Rieckmann, K.H., Heidrich, H.G., 1982. Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 5, 275290. Reynolds, M.G., Roos, D.S., 1998. A biochemical and genetic model for parasite resistance to antifolates. Toxoplasma gondii provides insights into pyrimethamine and cycloguanil resistance in Plasmodium falciparum. J. Biol. Chem. 273, 34613469. Reynolds, M.G., Oh, J., Roos, D.S., 2001. In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate reductase. Antimicrob. Agents Chemother. 45, 12711277. Roberts, F., Roberts, C.W., Johnson, J.J., Kyle, D.E., Krell, T., Coggins, J.R., et al., 1998. Evidence for the shikimate pathway in apicomplexan parasites. Nature 393, 801805. Roberts, S., Ullman, B., 2017. Parasite polyamines as pharmaceutical targets. Curr. Pharm. Des. 23, 33253341. Roberts, S.C., Tancer, M.J., Polinsky, M.R., Gibson, K.M., Heby, O., Ullman, B., 2004. Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania. Characterization of gene deletion mutants. J. Biol. Chem. 279, 2366823678. Robles lopez, S.M., Hortua Triana, M.A., Zimmermann, B. H., 2006. Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii. Mol. Biochem. Parasitol. 148, 9398. Romano, J.D., Sonda, S., Bergbower, E., Smith, M.E., Coppens, I., 2013. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole. Mol. Biol. Cell. 24, 19741995. Roos, D.S., 1993. Primary structure of the dihydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii. J. Biol. Chem. 268, 62696280. Roos, D.S., Donald, R.G., Morrissette, N.S., Moulton, A.L., 1994. Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol. 45, 2763. Roos, D.S., Sullivan, W.J., Striepen, B., Bohne, W., Donald, R.G., 1997. Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis. Methods 13, 112122. Ruiz, V., Czyzyk, D.J., Valhondo, M., Jorgensen, W.L., Anderson, K.S., 2019. Novel allosteric covalent inhibitors of bifunctional Cryptosporidium hominis TS-DHFR

from parasitic protozoa identified by virtual screening. Bioorg. Med. Chem. Lett. 29, 14131418. Sanders, K.L., Fox, B.A., Bzik, D.J., 2015. Attenuated Toxoplasma gondii stimulates immunity to pancreatic cancer by manipulation of myeloid cell populations. Cancer Immunol. Res. 3, 891901. Sanders, K.L., Fox, B.A., Bzik, D.J., 2016. Attenuated Toxoplasma gondii therapy of disseminated pancreatic cancer generates long-lasting immunity to pancreatic cancer. Oncoimmunology 5, e1104447. Sarkar, D., Ghosh, I., Datta, S., 2004. Biochemical characterization of Plasmodium falciparum hypoxanthine-guaninexanthine phosphorybosyltransferase: role of histidine residue in substrate selectivity. Mol. Biochem. Parasitol. 137, 267276. Sarwono, A.E.Y., Mitsuhashi, S., Kabir, M.H.B., Shigetomi, K., Okada, T., Ohsaka, F., et al., 2019. Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening. J. Enzyme Inhib. Med. Chem. 34, 171178. Satriano, J., 2003. Agmatine: at the crossroads of the arginine pathways. Ann. N.Y. Acad. Sci. 1009, 3443. Schatten, H., Ris, H., 2004. Three-dimensional imaging of Toxoplasma gondii-host cell interactions within the parasitophorous vacuole. Microsc. Microanal. 10, 580585. Schumacher, M.A., Carter, D., Ross, D.S., Ullman, B., Brennan, R.G., 1996. Crystal structures of Toxoplasma gondii HGXPRTase reveal the catalytic role of a long flexible loop. Nat. Struct. Biol. 3, 881887. Schumacher, M.A., Carter, D., Scott, D.M., Roos, D.S., Ullman, B., Brennan, R.G., 1998. Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding. EMBO J. 17, 32193232. Schumacher, M.A., Scott, D.M., Mathews, I.I., Ealick, S.E., Roos, D.S., Ullman, B., et al., 2000a. Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding. J. Mol. Biol. 298, 875893. Schumacher, M.A., Scott, D.M., Mathews, I.I., Ealick, S.E., Roos, D.S., Ullman, B., et al., 2000b. Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding. J. Mol. Biol. 296, 549567. Schumacher, M.A., Bashor, C.J., Song, M.H., Otsu, K., Zhu, S., Parry, R.J., et al., 2002. The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase. Proc. Natl. Acad. Sci. U.S.A. 99, 7883. Schwab, J.C., Beckers, C.J., Joiner, K.A., 1994. The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve. Proc. Natl. Acad. Sci. U.S.A. 91, 509513.

Toxoplasma Gondii

References

Schwab, J.C., Afifi afifi, M., Pizzorno, G., Handschumacher, R.E., Joiner, K.A., 1995. Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter. Mol. Biochem. Parasitol. 70, 5969. Schwartzman, J.D., Pfefferkorn, E.R., 1981. Pyrimidine synthesis by intracellular Toxoplasma gondii. J. Parasitol. 67, 150158. Schwartzman, J.D., Pfefferkorn, E.R., 1982. Toxoplasma gondii: purine synthesis and salvage in mutant host cells and parasites. Exp. Parasitol. 53, 7786. Schwertz, G., Witschel, M.C., Rottmann, M., Leartsakulpanich, U., Chitnumsub, P., Jaruwat, A., et al., 2018. Potent Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT) featuring a spirocyclic scaffold. ChemMedChem. 13, 931943. Sethi, K.K., Pelster, B., Piekarski, G., Brandis, H., 1973. Multiplication of Toxoplasma gondii in enucleated L cells. Nat. New Biol. 243, 255256. Seymour, K.K., Lyons, S.D., Phillips, L., Rieckmann, K.H., Christopherson, R.I., 1994. Cytotoxic effects of inhibitors of de novo pyrimidine biosynthesis upon Plasmodium falciparum. Biochemistry 33, 52685274. Sharling, L., Liu, X., Gollapalli, D.R., Maurya, S.K., Hedstrom, L., Striepen, B., 2010. A screening pipeline for antiparasitic agents targeting cryptosporidium inosine monophosphate dehydrogenase. PLoS Negl. Trop. Dis. 4, e794. Shaw, M.H., Freeman, G.J., Scott, M.F., Fox, B.A., Bzik, D.J., Belkaid, Y., et al., 2006. Tyk2 negatively regulates adaptive Th1 immunity by mediating IL-10 signaling and promoting IFN-gamma-dependent IL-10 reactivation. J. Immunol. 176, 72637271. Shi, W., Li, C.M., Tyler, P.C., Furneaux, R.H., Cahill, S.M., Girvin, M.E., et al., 1999a. The 2.0 A structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor. Biochemistry 38, 98729880. Shi, W., Li, C.M., Tyler, P.C., Furneaux, R.H., Grubmeyer, C., Schramm, V.L., et al., 1999b. The 2.0 a structure of human hypoxanthine-guanine phosphoribosyltransferase in complex with a transition-state analog inhibitor. Nat. Struct. Biol. 6, 588593. Shi, W., Ting, L.M., Kicska, G.A., Lewandowicz, A., Tyler, P.C., Evans, G.B., et al., 2004. Plasmodium falciparum purine nucleoside phosphorylase: crystal structures, immucillin inhibitors, and dual catalytic function. J. Biol. Chem. 279, 1810318106. Shivashankar, K., Subbayya, I.N., Balaram, H., 2001. Development of a bacterial screen for novel hypoxanthine-guanine phosphoribosyltransferase substrates. J. Mol. Microbiol. Biotechnol. 3, 557562.

447

Sibley, L.D., Niesman, I.R., Asai, T., Takeuchi, T., 1994. Toxoplasma gondii: secretion of a potent nucleoside triphosphate hydrolase into the parasitophorous vacuole. Exp. Parasitol. 79, 301311. Sibley, L.D., Niesman, I.R., Parmley, S.F., Cesbron-Delauw, M.F., 1995. Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J. Cell. Sci. 108 (Pt 4), 16691677. Srivastava, I.K., Morrisey, J.M., Darrouzet, E., Daldal, F., Vaidya, A.B., 1999. Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites. Mol. Microbiol. 33, 704711. Striepen, B., Kissinger, J.C., 2004. Genomics meets transgenics in search of the elusive Cryptosporidium drug target. Trends Parasitol. 20, 355358. Striepen, B., White, M.W., Li, C., Guerini, M.N., Malik, S.B., Logsdon JR., J.M., et al., 2002. Genetic complementation in apicomplexan parasites. Proc. Natl. Acad. Sci. U.S.A. 99, 63046309. Striepen, B., Pruijssers, A.J., Huang, J., Li, C., Gubbels, M.J., Umejiego, N.N., et al., 2004. Gene transfer in the evolution of parasite nucleotide biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 101, 31543159. Suarez, C.E., Bishop, R.P., Alzan, H.F., Poole, W.A., Cooke, B.M., 2017. Advances in the application of genetic manipulation methods to apicomplexan parasites. Int. J. Parasitol. 47, 701710. Sufrin, J.R., Meshnick, S.R., Spiess, A.J., Garofalo-Hannan, J., Pan, X.Q., Bacchi, C.J., 1995. Methionine recycling pathways and antimalarial drug design. Antimicrob. Agents Chemother. 39, 25112515. Sukhumavasi, W., Egan, C.E., Warren, A.L., Taylor, G.A., Fox, B.A., Bzik, D.J., et al., 2008. TLR adaptor MyD88 is essential for pathogen control during oral Toxoplasma gondii infection but not adaptive immunity induced by a vaccine strain of the parasite. J. Immunol. 181, 34643473. Sullivan JR, W.J., Chiang, C.W., Wilson, C.M., Naguib, F. N., El kouni, M.H., Donald, R.G., et al., 1999. Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus. Mol. Biochem. Parasitol. 103, 114. Sun, X.E., Sharling, L., Muthalagi, M., Mudeppa, D.G., Pankiewicz, K.W., Felczak, K., et al., 2010. Prodrug activation by Cryptosporidium thymidine kinase. J. Biol. Chem. 285, 1591615922. Sun, Z., Khan, J., Makowska-Grzyska, M., Zhang, M., Cho, J.H., Suebsuwong, C., et al., 2014. Synthesis, in vitro evaluation and cocrystal structure of 4-oxo-[1]benzopyrano[4,3-c]pyrazole Cryptosporidium parvum inosine 50 monophosphate dehydrogenase (CpIMPDH) inhibitors. J. Med. Chem. 57, 1054410550.

Toxoplasma Gondii

448

9. Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa

Suss-Toby, E., Zimmerberg, J., Ward, G.E., 1996. Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore. Proc. Natl. Acad. Sci. U.S.A. 93, 84138418. Tabor, C.W., Tabor, H., 1985. Polyamines in microorganisms. Microbiol. Rev. 49, 8199. Takashima, Y., Mizohata, E., Krungkrai, S.R., Fukunishi, Y., Kinoshita, T., Sakata, T., et al., 2012a. The in silico screening and X-ray structure analysis of the inhibitor complex of Plasmodium falciparum orotidine 50 -monophosphate decarboxylase. J. Biochem. 152, 133138. Takashima, Y., Mizohata, E., Tokuoka, K., Krungkrai, S.R., Kusakari, Y., Konishi, S., et al., 2012b. Crystallization and preliminary X-ray diffraction analysis of orotate phosphoribosyltransferase from the human malaria parasite Plasmodium falciparum. Acta Crystallogr. Sect. F: Struct. Biol. Cryst. Commun. 68, 244246. Tanaka, M., Gu, H.M., Bzik, D.J., Li, W.B., Inselburg, J., 1990a. Mutant dihydrofolate reductase-thymidylate synthase genes in pyrimethamine-resistant Plasmodium falciparum with polymorphic chromosome duplications. Mol. Biochem. Parasitol. 42, 8391. Tanaka, M., Gu, H.M., Bzik, D.J., Li, W.B., Inselburg, J.W., 1990b. Dihydrofolate reductase mutations and chromosomal changes associated with pyrimethamine resistance of Plasmodium falciparum. Mol. Biochem. Parasitol. 39, 127134. Teotia, P., Prakash dwivedi, S., Dwivedi, N., 2016. A QSAR model of benzoxazole derivatives as potential inhibitors for inosine 5ʹ-monophosphate dehydrogenase from Cryptosporidium parvum. Bioinformation 12, 119123. Terao, M., 1963. On A new antibiotic, Emimycin. II. Studies on the structure of emimycin. J. Antibiot. (Tokyo) 16, 182186. Terao, M., Karasawa, K., Tanaka, N., Yonehara, H., Umezawa, H., 1960. On a new antibiotic, emimycin. J. Antibiot. 13A, 401405. Tewari, S.G., Rajaram, K., Schyman, P., Swift, R., Reifman, J., Prigge, S.T., et al., 2019. Short-term metabolic adjustments in Plasmodium falciparum counter hypoxanthine deprivation at the expense of long-term viability. Malar. J. 18, 86. Thomas, A., Field, M.J., 2002. Reaction mechanism of the HGXPRTase from Plasmodium falciparum: a hybrid potential quantum mechanical/molecular mechanical study. J. Am. Chem. Soc. 124, 1243212438. Ting, L.M., Shi, W., Lewandowicz, A., Singh, V., Mwakingwe, A., Birck, M.R., et al., 2005. Targeting a novel Plasmodium falciparum purine recycling pathway with specific immucillins. J. Biol. Chem. 280, 95479554. Traut, T.W., 1994. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 122.

Triglia, T., Cowman, A.F., 1994. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S.A. 91, 71497153. Triglia, T., Wang, P., Sims, P.F., Hyde, J.E., Cowman, A.F., 1998. Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria. EMBO J. 17, 38073815. Tyler, P.C., Taylor, E.A., Frohlich, R.F., Schramm, V.L., 2007. Synthesis of 50 -methylthio coformycins: specific inhibitors for malarial adenosine deaminase. J. Am. Chem. Soc. 129, 68726879. Upston, J.M., Gero, A.M., 1995. Parasite-induced permeation of nucleosides in Plasmodium falciparum malaria. Biochim. Biophys. Acta 1236, 249258. Valdes, R., Elferich, J., Shinde, U., Landfear, S.M., 2014. Identification of the intracellular gate for a member of the equilibrative nucleoside transporter (ENT) family. J. Biol. Chem. 289, 87998809. Vasanthakumar, G., Davis JR., R.L., Sullivan, M.A., Donahue, J.P., 1989. Nucleotide sequence of cDNA clone for hypoxanthine-guanine phosphoribosyltransferase from Plasmodium falciparum. Nucleic Acids Res. 17, 8382. Vasanthakumar, G., Davis JR., R.L., Sullivan, M.A., Donahue, J.P., 1990. Cloning and expression in Escherichia coli of a hypoxanthine-guanine phosphoribosyltransferase-encoding cDNA from Plasmodium falciparum. Gene 91, 6369. Vasquez, J.R., Gooze, L., Kim, K., Gut, J., Petersen, C., Nelson, R.G., 1996. Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum. Mol. Biochem. Parasitol. 79, 153165. Vinayak, S., Pawlowic, M.C., Sateriale, A., Brooks, C.F., Studstill, C.J., Bar-Peled, Y., et al., 2015. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523, 477480. Wang, C.C., 1984. Parasite enzymes as potential targets for antiparasitic chemotherapy. J. Med. Chem. 27, 19. Wang, C.C., Aldritt, S., 1983. Purine salvage networks in Giardia lamblia. J. Exp. Med. 158, 17031712. Wang, C.C., Simashkevich, P.M., 1981. Purine metabolism in the protozoan parasite Eimeria tenella. Proc. Natl. Acad. Sci. U.S.A. 78, 66186622. Wang, P., Nirmalan, N., Wang, Q., Sims, P.F., Hyde, J.E., 2004a. Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 135, 7787. Wang, P., Wang, Q., Aspinall, T.V., Sims, P.F., Hyde, J.E., 2004b. Transfection studies to explore essential folate metabolism and antifolate drug synergy in the human

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Further reading

malaria parasite Plasmodium falciparum. Mol. Microbiol. 51, 14251438. Webster, H.K., Wiesmann, W.P., Pavia, C.S., 1984. Adenosine deaminase in malaria infection: effect of 20 deoxycoformycin in vivo. Adv. Exp. Med. Biol. 165 (Pt A), 225229. White, E.L., Ross, L.J., Davis, R.L., Zywno-Van ginkel, S., Vasanthakumar, G., Borhani, D.W., 2000. The two Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers. J. Biol. Chem. 275, 1921819223. White, J., Dhingra, S.K., Deng, X., El mazouni, F., Lee, M.C. S., Afanador, G.A., et al., 2018. Identification and mechanistic understanding of dihydroorotate dehydrogenase point mutations in Plasmodium falciparum that confer in vitro resistance to the clinical candidate DSM265. ACS Infect Dis. 5, 90101. Woods, K.M., Upton, S.J., 1998. Efficacy of select antivirals against Cryptosporidium parvum in vitro. FEMS Microbiol. Lett. 168, 5963. Wrenger, C., Luersen, K., Krause, T., Muller, S., Walter, R. D., 2001. The Plasmodium falciparum bifunctional ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, enables a well balanced polyamine synthesis without domain-domain interaction. J. Biol. Chem. 276, 2965129656. Wu, G., Morris, S.M., 1998. Arginine metabolism: nitric oxide and beyond. Biochem. J. 336, 117. Xu, L., Li, W., Diao, Y., Sun, H., Li, H., Zhu, L., et al., 2018. Synthesis, design, and structureactivity relationship of the pyrimidone derivatives as novel selective inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase. Molecules 23, E1254. Xu, P., Widmer, G., Wang, Y., Ozaki, L.S., Alves, J.M., Serrano, M.G., et al., 2004. The genome of Cryptosporidium hominis. Nature 431, 11071112.

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Yarlett, N., Martinez, M.P., Zhu, G., Keithly, J.S., Woods, K., Upton, S.J., 1996. Cryptosporidium parvum: polyamine biosynthesis from agmatine. J. Eukaryot. Microbiol. 43, 73S. Yuvaniyama, J., Chitnumsub, P., Kamchonwongpaisan, S., Vanichtanankul, J., Sirawaraporn, W., Taylor, P., et al., 2003. Insights into antifolate resistance from malarial DHFR-TS structures. Nat. Struct. Biol. 10, 357365. Zeiner, G.M., Cleary, M.D., Fouts, A.E., Meiring, C.D., Mocarski, E.S., Boothroyd, J.C., 2008. RNA analysis by biosynthetic tagging using 4-thiouracil and uracil phosphoribosyltransferase. Methods Mol. Biol. 419, 135146. Zhang, M., Wang, C., Otto, T.D., Oberstaller, J., Liao, X., Adapa, S.R., et al., 2018. Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis. Science 360, eaap7847. Zhou, Z., Metcalf, A.E., Lovatt, C.J., Hyman, B.C., 2000. Alfalfa (Medicago sativa) carbamoylphosphate synthetase gene structure records the deep lineage of plants. Gene 243, 105114.

Further reading Allison, A.C., Eugui, E.M., 2000. Mycophenolate mofetil and its mechanisms of action. Immunopharmacology 47, 85118. Pockros, P.J., Reindollar, R., Mchutchinson, J., Reddy, R., Wright, T., Boyd, D.G., et al., 2003. The safety and tolerability of daily infergen plus ribavirin in the treatment of naive chronic hepatitis C patients. J. Viral. Hepat. 10, 5560. Sierra pagan, M.L., Zimmermann, B.H., 2003. Cloning and expression of the dihydroorotate dehydrogenase from Toxoplasma gondii. Biochim. Biophys. Acta 1637, 178181.

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C H A P T E R

10 Metabolic networks and metabolomics Joachim Kloehn1,*, Aarti Krishnan1,*, Christopher J. Tonkin2,3, Malcolm J. McConville4,5 and Dominique Soldati-Favre1 1

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland 2Division of Infectious Disease and Immune Defence, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 3Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia 4Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia 5Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia

10.1 Introduction All members of the Apicomplexa, including Toxoplasma gondii, are obligate intracellular parasites and exhibit many metabolic specializations that reflect their intracellular lifestyle, niche, and dependency on their host’s metabolic capabilities. These parasites display specific or alternate metabolic networks, including those associated with the vestigial plastid (apicoplast) found in many Apicomplexans. Understanding the parasite’s requirements for intracellular replication and the contribution of uptake of essential metabolites versus de novo synthesis is crucial to identify new drug targets for these important human and animal pathogens. While the T. gondii genome sequence provides clues about the global metabolic

capabilities of these parasites, pathways and enzymes that are essential in one life cycle stage may be dispensable in another, highlighting the importance in developing a detailed understanding of the metabolic needs of each life cycle stage. Recent studies have advanced our knowledge of T. gondii metabolism and allow the delineation of central carbon metabolism as well as the synthesis and uptake of lipids, cofactors, and vitamins. These studies include genome-wide gene essentiality screens, transcriptomic analysis of T. gondii life cycle stages, detailed functional analyses of metabolic pathways employing molecular biology, and metabolomic methods as well as in silico approaches that can predict essential metabolites and synthetic lethalities. Fig. 10.1 provides

* These authors contributed equally to this work.

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00010-4

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FIGURE 10.1 Metabolic pathways in T. gondii. An overview of the main metabolic pathways and their subcellular localization in T. gondii is presented. The five main metabolic compartments are shown and the pathways occurring within these compartments are depicted. Pathways that take place across two or more organelles are highlighted in blue. ATP, adenosine triphosphate; BCAA, branched chain amino acids; FAS, fatty acid synthase; Fe
S, iron
sulfur; GPI, glycosylphosphatidylinositol; ISC, iron
sulfur cluster; NADH, nicotinamide adenine dinucleotide; SUF, sulfur utilization factor; TCA, tricarboxylic acid.

an overview of the major metabolic pathways and their localization in T. gondii. This chapter provides an overview of T. gondii tachyzoite and bradyzoite central carbon metabolism. We start with a description of a genome-scale model that has recently been developed using computational approaches, incorporating genome annotations and -omics data, providing meaningful and harmonized predictions that match with experimentally observed phenotypes. The second section includes a detailed review of the recent advances in central-carbon, carbohydrate, vitamin, and cofactormetabolism and their contributions for the development and differentiation of T. gondii during the acute and chronic stage of the infection. The last section concludes with an overview of the different metabolomics methodologies that have been used to dissect and understand the metabolic shifts upon perturbation of metabolic pathways by measuring the carbon fluxes using stable isotope labeling and mass spectrometry

(MS). In addition to this chapter, other metabolic networks taking place in the apicoplast and mitochondrion are discussed in Chapter 11 (The apicoplast and mitochondrion of Toxoplasma gondii), while Chapter 8 (Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake) and Chapter 9 (Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa) provide a comprehensive overview of the lipid metabolism and amino acid/ nucleotide metabolism, respectively.

10.2 Genome-scale metabolic modeling 10.2.1 Systems biology approaches for understanding metabolism The systems-wide analysis of parasite metabolism is now possible with the availability of many parasite genomes, and large-scale

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10.2 Genome-scale metabolic modeling

-omics datasets (Bordbar et al., 2014). In particular, the development of genome-scale models has great utility for identifying metabolic bottlenecks or transport mechanisms that might be viable targets for novel therapeutic interventions. Because model predictions greatly depend on the quality and completeness of the genome annotation, the scientific community has put a great emphasis on increasing gene identification and in curating genome annotation. To integrate all the available genome-wide datasets such as transcriptomics, proteomics, and metabolomics, genome-scale metabolic models (GEMs) serve as valuable platform and enable model-guided investigations. Following curation and incorporation of data, metabolic networks can illuminate context-specific differences, in an attempt to understand metabolic capabilities under a given state. The models can further elucidate how changes in one component affect other pathways and phenotypes, for example, growth, bioenergetics, fluxes, biosynthesis of biomass components, and metabolite and biproduct secretion, thus providing a mechanistic link between genotype and phenotype. In this section, we will review the process for metabolic reconstruction from the genome, integration of -omics data, imposition of constraints, and applications to better understand the biology of obligate intracellular parasites.

10.2.2 Metabolic modeling and analysis of T. gondii During its complex life cycle, T. gondii exists in both sexual (gametes) and asexual stages (tachyzoites, bradyzoites, merozoites, and sporozoites). The two forms found in intermediate hosts, including humans, are the tachyzoites and bradyzoites. While tachyzoites are generally characterized as fast-growing, metabolically active stages, bradyzoites are thought to be slow-growing or non-dividing and

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metabolically quiescent. However, it is likely that both stages exhibit a spectrum of growth and metabolic phenotypes over time and depending on the precise tissue context in which they are found. Understanding the parasites’ biology and the factors enabling parasite proliferation and persistence is vital for drug target identification. Given the complexity of metabolic pathways and the cross-talk between them, several approaches combining mathematical modeling and its integration with various large-scale data are needed. The two previously published genome-scale reconstructions for T. gondii [ToxoNet1 (Tymoshenko et al., 2015) and iCS382 (Song et al., 2013)] comprised 1089 and 400 metabolic reactions, respectively. These models attempted to capture major anabolic and catabolic pathways, as well as nutrient salvage processes, that are needed to generate defined biomass building blocks (BBBs). The main objective of a simulated metabolic network is typically the production of the BBBs that encompass proteins, lipids, nucleic acids, and carbohydrates and represent the essential components required for cell survival. A standard approach to analyze a metabolic network and simulate in silico gene knockouts (KOs) is flux-balance analysis (FBA). FBA is a constrained-based approach that enumerates all feasible phenotypic states, that is, flow of metabolites through the network, thereby making it possible to predict the growth rate of a cell or the rate of production of BBBs (Orth et al., 2010; Yilmaz and Walhout, 2017). Simple FBA models are constrained by the objective function selected (i.e., optimization for maximum growth and efficient use of nutrients) as well as the inclusion of other parameters such as compartmentalization, mass conservation (stoichiometry), and thermodynamic directionality. Fig. 10.2A describes a general workflow for the reconstruction of a genome-scale model, and its subsequent analysis. More recently, integration of -omics datasets has been used to

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FIGURE 10.2 Metabolic modeling of Toxoplasma gondii. (A) Workflow to reconstruct a metabolic network. (B) Components of a genome-scale model, integration of -omics data and analysis with FBA (constraint based) or kinetic modeling. FBA, Flux-balance analysis; GPR, gene
protein-reaction associations. Source: Adapted from (A) Tymoshenko, S., Oppenheim, R.D., Agren, R., Nielsen, J., Soldati-Favre, D., Hatzimanikatis, V., 2015. Metabolic needs and capabilities of Toxoplasma gondii through combined computational and experimental analysis. PLoS Comput. Biol. 11, e1004261. (B) Bordbar, A., Monk, J.M., King, Z.A., Palsson, B.O., 2014. Constraint-based models predict metabolic and associated cellular functions. Nat. Rev. Genet. 15, 107
120; Saa, P.A., Nielsen, L.K., 2017. Formulation, construction and analysis of kinetic models of metabolism: a review of modelling frameworks. Biotechnol. Adv. 35, 981
1003.

optimize and reduce the number of computed feasible states (Ebrahim et al., 2016; Kim et al., 2016). The mathematical representations of the reactions and of the objective function are

defined as a system of linear equations. These equations can then be solved using linear programming and software tools such as the Constraint-Based Reconstruction and Analysis

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(COBRA) Toolbox (Bordbar et al., 2014; Ebrahim et al., 2016; Lewis et al., 2012) for MATLAB (MathWorks Inc.). GEMs have been used to predict the consequences of individual gene KOs. Using this approach, ToxoNet1 identified 53 genes and 76 reactions essential for the production of biomass. FBA analysis was also used to identify gene KOs that are not essential individually but lead to synthetic lethality when combined with deletion of a second gene. Using ToxoNet1, 20 gene combinations were identified as synthetic lethal (Tymoshenko et al., 2015). To further improve the predictions, methods to infer thermodynamic parameters have also been developed. Algorithms to apply rigorous thermodynamic constraints by eliminating thermodynamically infeasible pathways or constraining flux according to Gibbs free energy calculations (Henry et al., 2007) are being used and optimized. Because constraints-based models are often under-determined, they may provide multiple mathematically feasible solutions, and therefore the predictions must always be validated with experimentally proven data.

10.2.3 Harmonization of metabolic models with experimental data In order to identify metabolic bottlenecks and potential drug targets, working with a harmonized model, which matches with experimentally observed data, is becoming increasingly important. Models can now be challenged or analyzed for their ability to provide “true-positive” predictions, by comparing the results with large-scale datasets, such as the CRISPR-Cas9-mediated loss-of-fitness screens. For T. gondii, the recently published genomewide screen scores genes as fitness-conferring or dispensable, assigning a fitness score (FS) between a highly negative (27.0, indicating essentiality) and a positive value (13, indicating dispensability) (Sidik et al., 2016).

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The first step in building a highly harmonized and robust network is careful manual curation. Gap-filling of pathways based on newly annotated genes and the inclusion of additional and missing biochemical data such as substrate uptake or secretion rates are needed to improve the accuracy of the model. Continuous data mining and literature search for the identification and integration of updated findings into the model is required. Once a model is well curated, workflows to impose further thermodynamics constraints, restrictions on substrate uptake and secretion, -omics data, and finally kinetic parameters can be applied (Fig. 10.2B). In order to generate a context-specific and harmonized model for T. gondii, recent studies (Krishnan et al., unpublished) have aimed at gap-filling ToxoNet1 with the most recent genome annotation and incorporating additional constraints on substrate availabilities from the host as well as transcriptomics data (Stanway et al., 2019). This workflow, known as PhenoMapping, led to the generation of a metabolic network that displays 80% consistency with experimentally observed phenotypes (Krishnan et al., unpublished). Overall, simulation of a harmonized network leads to a better understanding of mechanisms that govern parasite development and differentiation and can be used to postulate new hypotheses that can be tested experimentally. As complementary components of an iterative process, experimental and computational analysis must ultimately lead to the identification of novel genes, reactions, and pathways within the parasite and interactions between the parasite and its host.

10.2.4 Future perspectives Key challenges remain in building largescale metabolic models and understanding how they are regulated. In particular, a

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number of recent studies in other systems have shown that for the majority of metabolic reactions, the concentrations of the responsible enzyme (itself the product of upstream transcriptional control) have only a minor impact on the rate of the reaction. The major exceptions to this are enzymes that control essentially irreversible (rate limiting) reactions (Hackett et al., 2016). Therefore integration of transcriptomics and proteomic data into metabolic models may have variable validity. Conversely, many metabolic reactions within cells are regulated by the availability of key reactants (carbon sources) in the extracellular milieu. Therefore, determination of metabolic fluxes in intracellular parasites such as T. gondii will be critically dependent on an understanding of metabolite levels in the host cell, and more specifically in the vacuolar compartment. Another limitation of the simulation of a static network is that it represents only a snapshot of the metabolic state of a cell. Given that cells are highly dynamic and time-dependent changes occur, kinetic modeling of metabolism is a promising alternative. In kinetic models, biochemical reactions are represented by both the stoichiometry of metabolites and the reaction rates as functions of their concentrations. Reaction rates (or flux through a reaction) and stoichiometry together define differential equations that describe the evolution of metabolite concentrations over time. The rate equations therefore contain parameters that can be measured and validated by standard biochemical methods (Saa and Nielsen, 2017). Fully parametrized kinetic models can be used to simulate the time-dependent flux distribution and concentrations of metabolites under physiological or perturbation conditions resulting from KOs and knockdowns (KDs) of specific genes or enzymatic-inhibition with drugs. Kinetic models are more precise than static models and offer the possibility to integrate metabolic pathways with gene regulation

and protein
protein interactions, crucial for predicting adaptation and resistance to treatment of parasites. The main limitation in the generation of kinetic models is the lack of kinetic data, and its complexity at a genomewide level (Srinivasan et al., 2015). In conclusion, increasing amounts of largescale data, mathematical modeling coupled with data integration, aim to unravel parasite biology and identify the crucial factors involved in the proliferation and differentiation. Elucidating important aspects of parasite development and its interaction with diverse host environments can guide the research for novel drug discoveries and methodologies for efficient interventions. The virtuous cycle of experimental data feeding mathematical models, and hypothesis generation for future experiments, would allow for an in-depth analysis of biological systems and novel therapies targeting metabolism. With the emergence of drug resistance and the necessity for rapid solutions, analysis at a systems level with combined methods is fundamental.

10.3 Central carbon metabolism 10.3.1 Glycolysis Free glucose (Glc) is an abundant metabolite in most mammalian cells and is one of the major carbon sources used by intracellular tachyzoite or bradyzoite stages. Following its phosphorylation by hexokinase (HK), Glc is catabolized in glycolysis, to generate ATP, or channeled into the pentose phosphate pathway (PPP) to generate redox equivalents and enable the synthesis of pentose-phosphates (pentoseP) required for DNA and RNA synthesis. Hexose-P can also be channeled into the synthesis of amylopectin, the major reserve polysaccharide of these parasites, as well as into the synthesis of sugar nucleotides that act as precursors for several glycosylation pathways

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(see Section 10.4). Glc is primarily taken up by the plasma membrane Glc transporter, GT1 (Pomel et al., 2008), although T. gondii expresses three other putative hexose transporters (ST1-3). While ST2 localizes to the plasma membrane, ST1 and ST3 are present in intracellular organelles (Blume et al., 2009). While T. gondii tachyzoites preferentially utilize Glc they can survive and grow in Glc depleted medium and T. gondii mutants lacking GT1 and ST2 exhibit only a minor decrease in intracellular growth rate (Blume et al., 2009). Parasites lacking the main Glc transporter, GT1, survived and grew normally in fibroblasts by increasing their utilization of glutamine (Blume et al., 2009), which is catabolized in the tricarboxylic acid (TCA) cycle and converted to sugar phosphates via gluconeogenesis (MacRae et al., 2012). Significantly, parasites of the type I RH strain lacking GT1 were fully virulent in mice, demonstrating that glycolysis is dispensable during the acute phase of T. gondii infection. Similarly, RH parasites lacking HK also remained virulent in mice (Shukla et al., 2018). While residual Glc uptake through ST2 or other promiscuous transporters could not be entirely excluded in Δgt1 parasites, HK is solely responsible for Glc activation and essential to initiate glycolysis. The relatively low negative FS from the genome-wide CRISPR Cas9 screen for HK (FS: 21.37) is in accordance with the experimentally demonstrated dispensability of T. gondii HK under standard culture conditions (Sidik et al., 2016; Shukla et al., 2018). Importantly, type II parasites (Prugniaud strain) lacking HK were severely impaired in their ability to establish chronic infection, forming 10
100-fold less cysts in the brains of infected mice compared to wild type parasites (Shukla et al., 2018). It remains unclear whether bradyzoites are more dependent on the uptake of Glc (or other sugars) for intracellular survival because they are more dependent on glycolysis for ATP synthesis and/or whether these stages have less access

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to glutamine or other gluconeogenic sources within their host niche. Following Glc uptake and phosphorylation, Glc-6-phosphate (Glc6P) undergoes a series of metabolic reactions and is ultimately converted into two molecules of pyruvate. T. gondii expresses all glycolytic enzymes which localize to the cytosol, while some display a dual localization, residing both in the cytosol and apicoplast. Fig. 10.3 provides an overview of the enzymes and reactions in glycolysis and gluconeogenesis. The figure points out the FS for each enzyme using a heatmap color code and highlights the dual localization to the cytosol and apicoplast of some glycolytic enzymes discussed next. In contrast to the HK the FS of several other glycolytic enzymes suggest essentiality. As glycolysis per se has been shown to be dispensable, essentiality may be attributed to one or several of the following reasons: the accumulation of a toxic substrate, a second metabolic function of the targeted gene, or a nonmetabolic role which is essential. T. gondii aldolase, which catalyzes the conversion of fructose-1,6-bisphosphate (FBP) into two triose-phosphates, was previously believed to have an essential non-metabolic function as the F-actin binding protein able to bridge the adhesins cytoplasmic tails to the actin-myosinmotor during motility, invasion, and egress from infected cells (Jewett and Sibley, 2003). However, it was later shown that aldolase is not required for gliding but rather may be essential due to the accumulation of its toxic substrate FBP (Shen and Sibley, 2014). Other glycolytic enzymes such as glyceraldehyde-3phosphate (GA3P) dehydrogenase 1 (GAPDH1) have also been proposed to have moonlighting activity. GAPDH1 is found in the cytosol and catalyzes the conversion of GA3P into 1,3-bisphosphoglycerate, generating NADPH. Following egress of T. gondii, GAPDH1 translocates to the cortical membrane skeleton, likely mediated by N-terminal

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FIGURE 10.3 Glycolysis and gluconeogenesis. An overview of the reactions and enzymes in glycolysis and gluconeogenesis is given. Glycolysis describes the breakdown of Glc into two molecules of pyruvate to produce energy, while gluconeogenesis is the synthesis of Glc from non-carbohydrate sources. Most enzymes function bidirectionally in glycolysis and gluconeogenesis depending on the metabolic state of the cell. Metabolites are depicted in black, enzymes are depicted in purple. Reducing equivalents, cofactors, or energetic compounds produced or consumed are shown in gray. The FS for each enzyme and in some cases, both isoenzymes, are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. Other related pathways are encircled. The dual localization of some glycolytic enzymes and its central carbon metabolism reactions in the apicoplast are also depicted. Substrates, products, and cofactors: ATP/ADP, Adenosine tri/diphosphate; CoA, coenzyme A; NAD(P)H, nicotinamide adenine dinucleotide (phosphate). Metabolites: 1,3-DPGA/1,3-BP-glycerate, 1,3-bisphosphoglycerate; 3-PG, 3-phosphoglycerate; GA3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; OAA, oxaloacetate; PYR, pyruvate; PEP, phosphoenolpyruvate. Enzymes: ALD, aldolase; APT, apicoplast phosphate transporter; ENO, enolase; GT, Glc transporter; PDH, pyruvate dehydrogenase; FAS, fatty acid synthase; FBPase, fructose-1,6-bisphosphatase; FNT, formate
nitrite transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, Glc-6-phosphate isomerase; HK: hexokinase; LDH, lactate dehydrogenase; PEPCK, phosphoenolpyruvate-carboxykinase; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PYK, pyruvate kinase; TPI, triosephosphate isomerase.

palmitoylation (Dubey et al., 2017). The function it fulfills here is unclear. GAPDH1 was essential in glutamine-depleted, but not glutamine-replete medium, consistent with the notion that glycolysis can be compensated

through increased glutaminolysis (Dubey et al., 2017). However, the rescue of Δgapdh1 mutant parasites with glutamine was not as effective as in the case of the Δhk mutant parasites (Shukla et al., 2018), which may reflect the dual

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role of GAPDH1 in glycolysis and gluconeogenesis, the role of its product GA3P in the nonoxidative PPP, or an unknown nonmetabolic function associated with its translocation to the cortex. Previously, other groups have observed the translocation of several glycolytic enzymes to the pellicle following egress, presumably to provide ATP locally for the high energy demand of the molecular processes that drive motility (Pomel et al., 2008). Other glycolytic isoenzymes have also been shown to localize to the apicoplast and may be essential for normal apicoplast metabolic function. For example, T. gondii expresses a second GAPDH enzyme (GAPDH2), which localizes exclusively to the apicoplast (Pino et al., 2007). Its FS of 24.40 indicates essentiality; however, the role of GAPDH2 and other glycolysis and TCA cycle enzymes in the apicoplast remains unclear. GAPDH2 may provide reducing power in the form of NADPH required for the fatty acid (FA) synthesis by FA synthase II (FASII) located in apicoplast. The potential moonlighting activities, dual localizations to the apicoplast and cytosol, and the fact that most glycolytic enzyme also function in gluconeogenesis complicate the interpretation of the role of glycolysis and the involved enzymes. Under anaerobic conditions, pyruvate is converted to lactate which can be secreted from the parasite as a fermentation end-product. For the conversion of pyruvate, T. gondii encodes two lactate dehydrogenases (LDH1 and LDH2), both of which are individually dispensable according to their FS (LDH1-FS: 0.78; LDH2-FS: 0.49). The expression of the two genes is highly stage specific, with LDH1 being exclusively expressed in the tachyzoite stage and LDH2 being expressed predominantly in the bradyzoite stage (ToxoDB). Two research groups independently confirmed that deletion of LDH1 and LDH2 as well as simultaneous deletion of both genes is tolerated under standard culture conditions with no obvious fitness

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defect (Abdelbaset et al., 2017; Xia et al., 2018). However, loss of LDH1 was associated with attenuated virulence during the acute phase of infection and reduced cyst numbers during the chronic infection. While additional deletion of LDH2 further reduced the in vivo virulence during acute infection, it did not affect the cyst numbers. Hence, LDH1, although expressed at low levels in the bradyzoite stage, is a key virulence factor during chronic infection. Crucially, it was demonstrated that LDH1 becomes essential for tachyzoites and bradyzoites in vitro under physiological oxygen (O2) conditions (3% as opposed to ambient 21% O2) (Xia et al., 2018). These findings indicate that lactate fermentation is dispensable under aerobic conditions but becomes limiting under anaerobic conditions. Three formate
nitrite transporter (FNT)
type monocarboxylate/proton symporters, which potentially transport lactate across the plasma membrane, have been identified (Erler et al., 2018). While the low FS of the FNTs (1.1; 0.23; 1.31) suggest individual dispensability of these transporters, multiple KOs to assess potential functional redundancy have not been attempted to date. Under standard culture conditions, lactate is secreted from the parasite and host cell and accumulates in spent medium.

10.3.2 Gluconeogenesis Gluconeogenesis refers to the process of synthesizing Glc de novo from various noncarbohydrate carbon sources including amino acids, lactate, glycerol, propionate, and others. Under Glc-limiting conditions, gluconeogenesis becomes an essential pathway for T. gondii with glutamine being the essential carbon source, which cannot be replaced by other potential gluconeogenic substrates (Nitzsche et al., 2016). Gluconeogenesis requires the expression of phosphoenolpyruvate-carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FBPase), in

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addition to other enzymes in glycolysis that catalyze the reverse flux to their normal role. PEPCK converts oxaloacetate (OAA) generated in the TCA cycle (derived from glutamine under Glc deplete conditions) into PEP, which is subsequently converted to FBP that is dephosphorylated to fructose-6-phosphate by FBPase (Fig. 10.3). T. gondii encodes two isoforms of PEPCK. PEPCK1 is expressed in tachyzoites and targeted to the mitochondrion; PEPCK2 is thought to play a role in the oocyst stage (Nitzsche et al., 2017). T. gondii also expresses two isoforms of FBPase, although both are constitutively expressed in tachyzoites (Blume et al., 2015). While both enzymes exhibit FBPase activity in vitro, the deletion of FBPase1 had no effect on parasite fitness. On the other hand, inducible KD of FBPase2 led to complete loss of tachyzoite viability under both Glcreplete, as well as Glc-limited culture conditions and loss of virulence in mice (Blume et al., 2015). The catalytic activity of FBPase2 is essential for T. gondii survival and growth under Glc-replete conditions, and KD of expression was associated with altered central carbon metabolism. These findings suggest that FBPase may participate in a metabolic futile cycle with phosphofructokinase (PFK) to regulate glycolytic and PPP fluxes in T. gondii tachyzoites. This is further confirmed by the high negative FS of both enzymes, FBPase (24.77) and PFK (25.19), suggesting essentiality of PFK, which stands in contrast to the dispensability of the HK.

10.3.3 Pentose phosphate pathway The oxidative branch of the PPP generates NADPH reducing equivalents and ribose-5phosphate (R5P), a precursor for nucleotide synthesis, while the nonoxidative branch facilitates the interconversion and recycling of various sugar phosphates (triose-P, tetrose-P, pentose-P, hexose-P, and heptulose-P) back

into glycolysis or the oxidative PPP. The genome of T. gondii encodes all major PPP enzymes of the oxidative and non-oxidative branch. Fig. 10.4 summarizes the enzymes and reactions of the PPP and their FS as indicated by a heatmap color code. Unexpected insights came from the genome-wide CRISPR/Cas9 screen, which indicates that all PPP enzymes are dispensable, having low negative or positive FS, with the exception of phosphoribosyl pyrophosphate synthetase (PRPS) which appears to be essential. The PRPS catalyzes the activation of R5P to form phosphoribosyl pyrophosphate, which is required for the de novo synthesis of pyrimidines and the purine salvage pathway in T. gondii. These results suggest that T. gondii may salvage key intermediates, possibly including R5P from the host cell. Alternatively, several PPP enzymes have isoenzymes, particularly in the oxidative branch, such as the Glc6P dehydrogenase and the 6phosphogluconate dehydrogenase, which may compensate for the loss of single isoforms. The FS of transaldolase and transketolase of T. gondii are 21.14 and 0.33, respectively, suggesting that the nonoxidative branch may also be dispensable. A recent study showed that T. gondii encodes a sedoheptulose-1,7-bisphosphatase (SBPase) (Olson et al., 2018). SBPase participates in the Calvin cycle in plants but has been proposed to contribute to the nonoxidative branch of the PPP in some nonphotosynthetic organisms, including protozoan parasites (Hannaert et al., 2003; Kovarova and Barrett, 2016), while being absent in mammals. Consistent with this, overexpression of the SBPase resulted in increased carbon flux through the non-oxidative PPP (Olson et al., 2018). While the authors propose the SBPase as a potential drug target, it seems to be dispensable based on its low positive FS (0.65). The PPP and its role in synthesizing pentoses versus the uptake of intermediates from the host is poorly characterized in T. gondii and deserves further investigation.

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FIGURE 10.4 Oxidative and nonoxidative PPP. An overview of the reactions and enzymes involved in the oxidative and nonoxidative PPP is given. Enzymes in the PPP catalyze the synthesis of pentoses from Glc and produce reducing equivalents. All reactions occur in the cytosol and are connected to glycolysis as indicated. The FS for each enzyme and in some cases for both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. Metabolites are depicted in black, enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. Other related pathways are encircled. Substrates, products, and cofactors: ATP/ADP, adenosine tri/diphosphate; NAD(P)H, nicotinamide adenine dinucleotide (phosphate). Metabolites: DHAP, dihydroxyacetone phosphate; PRPP, 5-phospho-D-ribose α-1-pyrophosphate. Enzymes: G6PDH, Glc-6-phsosphate dehydrogenase; 6-PGL, 6-phosphogluconolactonase; 6-PGDH, 6-phosphogluconate dehydrogenase; RPI, ribose-5-phosphate isomerase; RPE, ribose-5-phosphate epimerase; RBK, ribokinase; PRPP, phosphoribosyl pyrophosphate; TKL, transketolase; TALD, transaldolase; SBPase, sedoheptulose-1,7-bisphosphatase; FB-ALD, fructosebisphosphate aldolase; dR-ALD, deoxyribose-phosphate aldolase.

10.3.4 Tricarboxylic acid cycle, 2-MCC, and the γ-aminobutyric acid shunt MacRae et al. (2012) were the first to show that T. gondii has a complete TCA cycle, which is fueled by both Glc and glutamine. Glcderived pyruvate enters the mitochondrion through the mitochondrial pyruvate carrier (MPC), a heterodimer composed of MPC1 and MPC2 (MacRae et al., 2012; Oppenheim et al., 2014), which are non-essential in T. gondii (Krishnan et al., unpublished). Pyruvate is subsequently converted to acetyl(C2)-coenzyme A (CoA) which then combines with OAA (C4) to generate citrate (C6) and initiate the canonical oxidative TCA cycle (Fig. 10.5). In other

eukaryotes, the conversion of pyruvate to acetyl-CoA is catalyzed by the pyruvate dehydrogenase (PDH) complex. T. gondii and other Apicomplexa express a PDH complex, but all of the subunits in this complex are targeted to the apicoplast and are thus unable to provide acetyl-CoA for the TCA cycle (Pino et al., 2007). The conundrum of how pyruvate is converted to acetyl-CoA in the mitochondrion was solved when Oppenheim et al. (2014) demonstrated that the branched chain α-keto acid dehydrogenase (BCKDH) complex fulfills the PDH function in the mitochondrion of T. gondii. While BCKDH typically acts in the catabolism of the branched chain amino acids (BCAAs—leucine, isoleucine, and valine),

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FIGURE 10.5 TCA cycle, 2-MCC, and the GABA shunt. A schematic of the reactions occurring in the TCA cycle, 2MCC, and GABA shunt is shown. The TCA cycle produces energy and reducing equivalents from acetyl-CoA. The GABA shunt can feed glutamate into the TCA cycle while bypassing two steps of the TCA cycle and allows the catabolism of GABA while the 2-MCC detoxifies propionate. All three pathways occur in the mitochondrial lumen and are interconnected. The FS for each enzyme and in some cases both isoenzymes is indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. Metabolites are depicted in black, enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. Other related pathways are encircled. Pathways: BCAA degradation, Branched chain amino acid degradation; ETC, electron transport chain; TCA cycle, tricarboxylic acid cycle. Substrates, products, and cofactors: ATP/ADP, adenosine tri/diphosphate; GTP, guanosine triphosphate; NAD(P)H, nicotinamide adenine dinucleotide (phosphate); CoA, coenzyme A. Enzymes: PrpE, propionyl-CoA synthetase; PrpC, 2-methylcitrate synthase; PrpD, 2-methylcitrate dehydratase; ACN, aconitase; PrpB, 2-methylcitrate lyase; SDH succinate dehydrogenase-; FH, fumarate hydratase; MDH, malate dehydrogenase; PyC, pyruvate carboxylase; MPC, mitochondrial pyruvate carrier; BCKDH, branched chain α-keto acid dehydrogenase; CS, citrate synthase; ICDH, isocitrate dehydrogenase; GDH, glutamate dehydrogenase; GAD, glutamate decarboxylase; GABA, γ-aminobutyric acid; AA, amino acid; GS, glutamine synthetase; α-KDH, ketoglutarate dehydrogenase; GAT, GABA transaminase; SSDH, succinatesemialdehyde dehydrogenase; S-CoA-S, succinyl coenzyme A synthetase.

the catalytic mechanism of reaction is similar to that of the α-KG dehydrogenase and the PDH. Hence, it was speculated that the BCKDH may have replaced the function of PDH in T. gondii and Plasmodium (Seeber et al., 2008;

Cobbold et al., 2013). Later, Oppenheim et al. demonstrated that deletion of BCKDH indeed results in the loss of flux from Glc derived carbons into the TCA cycle and is compensated for by increased glutaminolysis and

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gluconeogenesis. This rewired central carbon metabolism is accompanied by attenuated growth in culture and virulence in vivo of T. gondii as well as P. berghei. T. gondii possesses all TCA cycle enzymes allowing the recycling of OAA, while generating reducing equivalents (two NADH) and energy (two ATP). The NADH generated in the TCA cycle can enter the electron transport chain (ETC) in the inner mitochondrial membrane to facilitate the generation of further ATP and complete the full oxidation of Glc. Why the TCA cycle of T. gondii is essential is not fully understood. As for glycolytic enzymes, the interpretation of TCA cycle gene depletions is complicated by potential moonlighting activity, multiple localizations, and contribution of enzymes to other pathways (e.g., oxidative phosphorylation—OXPHOS, 2MCC). Oppenheim et al. (2014) have shown that loss of BCKDH results in a severe fitness defect but the complex is dispensable, as it is compensated for by increased glutaminolysis that fuels the lower TCA cycle. Under these conditions, OAA/aspartate are the endproduct, and the pathway does not function as a continuous cycle. According to these findings, it is anticipated that the citrate synthase (CS), aconitase (ACN), and isocitrate dehydrogenase (ICDH) activities are dispensable, as these enzymes function downstream of BCKDH and upstream of the glutamate dehydrogenase which fuels glutamine derived carbons into the TCA cycle as α-KG. Indeed, the FS for putative CSs indicates dispensability (putative CS1, TGME49_263130 FS: 1.86; CS1, TGME49_268890 FS: 1-2.40; putative CS2, TGME49_203110 FS: 1.72). The subsequent enzyme, ACN, however, has a high negative FS (24.73), indicating its essentiality. Consistent with this, pharmacological inhibition of ACN with sodium fluoroacetate was shown to be detrimental to T. gondii tachyzoite growth, and associated with a severe increase (150-fold) in isocitrate/citrate levels that are

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most likely toxic to the parasites (MacRae et al., 2012). The essentiality of ACN function may thus be attributed to the toxicity of its substrate rather than a lack of its product. Importantly, ACN localizes to the mitochondrion, cytosol, and apicoplast and possibly holds a non-metabolic function as an RNA binding protein participating in transcriptional regulation of genes involved in iron (Fe) metabolism and contains an Fe
sulfur (Fe
S) cluster. The Fe
S cluster may be involved in its metabolic catalytic mechanism and may also be required for Fe sensing and, hence the non-metabolic role of ACN. Finally, ACN also functions in the 2-MCC. The 2-MCC consists of five enzymes and catalyzes the conversion of toxic propionate, which accumulates during the breakdown of BCAAs, to pyruvate and succinate. The enzymes required for a complete 2-MCC have been identified in T. gondii and evidence was presented that the pathway was acquired through horizontal gene transfer (Limenitakis et al., 2013). Although the pathway is not essential, parasites lacking the rate-limiting enzyme 2methylisocitrate lyase exhibit an increased sensitivity to exogeneous propionate. Following the activity of ACN in the TCA cycle, the subsequent conversion of isocitrate to α-KG is catalyzed by ICDH. T. gondii encodes two ICDHs, one of which localizes to the apicoplast, while the other localizes to the mitochondrion (Pino et al., 2007). The function of the apicoplast resident ICDH1 is unclear, but, like GAPDH2, it may provide reducing power for the synthesis of FAs in the apicoplast. In contrast to the apicoplast resident GAPDH2, ICDH1 is dispensable according to its FS of 0.35, which was also confirmed experimentally (Oppenheim et al., unpublished). The mitochondrial ICDH2, on the other hand, has a high negative FS (23.04) suggesting essentiality. ICDH2 directly generates NADPH, which is utilized to generate ATP in the ETC, which may account for its essentiality. Following

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ICDH2 activity, α-KG is converted to succinyl CoA through the α-KG dehydrogenase. Based on its very high negative FS (24.80), it is expected that the α-KG dehydrogenase is essential for tachyzoites survival. Together, this can be interpreted as evidence that the loss of Glc contribution to the TCA cycle is tolerable for tachyzoites, while a partially functioning TCA cycle, operating from α-KG to OAA, is essential. This partial TCA cycle may be crucial to generate energy [guanosine triphosphate (GTP)—succinyl CoA synthetase], participate directly in the ETC (succinate dehydrogenase), generate reducing equivalents for the ETC (NADH—α-KG dehydrogenase, malate dehydrogenase), or enable gluconeogenesis from glutamine. Surprisingly though, downregulation of the succinyl-CoA synthetase only results in a 30% reduction in intracellular growth, indicating that these reactions in the TCA cycle may be non-essential (Fleige et al., 2007). The authors, however, cannot exclude residual levels of succinyl-CoA in the analyzed parasites. Further research is needed to dissect the role of the TCA cycle in T. gondii energy metabolism. Extracellular tachyzoites were found to accumulate very high millimolar levels of intracellular γ-aminobutyric acid (GABA), and 13 C-glutamine labeling studies indicated the presence of a GABA shunt that bypasses several steps in the TCA cycle (MacRae et al., 2012). This shunt is catalyzed by three enzymes that were all localized to the mitochondrion: glutamate decarboxylase, glutamate transamidase, and a succinic-semialdehyde dehydrogenase which convert glutamate to GABA and succinate, as well as GABA transporters which transport GABA out of the mitochondrion and across the plasma membrane. Inhibition of the GABA shunt resulted in reduced tachyzoite motility suggesting that this metabolite may function as a short-term energy reserve, while extracellular tachyzoites find a new host cell (MacRae et al., 2012). However, deletion of

GABA shunt enzymes only resulted in a modest decrease in intracellular growth, consistent with the low positive or low negative FS for these enzymes (MacRae et al., 2012).

10.3.5 Oxidative phosphorylation OXPHOS allows the complete oxidation of Glc under aerobic conditions, where reducing equivalents formed during the TCA cycle provide electrons for an ETC located in the inner mitochondrial membrane. The transport of electrons from NADH and FADH2 to O2 through several protein complexes enables the build-up of a proton gradient that is utilized by the ATP synthase (complex V) to generate ATP. T. gondii lacks the conventional complex I but possesses two genes that encode a nonproton pumping type-II NADH dehydrogenases which localizes to the mitochondrion (Lin et al., 2008). Aside from complex I, T. gondii codes for all enzymes required for a functional ETC. Fig. 10.6 provides an overview of the complexes involved in the ETC and provides the FS for each complex as indicated by a heatmap color scheme. Vercesi et al. (1998) were the first to demonstrate that T. gondii has a functional OXPHOS that is active in tachyzoites by using a range of metabolic inhibitors and measuring the O2 consumption rate in permeabilized parasite extracts. Recently, a number of uncharacterized subunits of the T. gondii complex V were identified, and shown to have orthologs that were restricted to Apicomplexa, Chromerida, and Dinoflagellates, suggesting they may be possible drug targets (Salunke et al., 2018). Another study has revealed two subunits of the T. gondii complex V lateral stalk or stator, ICAP2 and ICAP18 (Huet et al., 2018). Downregulation of ICAP2, one of the stator subunits, led to a marked decrease in O2 consumption and a stark growth defect, suggesting an essential role of OXPHOS in tachyzoites (Huet et al., 2018). This dependence on OXPHOS is increased when

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FIGURE 10.6

The electron transport chain (ETC). An overview of the complexes embedded in the inner mitochondrial membrane carrying out OXPHOS is shown. Electrons are transported across the complexes facilitating the translocation of protons into the intermembrane space. Flux of protons through complex V drives the synthesis of ATP. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. The FS for multiprotein complexes represents an average of the FS for each subunit. Substrates, products, and cofactors: ATP/ADP, adenosine tri/diphosphate; NAD(P)H, nicotinamide adenine dinucleotide (phosphate); FADH2, flavin adenine dinucleotide; Pi, inorganic phosphate; NDH, NADH dehydrogenase; SDH, succinate dehydrogenase; DHOD, dihydroorotate dehydrogenase; G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

glycolysis is inhibited, as in mutant parasites lacking LDH1 and LDH2, which become hypersensitive to oligomycin, an inhibitor of the complex V (Xia et al., 2018). Taken together, these results demonstrate that T. gondii is able to rapidly respond to changes in extracellular levels of carbon sources, by rerouting carbon flux through glycolysis and the TCA cycle, and generate most of their ATP from either glycolysis and/or mitochondrial OXPHOS, contributing to their remarkable ability to adapt to different host cell niches.

10.3.6 Fatty-acid biosynthesis T. gondii expresses two FAS complexes. The FASI is evolutionarily related to the animal/ fungal FAS complexes and is expressed in the cytosol, while the bacterial/archaea-like FASII complex is targeted to the apicoplast. Both FAS

complexes catalyze similar reactions but are structurally distinct. FASI comprises a single large multifunction protein, while FASII is a multiprotein complex. Both FAS pathways extend a priming acyl chain with an activated malonyl group, with both, acyl and malonyl linked to an acyl-carrier-protein (ACP). The growing chain is extended by two carbons with each cycle consisting of condensation, reduction, rehydration, and reduction reactions. The repetition of this process, initiated each time by malonyl condensation with the growing chain, leads to the formation of evennumbered FAs, typically palmitic (C16:0) or myristic acid (C14:0). Fig. 10.7 depicts the FA synthesis and elongation pathway in T. gondii. The FS for the involved enzymes is indicated by a heatmap color scheme. The role and importance of the cytosolic FASI in T. gondii remain unclear. We have recently depleted FASI in type II ME49 parasites and observed

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FIGURE 10.7 FA synthesis and elongation. A schematic of the FA synthesis and elongation pathway in Toxoplasma gondii is shown. FA synthesis occurs in the apicoplast through a bacterial- or archaea-like FASII or a cytosolic FASI. The de novo synthesized FAs can be further elongated or desaturated at the cytosolic leaflet of the ER. The enzymes implemented in the pathways are shown in purple and the FS associated with the loss of the enzyme function is indicated in a heatmap color code—blue indicating dispensability, while red indicates essentiality. Metabolites are depicted in black, enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. Substrates, products, and cofactors: ATP/ADP, adenosine tri/diphosphate; GTP, guanosine triphosphate; NAD(P)H, nicotinamide adenine dinucleotide (phosphate); CoA, coenzyme A; SAM, S-adenosyl methionine. Metabolites: PEP, phosphoenolpyruvate; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid. Enzymes: APT, apicoplast phosphate transporter; G3PDH, glycerol-3-phosphate dehydrogenase; G3PAT, glycerol-3-phosphate acyltransferase; Acyl-T, acyl transferase; ACP, acyl carrier protein; LipA, lipoyl synthetase; LipB, octanoyltransferase; ACCase, acetyl-CoA carboxylase; PDH, pyruvate dehydrogenase; ELO, elongase; PYK, pyruvate kinase.

no loss of fitness in the tachyzoite or bradyzoite stage in vitro or in vivo (unpublished data). Presumably, FASI is required for FA synthesis in T. gondii merozoites or sporozoites based on the higher expression of FASI in these stages (ToxoDB). In contrast, FASII is constitutively expressed and has been shown to be the main site of FA synthesis in tachyzoites.

Conditional depletion of the FASII protein ACP caused a severe fitness defect in tachyzoites and loss of virulence in mice, highlighting the importance of FASII (Mazumdar et al., 2006). FAs generated by FASII are required for membrane biosynthesis, apicoplast biogenesis, and the posttranslational lipoylation of the apicoplast PDH. In addition, parasites treated

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with the FASII inhibitor triclosan as well as tetracycline-inducible ACP mutant parasites were shown to display impaired pellicle formation during cytokinesis (Martins-Duarte et al., 2016). In accordance with these findings, we observed that the depletion of FabZ, the FASII 3-hydroxyacyl-[ACP] dehydratase, results in a severe fitness defect, yet ΔFabZ tachyzoites can survive and continue to propagate, albeit slowly, through increased uptake of hostderived FAs (Krishnan et al., unpublished). While Ramakrishnan et al. reported that loss of ACP was not rescued through provision of exogenous C14:0 and C16:0 (20 nM
2 μM), we observed a partial rescue of the fitness defect in parasite lacking FabZ when supplementing with very high concentrations of exogenous C14:0 and C16:0 (100 μM) (Ramakrishnan et al., 2012; Krishnan et al., unpublished). As observed for parasites lacking ACP, T. gondii depleted in FabZ showed partial loss of the apicoplast, suggesting that FASII participates in apicoplast biogenesis directly through its supply of FAs or through interaction with other proteins involved in the organelle biogenesis. While apicoplast resident FASII is required for the de novo synthesis of medium chain length saturated FAs (C14:0 and C16:0), synthesis of long-chain monounsaturated and saturated FAs occurs at the cytosolic leaflet of the endoplasmic reticulum (ER) through the action of three elongases (ELO-A, -B, and -C), two reductases, and a dehydratase. Depletion of the three ELOs, while individually dispensable, results in a severely altered FAs profile of these parasites with loss of long-chain saturated FAs (Ramakrishnan et al., 2012). Specifically, ELOA appears to be responsible for the generation of a range of saturated and monounsaturated C18-C26 FAs, while ELO-B elongates long unsaturated FAs (C20-C26) and ELO-C is specific for the generation of C26:1, which is present in T. gondii but absent in host cells. While the elongases are individually dispensable, the

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loss of the elongation pathway altogether is detrimental to T. gondii (Ramakrishnan et al., 2015). Parasites depleted of the hydroxyacylCoA dehydratase and enoyl-CoA reductase enzymes, which together with the ketoacylCoA reductase and ELO-A, ELO-B, and ELO-C function in the elongation pathway, were not viable. The mutant parasites displayed a loss in unsaturated long-chain FAs and related lipid species. Intriguingly, the genetic obstruction of the ELO pathway could be rescued by supplementing exogeneous unsaturated longchain FAs. As for FASII, this indicates de novo FAs synthesis and elongation are required for parasite fitness and uptake of exogenous FAs is inefficient and compensates poorly for the loss of synthesis. It was recently reported that the cytosolic acetyl-CoA synthetase (ACS) provides acetyl-CoA for FAs elongation as KD of this enzyme led to a mildly altered FA and lipid profile (Dubois et al., 2018). However, the authors observed no drastic phenotype or fitness defect, likely because cytosolic acetyl-CoA can also be provided by the ATP-citrate lyase (ACL). Indeed, dual depletion of ACS and ACL is detrimental to T. gondii (Tymoshenko et al., 2015), likely due to impaired FA elongation or due to impairment of histone and nonhistone protein acetylation. Another study elucidated the transport of acyl-CoA chains in T. gondii. The acyl-CoA-binding protein (TgACBP1) and a sterol carrier protein-2 (TgSCP2) were found to participate in the transport of long-chain-acyl-CoA esters in the cytosol (Fu et al., 2019). While the individual depletion of the carrier proteins does not cause any fitness defect, the simultaneous depletion of both enzymes cytosolic acyl-CoA transporters results in smaller plaque size, concomitant with reduced intracellular growth and an invasion defect. Parasites depleted in both enzymes show reduced levels of long-chain monounsaturated FAs as well as altered levels in several phospholipids. These results indicate that ACBP1 and SCP2 play a synergistic role in

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transporting acyl chains through the FA elongation pathway and through lipid synthesis pathways.

10.3.7 Beta-oxidation Most eukaryotic cells are able to catabolize FAs by β-oxidation, generating acetyl-CoA which can enter the TCA cycle and generate energy. T. gondii encodes all the genes required for β-oxidation, but it remains unclear under what conditions this pathway is active or whether it is essential for intracellular growth of different parasite stages. There is no evidence for active β-oxidation in T. gondii tachyzoites or bradyzoites. β-Oxidation can occur in the mitochondrion (typically FAs of shorter chain lengths) as well as in peroxisomes (typically long-chain FAs). And it has been proposed that a peroxisome located β-oxidation pathway may occur in the sporozoite stages of some coccidian parasites. These stages need to survive outside the host for extended periods of time, and may need to switch to catabolizing intracellular lipids reserves for energy generation (Moog et al., 2017).

major Glc transporter GT1 and phosphorylated by HK or galactokinase, prior to their conversion to their cognate sugar nucleotide. UDP-Glc and GDP-Man are also converted to the dolichol (Dol)-linked sugars, Dol-P-Glc and Dol-P-Man, which are used as sugar donors for the assembly of Dol-PP-oligosaccharides and glycosylphosphatidylinositol (GPI) anchors, respectively, in the lumen of the ER. Many of the enzymes involved in UDP-GlcNAc and GDP-Man synthesis are essential for intracellular growth based on their FSs in the genome-wide CRISPR/Cas9 phenotypic screen (Sidik et al., 2016). In contrast, parasite mutants with defects in galE (interconverts UDP-Glc/GlcNAc and UDP-Gal/GalNac) or the two isoforms of phosphoglucomutases (GPMs) (regulates flux of hexose-P into UDP-Glc and/or the recycling of Glc-1-phosphate (Glc1P) generated during amylopectin breakdown) exhibited little change in fitness (Saha et al., 2017; Sidik et al., 2016). The modest growth phenotypes of the latter mutants likely reflect the presence of alternative pathways (in the case of galE) or as yet uncharacterized enzyme activities (in the case of the two GPMs) that compensate for the loss of these proteins (Saha et al., 2017).

10.4 Carbohydrate metabolism 10.4.2 Glycosylation pathways in the secretory pathway

10.4.1 Sugar nucleotide synthesis Sugar nucleotides constitute an important class of anabolic intermediates that are used to synthesize the major T. gondii carbohydrate reserve material, amylopectin, as well as an array of different lipid- and protein-linked glycoconjugates. Genome annotations and experimental studies have demonstrated that T. gondii has de novo or salvage pathways for the synthesis of uridine diphosphate-Glc/N-acetylglucosamine (UDP-Glc/GlcNAc), UDP-galactose/Nacetylgalactosamine (UDP-Gal/GalNAc), guanosine diphosphate-mannose (GDP-Man), and GDP-fucose (GDP-Fuc) (Fig. 10.8). In the case of the salvage pathway, sugars are taken up by the

As in other eukaryotes, the majority of proteins that enter the secretory pathway of T. gondii are co- or posttranslationally modified with N-glycans, O-glycans, and/or a GPI-anchor (Bandini et al., 2019a). GPI glycolipids that are not attached to proteins also constitute the major class of glycolipids in these parasites (Striepen et al., 1997). The structures and biosynthesis of these glycans are briefly discussed next. 10.4.2.1 N-Glycans T. gondii proteins synthesized on ER ribosomes and translocated into the ER lumen are

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FIGURE 10.8 Sugar nucleotide biosynthesis in Toxoplasma gondii. Pathways for the synthesis of UDP-Glc/GlcNAc, UDP-Gal/GalNAc, and GDP-Man have been inferred from genome annotations and experimental studies. Glycosylation pathways that utilize different sugar nucleotides are indicated in red boxes. The enzymes implemented in the pathways are shown in purple and the FS associated with the loss of the enzyme function is indicated in a heatmap color code—blue indicating dispensability, while red indicates essentiality. Metabolites are depicted in black. Dol, Dolichol; Dol-Man-Syn, dolichol-phosphate-mannose synthase; DolPGlc-Syn, dolichol-phosphate-Glc synthase; Fuc, fucose; GalK, galactose-1-kinase; GalNAc, N-acetylglucosamine; GDP, guanosine diphosphate; GDP-L-Fuc-Syn, GDP-L-Fucose synthetase; GDP-Man-DH, GDP-Man 4,6-dehydratase; GDP-Man-PL, GDP-Man pyrophosphorylase; Glc, glucose; GlcN AcetylT/, glucosamine-phosphate N-acetyltransferase; GlcNAc, N-acetylglucosamine; GlcN-Fruc6P-AT, glucosamine:fructose-6-phosphate aminotransferase; GPM, phosphoglucomutase; Hyp, hydroxyproline; Man, mannose; ManPM, phosphomannose mutase; PFK, phosphofructokinase; UDP, uridine diphosphate; UDP-GlcNAc-PL, UDP-N-acetylglucosamine pyrophosphorylase; UTP:GalPUT, UTP hexose-1-phosphate uridyltransferase; UTP:Glc1P-UT, UTP:Glc-1-phosphate uridylylltransferase.

cotranslationally modified with relatively simple N-glycans that typically contain the canonical Glc3Man5GlcNAc2 core structure (Fig. 10.9) (Gas-Pascual et al., 2019). These glycans are initially assembled on a Dol-pyrophosphate carrier lipid on the cytoplasmic leaflet of the ER by UDP-GlcNAc or GDP-Man dependent Alg proteins (Alg-7, Alg-14, Alg-1, Alg-2, and Alg11), before being flipped into the lumen of the ER where they are modified with three Glc residues by two Dol-P-Glc-dependent glucosyltransferases (Alg8 and Alg10). T. gondii lacks the Dol-P-Man-dependent enzymes needed to make the larger Man9GlcNAc2 structures

found in fungi, plants, and animals, as well as the GDP-Glc-dependent glucosyltransferase that participates in the calnexin/calreticulin cycle in the ER lumen, suggesting that T. gondii lacks this protein quality control machinery (Joyce et al., 2013). Recent profiling studies suggest that protein linked N-glycans may be modified with an additional Man chain, but that they are otherwise minimally processed in the ER and Golgi during transit to the cell surface (Gas-Pascual et al., 2019). Indeed, despite expressing three ER/Golgi glucosidases (glul1, glullA, and glullB), many of the N-glycans on mature proteins retain the terminal Glc

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FIGURE 10.9 Structures of Toxoplasma gondii glycoconjugates. Glycans that are added to proteins in the secretory pathway and in the nucleus/cytoplasm are indicated in orange and blue colored boxes, respectively. Asn, Asparagine; EtN, ethanolamine; Fuc, fucose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; GlcN, glucosamine; GPI, glycosylphosphatidylinositol; Hyp, hydroxyproline; Man, mannose; PI, phosphatidylinositol; S/T, serine/threonine; W, tryptophan.

residues, demonstrating that removal of these sugars is not required for surface transport. Notwithstanding their relatively uncomplicated structures, N-glycosylation is essential for tachyzoite growth, as T. gondii CRISPR/ Cas9 mutants lacking genes involved in assembly of the Dol-linked oligosaccharide precursors (Alg1, Alg14, Alg11, Alg5, and Alg7) and a subunit of the oligosaccharide transferase (Ost) complex all exhibited reduced fitness in fibroblasts (Sidik et al., 2016). Similarly, tunicamycin treatment effectively inhibits protein Nglycosylation in T. gondii and results in complete loss of tachyzoite motility after egress and severely affects their capacity to invade and survive within new host cells (Luk et al., 2008). In contrast, disruption of enzymes involved in the addition or removal of the terminal Glc residues on the Dol-oligosaccharide precursor and protein-N-glycans, respectively, had little effect on tachyzoite growth (GasPascual et al., 2019). 10.4.2.2 Glycosylphosphatidylinositol glycolipids The cell surface of T. gondii tachyzoites and bradyzoites are dominated by GPI-anchored

proteins, including the surface antigen 1 (SAG1)related sequence and SAG-unrelated surface antigens. Early studies showed that the T. gondii GPI protein anchors contained a minimal canonical Man3GlcN1 glycan structure linked to a diacylglycerol-containing phosphatidylinositol (PI) moiety (Striepen et al., 1999). The glycan backbone of the protein anchors are modified with a single β1,4-linked GalNAc residue, but lack other elaborations, such as additional ethanolamine residues, despite expressing putative ethanolamine-phosphate transferases (Striepen et al., 1999). Similar to N-glycan precursors, the initial steps in GPI anchor biosynthesis occur on the cytoplasmic leaflet of the ER. These include the modification of PI with GlcNAc (catalyzed by pig-A) followed by the deacetylation of GlcNAc-PI to GlcN-PI (pig-L), which is subsequently flipped into the lumen of the ER. The assembly of the tri-Man backbone (catalyzed by pig-M, pig-V, and pig-B) and addition of terminal ethanolamine-phosphate occur in the ER lumen. Free GPI glycolipids have the same structure as the protein-linked GPIs but can have an additional α1-4-linked Glc cap on the GlcNAc side chain (Striepen et al., 1999). In tachyzoites, free GPIs are two- to fourfold more

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abundant than GPI-anchored proteins and can induce macrophages to produce TNF-α and Il12 following activation of pTLR2/TLR4/ MyD88-mediated pathways (Niehus et al., 2014). Interestingly, the glucosylated GPI side chains were only detected on egressed tachyzoites suggesting that expression of these epitopes might be staged to modulate host immune responses and/or facilitate parasite adhesion to host galectins (Debierre-Grockiego et al., 2006). T. gondii mutants with defects in different enzymes involved in formation of GlcN-PI (pig-A, pig-L) and assembly of the core Man backbone (pig-M, pig-V) all exhibit highly attenuated growth in fibroblasts indicating that protein and/or free GPIs are important for intracellular growth (Gas-Pascual et al., 2019). 10.4.2.3 O-Glycosylation Many T. gondii cell surface and secretory proteins are also modified with a single Olinked GalNAc residue or the disaccharide, GalNAcα1-3GalNAcα1- (Gas-Pascual et al., 2019) (Fig. 10.9). Addition of O-GalNAc is initiated in the Golgi by a family of polypeptide N-α-acetyl galactosaminyltransferases (ppGalNAc-Ts) that transfer GalNAc from UDP-GalNAc in Ser/Thr residues in the polypeptide backbone. ppGalNAc-T1, -T2, and -T3 are constitutively expressed in tachyzoites and bradyzoites and are involved in modifying cell surface and secreted protein that contain Ser/ Thr/Pro-rich mucin-like domains. These include the major bradyzoite cyst wall protein, CST1, which is extensively modified with OGalNAc chains that are recognized by the Dolichos biflorus (DBA) lectin that binds terminal GalNAcα1-3GalNAc epitopes. Weiss et al. have recently shown that T. gondii ppGalNAcT2 is required to initiate O-glycosylation of these mucin domains and that ppGal NAc-T1 and T3 subsequently recognize the priming GalNAc residue and modify adjacent Ser/Thr residues (Tomita et al., 2017). While individual ppGalAc-Ts, including ppGalNAc-T2, are not

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essential for tachyzoite growth in fibroblasts (Tomita et al., 2017), deletion of the sugar nucleotide transporter, TgNST1, which imports the sugar donor UDP-GalNAc into the lumen of the Golgi, leads to reduced bradyzoite fitness (Caffaro et al., 2013). O-Glycosylation of the mucin domains of CST-1, mediated by the ppGalNAc-Ts, therefore appears to be required to maintain the physicochemical properties of the cyst wall (Caffaro et al., 2013). A number of apicomplexan parasites, including T. gondii, are dependent on surface adhesions derived from micronemes for motility and host cell invasion. Many of these proteins contain one or more thrombospondin repeat (TSR) domains that are modified with both O-Fuc and C-Man (Gas-Pascual et al., 2019). Similar modifications occur on the TSR domains of metazoan organisms but have not been identified in other protists outside the Apicomplexa. As in animal cells, TSR-O-fucosylation in T. gondii is mediated by O-fucosyltransferase-2 (POFUT-2), a member of the CAZy GT68 family, which adds a single Fuc residue to Ser/Thr residues in the consensus sequon CXX(S/T)C (Bandini et al., 2019b; Khurana et al., 2019). This modification occurs in the lumen of the ER and POFUT-2 uses GDP-Fuc that is imported into the ER by the sugar nucleotide transporter NST2 (Bandini et al., 2019b). The O-linked Fuc can be further modified with a β1,3-linked Glc residue by an, as yet, unidentified glucosyltransferase. While O-fucosylation has been shown to stabilize the TSR domains of proteins such as MIC2, deletion of POFUT-2 or the GDP-Fuc transporter has only a modest (Bandini et al., 2019b) or negligible effect on tachyzoite motility and intracellular survival (Khurana et al., 2019). It is possible that the function of O-Fuc is partially compensated for by modification of neighboring tryptophan residues in the TRP domains with single α-Man residues (Hoppe et al., 2018). This atypical glycosylation involves a C
C link between Man and the

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indole carbon 2 of the tryptophan and is catalyzed by a homolog of metazoan C-mannosyltransferase, DPY-19 (Hoppe et al., 2018; Khurana et al., 2019). T. gondii DPY-19 is expressed in sporulated oocysts, tachyzoites, and bradyzoites and enhances parasite fitness during infection of human fibroblasts (Sidik et al., 2016). 10.4.2.4 Nucleocytoplasmic glycosylation Recent studies showed that nearly 70 nuclear-cytoplasmic proteins, including nucleoporins, mRNA processing enzymes, and cell signaling proteins are modified with a single O-linked Fuc (Bandini et al., 2016). This modification was initially detected using the Fuc-binding Aleuria aurantia lectin, which revealed the presence of fucosylated glycoconjugates in the nucleus of tachyzoites, bradyzoites, and sporozoites, but not oocysts. Nucleo-cytoplasmic O-fucosylation occurs within serine-rich domains of target proteins and is mediated by a second family of fucosyltransferases that are distinct from the POFUTs in the secretory pathway, but which share strong homology to a new family of protein POFUTs that have recently been characterized in plants (Zuther et al., 1999). Intriguingly, the plant POFUT, SPY, has a similar 3-D fold to the vertebrate O-GlcNAc transferase (OGT) that modify Ser/Thr residues with a single Olinked GlcNAc residue in many nuclear and cytoplasmic proteins (Banerjee et al., 2016). Vertebrate OGTs have been extensively studied and shown to regulate protein function directly or indirectly, by regulating complementary phosphorylation of the same or proximal Ser/ Thr residues (Banerjee et al., 2016). While the function of cytoplasmic O-fucosylation in T. gondii remains unknown, CRISPR/Cas9 deletion of the spy results in a significant loss of tachyzoite fitness in fibroblasts assays (GasPascual et al., 2019). T. gondii also expresses five cytoplasmic glycosyltransferases that selectively modify a

single hydroxyproline residue (Pro-143) in the cytoplasmic protein, Skp1, with a linear pentasaccharide containing Gal, Glc, Fuc, and GlcNAc (Fig. 10.9) (Florimond et al., 2019; West et al., 2004). Skp1 forms a complex with two other proteins, cullin-1 and F-box (to form the SCF complex) that binds to ubiquitin-E2 and regulates protein polyubiquitination and turnover. This unusual modification occurs in some other protists but is absent in animal cells (Florimond et al., 2019). The formation of the modified hydroxyproline residue in the Skp1 protein is dependent on an O2/α-ketoglutarate-dependent prolyl-4-hydroxylase (PDH), which may function as an O2-sensing protein. Parasite mutants lacking enzymes involved in Skp1 glycosylation exhibited only a modest decrease in virulence under normal growth conditions but are sensitive to hypoxic conditions. Interestingly, a second PDH has recently been characterized from T. gondii, which hydroxylates proline residues in other proteins and has a role in allowing these parasites to adapt to elevated O2 levels. The two prolyl hydroxylases may therefore act in concert to allow these parasites to grow in diverse anatomical sites (Florimond et al., 2019).

10.4.3 Amylopectin All stages of T. gondii synthesize amylopectin as their major carbohydrate reserve material (Coppin et al., 2005). Amylopectin is the major component of plant starch and is composed of α1,4-linked glucan chains with asymmetrically distributed α1,6-linked branches and forms large (107
109 Da in size) semicrystalline granules. Amylopectin granules grow by the successive deposition of concentric layers of highly branched glucan (amorphous rings) and intervening layers of nonbranched glucan chains that pack together in highly organized α-helices (crystalline rings) (Coppin et al., 2005). Amylopectin differs from hydrosoluble

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glycogen in having fewer branches and longer stretches of unbranched α1,4-glucans. In T. gondii, amylopectin granules accumulate in the cytoplasm, similar to the situation in red algae, glaucophytes, and related secondary endosymbionts, but different from the situation in green algae and plants (Chloropastida) where amylopectin is found primarily in plastids. Levels of amylopectin vary enormously within and between different T. gondii developmental stages (Coppin et al., 2003; Guerardel et al., 2005). While bradyzoites accumulate many large amylopectin granules that can occupy a significant proportion of the cytoplasm, tachyzoites generally synthesize microgranules that are distributed throughout the cytoplasm (Uboldi et al., 2015). However, larger amylopectin granules can form in tachyzoites during growth in some host cells (i.e., HepG cells), or when tachyzoites are suspended in rich media for extended periods of time, highlighting the influence of local nutrient levels of granule formation and size (Guerardel et al., 2005; Luder and Rahman, 2017). Other developmental stages, including macrogametes, oocysts, and sporozoites, also accumulate amylopectin granules (Guerardel et al., 2005). 10.4.3.1 Synthesis and turnover of amylopectin The T. gondii genome contains putative homologs for all of the enzymes needed for amylopectin synthesis and degradation (Dauvillee et al., 2009) (Fig. 10.10). Sugarphosphates are channeled into amylopectin synthesis via the interconversion of Glc6P to Glc1P and UDP-Glc by the enzymes GPM and UDP-Glc pyrophosphorylase, respectively. T. gondii express two isoforms of GPM, which are both enzymatically active, but may also have moonlighting functions in regulating calcium signaling and microneme secretion in tachyzoites stages (Saha et al., 2017). Deletion of individual or both gpm genes does not lead to attenuated virulence in mice (Saha et al., 2017),

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suggesting either that amylopectin synthesis is not essential in tachyzoite stages and/or that other hexose-P mutases can substitute for GPM. The α1,4-glucan chains of amylopectin are synthesized by a putative glycogen synthase that utilizes UDP-Glc as sugar donor and adds single Glc residues to the nonreducing ends of the growing chains. The T. gondii genomes also contain a homolog of mammalian glycogenin that may function as the primer for de novo amylopectin synthesis (Gas-Pascual et al., 2019; Sugi et al., 2017). Glucan-branching enzymes introduce branch points by the successive cleavage and reattachment of short α1,4-glucan chains to the same or neighboring glucan chains in α1,6-linkage. The glucan-debranching enzyme (which harbors α4-glucosyl transferase activity and amylo-1,6-glucosidase activity) is also involved in amylopectin synthesis, by removing excess branch chains within the crystalline growth rings to allow close packing of α1,4-glucan chains. Amylopectin degradation is predicted to be initiated by two polysaccharide kinases (a glucan, water dikinase, and a phosphoglucan water dikinase) that add phosphate groups to a small number of Glc residues (1 per B100 Glc residues) in the outer amylopectin chains, creating localized regions of disorder and thereby facilitating access to glucan hydrolases (Nitschke et al., 2013). These phosphate residues are subsequently removed, most likely by a glucan phosphatase with homology to vertebrate Laforin protein (Gentry et al., 2007) to allow the degradation of glucan chains by the concerted actions of the glucan debranching enzyme (iso-amylase), two α-amylases (α1,4glucanases) and a glycogen phosphorylase (GP) (Sugi et al., 2017). While the α-amylase and glucanases generate free Glc, which needs to be phosphorylated by HK before it can reenter central carbon metabolism, the GP generates Glc1P which can be converted directly to Glc6P without consumption of additional ATP.

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FIGURE 10.10

Amylopectin biosynthesis and turnover in Toxoplasma gondii. The enzymes implemented in the pathways are shown in purple and the FS associated with the loss of the enzyme function is indicated in a heatmap color code—blue indicating dispensability, while red indicates essentiality. Metabolites are depicted in black and energetic compounds produced or consumed are shown in gray. Steps: GPM—phosphoglucomutase; UTP:Glc1P-UT—UTP:Glc-1-phosphate uridylyltransferase; Glycogen Syn—glycogen synthase; (P)-glucan—phosphoglucan; α-amylase/amyloglucosidase.

Thus cycling of hexose-P through amylopectin results in the consumption of either one or two ATPs, depending on whether the amylopectin is mobilized as Glc or Glc1P, respectively. While the contribution of the α-amylases to amylopectin degradation has not been studied, loss of the GP leads to massive accumulation of amylopectin granules in both tachyzoites and bradyzoites (Sugi et al., 2017). Conversely, overexpression of T. gondii GP leads to loss of amylopectin granules, supporting a key role for this enzyme in regulating the dynamic turnover of the amylopectin granules (Sugi et al., 2017). 10.4.3.2 Regulation of amylopectin turnover Many of the enzymes involved in amylopectin synthesis/degradation contain carbohydratebinding domains (CBD) that target these

enzymes to amylopectin granules (Uboldi et al., 2015). Enzyme recruitment to the granules can change under different nutrient conditions and may be important for coordinated synthesis and mobilization of granule synthesis and degradation. In yeast, plants, and animal cells, key enzymes involved in starch/glycogen synthesis are also regulated through posttranslational modifications, as well as allosteric and metabolic processes. A recent study showed that CDPK2, a member of the calcium-dependent protein kinases, phosphorylates several key enzymes in amylopectin synthesis and degradation (Uboldi et al., 2015). In particular, CDPK2-dependent phosphorylation of serine (Ser)-25 in GP activates this enzyme (Sugi et al., 2017; Uboldi et al., 2015). KD of CDPK2 results in both increased synthesis and reduced degradation of amylopectin, consistent with this kinase having a role in coordinately

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regulating multiple enzymes in this pathway. Significantly, while Δcdpk2 tachyzoites only exhibit a modest loss of fitness in fibroblasts and mice, mutant bradyzoites accumulate amylopectin granules that occupy most of the cytoplasm, resulting in parasite death and a complete absence of cysts in the brains of mice (Uboldi et al., 2015). The amylopectin hyperaccumulation phenotype observed in both the Δgp and Δcdpk2 mutants suggests that there is very little allosteric or metabolic feedback inhibition of amylopectin synthesis (Sugi et al., 2017; Uboldi et al., 2015). Interestingly, the large amylopectin granules that accumulate in tachyzoite stages of these mutant lines are expelled into the residual body as daughter cells form (Uboldi et al., 2015). Bradyzoites divide asynchronously in cysts and lack residual bodies, which might contribute to the lethal phenotype in this stage in vivo. 10.4.3.3 Amylopectin function Analysis of the virulence phenotype of T. gondii GP or CDPK2 mutants indicates that the dynamic regulation of amylopectin stores is dispensable for tachyzoite growth, but critical for bradyzoite growth and formation of cysts (Sugi et al., 2017; Uboldi et al., 2015). Amylopectin might provide an important carbon and energy reserve when bradyzoites differentiate back to tachyzoites or during transition between replicating and nonreplicating bradyzoites stages in infected tissues (Watts et al., 2015). In other Apicomplexa, including Eimeria and Cryptosporidium, levels of amylopectin stores in sporozoites correlated with the infectivity and survival of these transmission stages (Harris et al., 2004; Karkhanis et al., 1993). Alternatively, the constitutive turnover of amylopectin could act as a rheostat, regulating the intracellular levels of sugarP and inorganic phosphate (both are used and consumed during amylopectin synthesis and degradation), and regulate fluxes into different pathways of carbohydrate metabolism. Constitutive turnover of amylopectin in

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tachyzoites stages is supported by 13C-Glc labeling studies (Uboldi et al., 2015) and the finding that enzymes involved in amylopectin synthesis are transcriptionally upregulated in tachyzoites despite the fact that these stages accumulate few amylopectin granules (Coppin et al., 2003, 2005). Similarly, the hyperaccumulation of amylopectin in bradyzoites stages of Δgp or Δcdpk2 is also consistent with constitutive synthesis in this stage (Uboldi et al., 2015). Finally, these studies indicate that amylopectin turnover is also regulated by nonnutritional signals, such as calcium. Changes in calcium fluxes, detected by the CDPKs, play a key role in regulating tachyzoite motility following egress from host cells. The activation of CDPK2 and mobilization of amylopectin stores in extracellular tachyzoites is therefore likely to be important in sustaining active motility, and powering parasite invasion.

10.5 Vitamins and cofactor metabolism 10.5.1 Overview of vitamins and cofactors Intracellular parasites commonly have a streamlined metabolism, reflecting the loss of metabolic pathways and enzymes that are required for survival in more diverse environments and/or that are no longer needed because key end-products can be salvaged directly from the host cell. In some cases, intracellular parasites have retained or acquired new pathways to compensate for the absence of requisite pathways in their host cell, making them excellent drug targets. Analysis of the T. gondii genome reveals several examples of pathway retention and loss, which provide meaningful insights into the nutritional availabilities during its multihost life cycle. All eukaryotes require a number of vitamins and cofactors, which function in a diverse array of cellular processes. Humans lack the capacity to synthesize many of these

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metabolites and must therefore be acquired through their diet. These include fat- (vitamin A, D, E, and K) and water-soluble (8 Vit B and Vit C) compounds. Vitamins display a wide range of biochemical functions: as regulators of cell and tissue growth and differentiation, as antioxidants and as coenzymes or the precursors for them. Cofactors, on the other hand, are non-protein compounds that facilitate biochemical reactions and are responsible for the activity of an enzyme. Cofactors can be classified into organic or inorganic, which can be transiently, tightly, or even covalently bound to the protein. Except for the inorganic metallic ions that need to be acquired from the diet, the human host can synthesize all of its organic cofactors de novo. Interestingly, T. gondii is capable of synthesizing vitamins such as pantothenate (PAN) (Vit B5), pyridoxal-5P (Vit B6), and folates (Vit B9), as a result of horizontal gene transfer of several enzymes which display high sequence homology to bacterial orthologs. Why the parasite has acquired and retained biosynthesis pathways, given the likely possibility to scavenge the vitamins from the host, is largely unknown. One hypothesis is the need for the pathway in a different life cycle stage, when nutrient availability from the host is limited. T. gondii, like its mammalian host, can also synthesize most of its cofactors de novo (except myo-inositol), although the utilization of the pathway under nutrient-rich conditions is minimal. Myo-inositol is synthesized from Glc6P in the host and is abundant in brain and other tissues. In the parasite, myo-inositol is used for de novo PI and GPI synthesis. Divergent biosynthesis pathways for some cofactors (heme) or cofactor precursors (shikimate and chorismate) can also be found in the parasite, but not in the host, making them potential pathways for drug intervention. Fig. 10.11 depicts the essential as well as synthesized vitamins and cofactors in humans and in T. gondii.

10.5.2 Vitamins: thiamine B1, flavins B2, niacin B3, pantothenate B5, pyridoxal B6, biotin B7, Myo-inositol B8, and folates B9 10.5.2.1 Thiamine biosynthesis Thiamine diphosphate (or pyrophosphate) is the metabolically active form of thiamine, also known as Vitamin B1. It is an important cofactor for enzymes of carbohydrate and amino acid metabolism such as PDH, 2-oxoglutarate dehydrogenase, pyruvate decarboxylase, and dihydrolipoamide dehydrogenase, all present in the genome of T. gondii. Plants, bacteria, and fungi can synthesize thiamine, whereas mammals must obtain it from their diet. Mammals possess the enzyme thiamine diphosphokinase (TPK) which catalyzes pyro-phosphorylation of thiamine to thiamine pyrophosphate (TPP). As in mammals, T. gondii harbors only the enzyme TPK while missing the enzymes for de novo thiamine biosynthesis. TPK has a high negative FS (23.28) suggesting essentiality of the enzyme and the inaccessibility of the phosphorylated form of thiamine from its host (Fig. 10.12). The transporters involved in uptake of thiamine in both the mammalian host, and across the parasite plasma membrane as well as the translocation of active TPP across organellar membranes have not yet been identified. 10.5.2.2 Flavins biosynthesis The metabolism of riboflavin is critical as numerous metabolic processes are flavindependent and occur in several subcellular compartments. Produced from riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) function as cofactors for a variety of enzymatic reactions. In T. gondii the enzymes for the synthesis of riboflavin are absent, indicating acquisition of vitamin B2 from the host. The two genes coding for the subsequent enzymes, riboflavin kinase and FAD synthase, are present and very likely

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FIGURE 10.11 Overview of the vitamins and cofactor pathways in both the host and Toxoplasma gondii. A network displaying the acquisition and generation of all known vitamins and cofactors from precursors emerging from the centralcarbon, amino-acid and nucleotide metabolism is displayed. Dotted arrows in pink describe the de novo synthesis capabilities absent in T. gondii, where the parasite must rely solely on the uptake from its host. Several vitamins and cofactors are taken up from the host, indicated with the transporters. All amino acids, also likely acquired from the host, are shown in dark purple. The transporters and uptake mechanisms for several of the vitamins and cofactors are largely unknown. 3-PG, 3-Phosphoglycerate; AIR, amino-imidazole ribonucleotide; AMP/ADP/ATP, adenosine mono/di/triphosphate; CoA, Coenzyme A; CTP, cytidine triphosphate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; G3P, Glc-3phosphate; GMP/GDP/GTP, guanosine mono/di/triphosphate; HCO3, bicarbonate; IMP, inosine monophosphate; NAD(P), nicotinamide adenine dinucleotide (phosphate); PEP, phosphoenolpyruvate; PRPP, phosphoribosyl-pyrophosphate; TPP, thiamine pyrophosphate; UDP/UTP, uracil di/tri-phosphate; XMP, xanthine monophosphate.

essential for parasite survival with an FS of (23.97) and (24.87), respectively (Fig. 10.12). In the mitochondrion, flavin-containing domain of succinate dehydrogenase, FMN for

Complex I and FAD for Complex II are essential for the functioning of ETC. In the cytosol the primary coenzyme form of pyridoxalphosphate (PLP) is FMN dependent and the

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FIGURE 10.12 Vitamins B1, B2, and B3: thiamine, flavins, and niacin. Metabolic map of the de novo synthesis and (or) scavenge of vitamin B1, B2, and B3 is shown. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. FAD, Flavin adenine dinucleotide; FMN, flavin mononucleotid; NAD(P), nicotinamide adenine dinucleotide (phosphate); NNAT, nicotinamide/nicotinate mononucleotide adenylyltransferase; NPPRT, nicotinamide phosphoribosyltransferase; PRPP, phosphoribosyl-pyrophosphate; TPP, thiamine pyrophosphate.

synthesis of an active form of folate (methyltetrahydrofolate) is also FADH2 dependent. In the apicoplast, oxidation of pyruvate, α-KG, and BCAAs requires FAD in the shared E3 portion of their respective dehydrogenase complexes (PDH). Further, fatty-acyl-CoA dehydrogenase for FA oxidation requires FAD. How FMN and FAD are translocated between these different subcellular compartments is largely unknown. The membrane transporter for riboflavin has also not yet been identified. 10.5.2.3 Niacin metabolism Nicotinic acid (anionic form: nicotinate) is also known as niacin or vitamin B3. Nicotinate and nicotinamide (the amide-form of the vitamin) are essential for organisms as the precursors for generation of coenzymes, NAD(1) and NADP(1), which are essential for redox reactions and as essential electron carriers. The coenzymes are crucial for many metabolic pathways including glycolysis, TCA cycle, PPP, FA biosynthesis, and many others. The genome of T. gondii lacks the genes coding for the enzymes that synthesize nicotinate and nicotinamide de novo, but have the ability to

salvage them from its host. T. gondii possesses all the genes coding for the enzymes that produce NAD(1) and NADP(1) from the scavenged vitamin B3 derivatives. All the genes in the pathway, based on their FS, are dispensable for tachyzoites survival except for the NAD(1) kinase, which phosphorylates NAD(1) into NADP(1) (Fig. 10.12), leaving the option of NAD(1) scavenge from the host. 10.5.2.4 Pantothenate biosynthesis for CoA production CoA is an important cofactor (acyl-chain carrier) for many cellular functions including the citrate cycle and FA biosynthesis and metabolism. Vitamin B5, also known as PAN, is a precursor for CoA biosynthesis. PAN synthesis takes place in plants, fungi, and some bacteria, whereas animals require PAN from their diet. On the other hand, CoA biosynthesis from PAN is present in most organisms. Interestingly, T. gondii possesses all the enzymes required for the generation of PAN from the BCAA, valine, and the non-proteinogenic amino acid β-alanine. Enzymes required for the interconversion of PAN to CoA are also present. Recently, the

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FIGURE 10.13 Vitamin B5: pantothenate and CoA. The presence of both PAN and CoA biosynthesis pathways with unknown transporters or transport mechanisms. The FS of the enzymes indicates the reliance of PAN uptake and its conversion to CoA for parasite survival. The presence of the PAN biosynthesis enzymes indicates its probable utilization in another life-cycle stage of the parasite. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. BCAT, Branched-chain-amino-acid-transaminase; CoA, coenzyme A; HMT, hydroxy-methyl-transferase; PAN, pantothenate; PBAL, pantoate-beta-alanine ligase; PPAT, pantetheine-phosphate adenylyl-transferase; PPCDC, phospho-pantothenoyl-cysteine decarboxylase; PPCS, phospho-pantothenoyl-cysteine synthetase; THF, tetrahydrofolate.

enzyme responsible for the last step in CoA biosynthesis, dephosphoCoA kinase (DPCK, EC 2.7.1.24), previously thought missing, was identified (Lunghi et al., unpublished). The FS of DPCK clearly indicates essentiality of the CoA biosynthesis pathway, via PAN, whereas the FS for enzymes involved in PAN biosynthesis suggests dispensability in the tachyzoite stage (Fig. 10.13). PAN is present in most media used for cell culture, as it is an essential vitamin for mammalian cells and can presumably also be taken-up efficiently by the parasite to fulfill its metabolic needs. The retention of the PAN biosynthesis pathway in T. gondii points to its utilization in another life-cycle stage of the parasite. 10.5.2.5 Pyridoxal-phosphate metabolism Pyridoxal-phosphate (PLP) is the active form of vitamin B6, whereas pyridoxamine,

pyridoxal, and pyridoxine and their phosphate esters form the vitamin B6 complex. PLP is a cofactor crucial for the functioning of many enzymes, mostly involved in amino acid metabolism. Two different routes for de novo PLP synthesis exist in various organisms: in the DOXP (1-deoxy-D-xylulose-5-phosphate)dependent first route the PLP precursor pyridoxine 50 -phosphate (PNP) is produced from 4-phosphohydroxyl-L-threonine (4PHT) and DOXP by the actions of the enzymes PDXA and PDXJ that are absent in T. gondii. PNP can then be converted to PLP by the enzyme PLP synthase. In the DOXP-independent second route, PLP synthesis is catalyzed by the actions of PDX1 and PDX2 with amino-acid glutamine, ribulose 5-phosphate and GA3P (or glycerone phosphate) as substrates. Of the two routes mentioned previously, DOXP-independent

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route is responsible for the de novo PLP biosynthesis in apicomplexans. The de novo biosynthesis of PLP via the action of PDX1 and PDX2 enzymes was also experimentally demonstrated in T. gondii (Krishnan et al., unpublished). In a third route, the pyridoxine, pyridoxamine, and pyridoxal can be phosphorylated with the action of the enzyme pyridoxal kinase (PLK), with the former two being converted to pyridoxal by the action of PLP synthase. The genome of T. gondii possesses both the PLP synthase enzyme and PLK which phosphorylates all the vitamers, pyridoxal, pyridoxamine, and pyridoxal (Fig. 10.14). FS for the genes coding for PDX1, PDX2, PLP synthase, and PLK indicate dispensability, with the parasite utilizing both routes (de novo synthesis and/or salvage) to meet its PLP requirements in vitro.

10.5.2.6 Folate and biopterins biosynthesis Folic acid, also known as Vitamin B9, is important for several biological functions. The folate-derivative 5,10-methylene-tetrahydrofolate is essential for the synthesis of deoxythymidine monophosphate from deoxyuridine monophosphate, and is therefore crucial for DNA replication and cell division. Tetrahydrofolate is an essential substrate in the biosynthesis of the amino acid glycine. The two essential precursors for folate biosynthesis are 4-aminobenzoate (a product of shikimate biosynthesis pathway) and GTP. In addition to the de novo folate biosynthesis pathway, T. gondii can salvage folate from the host (Fig. 10.15). The uptake of radio-labeled exogenous folic acid revealed the presence of a common folate transporter which has high affinity for folic acid (Massimine et al., 2005).

FIGURE 10.14

Vitamin B6: pyridoxal-phosphate. The presence of both de novo synthesis and scavenge pathways for PLP production indicates differential utilization of the pathways in different life-cycle stages of Toxoplasma gondii. PLP is synthesized from the amino-acid glutamine and precursors from glycolysis and the PPP. Vitamers of PLP—pyridoxal, pyridoxamine, and pyridoxine can be taken-up and phosphorylated to form PLP via PLK. The transporter or uptake mechanisms for the vitamers are unknown. Enzymes are depicted in purple and reducing equivalents, cofactors, or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. PLP synthase, pyridoxal-phosphate synthase; PDX1, subunit 1 of the synthase complex; PDX2, glutamine aminotransferase, subunit 2 of the synthase complex; PLK, pyridoxal kinase.

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FIGURE 10.15 Vitamin B9: folates. Folates (DHF and THF) can be both de novo synthesized and scavenged from the host. The precursors for folate biosynthesis are biopterins, also synthesized or scavenged from the host. Several folate transporters have been identified and characterized in Toxoplasma gondii that include transporters from the pterine, BT1 family. The scheme also shows the production of molybdopterin, an important cofactor for metabolic enzymes containing molybdenum and tungsten. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. 6PTPS, 6-Pyruvoyltetrahydro-pterin synthase; alkaline-P, alkaline phosphatase; DHFR, dihydrofolate reductase/ thymidylate synthase; DHFS, dihydrofolate synthase; DHNR, dihydroneopterin reductase; DHPR, 6,7-dihydropteridine reductase; DHPS, dihydropteroate synthase; Formyl-T, transferase; GTP, guanosine triphosphate; GTP-CH, GTP cyclohydrolase; HMDPPK, hydroxy-methyl-pterin pyro-phospho-kinase; MDTS, molybdopterin synthase; MTHD, methylenetetrahydrofolate dehydrogenase; MTHFCH, methenyl-tetrahydrofolate cyclohydrolase; PABA, para-amino-benzoic-acid; SPR, sepiapterin reductase.

The transporters (homologous to BT1 family proteins) are suggested to be bidirectional and concentration dependent. If folates can be taken-up efficiently in tachyzoites, the existence of the folate biosynthesis pathway is likely relevant for a different life-cycle stage of the parasite where it encounters environments with limited amounts of p-aminobenzoic acid or folates.

Tetrahydrobiopterin (the active form, BH4) and dihydrobiopterin (BH2) are cofactors for several hormones. In mammalian cells, BH4 acts as the cofactor in dopamine/serotonin synthesis via tyrosine/tryptophan hydroxylase. The precursors for folates and pteridines are GTP and dihydroneopterin triphosphate. T. gondii possesses all the genes coding for the enzymes synthesizing BH4 and BH2, although

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the entire pathway is dispensable for tachyzoite survival, indicating an uptake mechanism for the biopterins like for the folates. Finally, homology-based search has also led to the identification of five enzymes catalyzing biosynthesis of molybdopterin from GTP, with two enzymes, molybdopterin cofactor synthesis protein 1 and 3 showing an FS of (22.92) and (23.04), respectively, indicating the utilization of molybdopterin synthesis pathway in the tachyzoite stage. Enzymes that contain the molybdopterin cofactor include xanthine oxidase, sulfite oxidase, and nitrate reductase, all of which are encoded in the genome of T. gondii. 10.5.2.7 Myo-inositol and biotin uptake and utilization Myo-inositol (non-essential Vitamin B8) is an important signaling molecule and precursor for GPI-anchored proteins to the cell surface. Enzymes for the biosynthesis of myo-inositol are missing from the genome of T. gondii, and intracellular parasites likely salvage this metabolite directly from their host which typically contain large intracellular pools of free inositol and inositol-phosphate. Targeted inhibition of host cell myo-inositol biosynthesis and/or uptake of myo-inositol by intracellular parasites exhibit potential drug targets. Biotin: An important biotin-dependent enzyme is the acetyl-CoA carboxylase (ACCase), the target of several classes of herbicides (Nikolskaya et al., 1999; Zuther et al., 1999). T. gondii possesses two ACCases in the apicoplast for FA biosynthesis and in the cytosol, with the ACCase1 in the apicoplast predicted to be biotinylated and activated in the parasite. T. gondii and their mammalian host cells both lack enzymes for de novo biotin synthesis which is therefore scavenged exclusively from the medium or the host diet. How biotin is taken up and transported into the apicoplast is unknown.

10.5.3 Cofactors: shikimate and chorismate, ubiquinone, heme, lipoic-acid, S-adenosyl-methionine, and glutathione 10.5.3.1 Shikimate and chorismate biosynthesis Shikimic acid or shikimate is an important metabolite found in plants and microorganisms but is absent in animals. Shikimate is important for many biosynthetic processes including that of folate, aromatic amino acids, and ubiquinone. Shikimate is primarily synthesized and interconverted to chorismate in a seven-step reaction. Steps 2
6 of shikimate biosynthesis pathway are carried out by a penta-functional protein called AROM peptide starting with glycolytic and pentose-phosphate precursors, PEP, and erthyrose-5P. In T. gondii, the penta-functional activities of the protein are also annotated to a single gene, and the presence of all functional domains have been verified with bioinformatic analyses (Campbell et al., 2004; Peek et al., 2014; Richards et al., 2006). In another study, the presence of shikimate pathway and its active synthesis was demonstrated in T. gondii, by treating the parasites with the herbicide glyphosate, an inhibitor of the enzyme, 5-enolpyruvylshikimate-3phosphate synthase, resulting in the inhibition of growth of in vitro T. gondii tachyzoites. The effect was reversible with addition of para-aminobenzoic acid or folate in medium suggesting the role of shikimate pathway in providing precursors for folate biosynthesis (Roberts et al., 1998, 2002). The role of chorismate for folate biosynthesis has been demonstrated, but its importance for ubiquinone biosynthesis has not yet been defined. Since the mammalian host does not contain the pathway to produce chorismate from shikimate, the entire pathway displays an important role as a drug-target. The FS of all enzymes

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FIGURE 10.16 (A) Shikimate and chorismate biosynthesis. De novo synthesis of shikimate and chorismate, precursors for amino-acid, ubiquinone and folates biosynthesis. (B) Ubiquinone biosynthesis—The presence and essentiality of the ubiquinone de novo biosynthesis pathway. Enzymes are depicted in purple and reducing equivalents, cofactors, or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. ADCL, Amino-deoxy-chorismate lyase; ADCS, amino-deoxy-chorismate synthase; DAHP, deoxy-phospho-heptulonate; DH, dehydrogenase; DHQ, dehydroquinate; DMUbi, dimethylubiquinone; EPS, enoyl-pyruvate shikimate; HB, hydroxybenzoate; MP, methoxyphenol; OP, octaprenyl; PEP, phosphoenolpyruvate; SAM, S-adenosyl-methionine.

involved in the pathway are also highly-fitness conferring confirming essentiality in tachyzoites (Fig. 10.16A). 10.5.3.2 Ubiquinone biosynthesis Ubiquinone, also known as coenzyme Q is an integral component of the ETC. In the inner mitochondrial membrane, ubiquinone acts as an electron carrier transferring electrons from NADH dehydrogenase and succinate dehydrogenase

(complex II) to cytochrome bc1 complex (complex III). In most organisms, ubiquinone is synthesized from chorismate, the end product of the shikimate pathway, in nine enzymatic reactions, with two enzymes, oxo-acid lyase and 3octaprenyl-4-hydroxybenzoate carboxy-lyase missing from genome of T. gondii. The other important cosubstrates involved in this pathway are glutamine, isoprenoids (terpenoid metabolism), and S-adenosyl methionine (SAM)

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FIGURE 10.17 Heme biosynthesis. The scheme of de novo biosynthesis of heme as an essential cofactor for several metabolic processes is depicted. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. ALAD, Aminolevulinate dehydratase; ALAS, aminolevulinate synthase; CPO, coproporphyrinogen oxidase; CPOH, oxygen-independent coproporphyrinogen-III oxidase; FC, ferrochelatase; PBGD, porphobilinogen deaminase; PPO, protoporphyrinogen oxidase; UROD, uroporphyrinogen decarboxylase; UROS, uroporphyrinogen-III synthase.

(methionine metabolism). The majority of the pathway displaying high negative FS indicates essentiality of the de novo synthesis of ubiquinone for tachyzoites (Fig. 10.16B). 10.5.3.3 Heme biosynthesis Porphyrins are essential cofactors of many proteins, including cytochrome proteins required for proper functioning of the ETC. The first step in the production of porphyrin is the mitochondrial formation of δ-aminolevulinate from glycine and succinyl-CoA. In apicomplexans, δ-aminolevulinate is then transported to the apicoplast where the 4-step conversion into coproporphyrinogen III occurs. The remaining processing of this intermediate to protoporphyrin IX and then to heme, with the addition of Fe21 takes place in mitochondrion. Various enzymes of this pathway in T. gondii have been biochemically characterized (Shanmugam et al., 2010). The crystal structure of T. gondii aminolevulinate dehydratase was elucidated by Jaffe et al. (2011) which

demonstrated that the T. gondii enzyme behaves as an octamer and does not contain any metal ions in the active site, although Mg21 ions are present at the intersections between prooctamer dimers. The nondependence of metal for catalysis is unique to the apicomplexans and could be exploited for drug development. All enzymes required for the catalysis of heme synthesis from glycine and succinyl-CoA are present in T. gondii and are highly fitness conferring as seen by the FS (Fig. 10.17). Interestingly, two different types of Coproporphyrinogen oxidase (CPO and CPOH) were found that catalyze the reactions in an oxygen-dependent and oxygen-independent manner, respectively. The oxygen-independent CPOH is the only dispensable enzyme in the pathway and catalyzes the reaction with S-adenosyl-methionine (SAM) as a cosubstrate instead of O2. Its role in the heme synthesis of T. gondii is still unknown and possibly functions as an active enzyme in a different life-cycle stage where O2 conditions are limiting. Lastly, the

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enzymes for heme degradation and detoxification, heme oxygenase and biliverdin reductase, are absent in T. gondii suggesting the parasite does not encounter host environments or circumstances where heme levels are high. This stands in contrast to intraerythrocytic stages of Plasmodium spp. which are exposed to very high concentrations of heme during digestion of hemoglobin which these stages need to detoxify. The existence of heme uptake and its importance compared to heme synthesis in T. gondii remains unclear. 10.5.3.4 Lipoic acid metabolism Lipoic acid is an essential cofactor of dehydrogenase enzymes, mainly the E2 subunit of PDH, BCKDH, and 2-oxoglutarate dehydrogenase complexes (Mazumdar et al., 2006). Lipoic acid is an organic sulfur compound derived from the 8-carbon FA, octanoic acid. In most eukaryotes, lipoic acid is synthesized in the mitochondrion and transported to other subcellular compartments. In several apicomplexan parasites the lipoylated PDH localizes to the

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apicoplast, where de novo FA biosynthesis occurs. Hence, T. gondii is likely able to synthesize lipoic acid in the apicoplast, as supported by the localization of LipA (Thomsen-Zieger et al., 2003). The genome of T. gondii possesses two enzymes for lipoic acid biosynthesis, LipA and LipB. In a study by Crawford et al., lipoylation of mitochondrial proteins was reduced when the parasites were grown in lipoic acid
deficient medium without affecting the lipoylation of apicoplast proteins, confirming the synthesis of lipoic acid in the plastid (Crawford et al., 2006). In contrast, lipoylation of mitochondrial proteins was affected by the absence of exogeneous lipoic acids and could be reversed through lipoic acid supply, confirming that the salvaged lipoic acid is used primarily in the mitochondrion. The pathway for lipoylation in the mitochondrion is essential for survival in the tachyzoite stage, as seen by the FS of the LPL enzyme, but lipoylation in the apicoplast can be circumvented, possibly due to the uptake of FAs from the host under perturbation conditions (Fig. 10.18).

FIGURE 10.18

Lipoic-acid biosynthesis and metabolism. The presence of de novo synthesis of lipoic acid in the apicoplast and utilization of scavenged lipoic acid for mitochondrial enzymes. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. KADH, 2-Oxoglutarate dehydrogenase; LipA, lipoyl synthase; LipB, N-lipoyl (octanoyl) transferase; LPL, lipoate-protein ligase; PDH, pyruvate dehydrogenase.

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FIGURE 10.19

SAM biosynthesis. De novo synthesis of SAM from scavenged methionine. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. 5-MTHF—Methyl-tetrahydrofolate; AHC—adenosylhomocysteinase; HC-S-MT—homocysteine-S-methyltransferase; Met-adoT—adenosyl-transferase; SAM, S-adenosyl-methionine.

The plasma membrane and mitochondrial transporter which are involved in lipoic acid salvage and mitochondrial localization have not yet been identified. 10.5.3.5 S-Adenosyl-methionine biosynthesis Methionine is an essential amino acid in all apicomplexan parasites, as well as in mammalian cells, and is therefore salvaged from the host. The methionine metabolism pathway in T. gondii is essential for providing two important substrates: homocysteine, which is essential for cysteine biosynthesis from serine and SAM, which plays an important role as the methyl-group donor for various enzymatic reactions in several metabolic pathways. The enzymes for the synthesis of SAM and S-adenosyl-homocysteine are present in the T. gondii genome and are predicted as essential for tachyzoites based on their FS (Fig. 10.19). The mechanism of translocation of SAM from the cytosol, where it is predicted to be produced, to other intracellular compartments where it is needed for methylation, is not known. 10.5.3.6 Glutathione biosynthesis and redox metabolism T. gondii is able to maintain redox balance in the face of oxidative stress induced by superoxide anions (O2 2 ), hydrogen peroxide (H2O2), and other toxic nucleophiles produced from

different metabolic reactions. These reactive oxygen species cause oxidative damage to DNA, lipids and proteins, and several antioxidant systems are present in cells to protect them from these damages. Parasite enzymes involved in the detoxification of these species include superoxide dismutase (SOD) which reduces O2 2 to H2O2 and catalase which can reduce H2O2 to water and oxygen. T. gondii possesses three SOD enzymes and one catalase, which are differentially localized in the mitochondrion and cytosol. The T. gondii enzyme, peroxiredoxin, also reduces H2O2 to H2O and O2 (Kwok et al., 2004). The predominant redox thiol in T. gondii is glutathione, a tripeptide of glutamine
cysteine
glycine which is synthesized de novo in two reactions steps. The balance between reduced and oxidized glutathione is maintained by the enzymes, glutathione peroxidase, glutathione reductase, and glutathione S-transferase. A final redox protein in the pathway, thioredoxin, is important in reduction of ribonucleotides to deoxyribonucleotides (XTP to dXTP) and its partner thioredoxin reductase catalyzes reduction of oxidized thioredoxin using NADPH as electron donor (Biddau et al., 2018; Xue et al., 2017). All the enzymes are present in the genome of T. gondii, with most enzymes being dispensable for parasite survival during the tachyzoite stage as seen by their FS (Fig. 10.20). The synthesis of

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FIGURE 10.20 Glutathione biosynthesis and redox metabolism. The presence of de novo synthesis and scavenge pathways for glutathione production. FS of the enzymes indicates uptake of glutathione in tachyzoites. Enzymes are depicted in purple and reducing equivalents, cofactors or energetic compounds produced or consumed are shown in gray. The FS related to the loss of enzyme and in some cases both isoenzymes are indicated by a heatmap color code—blue indicating dispensability, while red indicates essentiality. GCS, Glutamyl-cysteine synthase; GrxS/GrxSH, glutaredoxin; GSH, glutathione; GSH-ST, glutathione S-transferase; GSSG, glutathione disulfide; H2O2, hydrogen peroxide; O22, radical oxygen; PRxin, peroxiredoxin; RNR, ribonucleotide reductase (SS, small subunit; LS, large subunit); SOD, superoxide dismutase; TrxS/TrxSH, thioredoxin; XTP, xanthine triphosphate.

glutathione is also dispensable, suggesting that this metabolite can also be scavenged from the host.

10.6 Metabolomics approaches Current genomic, transcriptomic, and proteomic studies of protists, such as T. gondii, are limited by the fact that a significant number of protein-encoding genes remain functionally uncharacterized and/or encode proteins with functions distinct from those in other eukaryotes. Metabolomic analysis allows direct analysis of all primary and secondary metabolites (typically those under 1500 Da in size) and thus provides complementary and in many cases, unique insights into the metabolic potential of these organisms and functional characterization of their genomes. Key challenges in metabolomics include the chemical diversity of different metabolites, enormous differences in intracellular concentrations (pM or mM), the lack of amplification methods for increasing

sensitivity of detection and the fact that many metabolites and cofactors directly or indirectly regulate many metabolic pathways, complicating data interpretation (Matsuda, 2014). As a result, metabolomics analyses commonly utilize multiple analytical platforms, including direct infusion-MS, hyphenated MS [i.e., gaschromatography (GC)
MS or liquid chromatography (LC)
MS] and nuclear magnetic resonance (NMR) spectroscopy (Boccard and Rudaz, 2014; Gowda and Djukovic, 2014; Johnson and Gonzalez, 2012). Recent advances in MS include the introduction of ion-mobility and ultra-high mass resolution MS which allow the separation of molecules with different structures but identical masses and determination of the elemental composition of known and unknown metabolites, respectively. We briefly summarize some of the metabolomic approaches that have been used in T. gondii research. Lipidomic analyses are covered in more detail in Chapter 8, Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake.

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10.6.1 Intracellular metabolite levels Early metabolite profiling studies on extracellular and purified intracellular T. gondii tachyzoites provided important new insights into the metabolism of each of these stages. In particular, they provided the first direct evidence for the presence of a canonical TCA cycle in these stages as well as revealing the operation of a GABA shunt that had not been anticipated from genome annotations (MacRae et al., 2012). These analyses required the optimization of the steps involved in rapidly isolating different parasite stages, quenching parasite (and host cell) metabolism, and efficient extraction of polar and apolar metabolites in a single extraction protocol (MacRae et al., 2012). The most commonly used procedure for quenching parasite/host cell metabolism involves rapid chilling of cultures in situ. As T. gondii grow within adherent cells, the culture supernatant can be quickly aspirated, and the tissue culture plates placed on ice and rinsed with ice-cold phosphate buffer saline (PBS). While this protocol will not halt all metabolic reactions, it is sufficient to stabilize the levels of the majority of metabolites in central carbon metabolism (i.e., glycolysis, PPP, and TCA cycle). Tachyzoites are subsequently extracted from their host cell by passage through a fine needle and released parasites purified from host cell debris by passage through different filter membranes. Purified parasites are washed with PBS or an equivalent buffer solution to remove contaminating host metabolites and intact parasites extracted in single phase or biphasic aqueous
solvent mixtures. Monophasic extractions are simpler and more reproducible and commonly used for untargeted analysis, while biphasic mixture during or after extraction allows for separation and enrichment of polar and apolar metabolites. Extracted polar/apolar metabolites can be analyzed directly by LC
MS (without

derivatization), but generally need to be derivatized to generate volatile analytes for GC
MS analysis. LC
MS has been used in various studies of T. gondii metabolism to measure polar metabolites in central carbon metabolism of T. gondii (Limenitakis et al., 2013; Shukla et al., 2018; Xia et al., 2018; Olson et al., 2018) as well as in global lipid analyses (Amiar et al., 2016; Besteiro et al., 2008; Fu et al., 2019; Kong et al., 2017; Welti et al., 2007) and to identify novel/unanticipated metabolic pathways (Olson et al., 2018). Similarly, GC
MS has been used extensively to measure the abundance of polar central carbon metabolites, typically as their trimethylsilyl derivatives (Blume et al., 2015; MacRae et al., 2012; Nitzsche et al., 2016, 2017; Oppenheim et al., 2014) or of FAs as their methyl esters (Amiar et al., 2016; Dubois et al., 2018; Fu et al., 2019; MacRae et al., 2012; Ramakrishnan et al., 2012, 2015). Other techniques used to profile specific classes of intracellular metabolites, such as sugar phosphates and sugar nucleotides include high pH anion exchange chromatography (Uboldi et al., 2015).

10.6.2 Metabolic foot-printing Further insights into the metabolic needs and operation of specific pathways of purified parasites and/or infected host cells, can be gained by analyzing changes in metabolite levels in the culture medium (metabolomic footprinting). Samples of culture medium can be taken over time providing insights into the consumption and secretion of metabolites. NMR Analysis of the culture medium of T. gondii tachyzoites by NMR demonstrated that these stages utilized both Glc and glutamine, with concomitant secretion of lactate and glutamate (MacRae et al., 2012). Other secreted metabolites that were detected included CO2, (measured as bicarbonate), aspartate, alanine, and succinate. Interestingly, the majority of

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10.6 Metabolomics approaches

Glc and glutamine were catabolized to secreted end-products, indicating minimal incorporation of carbon into biomass in nondividing extracellular tachyzoites. The Seahorse extracellular flux analyzer was used to measure the consumption of O2 in wild-type parasites and in cells depleted in subunits of complex V and found significantly reduced O2 consumption in the mutant parasites, due to the disruption of the mitochondrial respiration (Huet et al., 2018). In another study, the authors used metabolic foot-printing to measure differences in T. gondii host cell metabolism which were found to impact T. gondii stage conversion. The authors noted that some host cells were permissive, enabling T. gondii differentiation from tachyzoites to bradyzoites (HFF, Vero), while others were resistant, preventing stage conversion (NIH3T3, 293T cells). Curiously, the transfer of supernatant from resistant cells was sufficient to inhibit stage conversion in otherwise permissive host cells and hydrophilic interaction chromatography MS was used to identify lactate as an inhibitory compound in the culture medium (Weilhammer et al., 2012). While lactate alone was sufficient to prevent T. gondii differentiation in Vero cells, it did not prevent stage conversion in HFFs indicating that the parameters enabling or disabling differentiation are more complex.

10.6.3 Stable isotope labeling approaches Stable isotope labeling approaches are increasingly being used to further define the metabolic potential of T. gondii and measure dynamic changes in parasite metabolism under different growth conditions. These approaches involve incubation of intracellular or isolated parasites with a stable isotope (13C/15N/2H) labeled metabolic precursor and analysis of the time-dependent uptake and incorporation of label into different parasite metabolites using the same analytical platforms used in

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metabolite profiling studies. Labeling studies have also been undertaken on infected host cells, which have demonstrated that extracellular metabolites are rapidly transported from the host cytoplasm to intracellular parasites across the parasitophorous vacuole membrane. Early 13C-Glc and 13C-glutamine labeling studies on purified intracellular tachyzoites and egressed tachyzoites demonstrated that both stages coutilize these abundant carbon sources and consequently derive their ATP from glycolysis and mitochondrial OXPHOS (MacRae et al., 2012). Subsequent studies used similar approaches to demonstrate that tachyzoites are able to compensate for loss of key transporters, such as the Glc transporter (Nitzsche et al., 2016), and enzymes involved in central carbon metabolism, such as HK (Shukla et al., 2018), LDHs (Xia et al., 2018), FBPase (Blume et al., 2015), PEPCK (Nitzsche et al., 2017), BCKDH (Oppenheim et al., 2014), by rerouting carbon fluxes. Similarly, 13C-Glc and 13Cacetate labeling have been employed to measure rates of FA synthesis in wild type T. gondii and in parasites disrupted in de novo synthesis or elongation (Amiar et al., 2016; Dubois et al., 2018; Fu et al., 2019; MacRae et al., 2012; Ramakrishnan et al., 2012, 2015), while 13C-labeled amino acids, such as tyrosine and BCAAs, have been used to identify the transporters and downstream pathways of catabolism of these amino acids (Limenitakis et al., 2013; Parker et al., 2019; Wallbank et al., 2018). Fig. 10.21 summarizes the workflow of a stable isotope labeling experiment combined with GC
MS analysis. In contrast to these MS approaches, Naemat et al. used deuterated phenylalanine and Raman microspectroscopy to monitor the uptake of phenylalanine by individual tachyzoites (Naemat et al., 2016). Such approaches have high spatial resolution and are very promising, providing insights into host-parasite interactions at the single-cell level.

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FIGURE 10.21 Metabolomics—stable isotope labeling workflow scheme. Infected host cells are cultivated in culture media containing 13C6-Glc (labeled carbons are highlighted in red) (1). After the desired time of labeling, cells are rapidly chilled to quench the metabolism in order to obtain a snapshot of the metabolism (2). Infected host cells are harvested, lysed through syringe passage and extracted parasites are purified through filtration (3). Metabolites are extracted and separated in a biphasic extraction (4). The polar phase contains various unlabeled, partially labeled and fully labeled intermediates of glycolysis, PPP, TCA cycle, amino acid metabolism, and others. Extracted metabolites are subsequently derivatized and analyzed by GC
MS (5). The exemplary total ion chromatogram highlights citrate (red triangle) and shows the corresponding ion spectrum. Due to the incorporation of heavy carbon derived from 13C6-Glc, the predominant ion is 467 m/z (red triangle) rather than the unlabeled mass isotopologue of 465 m/z (blue triangle) indicating the incorporation of one 13C2-acetyl-CoA during a single turn of the TCA-cycle. TCA, Tricarboxylic acid.

10.6.4 Metabolomic analysis of host tissues T. gondii infection results in many changes in host cell processes, including metabolism, which have been revealed using metabolomics approaches. In particular, Milovanovic et al. (2017) report changes in the host lipid metabolism during chronic and acute infection of mice with T. gondii, specifically, a decrease in cholesterol levels in the liver, brain, and peripheral lymphocytes. The authors postulate that the depletion of cholesterol may be a host defense mechanism to deprive T. gondii of this essential

metabolite. Other studies probed various tissues during T. gondii infection and found significant changes in the level of lysophospholipids and some amino acids in the serum and liver of infected mice as well as in arachidonic acid metabolism and other pathways in the spleen (Chen et al., 2017, 2018; Zhou et al., 2017). Similarly, untargeted metabolomics analyses were carried out on brain extracts of mice infected with the T. gondii Pru (type II) strain at different time points of infection (Zhou et al., 2015). Several metabolites were found to be altered in abundance between uninfected brains as well as between different

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10.7 Discussion and outlook

stages of the infection including the acute phase and chronic phase of the infection (7, 14, and 21 days post infection). However, it remains unclear how these alterations in host metabolism are induced and if they impact on the host pathogenicity.

10.7 Discussion and outlook A major challenge in effectively treating and eventually eradicating toxoplasmosis is to develop drugs against the quiescent bradyzoite stage. Bradyzoite cysts act as a source of transmission to the definitive host and between intermediate hosts. Hence, bradyzoites act as a reservoir of parasites and can potentially revert to tachyzoites in immunocompromised individuals, causing disease. Bradyzoites are expected to have metabolic needs which are vastly different from those of tachyzoites considering their altered physiology including encystation, slowed growth rate, and the distinct tissue environment. In order to develop drugs that target bradyzoites, we have to understand the metabolic needs and capabilities of this stage. However, to date, most studies focus on the tachyzoite stage which grows rapidly and is hence easier to culture, propagate and characterize. Consequently, relatively little is known about bradyzoite metabolism. New approaches in computational modeling, molecular biology, and metabolomics are needed to advance our knowledge of the persistent stage.

10.7.1 Computational modeling Metabolic modeling of the latent bradyzoite stage can provide insightful predictions on the growth and nutritional requirements of parasites during dormancy. Several metabolic switches are thought to occur when parasites transition from the acute proliferative stage to

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latency and enable parasite persistence and transmission. The ability of the pathogen to adjust to the varying host environments is often associated with high metabolic plasticity and rapid rewiring of metabolic pathways to produce the building blocks needed for survival. Constraint-based metabolic models that incorporate stage-specific information have been used to better understand the transitions and pathogen adaptation to its selective niche. One approach to generate models for latency is to adjust the growth requirement parameters and integrating high-throughput (transcriptomics, proteomics, and metabolomics) data, when available. Integrating transcriptomics data and simulating for gene and reaction KOs can provide insights into the utilization of specific routes for biomass production, based on the expression of an enzyme catalyzing a particular reaction. However, the predictions must be carefully validated through biological experiments. Computational predictions and experiments function in tandem, providing useful information in an iterative fashion. In the future, increasing biological information on latent stages will enable mathematical models to make reliable hypotheses and provide us with a greater understanding of the metabolic shifts and subsequent identification of drug targets.

10.7.2 Molecular biology The few findings on bradyzoite metabolism made so far have been discussed above and were made by deleting genes which are dispensable in tachyzoites but were found to impair the chronic infection in vivo such as the LDH and HK (Abdelbaset et al., 2017; Shukla et al., 2018; Xia et al., 2018). However, a major hurdle in characterizing genes that are essential in bradyzoites is that these genes may also (or exclusively) be essential in tachyzoites and current efforts have not yet led to genetic tools able to address the role of genes in bradyzoites

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specifically. However, new strategies may overcome this hurdle. For example, the function of a gene in the bradyzoite stage may be assessed following promoter swapping to a tachyzoite specific gene promoter such as the SAG1 promoter. Promoter swapping is commonly used in other pathogens such as Plasmodium to investigate the role of genes in different life cycle stages (De Niz et al., 2015; Laurentino et al., 2011). Following stage conversion, the gene is expected to be no longer expressed and its function/role in bradyzoite can be assessed. Alternatively, a gene can be conditionally deleted using the dimerizable Cre recombinase or DiCRe system (Andenmatten et al., 2013; Jullien et al., 2007). The Cre system allows the conditional deletion of a gene following rapamycin treatment. Previous studies have suggested that rapamycin can cross the blood
brain barrier and should thus be suitable to allow the depletion of genes in bradyzoites after the successful infection and state conversion (Majumder et al., 2011). Rapamycin analogs have also been reported to cross the blood
brain barrier (Mita et al., 2008). Finally, the advent of whole genome CRISPR/Cas9 screening has already provided a wealth of knowledge about the essentiality of different metabolic pathways for tachyzoite virulence. The initial screen by Sidik et al. (2016) focused on deriving FS across all genes in standard, nutrient-rich medium. Future screens, under growth conditions that more closely mimic the microenvironment in terms of nutrient, oxygen levels, etc., will highlight the metabolic flexibility or restrictions of T. gondii and perhaps reveal how they may adapt during infection of different tissue types or during development and persistence of latent stages.

10.7.3 Metabolomics The metabolomic approaches discussed previously have been crucial to characterizing

several metabolic pathways in T. gondii. However, these approaches typically measure assemblages of millions of parasites and/or host cells, which disguise heterogeneous responses at the single-cell level. There is increasing evidence that heterogeneity in the growth rate and metabolism of other microbial pathogens in response to changes in their local microenvironment plays an important role in determining host-pathogen interactions and the outcome of acute and persistent infections (Bumann and Cunrath, 2017; Martins and Locke, 2015) as well as susceptibility to antibiotic treatments (Claudi et al., 2014; Helaine et al., 2014). Similarly, a recent study identified significant heterogeneity in the growth rates of individual T. gondii bradyzoite cysts in brain tissues (Watts et al., 2015). While technically challenging, few approaches are able to measure the growth rate and metabolism of parasites at the single-cell level. Most promising is the combination of stable isotope labeling combined with imaging MS, secondary ion MS or Raman spectroscopy (Kopf et al., 2016; Louie et al., 2013; Naemat et al., 2016). These approaches allow quantitative measurements of growth rates and metabolic activity. Ultimately, it will be crucial to identify distinct populations of T. gondii and develop treatments which target cells with distinct phenotypes which likely occur in vivo.

References Abdelbaset, A.E., Fox, B.A., Karram, M.H., Abd Ellah, M. R., Bzik, D.J., Igarashi, M., 2017. Lactate dehydrogenase in Toxoplasma gondii controls virulence, bradyzoite differentiation, and chronic infection. PLoS One 12. Available from: https://doi.org/10.1371/journal. pone.0173745. Amiar, S., Macrae, J.I., Callahan, D.L., Dubois, D., Van dooren, G.G., Shears, M.J., et al., 2016. Apicoplastlocalized lysophosphatidic acid precursor assembly is required for bulk phospholipid synthesis in Toxoplasma gondii and relies on an algal/plant-like glycerol 3phosphate acyltransferase. PLoS Pathog. 12, e1005765.

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References

Andenmatten, N., Egarter, S., Jackson, A.J., Jullien, N., Herman, J.P., Meissner, M., 2013. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods 10, 125
127. Bandini, G., Haserick, J.R., Motari, E., Ouologuem, D.T., Lourido, S., Roos, D.S., et al., 2016. O-Fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 113, 11567
11572. Bandini, G., Albuquerque-Wendt, A., Hegermann, J., Samuelson, J., Routier, F.H., 2019a. Protein O- and Cglycosylation pathways in Toxoplasma gondii and Plasmodium falciparum. Parasitology 1
12. Bandini, G., Leon, D.R., Hoppe, C.M., Zhang, Y., AgopNersesian, C., Shears, M.J., et al., 2019b. O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites. J. Biol. Chem. 294, 1967
1983. Banerjee, P.S., Lagerlof, O., Hart, G.W., 2016. Roles of OGlcNAc in chronic diseases of aging. Mol. Aspects Med. 51, 1
15. Besteiro, S., Bertrand-Michel, J., Lebrun, M., Vial, H., Dubremetz, J.F., 2008. Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries: further insights into the role of cholesterol. Biochem. J. 415, 87
96. Biddau, M., Bouchut, A., Major, J., Saveria, T., Tottey, J., Oka, O., et al., 2018. Two essential thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii. PLoS Pathog. 14, e1006836. Blume, M., Rodriguez-Contreras, D., Landfear, S., Fleige, T., Soldati-Favre, D., Lucius, R., et al., 2009. Hostderived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis. Proc. Natl. Acad. Sci. U.S.A. 106, 12998
13003. Blume, M., Nitzsche, R., Sternberg, U., Gerlic, M., Masters, S.L., Gupta, N., et al., 2015. A Toxoplasma gondii gluconeogenic enzyme contributes to robust central carbon metabolism and is essential for replication and virulence. Cell Host Microbe 18, 210
220. Boccard, J., Rudaz, S., 2014. Harnessing the complexity of metabolomic data with chemometrics. J. Chemom. 28, 1
9. Bordbar, A., Monk, J.M., King, Z.A., Palsson, B.O., 2014. Constraint-based models predict metabolic and associated cellular functions. Nat. Rev. Genet. 15, 107
120. Bumann, D., Cunrath, O., 2017. Heterogeneity of Salmonella-host interactions in infected host tissues. Curr. Opin. Microbiol. 39, 57
63. Caffaro, C.E., Koshy, A.A., Liu, L., Zeiner, G.M., Hirschberg, C.B., Boothroyd, J.C., 2013. A nucleotide sugar transporter involved in glycosylation of the

493

Toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites. PLoS Pathog. 9, e1003331. Campbell, S.A., Richards, T.A., Mui, E.J., Samuel, B.U., Coggins, J.R., Mcleod, R., et al., 2004. A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation. Int. J. Parasitol. 34, 5
13. Chen, X.Q., Zhou, C.X., Elsheikha, H.M., He, S., Hu, G.X., Zhu, X.Q., 2017. Profiling of the perturbed metabolomic state of mouse spleen during acute and chronic toxoplasmosis. Parasites Vectors 10, 339. Chen, X.Q., Elsheikha, H.M., Hu, R.S., Hu, G.X., Guo, S.L., Zhou, C.X., et al., 2018. Hepatic metabolomics investigation in acute and chronic murine toxoplasmosis. Front. Cell. Infect. Microbiol. 8, 189. Claudi, B., Sprote, P., Chirkova, A., Personnic, N., Zankl, J., Schurmann, N., et al., 2014. Phenotypic variation of Salmonella in host tissues delays eradication by antimicrobial chemotherapy. Cell 158, 722
733. Cobbold, S.A., Vaughan, A.M., Lewis, I.A., Painter, H.J., Camargo, N., Perlman, D.H., et al., 2013. Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum. J. Biol. Chem. 288, 36338
36350. Coppin, A., Dzierszinski, F., Legrand, S., Mortuaire, M., Ferguson, D., Tomavo, S., 2003. Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii. Biochimie 85, 353
361. Coppin, A., Varre, J.S., Lienard, L., Dauvillee, D., Guerardel, Y., Soyer-Gobillard, M.O., et al., 2005. Evolution of plant-like crystalline storage polysaccharide in the protozoan parasite Toxoplasma gondii argues for a red alga ancestry. J. Mol. Evol. 60, 257
267. Crawford, M.J., Thomsen-Zieger, N., Ray, M., Schachtner, J., Roos, D.S., Seeber, F., 2006. Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast. EMBO J. 25, 3214
3222. Dauvillee, D., Deschamps, P., Ral, J.P., Plancke, C., Putaux, J.L., Devassine, J., et al., 2009. Genetic dissection of floridean starch synthesis in the cytosol of the model dinoflagellate Crypthecodinium cohnii. Proc. Natl. Acad. Sci. U.S.A. 106, 21126
21130. Debierre-Grockiego, F., Schofield, L., Azzouz, N., Schmidt, J., Santos de Macedo, C., Ferguson, M.A., et al., 2006. Fatty acids from Plasmodium falciparum down-regulate the toxic activity of malaria glycosylphosphatidylinositols. Infect. Immun. 74, 5487
5496. De Niz, M., Helm, S., Horstmann, S., Annoura, T., Del portillo, H.A., Khan, S.M., et al., 2015. In vivo and in vitro characterization of a Plasmodium liver stage-specific promoter. PLoS One 10, e0123473. Dubey, R., Staker, B.L., Foe, I.T., Bogyo, M., Myler, P.J., Ngo, H.M., et al., 2017. Membrane skeletal association

Toxoplasma Gondii

494

10. Metabolic networks and metabolomics

and post-translational allosteric regulation of Toxoplasma gondii GAPDH1. Mol. Microbiol. 103, 618
634. Dubois, D., Fernandes, S., Amiar, S., Dass, S., Katris, N.J., Botte, C.Y., et al., 2018. Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages. J. Lipid Res. 59, 994
1004. Ebrahim, A., Brunk, E., Tan, J., O’brien, E.J., Kim, D., Szubin, R., et al., 2016. Multi-omic data integration enables discovery of hidden biological regularities. Nat. Commun. 7. Available from: https://doi.org/10.1038/ ncomms13091. Erler, H., Ren, B., Gupta, N., Beitz, E., 2018. The intracellular parasite Toxoplasma gondii harbors three druggable FNT-type formate and L-lactate transporters in the plasma membrane. J. Biol. Chem. 293, 17622
17630. Fleige, T., Fischer, K., Ferguson, D.J., Gross, U., Bohne, W., 2007. Carbohydrate metabolism in the Toxoplasma gondii apicoplast: localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator. Eukaryot. Cell 6, 984
996. Florimond, C., Cordonnier, C., Taujale, R., Van Der wel, H., Kannan, N., West, C.M., et al., 2019. A Toxoplasma prolyl hydroxylase mediates oxygen stress responses by regulating translation elongation. mBio 10. Available from: https://doi.org/10.1128/mBio.00234-19. Fu, Y., Cui, X., Liu, J., Zhang, X., Zhang, H., Yang, C., et al., 2019. Synergistic roles of acyl-CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism. Cell Microbiol. 21, e12970. Gas-Pascual, E., Ichikawa, H.T., Sheikh, M.O., Serji, M.I., Deng, B., Mandalasi, M., et al., 2019. CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology. J. Biol. Chem. 294, 1104
1125. Gentry, M.S., Dowen III, R.H., Worby, C.A., Mattoo, S., Ecker, J.R., Dixon, J.E., 2007. The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease. J. Cell Biol. 178, 477
488. Gowda, G.A., Djukovic, D., 2014. Overview of mass spectrometry-based metabolomics: opportunities and challenges. Methods Mol. Biol. 1198, 3
12. Guerardel, Y., Leleu, D., Coppin, A., Lienard, L., Slomianny, C., Strecker, G., et al., 2005. Amylopectin biogenesis and characterization in the protozoan parasite Toxoplasma gondii, the intracellular development of which is restricted in the HepG2 cell line. Microbes Infect. 7, 41
48. Hackett, S.R., Zanotelli, V.R., Xu, W., Goya, J., Park, J.O., Perlman, D.H., et al., 2016. Systems-level analysis of mechanisms regulating yeast metabolic flux. Science 354, aaf2786.

Hannaert, V., Bringaud, F., Opperdoes, F.R., Michels, P.A., 2003. Evolution of energy metabolism and its compartmentation in Kinetoplastida. Kinetoplastid. Biol. Dis. 2, 11. Harris, J.R., Adrian, M., Petry, F., 2004. Amylopectin: a major component of the residual body in Cryptosporidium parvum oocysts. Parasitology 128, 269
282. Helaine, S., Cheverton, A.M., Watson, K.G., Faure, L.M., Matthews, S.A., Holden, D.W., 2014. Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science 343, 204
208. Henry, C.S., Broadbelt, L.J., Hatzimanikatis, V., 2007. Thermodynamics-based metabolic flux analysis. Biophys. J. 92, 1792
1805. Hoppe, C.M., Albuquerque-Wendt, A., Bandini, G., Leon, D.R., Shcherbakova, A., Buettner, F.F.R., et al., 2018. Apicomplexan C-mannosyltransferases modify thrombospondin type I-containing adhesins of the TRAP family. Glycobiology 28, 333
343. Huet, D., Rajendran, E., van Dooren, G.G., Lourido, S., 2018. Identification of cryptic subunits from an apicomplexan ATP synthase. eLife 7. Available from: https:// doi.org/10.7554/eLife.38097. Jaffe, E.K., Shanmugam, D., Gardberg, A., Dieterich, S., Sankaran, B., Stewart, L.J., et al., 2011. Crystal structure of Toxoplasma gondii porphobilinogen synthase. J. Biol. Chem. 286, 15298
15307. Jewett, T.J., Sibley, L.D., 2003. Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites. Mol. Cell 11, 885
894. Johnson, C.H., Gonzalez, F.J., 2012. Challenges and opportunities of metabolomics. J. Cell. Physiol. 227, 2975
2981. Joyce, B.R., Tampaki, Z., Kim, K., Wek, R.C., Sullivan Jr., W.J., 2013. The unfolded protein response in the protozoan parasite Toxoplasma gondii features translational and transcriptional control. Eukaryot. Cell 12, 979
989. Jullien, N., Goddard, I., Selmi-Ruby, S., Fina, J.L., Cremer, H., Herman, J.P., 2007. Conditional transgenesis using Dimerizable Cre (DiCre). PLoS One 2, e1355. Karkhanis, Y.D., Allocco, J.J., Schmatz, D.M., 1993. Amylopectin synthase of Eimeria tenella: identification and kinetic characterization. J. Eukaryot. Microbiol. 40, 594
598. Khurana, S., Coffey, M.J., John, A., Uboldi, A.D., Huynh, M.H., Stewart, R.J., et al., 2019. Protein O-fucosyltransferase 2-mediated O-glycosylation of the adhesin MIC2 is dispensable for Toxoplasma gondii tachyzoite infection. J. Biol. Chem. 294, 1541
1553. Kim, M., Rai, N., Zorraquino, V., Tagkopoulos, I., 2016. Multi-omics integration accurately predicts cellular state in unexplored conditions for Escherichia coli. Nat. Commun. 7, 13090.

Toxoplasma Gondii

References

Kong, P., Ufermann, C.M., Zimmermann, D.L.M., Yin, Q., Suo, X., Helms, J.B., et al., 2017. Two phylogenetically and compartmentally distinct CDP-diacylglycerol synthases cooperate for lipid biogenesis in Toxoplasma gondii. J. Biol. Chem. 292, 7145
7159. Kopf, S.H., Sessions, A.L., Cowley, E.S., Reyes, C., Van sambeek, L., Hu, Y., et al., 2016. Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum. Proc. Natl. Acad. Sci. U.S.A. 113, E110
E116. Kovarova, J., Barrett, M.P., 2016. The pentose phosphate pathway in parasitic trypanosomatids. Trends Parasitol. 32, 622
634. Kwok, L.Y., Schlu¨ter, D., Clayton, C., Soldati, D., 2004. The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury. Mol. Microbiol. 51, 47
61. Laurentino, E.C., Taylor, S., Mair, G.R., Lasonder, E., Bartfai, R., Stunnenberg, H.G., et al., 2011. Experimentally controlled downregulation of the histone chaperone FACT in Plasmodium berghei reveals that it is critical to male gamete fertility. Cell Microbiol. 13, 1956
1974. Lewis, N.E., Nagarajan, H., Palsson, B.O., 2012. Constraining the metabolic genotype-phenotype relationship using a phylogeny of in silico methods. Nat. Rev. Microbiol. 10, 291
305. Limenitakis, J., Oppenheim, R.D., Creek, D.J., Foth, B.J., Barrett, M.P., Soldati-Favre, D., 2013. The 2methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii. Mol. Microbiol. 87, 894
908. Lin, S.S., Kerscher, S., Saleh, A., Brandt, U., Gross, U., Bohne, W., 2008. The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2alkyl-4(1H)quinolones. Biochim. Biophys. Acta 1777, 1455
1462. Louie, K.B., Bowen, B.P., Mcalhany, S., Huang, Y., Price, J. C., Mao, J.H., et al., 2013. Mass spectrometry imaging for in situ kinetic histochemistry. Sci. Rep. 3, 1656. Luder, C.G.K., Rahman, T., 2017. Impact of the host on Toxoplasma stage differentiation. Microb. Cell 4, 203
211. Luk, F.C., Johnson, T.M., Beckers, C.J., 2008. N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii. Mol. Biochem. Parasitol. 157, 169
178. MacRae, J.I., Sheiner, L., Nahid, A., Tonkin, C., Striepen, B., McConville, M.J., 2012. Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii. Cell Host Microbe 12, 682
692. Majumder, S., Richardson, A., Strong, R., Oddo, S., 2011. Inducing autophagy by rapamycin before, but not after,

495

the formation of plaques and tangles ameliorates cognitive deficits. PLoS One 6, e25416. Martins, B.M., Locke, J.C., 2015. Microbial individuality: how single-cell heterogeneity enables population level strategies. Curr. Opin. Microbiol. 24, 104
112. Martins-Duarte, E.S., Carias, M., Vommaro, R., Surolia, N., De souza, W., 2016. Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii. J. Cell. Sci. 129, 3320
3331. Massimine, K.M., Doan, L.T., Atreya, C.A., Stedman, T.T., Anderson, K.S., Joiner, K.A., et al., 2005. Toxoplasma gondii is capable of exogenous folate transport. Mol. Biochem. Parasitol. 144, 44
54. Matsuda, F., 2014. Rethinking mass spectrometry-based small molecule identification strategies in metabolomics. Mass Spectrom. (Tokyo) 3, S0038. Mazumdar, J., Wilson, E.H., Masek, K., Hunter, C.A., Striepen, B., 2006. Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103, 13192
13197. Milovanovic, I., Busarcevic, M., Trbovich, A., Ivovic, V., Uzelac, A., Djurkovic-Djakovic, O., 2017. Evidence for host genetic regulation of altered lipid metabolism in experimental toxoplasmosis supported with gene data mining results. PLoS One 12, e0176700. Mita, M., Sankhala, K., Abdel-Karim, I., Mita, A., Giles, F., 2008. Deforolimus (AP23573) a novel mTOR inhibitor in clinical development. Expert. Opin. Investig. Drugs 17, 1947
1954. Moog, D., Przyborski, J.M., Maier, U.G., 2017. Genomic and proteomic evidence for the presence of a peroxisome in the apicomplexan parasite Toxoplasma gondii and other Coccidia. Genome Biol. Evol. 9, 3108
3121. Naemat, A., Elsheikha, H.M., Boitor, R.A., Notingher, I., 2016. Tracing amino acid exchange during hostpathogen interaction by combined stable-isotope timeresolved Raman spectral imaging. Sci. Rep. 6, 20811. Niehus, S., Smith, T.K., Azzouz, N., Campos, M.A., Dubremetz, J.F., Gazzinelli, R.T., et al., 2014. Virulent and avirulent strains of Toxoplasma gondii which differ in their glycosylphosphatidylinositol content induce similar biological functions in macrophages. PLoS One 9, e85386. Nikolskaya, T., Zagnitko, O., Tevzadze, G., Haselkorn, R., Gornicki, P., 1999. Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain. Proc. Natl. Acad. Sci. U.S.A. 96, 14647
14651. Nitschke, F., Wang, P., Schmieder, P., Girard, J.M., Awrey, D.E., Wang, T., et al., 2013. Hyperphosphorylation of glucosyl C6 carbons and altered structure of glycogen in the neurodegenerative epilepsy Lafora disease. Cell Metab. 17, 756
767.

Toxoplasma Gondii

496

10. Metabolic networks and metabolomics

Nitzsche, R., Zagoriy, V., Lucius, R., Gupta, N., 2016. Metabolic cooperation of glucose and glutamine is essential for the lytic cycle of obligate intracellular parasite Toxoplasma gondii. J. Biol. Chem. 291, 126
141. Nitzsche, R., Gunay-Esiyok, O., Tischer, M., Zagoriy, V., Gupta, N., 2017. A plant/fungal-type phosphoenolpyruvate carboxykinase located in the parasite mitochondrion ensures glucose-independent survival of Toxoplasma gondii. J. Biol. Chem. 292, 15225
15239. Olson, W.J., Stevenson, D., Amador-Noguez, D., Knoll, L. J., 2018. Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability. bioRxiv. Available from: https://doi.org/10.1101/ 463075. Oppenheim, R.D., Creek, D.J., Macrae, J.I., Modrzynska, K. K., Pino, P., Limenitakis, J., et al., 2014. BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei. PLoS Pathog. 10, e1004263. Orth, J.D., Fleming, R.M., Palsson, B.O., 2010. Reconstruction and use of microbial metabolic networks: the core Escherichia coli metabolic model as an educational guide. EcoSal Plus 4. Available from: https://doi.org/10.1128/ecosalplus.10.2.1. Parker, K.E.R., Fairweather, S.J., Rajendran, E., Blume, M., McConville, M.J., Broer, S., et al., 2019. The tyrosine transporter of Toxoplasma gondii is a member of the newly defined apicomplexan amino acid transporter (ApiAT) family. PLoS Pathog. 15, e1007577. Peek, J., Castiglione, G., Shi, T., Christendat, D., 2014. Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex. Mol. Biochem. Parasitol. 194, 16
19. Pino, P., Foth, B.J., Kwok, L.Y., Sheiner, L., Schepers, R., Soldati, T., et al., 2007. Dual targeting of antioxidant and metabolic enzymes to the mitochondrion and the apicoplast of Toxoplasma gondii. PLoS Pathog. 3, e115. Pomel, S., Luk, F.C., Beckers, C.J., 2008. Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites. PLoS Pathog. 4, e1000188. Ramakrishnan, S., Docampo, M.D., Macrae, J.I., Pujol, F.M., Brooks, C.F., Van dooren, G.G., et al., 2012. Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 287, 4957
4971. Ramakrishnan, S., Docampo, M.D., Macrae, J.I., Ralton, J.E., Rupasinghe, T., McConville, M.J., et al., 2015. The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell. Mol. Microbiol. 97, 64
76.

Richards, T.A., Dacks, J.B., Campbell, S.A., Blanchard, J.L., Foster, P.G., Mcleod, R., et al., 2006. Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements. Eukaryot. Cell 5, 1517
1531. Roberts, F., Roberts, C.W., Johnson, J.J., Kyle, D.E., Krell, T., Coggins, J.R., et al., 1998. Evidence for the shikimate pathway in apicomplexan parasites. Nature . Available from: https://doi.org/10.1038/31723. Roberts, C.W., Roberts, F., Lyons, R.E., Kirisits, M.J., Mui, E.J., Finnerty, J., et al., 2002. The shikimate pathway and its branches in apicomplexan parasites. J. Infect. Dis. Available from: https://doi.org/10.1086/338004. Saa, P.A., Nielsen, L.K., 2017. Formulation, construction and analysis of kinetic models of metabolism: a review of modelling frameworks. Biotechnol. Adv. 35, 981
1003. Saha, S., Coleman, B.I., Dubey, R., Blader, I.J., Gubbels, M. J., 2017. Two phosphoglucomutase paralogs facilitate ionophore-triggered secretion of the Toxoplasma micronemes. mSphere 2. Available from: https://doi.org/ 10.1128/mSphere.00521-17. Salunke, R., Mourier, T., Banerjee, M., Pain, A., Shanmugam, D., 2018. Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii. PLoS Biol. 16, e2006128. Seeber, F., Limenitakis, J., Soldati-Favre, D., 2008. Apicomplexan mitochondrial metabolism: a story of gains, losses and retentions. Trends Parasitol. 24, 468
478. Shanmugam, D., Wu, B., Ramirez, U., Jaffe, E.K., Roos, D. S., 2010. Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential. J. Biol. Chem. 285, 22122
22131. Shen, B., Sibley, L.D., 2014. Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion. Proc. Natl. Acad. Sci. U.S.A. 111, 3567
3572. Shukla, A., Olszewski, K.L., Llinas, M., Rommereim, L.M., Fox, B.A., Bzik, D.J., et al., 2018. Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii. Int. J. Parasitol. 48, 955
968. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in toxoplasma identifies essential apicomplexan genes. Cell 166, 1423
1435.e12. Song, C., Chiasson, M.A., Nursimulu, N., Hung, S.S., Wasmuth, J., Grigg, M.E., et al., 2013. Metabolic reconstruction identifies strain-specific regulation of virulence in Toxoplasma gondii. Mol. Syst. Biol. 9, 708.

Toxoplasma Gondii

References

Srinivasan, S., Cluett, W.R., Mahadevan, R., 2015. Constructing kinetic models of metabolism at genomescales: a review. Biotechnol. J. 10, 1345
1359. Stanway et al., 2019. Cell; Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage. Available from: https://doi. org/10.1016/j.cell.2019.10.030. Striepen, B., Zinecker, C.F., Damm, J.B., Melgers, P.A., Gerwig, G.J., Koolen, M., et al., 1997. Molecular structure of the “low molecular weight antigen” of Toxoplasma gondii: a glucose alpha 1-4 N-acetylgalactosamine makes free glycosyl-phosphatidylinositols highly immunogenic. J. Mol. Biol. 266, 797
813. Striepen, B., Dubremetz, J.F., Schwarz, R.T., 1999. Glucosylation of glycosylphosphatidylinositol membrane anchors: identification of uridine diphosphateglucose as the direct donor for side chain modification in Toxoplasma gondii using carbohydrate analogues. Biochemistry 38, 1478
1487. Sugi, T., Tu, V., Ma, Y., Tomita, T., Weiss, L.M., 2017. Toxoplasma gondii requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of brain cysts. mBio 8. Available from: https://doi. org/10.1128/mBio.01289-17. Thomsen-Zieger, N., Schachtner, J., Seeber, F., 2003. Apicomplexan parasites contain a single lipoic acid synthase located in the plastid. FEBS Lett. 547, 80
86. Tomita, T., Sugi, T., Yakubu, R., Tu, V., Ma, Y., Weiss, L. M., 2017. Making home sweet and sturdy: Toxoplasma gondii ppGalNAc-Ts glycosylate in hierarchical order and confer cyst wall rigidity. mBio 8. Available from: https://doi.org/10.1128/mBio.02048-16. Tymoshenko, S., Oppenheim, R.D., Agren, R., Nielsen, J., Soldati-Favre, D., Hatzimanikatis, V., 2015. Metabolic needs and capabilities of Toxoplasma gondii through combined computational and experimental analysis. PLoS Comput. Biol. 11, e1004261. Uboldi, A.D., Mccoy, J.M., Blume, M., Gerlic, M., Ferguson, D.J., Dagley, L.F., et al., 2015. Regulation of starch stores by a Ca(2 1 )-dependent protein kinase is essential for viable cyst development in Toxoplasma gondii. Cell Host Microbe 18, 670
681. Vercesi, A.E., Rodrigues, C.O., Uyemura, S.A., Zhong, L., Moreno, S.N., 1998. Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 273, 31040
31047. Wallbank, B.A., Dominicus, C.S., Broncel, M., Legrave, N., Macrae, J.I., Staines, H.M., et al., 2018. Characterisation

497

of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3. Mol. Microbiol. . Available from: https://doi. org/10.1111/mmi.14156. Watts, E., Zhao, Y., Dhara, A., Eller, B., Patwardhan, A., Sinai, A.P., 2015. Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. mBio 6, e01155-15. Weilhammer, D.R., Iavarone, A.T., Villegas, E.N., Brooks, G.A., Sinai, A.P., Sha, W.C., 2012. Host metabolism regulates growth and differentiation of Toxoplasma gondii. Int. J. Parasitol. 42, 947
959. Welti, R., Mui, E., Sparks, A., Wernimont, S., Isaac, G., Kirisits, M., et al., 2007. Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids. Biochemistry 46, 13882
13890. West, C.M., Van Der Wel, H., Sassi, S., Gaucher, E.A., 2004. Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways. Biochim. Biophys. Acta 1673, 29
44. Xia, N., Yang, J., Ye, S., Zhang, L., Zhou, Y., Zhao, J., et al., 2018. Functional analysis of Toxoplasma lactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo. Cell Microbiol. 20. Available from: https://doi.org/10.1111/cmi.12794. Xue, J., Jiang, W., Chen, Y., Gong, F., Wang, M., Zeng, P., et al., 2017. Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function. FASEB J. . Yilmaz, L.S., Walhout, A.J., 2017. Metabolic network modeling with model organisms. Curr. Opin. Chem. Biol. 36, 32
39. Zhou, C.X., Zhou, D.H., Elsheikha, H.M., Liu, G.X., Suo, X., Zhu, X.Q., 2015. Global metabolomic profiling of mice brains following experimental infection with the cyst-forming Toxoplasma gondii. PLoS One 10, e0139635. Zhou, C.X., Cong, W., Chen, X.Q., He, S.Y., Elsheikha, H. M., Zhu, X.Q., 2017. Serum metabolic profiling of oocyst-induced Toxoplasma gondii acute and chronic infections in mice using mass-spectrometry. Front. Microbiol. 8, 2612. Zuther, E., Johnson, J.J., Haselkorn, R., Mcleod, R., Gornicki, P., 1999. Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase. Proc. Natl. Acad. Sci. U.S.A. 96, 13387
13392.

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C H A P T E R

11 The apicoplast and mitochondrion of Toxoplasma gondii Frank Seeber1, Jean E. Feagin2,3, Marilyn Parsons2,4,5 and Giel G. van Dooren6 1

FG16: Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute, Berlin, Germany Department of Global Health, University of Washington, Seattle, WA, United States 3Department of Pharmacy, University of Washington, Seattle, WA, United States 4Department of Pediatrics, University of Washington, Seattle, WA, United States 5Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA, United States 6Research School of Biology, Australian National University, Canberra, ACT, Australia 2

11.1 Introduction One hallmark of eukaryotic cells, although not necessarily exclusive to them, is the division of cellular contents into specialized membrane-bounded compartments. Organelles provide multiple benefits to the cell, including protecting the rest of the cell from dangerous reaction products, generating gradients that can be exploited for biological processes, and separating potentially interfering metabolic pathways. The partitioning of the eukaryotic cell led I. E. Wallin to suggest in 1927 that it is a collection of symbiotic microorganisms. Of the numerous organelles in eukaryotic cells, two provide evidence of endosymbiosis since they contain small genomes and are bounded by Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00011-6

multiple membranes: the mitochondrion and the chloroplast (or more generically, the plastid; to prevent confusion, we will use the term “plastid” throughout this chapter, except when referring specifically to photosynthetic plastids, “chloroplasts”). Their genomes encode key proteins needed for the specialized function of these organelles, including some components of a separate translation system, and a variable phalanx of other genes. By 1970 these oddities spurred Lynn Margulis to propose the endosymbiont theory, which postulates that the present-day eukaryote originated from multiple, interacting organisms and more specifically that these organelles are the remnants of engulfed prokaryotic cells. Initially viewed as outlandish, today this idea is firmly entrenched in biological doctrine (Gray, 2017). Along with

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the endosymbiont theory, students learn that the mitochondrion is the powerhouse of the cell and that photosynthesis occurs in chloroplasts, but they generally know little regarding the other vital metabolic activities performed by these organelles. In the origins of these organelles lie new possibilities for intervention in diseases caused by apicomplexan parasites, including birth defects, blindness, and encephalitis due to Toxoplasma gondii, malaria due to Plasmodium species, and numerous veterinary diseases. The intense interest in the endosymbiont organelles of apicomplexans began with the realization that these parasites have not one but two extrachromosomal DNAs, each residing in its own organelle. The single mitochondrion has a minimal genome with unique rRNA genes and ribosomes. The second organelle is the apicoplast (apicomplexan plastid), a leftover from a photosynthetic past. There is only one apicoplast per cell in tachyzoites and its genome resembles the chloroplast DNA of plants and algae. The totally unanticipated finding of this novel organelle suggested the presence of plant-like metabolic pathways quite different from those in the vertebrate hosts. Such pathways were anticipated to provide a variety of new chemotherapeutic targets. Indeed, while T. gondii can survive temporarily without an apicoplast, such cells are incapable of proliferation following the invasion of new host cells (see Section 11.2.8). The unique characteristics of apicomplexan mitochondria also present possibilities for intervention. Studying the origins and activities of the DNA-containing organelles of T. gondii and of the malaria parasite Plasmodium falciparum has paid big dividends already and there are undoubtedly more discoveries to come. Work on the apicoplast and mitochondrion of T. gondii is inextricably intertwined with studies of these organelles in Plasmodium. P. falciparum has been intensively studied due to its major health relevance. While the sections that follow will focus on T. gondii, work with

other apicomplexans will be noted. For some topics, work on P. falciparum predominates and will be described in greater detail. Given the common ancestry of the apicoplast, this will provide a framework for also understanding the T. gondii organelles. However, despite the many similarities, there are also some surprising differences between T. gondii and P. falciparum. Consequently, predictions of apicoplast or mitochondrial functions based on data from just one organism must be tempered with caution. Topics include a brief history of the identification and origins of the organelles, genome content and gene expression, replication, and trafficking of proteins to the organelles. We also discuss insights from antibiotic sensitivity studies, organelle metabolism, and the potential for further drug development. Additional details on topics in this chapter can be found in several reviews (McFadden and Yeh, 2017; Seeber and Soldati-Favre, 2010; van Dooren and Hapuarachchi, 2017). See also Chapters 8
10 for more details on metabolic aspects.

11.2 The apicoplast 11.2.1 History Electron micrographs provided the first indication of the variety of subcellular organelles in T. gondii. Some appear quite conventional, such as the endoplasmic reticulum (ER), Golgi, and mitochondrion (Fig. 11.1). Others are novel to apicomplexans. Prominent examples include rhoptries, micronemes, dense granules, and the conoid, comprising the apical complex from which the phylum takes its name (described in Chapter 14, Toxoplasma secretory proteins and their roles in parasite cell cycle and infection, and Chapter 15, Endomembrane trafficking pathways in Toxoplasma). Among the novel organelles described early in the study of apicomplexans was a small structure surrounded by multiple

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FIGURE 11.1 Ultrastructural appearance of the apicoplast and mitochondrion in T. gondii. (A) Electron micrograph of a section through an intracellular tachyzoite, showing the Ap and a region of the Mi, anterior to the Nu. (B) is an enlargement of the boxed area from panel (A). Note the multiple membranes bounding the apicoplast and the tubular cristae of the mitochondrion. Ap, Apicoplast; Mi, mitochondrion; Nu, nucleus. Source: Courtesy Dr. Michael Laue, Robert KochInstitute, Berlin, Germany.

membranes (Fig. 11.1B), which had no known function. It did not even have the same name in different apicomplexans, being known, for example, as the spherical body in Plasmodium, the “Lamella¨rer Ko¨rper,” “Hohlzylinder,” “Golgi adjunct,” and “ve´sicule plurimembranaire” in T. gondii, and the “grosse Vakuole mit kra¨ftiger Wandung” in Eimeria (reviewed in Siddall, 1992). The multiple membranes provided the first clue to the organelle’s unusual identity, but the relevance of this observation was overlooked, even though secondary endosymbiosis had already been invoked as the origin of some chloroplasts with more than two membranes (Gibbs, 1981). The next clue came from studies of parasite genomes (reviewed in Feagin, 1994). In 1984, 12 and 23 μm circular extrachromosomal DNAs from T. gondii were reported, the latter being head-to-tail dimers of the former. When spread for electron microscopy, many of the molecules adopted a cruciform structure. These data echoed earlier reports of similarly sized circular extrachromosomal DNAs in Plasmodium lophurae and Plasmodium berghei. These molecules matched the size range and conformation expected for mitochondrial genomes of unicellular eukaryotes and so were immediately labeled as such. It was, of course, the logical conclusion. It was also wrong. Who

would have suspected these were plastid genomes? The only clue was the cruciform structure, typical of plastid but not mitochondrial genomes. Certainly, no one connected the circular genomes with the multimembraned organelle. Research on organellar DNA in apicomplexans was initially pursued exclusively in Plasmodium. Three bands in isopycnic sucrose density gradients of Plasmodium knowlesi and P. falciparum were identified in lysates. One was lighter than the main band of nuclear DNA, as is usually the case for mitochondrial genomes. It proved to be a B35 kb circular DNA, and when spread for electron microscopy, it demonstrated a cruciform structure. It thus displayed the characteristics of the previously reported “mitochondrial” DNAs of apicomplexans. The lowest band on the P. falciparum gradient migrated just below the nuclear DNA band and proved to contain tandem repeats of a 6 kb DNA sequence. Upon sequencing, the “6 kb element” in three Plasmodium species was found to encode classic mitochondrial proteins (apocytochrome b, COB; cytochrome c oxidase subunit I, COX1; and cytochrome c oxidase subunit III, COX3), and small, fragmented rRNAs (Feagin, 1994) (see Section 11.3.2). Despite its minute size, this repeated element

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has the requisite minimum of genes expected in mitochondrial genomes. But if the 6 kb element was the mitochondrial genome, what was the 35 kb DNA? Analysis of the 35 kb DNA revealed that it contains a large inverted repeat composed of two copies of a small and large subunit (LSU) rRNAs, arranged tail to tail. The rRNAs are similar to those of prokaryotes, as expected for both mitochondrial and plastid rRNAs. But mitochondrial genomes do not typically have duplicated rRNAs, while those of plastids do. Further sequencing showed that the 35 kb DNA also encodes subunits of a eubacterial-like RNA polymerase. This is unequivocally a plastid characteristic; all plastid genomes studied thus far encode and are transcribed by such RNA polymerases, although some import additional RNA polymerases. Indeed, the subunits encoded by RPOB and RPOC1 were determined to be more like plastid counterparts than bacterial ones (Feagin, 1994). In contrast, almost all mitochondria employ a single subunit RNA polymerase most closely related to phage RNA polymerases. Further analysis of the P. falciparum 35 kb DNA identified genes encoding components of an organelle translation system but no photosynthesis-related genes. Serendipitously, the plastid genomes of Epifagus virginiana, a nongreen plant, and Astasia longis, a nongreen alga, were under analysis at the same time. These are both much reduced in size compared to those of green plants. While plastid-encoded genes related to photosynthesis were missing, those needed for gene expression were present. The parallels with the P. falciparum 35 kb DNA were striking. With the accumulating data, the formerly implausible explanation that the 35 kb DNA was derived from chloroplast DNA became increasingly believable. It is now well established that apicomplexans have algal ancestors (see Section 11.2.2).

The T. gondii apicoplast genome is strikingly similar to its P. falciparum counterpart in size, gene content, and organization (see Section 11.2.3). Complete or near-complete apicoplast genome sequences are now available for multiple apicomplexans (Fig. 11.2; for details see Arisue and Hashimoto, 2015). Conservation of size, gene content, and genome organization is strong (Arisue and Hashimoto, 2015); the principal difference is that the piroplasms Babesia and Theileria have only one copy of the rDNA transcription unit. This high degree of genome similarity is matched by functional conservation that is largely dependent on the import of nuclearencoded proteins (see Sections 11.2.7 and 11.2.9). A lingering question was the subcellular location of the 35 kb genome. It did not colocalize with the mitochondrial genome in sucrose gradient fractionation of P. falciparum organelles, so a mitochondrial location appeared unlikely. As a remnant plastid genome, it should reside in an organelle with more than one bounding membrane. The 35 kb DNA was a genome without a home, and the spherical body was an organelle without a role—might they intersect? The well-defined subcellular structure of T. gondii makes it more amenable for cell biological studies than P. falciparum, so it is unsurprising that the localization question was first answered for T. gondii. In the mid1990s, in situ hybridization studies using probes derived from the T. gondii 35 kb DNA showed that this genome resides in an organelle located just apical to the nucleus, the hitherto mysterious multimembraned organelle (McFadden et al., 1996; Ko¨hler et al., 1997). The plethora of names for the organelle has been replaced with a single term: the apicoplast, for apicomplexan plastid. Solving the initial mysteries of the homeless genome and the unexplained organelle has generated a number of fascinating questions. How does a group of obligate intracellular parasites get a plastid?

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FIGURE 11.2 Comparison of apicomplexan plastid genomes. Schematic depiction of the plastid genome size (black/ blue, in kb), its GC content (solid gray/red, in %) and, for comparison, nuclear GC content (dotted gray/green, in %) of the indicated organisms. For comparison, the data for two chromerids (bottom) are also given. Source: Based on data from EuPathDB and Cinar, H.N., Qvarnstrom, Y., Wei-Pridgeon, Y., Li, W., Nascimento, F.S., Arrowood, M.J., et al., 2016. Comparative sequence analysis of Cyclospora cayetanensis apicoplast genomes originating from diverse geographical regions. Parasites Vectors 9, 611.

Why has it been maintained in nonphotosynthetic organisms? What role does it play for the cell? What possibilities for disease intervention result from the presence of “plant” genes in protozoan pathogens?

11.2.2 Evolution Despite earlier suggestions that there might be more than one origin of plastids, current data point to a single endosymbiotic event, with the engulfed organism being a photosynthetic cyanobacterium (reviewed in Archibald, 2015). The characteristic double membranes of both mitochondria and plastids are believed to derive from double membranes of the

Gram-negative bacteria from which they are descendent, reflecting the endosymbiotic event that produced them. As the symbiotic relationship evolved, many genes in the engulfed partner were transferred to the nucleus, and those gene products essential to organellar function were translated in the cytosol and subsequently imported across two membranes into the organelle. But a number of organisms have not just two, but three or four bounding membranes around their plastids. To explain this, the secondary endosymbiont hypothesis was formulated (reviewed in Archibald, 2015). All cases known to date involve engulfment of a photosynthetic alga that already bears a plastid. The two inner membranes are believed to be the original plastid membranes, the third

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membrane representing the plasma membrane of the algal cell, and the outermost membrane deriving from the host endomembrane system (Fig. 11.3). Prominent examples of organisms with secondary plastids with four membranes are chlorarachniophyte, chromist, and cryptomonad algae. Loss of one membrane has occurred in organisms such as euglenoids and many dinoflagellates (both with three membranes). Numerous electron micrographs show four membranes surrounding the T. gondii apicoplast (Fig. 11.1B). That number is now

generally accepted as well for Plasmodium (Lemgruber et al., 2013). The secondary plastids of cryptomonad and chlorarachniophyte algae retain a reduced nucleus (termed nucleomorph) from the algal symbiont, providing firm proof of secondary endosymbiosis involving a red and green alga, respectively (McFadden, 2017). In contrast to these algae, apicomplexans have lost the endosymbiont nucleus and photosynthesis-related genes, which they no longer need as intracellular parasites. However, they continue to

FIGURE 11.3 Secondary endosymbiosis and current scheme for protein import into the apicoplast. (A) Primary endosymbiosis entailed engulfment of a cyanobacterium by a eukaryote (bottom), whereas secondary endosymbiosis consists of engulfment of a plastid-bearing alga (middle). Loss of genes from the algal cell, with some transferred to the host nucleus, and final loss of the algal nucleus leaves behind an organelle bounded by four membranes (top). Light red/light gray, host cell cytosol; green/medium gray, algal cell cytosol (periplastid compartment); dark red/dark gray, plastid lumen. AP, Apicoplast; CB, cyanobacterium; M, mitochondrion; N, nucleus; P, plastid. (B) Enlarged view of the apicoplast’s four membranes and its resulting subcompartments. For details on the role of the indicated proteins (dark gray), see text. Structures/proteins with a “?” have not yet been identified. SPP, Signal peptide peptidase. Source: Reproduced unchanged from Mallo, N., Fellows, J., Johnson, C., Sheiner, L., 2018. Protein import into the endosymbiotic organelles of apicomplexan parasites. Genes (Basel) 9, E412 (Mallo et al., 2018) under CC BY 4.0 license.

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employ the apicoplast as a synthetic compartment (see Section 11.2.9). In many lineages, it appears that secondary plastids were acquired by a common ancestor and then lost by some extant representatives (Archibald, 2015). For example, gregarine apicomplexans such as Cryptosporidium lack an apicoplast as well as the enzymes typically found in the organelle, but it is presumed that the ancestral apicomplexan possessed a plastid. It should be noted that the involvement of tertiary endosymbiosis—that is, plastid acquisition from an alga containing a secondary plastid—has also been proposed for apicomplexans, although clear evidence for this is lacking (CavalierSmith, 2018). The movement of foreign genes into a host’s nucleus is called “lateral gene transfer” (LGT) (reviewed in Sieber et al., 2017). The increasing pool of genome sequences has greatly accelerated our understanding of LGT, showing it to be widespread and of considerable scope. Secondary endosymbiosis permits additional LGT, from the nucleus and the organelles of the endosymbiont to the nucleus and organelles of the host. Importantly, this means that genes from the endosymbiont’s nucleus, which may have no relationship to plastid function, may still provide insights useful for deciphering the history of secondary endosymbionts. Phylogenetic analyses of apicomplexans have considered genes encoded by the apicoplast genome, nuclear genes encoding apicoplasttargeted proteins, and genes not related to apicoplast function. A recurring question in analyses of apicomplexan evolution has been the identity of the secondary endosymbiont: was it a red or a green alga or is it of chimeric origin? While the imprint of the algal ancestor could potentially be seen in the nuclear genome, this provides little clarity because some genes have been proposed to be of red and others to be of green algal origin. Phylogenetic studies of numerous nuclear genes unrelated to the endosymbiont show that

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apicomplexans cluster most closely with dinoflagellates and ciliates; together, these comprise the alveolate clade. Since the secondary plastids of dinoflagellates are widely considered to derive from a red algal lineage, this relationship suggests a red algal lineage for apicomplexan plastids. Gene loss comparisons also favor a red algal ancestry for apicoplasts, since their genomes encode seven proteins common in the red algal lineage but missing in the green algal lineage (Janouskovec et al., 2010; Ko¨hler et al., 1997). Given the findings described previously, it was surprising that phylogenetic analyses of apicoplast-encoded genes, considered singly or in groups, usually favored a green algal origin (Ko¨hler et al., 1997). However, the plastid and nuclear genomes of two alveolate algae bridge the gap (Woo et al., 2015). These photosynthetic organisms, the chromerids, Chromera velia and Vitrella brassicaformis (formerly known as CCMP3155), have larger plastid genomes than apicomplexans (Fig. 11.2), with highly similar gene content. In sum, they encode orthologs of all protein-coding genes on the plastid genomes of both dinoflagellates and apicomplexans. Phylogenetic analysis using these sequences supports a close relationship of the apicomplexan genes to these red alga
derived plastids. Taken together, current evidence favors a red algal origin for the apicoplast (Arisue and Hashimoto, 2015). However, the possibility that apicomplexan ancestors once housed a green alga
derived plastid that was lost and replaced by the current red alga
derived plastid, as recently proposed for some ochrophyte algae possessing a red alga
derived plastid (Dorrell et al., 2017), is still not entirely ruled out.

11.2.3 The apicoplast genome The number of plastid genomes per cell is contested. Based on nucleic acid hybridization, Ko¨hler et al. (1997) reported 5
6 copies per cell for the T. gondii plastid genome and a

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single plastid genome per cell for P. falciparum. More recent studies in T. gondii have examined genome copy number by determination of the ratio of average numbers of nuclear and apicoplast genomes by qPCR (Reiff et al., 2012). It arrived at 22
25 copies of the apicoplast genome in T. gondii. Multiple copies of a genome would facilitate repair of mutations by gene conversion, making the higher numbers attractive from a functional point of view, but the matter requires further investigation. When photosynthetic capability is not needed, the loss of photosynthesis-related genes from the plastid genome is profound (reviewed in Hadariova et al., 2018). Plastid genomes average 150
200 kb in size but those of nonphotosynthetic plants are B70 kb and the T. gondii plastid genome is B35 kb. Apicoplast genomes in general are quite similar in length (Cinar et al., 2016), but differ substantially in GC content (ranging from 13.2% to 52.5%; Fig. 11.2). The apicoplast genomes of many apicomplexans contain a large inverted repeat. The repeat unit consists of small subunit (SSU) and LSU rRNAs encoded head-tohead and separated by seven tRNA genes. A single tRNA gene is found at the 30 ends of both rRNAs. As noted previously, this organization is highly reminiscent of plastid genomes from algae and plants. Curiously, the 39.4 and 33 kb apicoplast genomes of the piroplasms Theileria parva and Babesia bovis, respectively, lack the repeat rRNA structure and have only a single set of rRNAs with no intervening tRNAs (Cinar et al., 2016). The apicoplast genomes of both these species also differ from the respective P. falciparum, T. gondii, and Eimeria tenella genomes by being unidirectionally transcribed. The gene content of apicoplast genomes is highly conserved, although the relative location of genes can differ (for details see Cinar et al., 2016). The similarities of these genomes to each other are greatest within each taxonomic group, as anticipated. On the other hand,

Cryptosporidium parvum and Cryptosporidium hominis lack an apicoplast genome as well as the organelle and its metabolic pathways (see https://cryptodb.org). Most apicoplast genes encode components needed for expression of the apicoplast genome (Fig. 11.4). All of the tRNAs needed for apicoplast protein synthesis are encoded by the apicoplast genome. The protein-coding genes include three subunits of a multisubunit, eubacterial-like RNA polymerase—RPOB, RPOC1, and RPOC2 (Wilson et al., 1996)—as is the case for plastid genomes from plants and algae. The RPOA subunit of the eubacteriallike RNA polymerase is a nuclear-encoded gene that has a predicted apicoplast targeting sequence in both P. falciparum (PlasmoDB) and T. gondii (TGME49_249560). The other proteincoding genes on the apicoplast genome include 17 ribosomal proteins and the translation elongation factor Tu (Wilson et al., 1996). There are only two apicoplast-encoded genes that have predicted functions other than in gene expression, CLPC and SUFB (see Section 11.2.9). However, one additional apicoplast-encoded protein called YCF93 (Orf105) has been characterized to some extent (Goodman and McFadden, 2014). It is described as a small, potentially dimeric, inner apicoplast membrane
resident protein in P. falciparum and has a homolog in all the other apicoplast genomes. Finally, a handful of unidentified open reading frames are modestly conserved between T. gondii, P. falciparum, and other Apicomplexa (Cinar et al., 2016). They encode small basic proteins which may be components of the apicoplast ribosome that are difficult to identify due to limited sequence conservation. This possibility is particularly attractive, given that the total number of identified plastid ribosomal proteins, including those predicted to be imported from the cytosol, is less than that needed for functional ribosomes. More details on protein translation will be given in Section 11.2.8 in the context of drugs targeting these processes.

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FIGURE 11.4 Schematic map of the T. gondii apicoplast genome. Genes above or below the line, depending on direction of transcription (left to right above the line), are indicated. Protein-coding genes are gray; those marked with asterisks contain internal TGA codons. ORFs of unknown significance are identified with a single lower case letter. Large and small subunit ribosomal protein genes (RPL and RPS) are designated L and S. Other protein-coding genes are named as in the text. Noncoding RNAs are clear. tRNAs are identified by the single letter code above or below their location. LSU rRNA and SSU rRNA, large and SSU rRNAs, respectively. The two IR are indicated by thickened lines. They include rRNAs, tRNAs, and some protein-coding genes. IR, Inverted repeats; LSU, large subunit; SSU, small subunit. Source: Data derived from GenBank reference sequence NC_001799.

11.2.4 Expression and translation of the apicoplast genome Apicoplast gene expression data derive primarily from P. falciparum (reviewed in Nisbet and McKenzie, 2016). Individual transcripts exist for the rRNAs and tRNAs but transcription appears to be polycistronic. Although a precursor/mature RNA relationship has not been definitively shown, primer extension analysis of the plastid rRNAs, corroborated by RNase protection experiments, provided evidence for longer, less abundant RNAs (Feagin, 1994). Antisense transcripts are also detectable in both T. gondii (Bahl et al., 2010) and P. falciparum (Nisbet et al., 2016), but it is not known whether they are simply a result of read-through transcription due to the opposing orientation of the two major operons or if they are involved in transcriptional regulation (Nisbet et al., 2016).

In contrast to the rapidly processed rRNA/ tRNA transcripts, transcripts from RPOB and RPOC1 remain on long polycistronic transcripts, 10
12 kb in length (Feagin and Drew, 1995). Wilson et al. (1996) detected long transcripts for additional P. falciparum apicoplast genes and it is now assumed to be a universal property of apicoplast transcription, at least in the malaria parasite (Nisbet and McKenzie, 2016). The near identity of the expression patterns for adjacent genes is likely strongly affected by polycistronic transcription. Sequence analyses in Plasmodium have identified a conserved motif (UUAUA) adjacent to processed transcripts, raising the possibility that a specific protein is responsible for the cleavage of the majority of these polycistronic transcripts into mRNA/tRNA/rRNA. A substantial proportion of the apicoplast genome is devoted to genes for translation and several lines of evidence point to

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functional protein synthesis in the organelle. Ultrastructural imaging of the P. falciparum (Lemgruber et al., 2013) and T. gondii (McFadden et al., 1996) apicoplasts show granular structures of a size consistent with organellar ribosomes. A number of compounds known to inhibit protein synthesis on eubacterial and organelle ribosomes have negative effects on growth of T. gondii and P. falciparum in culture (see Section 11.2.8). However, the most convincing evidence for apicoplastresident protein synthesis is the detection by antibody staining of the apicoplast-encoded proteins TUFA (Chaubey et al., 2005) and YCF93 (Goodman and McFadden, 2014) in P. falciparum. The T. gondii apicoplast genome sequence has 33 TGA stop codons embedded in 17 of the 28 predicted protein-coding genes. It has long been known that many mitochondrial genomes encode TGA as a tryptophan codon rather than a stop codon, although this alternate codon usage has not been reported for plastid genomes (Smith and Keeling, 2015). The apicoplast genomes are an exception; many of the TGA codons occur at sites of conserved tryptophans, and T. gondii apicoplast genes end with TAA or TAG. Interestingly, there are no TGA codons in Plasmodium apicoplast genes, either internally or terminally. This suggests that Plasmodium lost the UGA decoding mechanism or that the mechanism developed after Plasmodium branched from other apicomplexans. The tryptophan tRNA encoded by the T. gondii plastid genome has an anticodon (CCA) that is incapable of pairing with UGA. Other organisms have developed several mechanisms to overcome this difficulty (reviewed in Gray et al., 2004). The mechanism employed by T. gondii is not yet known. Some apicoplast genes have internal TAA or TAG stop codons. T. gondii RPOC2 has one of each, both located toward the middle of the gene, as well as a TAA within RPS8. It is unclear whether these are pseudogenes or whether a

mechanism exists to allow translation through the internal stop codons. Both the T. gondii and P. falciparum apicoplast genomes have only a single gene with an intron: a conserved tRNAleu (see ToxoDB).

11.2.5 Apicoplast genome replication Although the T. gondii and P. falciparum apicoplast DNAs are strikingly similar in gene content and organization, they differ in an important element of physical structure: about 90% of the P. falciparum plastid genomes are circular, while the T. gondii apicoplast DNA is in linear tandem arrays (Williamson et al., 2001, 2002). This observation strongly suggests that the T. gondii apicoplast genome is replicated via a rolling circle mechanism. The progression of DNA polymerase around a circular genome can produce a displaced linear DNA. If it is not cleaved and recircularized, a linear concatemer of genomes is produced. The proportion of linear to circular molecules is reversed in P. falciparum, with circular DNAs predominating. Using two-dimensional gel analysis to track branch points, Williamson and colleagues (Williamson et al., 2002) showed that most P. falciparum plastid DNA replication initiates at two sites mirrored in the inverted repeat and then proceeds via D-loop intermediates, eventually yielding a circular replicate. The remaining DNA appears to replicate via a rolling circle mechanism, as has been predicted for T. gondii. Replication in other apicomplexans has not been studied, so it is unclear whether D-loop or rolling circle replication will be most common. The importance of apicoplast functions as potential drugs targets (see Section 11.2.8) has fostered interest in replication of its genome. The DNA replication machinery for the apicoplast appears largely conventional for a plastid genome (reviewed in Milton and Nelson, 2016). An apicoplast-localized ortholog of the

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bacterial histone-like DNA binding protein HU has been identified in T. gondii (Reiff et al., 2012). The corresponding gene complemented an Escherichia coli HU mutant. Gene knockout was achieved in T. gondii, but it produced greatly reduced growth kinetics and was associated with mis-segregation of the apicoplast genome and loss of the apicoplast from many cells. P. falciparum is sensitive to treatment with bacterial topoisomerase II inhibitors such as ciprofloxacin (Dahl and Rosenthal, 2007) and protein products of nuclear-encoded genes similar to the A and B subunits of DNA gyrase, a bacterial topoisomerase, are apicoplastlocalized. T. gondii encodes putative A and B subunits of gyrase (Sheiner et al., 2011) and the recombinant enzymes have been expressed and reconstituted as a functional complex (Lin et al., 2015). It was shown to possess supercoiling and also decatenating activity. Lin et al. attribute this surprising dual functionality to compensation for the apparent lack of topoisomerase IV in the apicoplast. Sheiner et al. (2011) have identified additional T. gondii proteins expected to function in plastid genome repair and replication. One is a putative ATPdependent helicase similar to a bacterial DNA replication and repair enzyme, and another has helicase-like domains. The third has a domain similar to phage integrases and DNA breakrejoining enzymes. The PREX gene, most studied in Plasmodium, but present in many apicomplexans, encodes a multifunctional, apicoplast-localized protein with primase, helicase, exonuclease, and polymerase domains, the last being similar to bacterial DNA polymerase I (reviewed in Milton and Nelson, 2016). The primase/helicase and the exonuclease/polymerase domains, and the primase domain with an adjacent zinc-binding domain, have been expressed recombinantly and the expected activities confirmed. Further enzymatic characterization showed that its fidelity, when the adjacent 30
50 exonuclease

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is included, is on par with other high fidelity DNA polymerases. Antibodies raised against each of these domains detect band sizes consistent with proteolytic processing to yield individual primase and exonuclease/polymerase proteins. A T. gondii PREX ortholog has been identified and its polymerase domain shown to be active (Mukhopadhyay et al., 2009). Genome-wide CRISPR gene disruption screens predict the T. gondii PREX gene to be essential (Sidik et al., 2016).

11.2.6 Apicoplast division To ensure perpetuation of their genomes, mitochondria and plastids must be present at all times, even if metabolically inactive. That means that both must divide and be partitioned during each cell cycle to provide organelles for the daughter cells. In unicellular eukaryotes the events of organelle division tend to be coordinated with the cell cycle. This coordination is especially important since only a single mitochondrion and apicoplast are present in Apicomplexa. Until recently, methods for synchronizing T. gondii have been lacking, leading to a paucity of data on some aspects of analysis of coordination of events with the cell cycle. For example, it is unclear whether replication of plastid DNA is coordinated with nuclear DNA replication in T. gondii, as it is in P. falciparum (Williamson et al., 2002). However, analysis of events in single T. gondii cells using fluorescent reporters has produced insights into organelle division. Division of the apicoplast is coordinated with the cell cycle. In a seminal study of apicoplast division in T. gondii (Striepen et al., 2000) a number of important observations were made (Fig. 11.5). In apicomplexans, the centrosome is extranuclear and located close to the apicoplast, which is itself apical to the nucleus. Before daughter cell formation, the centrosome duplicates and the two daughter centrosomes

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FIGURE 11.5 Apicoplast division during endodyogeny. A schematic depiction of endodyogeny is shown. Cell components are labeled in panel (A): A, C, Co, IMC, N, and Nu. In panel (B) the nucleoid and centrosome have both been duplicated and the apicoplast lengthens as the centrosomes and nucleoids move outward. In panel (C), new conoids have formed and new IMC is developing. The nucleus and apicoplast are beginning to move into the forming daughter cells, both becoming U-shaped. The relative position of the centrosomes and apicoplast has reversed. In panel (D), daughter cell formation is nearing completion. The organelles will return to the positions shown in panel (A) as cytokinesis is completed. See Nishi et al. (2008) and Striepen et al. (2000) for additional details. A, Apicoplast; C, centrosome; Co, conoid; IMC, inner membrane complex; N, nucleus; Nu, apicoplast nucleoid.

attach to the apicoplast (Leveque et al., 2015). The centrosomes then move apart and the ends of the apicoplast follow. Apicoplast DNA is localized in a nucleoid and as the organelle lengthens into a dumbbell shape before division, two nucleoids can be seen, one at each end, adjacent to a centrosome. Coordinately, the parasite nucleus is repositioned to the basal end of the cell and develops two arms that

move toward the centrosomes in the forming daughter cells. Centrosomes remain attached to the dividing apicoplast until cell division is finished, thereby ensuring correct organelle segregation. The timing of apicoplast growth, division, and segregation was further placed into the context of other subcellular organelles in studies that took advantage of parasites expressing compartment-specific tagged proteins (Nishi et al., 2008). The Golgi body duplicates around the time that the apicoplast begins to elongate and as the inner membrane complex (IMC) forms. Next, the ER begins to segregate with the nucleus. Mitochondrial division and partitioning to the daughter cells is completed well after apicoplast segregation, just prior to daughter cell emergence. The secretory organelles begin to form at this late stage. The close juxtaposition of the centrosomes and apicoplast at the time of division led to the hypothesis that centrosome movement elongates the apicoplast and that linkages with the newly forming IMC are responsible for the division, mechanically pulling the centrosomes and the linked apicoplasts into the daughter cell (Leveque et al., 2015; Striepen et al., 2000). Several proteins have been implicated in this interaction with centrosomes in recent years. Three essential players are F-actin (Andenmatten et al., 2013), the apicomplexanspecific myosin F (MyoF) (Jacot et al., 2013), and the actin polymerizing protein formin 2 (FRM2) (Tosetti et al., 2019). Individual depletion of the three proteins leads to defects in apicoplast segregation, and the current model is that actin, together with MyoF and FRM2, ensures the centrosomal positioning and inheritance of the apicoplast. Rather surprising contributors to the process of apicoplast segregation are autophagyrelated proteins 8 and 18 (ATG8 and ATG18). Individual depletion of these proteins leads to apicoplast loss in both T. gondii and

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P. falciparum (Bansal et al., 2017; Leveque et al., 2015; Walczak et al., 2018). ATG8 and ATG18 are best known for their central roles in autophagy, and their presumed role in tethering the apicoplast to the centrosome was not anticipated. At least in sexual blood stages of P. falciparum this role in apicoplast segregation appears to be the only essential role of ATG8, as shown by biochemical rescue of the death phenotype (Walczak et al., 2018) (see Section 11.2.8). Super-resolution microscopy revealed the presence of ATG8 in the peripheral membrane of the apicoplast and its enrichment at the ends of dividing organelles (Leveque et al., 2015). It is unknown whether T. gondii ATG8, as in other systems, is membrane-bound via phosphatidylethanolamine linkage. However, TgATG18 binds to the negatively charged membrane lipids phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate, and PI3P interaction is critical for apicoplast inheritance. Further experiments by Bansal et al. (2017) imply that ATG18 regulates ATG8 localization and membrane conjugation. Whether additional proteins are involved in the interaction with the centrosome needs to be determined. Plastid division in many other organisms involves a division ring that constricts around the middle of an elongated organelle until it splits in two. However, genes encoding the proteins of the ring or proteins important for its formation, such as FTSZ (a tubulin-like GTPase derived from the prokaryotic ancestor) and the dynamin-related protein ARC5 (a eukaryotic invention), have not been detected in apicomplexan or chromerid genomes. Further studies showed that a novel dynamin-related protein, DRPA, has taken over their function, at least in T. gondii (van Dooren et al., 2009). The dynamin family of GTPases is involved in the fission of various organelles including mitochondria. DRPA localizes to the outermost apicoplast membrane at the midpoint of the organelle immediately

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before division. Expression of a dominantnegative mutant of DRPA blocked plastid division even though elongation occurred (van Dooren et al., 2009). Consequently, many daughter cells lacked an apicoplast. Curiously, even though the only known function of DRPA is in apicoplast scission, phylogenetic analysis indicates that DRPA is present in Cryptosporidium species, which lack an apicoplast, suggesting it may have other functions as well. PI3P has been implicated as an attachment point for DRPA in the apicoplast membrane by studies that identified this phospholipid in the outermost apicoplast membrane of T. gondii (Tawk et al., 2011). However, the phenotypes of DRPA-depleted cells and those where PI3P synthesis was inhibited are not congruent (Daher et al., 2015). The role of phosphoinositides in apicomplexan dynamin biology needs further investigation. Genetic depletion of another, atypical dynamin, DRPC, has a drastic overall impact on T. gondii division and apicoplast segregation (Heredero-Bermejo et al., 2019). Following division, parasites either lacked the organelle or had two per cell. Similarly, the MORN1 protein has a general effect on assembly of a basal complex and cytokinesis and is essential for apicoplast division (Lorestani et al., 2010). These two examples further illustrate the connection between proper coordination of the cell cycle with apicoplast division and segregation. Apicoplast structure has also been examined in bradyzoites and in sexual stage T. gondii (Dzierszinski et al., 2004; Ferguson et al., 2005). Using fluorescent reporters localized to the apicoplast, Dzierszinski et al. (2004) found that 10%
20% of in vitro
induced bradyzoites apparently lack plastids. Both mis-segregation and loss of signal without cell division were observed. In contrast, Ferguson et al. (2005), performing a comprehensive examination of the apicoplast during in vivo infection in bradyzoites and the asexual and sexual forms (see Chapter 18: Bradyzoite and sexual stage

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development, for details) by electron and immunofluorescence microscopy, concluded that bradyzoites have an apicoplast adjacent to each nucleus in mature cysts. They attribute this discrepancy to possible differences between in vivo and in vitro cysts. The plastid in T. gondii microgametocytes, macrogametocytes, and macrogametes appears condensed and almost globular. Microgametes lack apicoplasts (Ferguson et al., 2005), which indicates that the apicoplast is maternally inherited in T. gondii.

11.2.7 Protein trafficking to the apicoplast 11.2.7.1 Targeting sequences With the identification of the apicoplast genome, it became apparent that any apicoplast-specific function would require the collaboration of additional proteins. These proteins are encoded in the nucleus and hence are called nucleus-encoded apicoplast-targeted (NEAT) proteins. Prior to the availability of the complete genome sequence, two such proteins, acyl carrier protein (ACP) and ribosomal protein S9 (S9), were identified and confirmed to be localized to the apicoplast by microscopic analysis using specific antisera (Waller et al., 1998). These genes, in turn, provided tools for dissecting the manner in which proteins are targeted to the apicoplast. Sequence analysis showed that these proteins, which were predicted to reside within the lumen of the apicoplast, possessed N-terminal extensions as compared to their bacterial orthologs, reminiscent of primary plastid proteins that are usually targeted directly from the cytosol via an N-terminal transit peptide. Localization to the secretory system usually involves an N-terminal signal sequence. These presequences are rapidly removed by specific processing enzymes upon import. In organisms with secondary plastids, N-terminal extensions appear to contain a

signal sequence followed by a transit peptide (Patron and Waller, 2007). This organization is exactly what is observed for both T. gondii and P. falciparum NEAT proteins predicted to reside in the apicoplast lumen (Patron and Waller, 2007; Waller et al., 1998). Using gene fusions, Waller et al. (1998) showed that the N-terminal extension of T. gondii ACP was able to target green fluorescent protein (GFP) to the T. gondii apicoplast, as were presequences of P. falciparum NEAT proteins. These data suggest that at least some mechanisms of targeting are conserved across the Apicomplexa (Patron and Waller, 2007). Both the signal and transit regions of the N-terminal extension of NEAT proteins are required to target a reporter to the apicoplast (Fig. 11.6); deletion of either region results in mis-targeting. Without a transit sequence the ribosomal protein S9 signal sequence targets GFP for secretion (DeRocher et al., 2000), indicating that the reporter protein had entered the secretory system. The signal sequences for these proteins do not appear different from those of proteins targeted to other destinations in the secretory system. Indeed, replacing the endogenous signal sequence with one from a heterologous secretory protein does not alter targeting (Tonkin et al., 2006). Taken together, these studies indicate that the first step in protein targeting to the apicoplast lumen is entry into the secretory system. The transit peptides of NEAT proteins vary in length, from about 50 to 200 aa, and are very diverse in sequence. Diversity is also reflected by the consequences of deleting signal sequences. Without a signal sequence, the S9 transit peptide directs GFP to the mitochondrion (DeRocher et al., 2000) (Fig. 11.6), while a cytosolic localization is seen with GFP fusions to the transit peptide of ferredoxin (FD) NADP1 reductase (FNR) in T. gondii (Harb et al., 2004). Like plant plastid transit peptides, these peptides have few acidic or hydrophobic residues (Tonkin et al., 2006). In fact, the transit peptide of T. gondii S9, when fused to GFP,

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FIGURE 11.6 Both domains of the N-terminal extension are required for targeting of NEAT proteins. GFP fusions containing the entire N-terminal extension of ribosomal protein S9 (aa 1-159, S 1 T-GFP), its signal sequence (aa 1-42, S-GFP) or its transit sequence (aa 33-159, T-GFP) were expressed in Toxoplasma gondii. The left hand panels show GFP fluorescence, while the right hand panels show DIC images of the same cells within a parasitophorous vacuole in host fibroblasts. Colocalization with apicoplast markers (DNA or acetyl CoA carboxylase) demonstrated that the single dot observed upon expression of S 1 T-GFP corresponds to the apicoplast. Colocalization with mitochondrial markers HSP60 and MitoTracker showed that T-GFP is found in the mitochondrion. S-GFP is found primarily in the parasitophorous vacuole, although some material can be seen within the endomembrane system of the parasite. GFP, green fluorescent protein; NEAT, nucleus-encoded apicoplast-targeted. Source: Courtesy Dr. Amy DeRocher.

allows it to be imported into isolated pea chloroplasts (DeRocher et al., 2000). Apicoplast transit peptides have a net positive charge. T. gondii and P. falciparum transit sequences show

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different amino acid biases; these appear to reflect the different nucleotide composition of their genomes (that of P. falciparum is very ATrich). The T. gondii transit sequences are enriched for serine and threonine, amino acids shown to be important in plant transit peptides (Patron and Waller, 2007). A recent study identified phenylalanine in the 11 position of the transit peptide of a significant number of luminal apicoplast proteins, suggesting a potential role for this residue in targeting proteins from the periphery to the lumen (Sheiner et al., 2015). Several studies have shown that T. gondii apicoplast transit peptides contain redundant information, since nonoverlapping segments can still mediate targeting (DeRocher et al., 2000; Harb et al., 2004). Detailed mapping of transit peptide functions of T. gondii FNR suggests that release from the ER, localization to the apicoplast, binding to chaperones, and processing are specified by discrete domains (Harb et al., 2004). Processing of plant plastid transit peptides is rapid, such that only the mature protein is seen under steady state conditions. In contrast, both the mature form and the precursor protein containing the transit peptide are observed for NEAT proteins, whether native proteins or artificial gene fusions (DeRocher et al., 2005; Waller et al., 1998). Indeed, this can be used as an experimental indicator for apicoplast localization and even for the presence or absence of the organelle since it is required for processing of the transit peptide (He et al., 2001). Pulsechase studies indicate that little processing is seen until 45
120 minutes after synthesis for GFP fused to signal and transit sequences of ACP in both T. gondii and P. falciparum (DeRocher et al., 2005; van Dooren et al., 2002). Whether this delay (as compared to chloroplast protein processing) reflects the time required for complete import or relative inefficiency of processing is not yet clear. A putative ortholog of the plant plastid stromal processing peptidase has been identified in both P. falciparum (van Dooren et al., 2002) and T. gondii

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(TGME49_253890). The predicted protein includes a bipartite targeting sequence that is shared with an upstream, apicoplast-targeted heme biosynthesis enzyme by a process of alternative splicing (van Dooren et al., 2002), suggesting that it is targeted to the apicoplast. The identification of NEAT proteins and characterization of bipartite apicoplast targeting sequences have allowed development of bioinformatic models that predict which proteins may be localized to the apicoplast of apicomplexan parasites. Early work in Plasmodium used a neural network (PATS; Zuegge et al., 2001) and rule-based algorithms (PlasmoAP; Foth et al., 2003) to identify candidate NEAT proteins, which have been useful in predicting apicoplast metabolic pathways. Differences in the amino acid biases of P. falciparum and T. gondii transit peptides precluded direct application of the algorithms to T. gondii. A rulebased algorithm, based on the known characteristics of the bipartite targeting sequence and generally applicable to different apicomplexans, has been described for both luminal and transmembrane apicoplast proteins (Cilingir et al., 2012, 2013). After training on a limited

set of known apicoplast proteins of T. gondii, approximately 400 proteins were predicted to localize to the apicoplast lumen. While the identification of these candidates represents a good starting point, it is important to use experimental methods to identify proteins that may lack or may deviate from the types of targeting sequences detected by these algorithms. Recently, using a proximity-dependent labeling approach based on targeting a promiscuous biotin ligase (BirA*) to the apicoplast (BioID method), together with a new neuronal network-based apicoplast-localization prediction algorithm (PlastNN), Boucher et al. (2018) were able to allocate 346 P. falciparum proteins to this organelle. About 50% of these proteins are of unknown function. Only 197 proteins are found by all four predictors plus the BioID experimental approach (Fig. 11.7), illustrating that all approaches have strengths and weaknesses. It is expected that more NEAT proteins, in particular apicoplast membrane proteins, remain to be identified. A systematic comparison of these P. falciparum NEAT proteins with all the T. gondii predicted proteins has not been reported in the literature. FIGURE 11.7 Number of Plasmodium falciparum NEAT proteins identified experimentally or predicted by different computer algorithms. The Venn diagram shows in its center that 197 proteins are identified by all approaches. For description of the four algorithms PATS, PlasmoAP, ApicoAP, and PlasNN, as well as for the experimental BioID approach see text. NEAT, Nucleus-encoded apicoplast-targeted. Source: Raw data were taken from Boucher, M.J., Ghosh, S., Zhang, L., Lal, A., Jang, S.W., Ju, A., et al., 2018. Integrative proteomics and bioinformatic prediction enable a high-confidence apicoplast proteome in malaria parasites. PLoS Biol. 16, e2005895.

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Not all proteins that reside in the apicoplast lumen bear bipartite targeting sequences. A screen for apicoplast proteins based on phylogenetic distribution and timing of expression during the cell cycle identified four genes encoding apicoplast luminal proteins that apparently lack a signal sequence or a transmembrane domain (Sheiner et al., 2011). This discrepancy could reflect mis-identification of the start codon or perhaps biological phenomena such as processing to reveal a recessed bipartite sequence or import by piggybacking with another protein. Yet another twist is that several proteins are dually targeted to the apicoplast and mitochondrion; the mechanisms underlying this phenomenon are discussed in Section 11.3.4. In addition, Apicomplexa lack enough genes to encode individual aminoacyl-tRNA synthetases for all translational compartments, so some of these enzymes are dually targeted to the cytosol and apicoplast in T. gondii (Pino et al., 2010). The cytosolic forms predominate, as expected, and may arise from use of alternative initiation codons that eliminate the targeting information. Indeed, in the case of Plasmodium CysRS (cysteinyl-tRNA synthetase), it was shown that alternative splicing is responsible for dual targeting (Pham et al., 2014). A substantial membrane proteome is presumably required for the import of proteins and substrates into, and the export of products from, the apicoplast. Several such proteins

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have been identified in apicomplexans, including some functioning in protein import (see below). These membrane proteins show a characteristic ring-like staining surrounding a luminal apicoplast marker upon immunofluorescence analysis, and localization to the outer compartments of the apicoplast by immunoelectron microscopy (Fig. 11.8A). Targeting of proteins to the inner membrane of plant plastids is often mediated by a transit peptide. The picture with outer membrane proteins is more mixed, with some bearing transit sequences, others having an N-terminal region resembling a signal sequence, and yet others apparently lacking either type of sequence (reviewed in Chotewutmontri et al., 2017). The findings regarding the apicoplast are somewhat reminiscent of plant plastids, with the caveat that no robust experimental technique is currently available that allows apicoplast membrane proteins to be assigned unequivocally to particular membranes. Several T. gondii proteins targeted to the outer compartments of the apicoplast have a recognizable bipartite sequence, indicating entry into the ER followed by sorting to the apicoplast. Among these are TIC20 and TIC22, which are likely localized to the innermost membrane and the adjacent intermembrane space, and DER1Ap, which is likely localized to the periplastid membrane. In contrast, other membrane proteins lack the canonical bipartite extension, although all appear to possess sequences that FIGURE 11.8 Localization of the thioredoxin ATRX1 to vesicles and the outer compartments of the apicoplast. Immunogold electron microscopy was used to detect epitope-tagged ATRX1 in Toxoplasma gondii. (A) Close view of the apicoplast and its multiple membranes. Triple arrows mark the apparent localization of the molecule to different compartments. (B) Presence of ATRX1 at the ap and abundant small v. Note the apparent fusion of a vesicle with the apicoplast, marked by arrows. ap, Apicoplast; v, vesicles.

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mediate entry into the ER. The transmembrane protease FTSH1 utilizes a signal anchor sequence to enter the endomembrane system (Karnataki et al., 2007b), as does the peripheral membrane thioredoxin ATRX1 (DeRocher et al., 2008). Notably, neither FTSH1 nor ATRX1 possesses an obvious transit peptide. The T. gondii apicoplast sugar phosphate transporter APT1 (Karnataki et al., 2007b) and its P. falciparum ortholog PfoTPT (outer triose phosphate transporter) that resides in the outer membrane (Mullin et al., 2006) lack any recognizable signal or transit sequence, although it is likely that one of the transmembrane domains functions in localization to the ER. Interestingly, the paralogous P. falciparum iTPT (inner triose phosphate transporter), predicted to reside in the innermost membrane, has a bipartite sequence (Mullin et al., 2006). In addition to the signal anchor sequence, ATRX1 requires some information in the 200 amino acids following the anchor for proper localization (DeRocher et al., 2008). For APT1 an N-terminal segment is essential for apicoplast localization (DeRocher et al., 2012). By testing a series of mutations, an YG motif facing the cytosol was identified as essential for correct targeting. Similar results were obtained for PfoTPT (Lim et al., 2016). Tyrosine-based motifs (with the consensus of YXXφ, where φ represents a hydrophobic amino acid) function in other pathways within eukaryotic secretory systems. One model consistent with the observed T. gondii data is that the cytosolic YG motif interacts with a protein that selects cargo for transport to the apicoplast. However, other apicoplast membrane proteins do not have such motifs, implying that proteins may employ distinct determinants for targeting to apicoplast membranes, possibly related to their final destination. 11.2.7.2 Trafficking mechanisms Many apicoplast-targeted proteins show similar cell cycle
specific transcript expression (Sheiner et al., 2011). Once synthesized, the path that NEAT proteins follow from the ER to

the apicoplast lumen is not yet clear, although several models have been proposed (Fig. 11.3). One hypothesis is that the apicoplast lies within the secretory system, with its outer membrane contiguous with ER. All proteins would pass it on their way to other destinations and the transit peptides of NEAT proteins would be recognized and bound by apicoplast receptors, then be imported through the inner three membranes. Some organisms with secondary plastids have ribosomes studding the outer membrane, suggesting continuity with the ER (Gibbs, 1981). There is no specific evidence supporting this mechanism in Apicomplexa: ribosomes have not been detected on the apicoplast surface and, although the apicoplast lies very close to the nuclear envelope, which is an extension of the ER in T. gondii, there is no evidence for fusion of the membranes (Tomova et al., 2009). Another possibility is that proteins move by vesicular trafficking from the ER to the apicoplast, either directly or indirectly. In this case the transit peptide would be responsible for packaging proteins into appropriate vesicles. Specificity of vesicle trafficking would likely be conveyed by an additional component, possibly proteins such as RAB family GTPases or SNAREs at the vesicle and apicoplast surfaces, as occurs for targeting to other destinations in the secretory system. Notably, expression of a dominant-negative αSNAP, a protein that interacts with SNARE proteins during vesicle fusion, leads to defects in apicoplast biogenesis (Stewart et al., 2016), although a direct role for SNARE proteins in trafficking of NEAT proteins has not been shown. The question of how proteins destined for the apicoplast lumen are sorted in the secretory system is complex and has been approached by use of Golgi body disruptors and analysis of ER retrieval (which canonically depends on a receptor in the Golgi body). Addition of an ER-retrieval sequence to an apicoplast-targeted marker protein did not affect targeting in

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T. gondii, suggesting the protein did not intersect with the Golgi receptor (DeRocher et al., 2005). In addition, there is little evidence that either brefeldin A (BFA) or low temperature (15 C), which both inhibit Golgi trafficking, block localization of luminal proteins to the T. gondii apicoplast (DeRocher et al., 2005). To circumvent potential confusion arising from preexisting marker proteins in the apicoplast, a conditional aggregation domain fusion was used to trap apicoplast-targeted GFP in the ER (DeRocher et al., 2005). After the apicoplast was depleted of the marker protein (Fig. 11.9), addition of a specific ligand released the fusion protein, which rapidly localized to the plastid region (DeRocher et al., 2005). This localization was not blocked by BFA or by incubation at 15 C. However, a BFA-sensitive step is involved in protein maturation or localization within the organelle in T. gondii, as no transit peptide cleavage was observed in pulse-chase experiments

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in the presence of BFA. These results were corroborated by studies in P. falciparum, in which BFA treatment did not prevent apicoplast targeting (Chaudhari et al., 2017; Tonkin et al., 2006). However, a more recent paper studying P. falciparum argues that BFA can inhibit apicoplast targeting and that ERretrieval sequences can compete with transit peptides (Heiny et al., 2014). More work is needed to understand this step of protein trafficking to the plastid. Large vesicles bearing membrane proteins that are also present in the outer apicoplast compartments have been detected in T. gondii (Bouchut et al., 2014; DeRocher et al., 2008; Karnataki et al., 2007a,b; Tawk et al., 2011). In addition to residing at the apicoplast, tagged versions of APT1, FTSH1, and ATRX1 were seen in “dots” and “tubules” upon immunofluorescence. These structures were resolved as electron-dense vesicles upon immunoelectron

FIGURE 11.9 Apicoplast protein targeting studied by conditional aggregation. A GFP fusion protein bearing the bipartite extension of ribosomal protein S9, plus a tandem array of four CAD domains, was expressed in Toxoplasma gondii. Removal of ligand causes aggregation of the CAD domains, while addition of ligand yields monomerization. When ligand is removed from the stable transfectants, GFP is detected in the ER. When ligand is added for 4 h, the protein traffics to the apicoplast. This tracking occurs even in the presence of BFA. GFP was detected using anti-GFP antibodies. DAPI staining reveals the DNA in the parasite nucleus and apicoplast, and in the upper and lower images, a portion of the host cell nucleus is visible (asterisk). BFA, Brefeldin A; ER, endoplasmic reticulum; CAD, conditional aggregation domain; DAPI, 40 ,6-diamidino-2-phenylindole; GFP, green fluorescent protein. Source: Courtesy Dr. Amy DeRocher.

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microscopy (Fig. 11.8B). The vesicles become increasingly abundant as the apicoplast enlarges prior to division, and are lacking in newly formed daughter cells. Some images show what appears to be recent or ongoing fusion of vesicles with the outer membrane of the apicoplast. The vesicles themselves bear PI3P, as does the apicoplast (Tawk et al., 2011). Overexpression of a PI3P-binding protein led to the eventual loss of the apicoplast in many cells, as well as accumulation of electron-lucent vesicles (Tawk et al., 2011). Thus PI3P is important for the biogenesis and maintenance of the apicoplast but the specific role it plays has not been dissected. It is tempting to view these vesicles as the carriers of membrane proteins to the apicoplast, but direct evidence for this is lacking. Interestingly, proteins destined for the apicoplast lumen appear not to traffic via these vesicles, suggesting that peripheral and luminal apicoplast proteins may traffic via different targeting routes (Bouchut et al., 2014), as was also suggested for P. falciparum (Chaudhari et al., 2017). Insights into the apicoplast import apparatus have increased in recent years. It is assumed, based on phylogenetic origins and similarity of targeting sequences, that the translocon spanning the inner membranes of apicoplasts resembles that of chloroplasts. The chloroplast translocon is composed of multiple components in the inner (TIC) and outer (TOC) membranes. Among the functions provided in the complex are specific binding to the transit sequence, channel formation, energy generation, and chaperone activity. Two components of the apicoplast inner translocon, TIC20 and TIC22, have been identified in T. gondii and shown to be important for import of apicoplast luminal proteins (Glaser et al., 2012; van Dooren et al., 2008). TIC20 is integral to the inner apicoplast membrane and a candidate to form the TIC complex channel (van Dooren et al., 2008). TIC22 likely localizes to the space between the two inner apicoplast membranes

and functions as a chaperone that may facilitate transfer of apicoplast luminal proteins from the translocon of the second innermost membrane to the TIC complex (Glaser et al., 2012); this translocon is thought to be homologous to the TOC complex of plants. Indeed, a homolog of TOC75, the candidate pore of the TOC complex, is encoded in the genomes of plastid-bearing apicomplexans. The T. gondii TOC75 protein localizes to the apicoplast and is important for apicoplast protein import (Sheiner et al., 2015). Insights into the means of protein translocation across the periplastid membrane came from studies of cryptomonads in which the nucleomorph genome was scanned for candidate proteins that might mediate protein translocation. These analyses identified paralogs of several proteins associated with the ERassociated degradation (ERAD) pathway (Sommer et al., 2007). The ERAD pathway extrudes misfolded proteins from the ER through a multicomponent translocon into the cytosol for degradation. Thus by analogy, the ERAD-like machinery was proposed to function in importing proteins from the outermost plastid space across the periplastid membrane. This nucleomorph-encoded ERAD-like machinery exists in parallel with canonical ERAD machinery, which is nuclearly encoded. Extending these studies to T. gondii, Agrawal et al. (2009) identified two sets of ERAD proteins encoded in the nuclear genome, with one set apparently derived from the algal symbiont that gave rise to the apicoplast. Key features of the ERAD machinery are the pore (possibly formed by the multimembrane spanning protein DER1), ubiquitination machinery (important for translocation, as well as degradation in canonical ERAD), and an AAA ATPase mechanoprotein CDC48 that assists in pulling ubiquitinated proteins through the pore. Components of the symbiont-derived ERADlike machinery were demonstrated to localize to the apicoplast of T. gondii and include

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homologs to DER1, CDC48, and several components of the ubiquitination machinery, including a ubiquitin-like protein, PUBL (Agrawal et al., 2009, 2013; Fellows et al., 2017). These studies showed that knockdown of DER1, an E2 ubiquitin conjugating enzyme, the CDC48 homolog, and PUBL all led to defects in the import of NEAT proteins. Whether these proteins must be ubiquitinated before or during translocation across the periplastid membrane is unknown, although the PUBL protein contains an essential diglycine motif that in other ubiquitin-like proteins plays a key role in conjugating to target proteins (Fellows et al., 2017). The ERAD-like machinery, therefore, represents a protein translocon that has been repurposed for protein import across the periplastid membrane. A more mysterious molecule involved in apicoplast biogenesis is FTSH1, an ATPdependent metalloprotease. A screen of a small molecule library with activity against P. falciparum showed that one, actinonin (previously known as an inhibitor of eubacterial peptide deformylases), caused a delayed death phenotype (see Section 11.2.8) and apicoplast loss. By selecting T. gondii resistant to the drug, Amberg-Johnson et al. (2017) were able to identify FTSH1 as the target. The exact role of this protease in apicoplast biogenesis remains to be studied, but it is worth noting that other FTSH paralogs play a role in quality control of organellar membrane proteins and assembly/stability of protein complexes. With four bounding membranes, the apicoplast offers multiple options for localization. The mechanisms by which membrane proteins are shuttled from one membrane to another are not understood nor are the determinants that specify their final localization. Membrane composition may differ between them and may convey some specificity to the targeting of membrane proteins. The mechanisms by which soluble proteins localize to particular intermembrane space compartments are also not

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known. Knockdown of ATRX1, a protein that localizes to the apicoplast periphery (as mentioned previously), leads to selective defects in the trafficking of NEAT proteins (Biddau et al., 2018). This raises the possibility that ATRX1, a thioredoxin enzyme, mediates the formation of disulfide bridges on target proteins, thereby regulating protein trafficking by, for example, trapping folded proteins in intermembrane spaces.

11.2.8 Drug sensitivities and the phenomenon of “delayed death” The potential of the apicoplast as a drug target relates to its algal, hence cyanobacterial, origin, with many proteins and pathways not shared by the human host. Indeed, several prokaryotic-like features of apicoplast functions can be inhibited by existing antimicrobials. These have been important research tools and some are important clinically as well. Recent reviews discuss various aspects of the apicoplast as a drug target in apicomplexan diseases (Kadian et al., 2018; Sharma et al., 2018) and the topic is also covered in Chapter 7, Toxoplasma animal models and therapeutics, of this volume. The ability of inhibitors of organellar DNA replication, transcription, and translation to kill T. gondii and P. falciparum indicates that organellar functions are essential. For example, inhibitors of the apicoplast-localized DNA gyrase are toxic to T. gondii and the parasite is sensitive to the macrolide antibiotic clindamycin, an inhibitor of prokaryotic-like translation (Fichera and Roos, 1997). Two clindamycinresistant T. gondii mutants were found to have a point mutation in the plastid LSU rRNA that mapped close to known clindamycinspecificity determinants (Camps et al., 2002). Tetracyclines, which inhibit translation by prokaryotic ribosomes, bind to the small subunit of the ribosome, which suggested initially that

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the mitochondrial ribosome was the main site of action in apicomplexans. This was consistent with antibiotic effects on mitochondrial morphology, functions, and the pattern of protein synthesis inhibition in T. gondii (Beckers et al., 1995). However, Camps et al. (2002) reported that the kinetics of T. gondii growth inhibition by clindamycin and tetracycline were quite similar, including a delayed death phenotype (see next). As noted previously, the antibiotic actinonin, which targets the metalloprotease FTSH1, also causes apicoplast loss and delayed death (Amberg-Johnson et al., 2017). Although an effect on the mitochondrion often cannot be ruled out, many of the drugs discussed previously, including clindamycin, clearly affect the apicoplast. They show an intriguing phenotype, called “delayed death” (Fichera et al., 1995). After drug is added, parasite multiplication continues normally within the first vacuole. However, when establishing the second vacuole, the parasites fail to replicate. This effect occurs even if the drug is removed at the second cycle. A characteristic indication of a delayed death effect by a compound is that much less drug is necessary to kill 50% of parasites (IC50) when replication is measured in the second as compared to the first growth cycle (Dahl and Rosenthal, 2007). The initial model to explain the delayed death phenotype was that apicoplast metabolites are necessary for the establishment of a parasitophorous vacuole but not during replication (Fichera et al., 1995). Another proposed mechanism was based on the projected need for a formylated methionine-tRNA for translation initiation in the mitochondrion of T. gondii. The apicoplast but not the mitochondrial genome encodes a methionine tRNA and there is only a single gene each for the methioninetRNA formyltransferase and deformylase in T. gondii, the products of which localize to the apicoplast (Pino et al., 2010). Howe and Purton (2007) have invoked a requirement for transfer of formylated tRNAMet from the site of its

synthesis within the apicoplast to the closely associated mitochondrion to enable mitochondrial translation. However, the lack of both genes in the genomes of Babesia sp. and Theileria sp. argues against a requirement of formylated methionine-tRNA for mitochondrial translation initiation in T. gondii (Pino et al., 2010), and thus also as an explanation for the delayed death phenotype. A third hypothesis derives from the observation that the delayed death phenomenon is not only seen under drug treatment. He et al. (2001) generated apicoplast-depleted T. gondii by transient expression of a protein that interfered with apicoplast segregation. After a series of divisions, the parasitophorous vacuole contained many parasites but only one with an apicoplast. Within the original vacuole, the cells remained healthy, whether they contained an apicoplast or not, and all were capable of invading a new host cell. However, only those with an apicoplast could proliferate in the new host. Hence, it appears that some molecules produced directly or indirectly by the apicoplast in the preceding cycle are required in the next round of infection. A similar loss of apicoplast inheritance with subsequent delayed death was described in other T. gondii mutants (Fig. 11.10), like parasites expressing a dominant-negative mutant form of MyoF (TgMYOF tail) (Jacot et al., 2013). In subsequent work the TgMYOF mutant was instrumental to show that survival during the first intracellular replication cycle was dependent on the intravacuolar connection between parasites at the basal constriction within a vacuole (Frenal et al., 2017). Disrupting this connection by deleting either MYOI or MYOJ myosins in the apicoplast-lacking TgMYOF tail mutant led to cell death in the first lytic cycle. The conclusion is that one or more apicoplast-derived factor (likely a metabolite) is sufficient to maintain the vacuolar proliferation of all the other apicoplast-deficient parasites as long as its diffusion is maintained via cell
cell

Toxoplasma Gondii

11.2 The apicoplast

521

FIGURE 11.10

Replication without apicoplast division. (A) An immunofluorescence image of an inducible knockdown of profilin (PRF), stained for apicoplast-localized CPN60 (green/white) and cytosolic actin (red/gray) (Jacot et al., 2013). In the absence of the inducer anhydrotetracycline (5presence of PRF), complete apicoplast segregation is seen and each parasite contains a single apicoplast. (B) Upon PRF depletion (by the addition of the inducer), apicoplast missegregation occurs and only two of the eight parasites retain an apicoplast. Source: Original image courtesy Dr. Damien Jacot (CMU, University of Geneva, Switzerland), edited by contrast and color enhancement.

communication at the basal complex within the vacuole. But what are these factor(s)? In vitro studies in P. falciparum blood stages by Yeh and DeRisi (2011) revealed a surprising aspect of the delayed death caused by drugs. They showed that much higher concentrations of drugs were required to kill parasites supplemented with isopentenyl pyrophosphate (IPP). As will be detailed in Section 11.2.9, IPP is the end product of the isoprenoid biosynthesis pathway in the apicoplast. These observations indicate that, at least in P. falciparum blood stages, the isoprenoid pathway is a central player for understanding the basis of the delayed death phenotype. More recently, it was shown that when tachyzoites were treated with drugs targeting the apicoplast and the host cell isoprenoid pathway was concurrently inhibited, parasite death was no longer delayed (Amberg-Johnson and Yeh, 2019). Thus during T. gondii’s first lytic cycle, host isoprenoids appear to partially compensate for apicoplast loss, delaying death to the next cycle. This implicates IPP (or subsequent metabolic products) as a key metabolite in the delayed death phenomenon in this parasite. However, it remains to be determined if IPP is the only apicoplast-derived factor that is required to delay death (see Section 11.2.9).

During the first lytic cycle, the level of apicoplast DNA is dramatically reduced upon drug treatment, as shown for clindamycin (Fichera et al., 1995) and similar inhibitors (Amberg-Johnson and Yeh, 2019), and by mutants affecting apicoplast replication and segregation (Reiff et al., 2012). This differs from P. falciparum, where disruption of apicoplast biogenesis is not necessarily reflected by delayed death (Amberg-Johnson et al., 2017), indicating that results from one parasite cannot always be transferred to the other, and that the explanation for delayed death is complex and might differ between the two apicomplexans (Amberg-Johnson and Yeh, 2019). Evidently, the inability to translate certain proteins encoded by the apicoplast genome prevents the proper replication of the apicoplast DNA, either directly or indirectly, potentially causing delayed death. Which apicoplastencoded proteins are important for replication of the apicoplast genome are not known. One of the few apicoplast-encoded proteins not involved in transcription/translation is a putative chaperone of the caseinolytic protease (CLP) family of proteins, which may be required for import of NEAT proteins necessary for apicoplast DNA replication. SUFB, a protein required for iron
sulfur cluster (ISC) synthesis,

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11. The apicoplast and mitochondrion of Toxoplasma gondii

is also encoded on the apicoplast genome. It could be that enzymes important for genome replication require ISCs. ISCs are also essential cofactors for two enzymes involved in isoprenoid biosynthesis (see Section 11.2.9), and it is conceivable that derivatives of the isoprenoid synthesis pathway may have a role in genome replication.

11.2.9 Apicoplast metabolism What functions are potentially localized to the apicoplast? Most of the clues come from the predicted NEAT proteins (see Section 11.2.7). Due to the cyanobacterial origin of the apicoplast, it was possible early on to map several known metabolic pathways to the apicoplast, and these have been compared between several sequenced apicomplexan genomes (Table 11.1; see Seeber and Soldati-Favre, 2010 for review). Because apicoplast pathways have an evolutionary history that does not overlap with the human host, the organelle has long been considered to harbor promising

potential drug targets (Fichera and Roos, 1997). Some of the functions mapped to the apicoplast have already been experimentally demonstrated to be essential through the use of inhibitors (as mentioned previously) or gene disruptions (Blume and Seeber, 2018; Sidik et al., 2016). A summary of metabolic pathways predicted or demonstrated to reside in the apicoplast is given in Fig. 11.11, together with their interrelationship with those of the mitochondrion. For detailed descriptions of the biosynthesis of fatty acids plus important cofactors and of heme, see Chapters 8
10. Judged by its presence in all apicomplexan parasites containing the organelle (Table 11.1), isoprenoid biosynthesis seems to be the least dispensable apicoplast-localized pathway (reviewed in Imlay and Odom, 2014; Seeber and Soldati-Favre, 2010). This is not too surprising as isoprenoids are a large class of natural compounds that fulfill important cellular functions in signaling processes and protein modifications like prenylation. They are also required for synthesis of coenzyme Q (ubiquinone) in the mitochondrion and for tRNA

TABLE 11.1 Comparison of genome size, predicted protein-coding sequences and known metabolic pathways of the apicoplast of some apicomplexan parasites. Genome size (Mb)

No. protein-coding sequences

FD/FNRa

ISCa

MEPa

FAS IIa

Hemea

Toxoplasma gondii ME49

65.67

8322

X

X

X

X

X

Neospora caninum

59.1

7122

X

X

X

X

X

Eimeria tenella

51.86

8597

X

X

X

X

X

Cyclospora cayetanensis

44.03

7455

X

X

X

X

X

Plasmodium falciparum 3D7

23.33

5460

X

X

X

X

X

Babesia bovis

8.18

3706

X

X

X

Theileria parva

8.35

4082

X

X

X

a

FAS II, Fatty acid biosynthesis type II; FD/FNR, ferredoxin redox system; heme, heme biosynthesis; ISC, iron
sulfur cluster biosynthesis; MEP, isoprenoid biosynthesis. X indicates the presence of the pathway. Assembled from data obtained from EuPathDB Gene Metrics (http://eupathdb.org), March 2019 and from Liu, S., Wang, L., Zheng, H., Xu, Z., Roellig, D.M., Li, N., et al., 2016. Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. BMC Genomics 17, 316 (Liu et al., 2016).

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11.2 The apicoplast

523

FIGURE 11.11 Overview of the metabolism of the apicoplast and mitochondrion. Metabolic pathways or important reactions in both compartments are shown (rounded white boxes) and their interactions indicated by arrows. The pathways detailed in this chapter are indicated with a dark border; those covered predominantly in Chapter 10, Metabolic networks and metabolomics, are without border. Compounds that are imported into the organelles (gray boxes) or produced inside them (black boxes) are also drawn. Their import (large gray arrows) or export (large black arrows) next to the compound indicates flux of the metabolites. Heme synthesis takes place in three compartments—apicoplast, mitochondrion, and cytosol. For more details, see the text and cited reviews. BCAA, Branched-chain amino acid; BCKDH, branched-chain α-keto acid dehydrogenase; DHODH, dihydroorotate dehydrogenase; DMAPP, dimethylallyl diphosphate; FNR, ferredoxin NADP1 reductase; IPP, isopentenyl pyrophosphate; ISC, iron
sulfur cluster; N5,N10-MTF, N5,N10-methylenetetrahydrofolate; PDH, pyruvate dehydrogenase.

modifications (reviewed in Holstein and Hohl, 2004). The enzymes mediating isoprenoid synthesis in the apicoplast are distinct from those of the mevalonate pathway found in the human host, although the two isomeric end products, IPP and dimethylallyl diphosphate (DMAPP), are identical (Fig. 11.12). The methylerythritol phosphate (MEP) pathway (also called nonmevalonate pathway or DOXP

pathway, for its early intermediate metabolite 1-deoxy-D-xylulose 50 phosphate) reflects the cyanobacterial origin of this synthesis pathway. It consists of seven enzymatic steps, ending in IPP and DMAPP (Fig. 11.12). T. gondii contains all the necessary enzymes for IPP synthesis, although the IPP isomerase required to interconvert IPP and DMAPP remains to be identified (F. Seeber, unpublished data). All genes

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11. The apicoplast and mitochondrion of Toxoplasma gondii

FIGURE 11.12

Isoprenoid biosynthesis pathways. Mammalian mevalonate (left) and Toxoplasma gondii MEP pathway (right) for the generation of the isoprenoid precursor IPP and DMAPP are juxtaposed, illustrating the fundamental enzymatic differences between both organisms despite identical end products. For each T. gondii enzyme, the ToxoDB accession number and the respective fitness score (Sidik et al., 2016) is given. The more negative the value, the more this gene is considered essential under the experimental conditions tested. No gene for an IPP isomerase has been annotated so far (indicated by “?”). IPP, isopentenyl pyrophosphate; MEP, methylerythritol phosphate.

Toxoplasma Gondii

11.2 The apicoplast

encoding enzymes in the MEP pathway exhibited negative scores in a genome-wide CRISPRbased screen to identify fitness-conferring genes (Sidik et al., 2016), indicating that their disruption results in greatly impaired tachyzoite growth (Fig. 11.12). The antibiotic fosmidomycin was shown to kill the malaria parasite by inhibiting DOXP reductase, the second enzyme in the MEP pathway, which is the only essential apicoplastlocalized metabolic pathway in the erythrocytic stages of P. falciparum (Yeh and DeRisi, 2011). The situation is more complex for T. gondii, which has a fully functional MEP pathway that is essential for its survival. However, fosmidomycin and derivatives show little activity against T. gondii and other coccidians (Baumeister et al., 2011). Fosmidomycin’s low activity is due to poor bioavailability in T. gondii, and open questions remain regarding the transport of the highly charged drug through the various cellular membranes and compartments (Baumeister et al., 2011; Nair et al., 2011). This precludes the simple IPP complementation assays used in Plasmodium that are so useful to assess apicoplast essentiality (as mentioned previously). Moreover, host cell isoprenoids salvaged by tachyzoites can to some extent complement genetic disruptions in downstream MEP pathway enzymes like farnesyl diphosphate/geranylgeranyl diphosphate synthase (Li and Teng, 2013), making analysis of this pathway in T. gondii even more challenging. How do the initial substrates for these pathways (phosphoenolpyruvate and dihydroxyacetone phosphate) enter the apicoplast? Due to the charged nature of the compounds, it is likely that specific carriers are required. In T. gondii the task appears to be fulfilled by a single sugar phosphate translocator, APT1 (Karnataki et al., 2007a), that likely localizes to multiple membranes of the apicoplast and that is able to transport different compounds such as triose phosphates and phosphoenolpyruvate (Brooks et al., 2010; Karnataki et al., 2007a).

525

In contrast, next to nothing is known about how the products, IPP and DMAPP, leave the apicoplast. The mitochondrion is a likely “consumer” of the isoprenoids from the apicoplast, for example, for condensation of IPP and DMAPP by mitochondrial farnesyl pyrophosphate synthase (Ling et al., 2007) as a precursor for coenzyme Q modification. The close association of both organelles might simplify metabolite exchange through membrane contact sites like those described between the apicoplast and ER (Tomova et al., 2009), although the transporters that facilitate isoprenoid exchange are unknown. In the plastid itself, tRNA modification by isoprenylation via an IPP transferase (MIAA) is a likely activity that requires IPP/DMAPP. Such modification is presumably required to suppress stop codons and frame shifts known to occur in some apicoplast genes (see Section 11.2.4). Connected to the MEP pathway is a redox system comprised of the small iron-sulfur protein FD and its partner FNR (Seeber et al., 2005). It is an accessory system required for the functions of the last two enzymes of the MEP pathway. The FD redox system is most similar to those of nonphotosynthetic tissues of plants and donates electrons derived from NADPH to acceptor proteins via protein
protein interactions (Seeber et al., 2005). NADPH, in turn, can potentially be generated via apicoplast-resident aconitase and isocitrate dehydrogenase and/or by glyceraldehyde 3-phosphate dehydrogenase isoenzymes, respectively (Pino et al., 2007). The last two enzymes of the MEP pathway (ISPH/ LYTB and ISPG/GCPE; see Fig. 11.12 for enzyme names) are known to be FD-interacting proteins and thus presumably obtain their electrons from this redox system (Seeber and Soldati-Favre, 2010). A further putative electron acceptor for FD is the lipoic acid (LA) synthase (LIPA) protein involved in the synthesis of the cofactor LA and thus also indirectly involved in fatty acid biosynthesis (FAS) II synthesis (see Chapter 8: Biochemistry and

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11. The apicoplast and mitochondrion of Toxoplasma gondii

metabolism of Toxoplasma gondii: lipid synthesis and uptake, for details). A role for FD/FNR in ISC synthesis, in analogy to the mitochondrial system, is predicted but unproven. FD, a [2Fe
2S]-containing protein, pointed very early to a requirement for the biosynthesis of these essential prosthetic groups in the apicoplast (Seeber et al., 2005). The labile nature (both chemically and structurally) of preassembled ISC proteins would likely not survive the unfolding thought to be required for import of NEAT proteins into the plastid. ISCs fulfill a variety of functions in proteins, most notably facilitating electron transfer processes. Three of the four currently known ISC proteins in the apicoplast, besides FD, are the enzymes noted above to interact with FD (i.e., LIPA, LYTB, and GCPE). The fourth is the putative methylthiotransferase MIAB, which has been implicated in tRNA modification in the apicoplast (Seeber and Soldati-Favre, 2010) but which has not been investigated so far. Likewise, it is unknown whether other ISC proteins known to assist in RNA modifications are localized to this organelle. Synthesis of ISCs occurs through a series of reactions that are predominantly conserved in prokaryotes and eukaryotes, with different players in different cellular compartments (see Table 11.2). In this respect, looking at plants is particularly instructive since they contain both organelles that house ISC synthesis: mitochondria and plastids (reviewed in PrzybylaToscano et al., 2018). The compartmentation of ISC synthesis within mitochondria and plastids is beneficial, segregating the generation of reactive intermediates and thereby avoiding damage to other cellular components. The first hint that ISC synthesis occurs in the apicoplast was the finding that the apicoplast-encoded gene SUFB is related to bacterial sufB. In bacteria sufB lies in the same operon as other genes that are involved in ISC biosynthesis via the so-called SUF (sulfur mobilization) pathway (reviewed in Perard and Ollagnier de

Choudens, 2018), present in many bacteria and plastids. Subsequently, genes encoding all the other homologous proteins of the SUF pathway, in addition to other accessory proteins, have been identified in T. gondii, P. falciparum (see Table 11.2 for details), and other Apicomplexa (Seeber and Soldati-Favre, 2010). Most experimental data come from the investigation of this pathway in Plasmodium spp. Apicoplast ISC synthesis basically follows the bacterial SUF system (for details see Pala et al., 2018; Seeber and Soldati-Favre, 2010). It starts with two redox processes: the generation of elemental sulfur from cysteine, mediated by the action of a cysteine desulfurase (SUFS), and the oxidation of iron (possibly with involvement of FD). Fe21 is imported into the apicoplast by still undefined iron transporter(s), and cysteine could derive though proteasemediated degradation of apicoplast proteins and/or via amino acid import. ISCs are then assembled on a specific protein scaffold complex consisting of SUFB, SUFC, and SUFD/E. Additional proteins are required for the release of ISCs from the scaffold and for their subsequent transfer to apo-proteins (Table 11.2). The apicoplast localization of all SUF proteins has been experimentally verified (Table 11.2). Curiously, with one exception, none of the ISC synthesis proteins has a predicted signal peptide, using SignalP 5.0, while another predictor for protein subcellular localization (including plants), DeepLoc, indicates plant plastid localization for most of these proteins (Table 11.2). Only FNR possesses a classical signal peptide, which is required for apicoplast localization (Harb et al., 2004), implicating alternate sorting mechanisms for the other proteins (see Section 11.2.7). Since the essential MEP pathway enzymes GCPE and LYTB both contain [4Fe
4S] clusters, all proteins involved in ISC synthesis should be essential, and this is borne out by a genomewide CRISPR/Cas9 screen in the tachyzoite stage of T. gondii (Sidik et al., 2016).

Toxoplasma Gondii

TABLE 11.2 Putative proteins involved in the apicoplast iron
sulfur cluster (ISC) synthesis of Toxoplasma gondii. Designation/ ToxoDB ID

Function

NFU-api

Transfer protein; transfer of ISC to apo-proteins TGME49_221818 such as FD or LipA

Fitness scorea 0.07

DeepLoc localizationb/ SignalP type 5.0b

Experimental localization in apicoplastc

Cytoplasm/ soluble

N

Tg Pb

Haussig et al. (2013), Sheiner et al. (2011)

Plastid/ membrane

N

Pb

Haussig et al. (2014)

Cytoplasm/ soluble

N

Tg Pf

On genome

Reference

Transfer protein; transfer of ISC to apo-proteins

2 4.01

Part of SUF BCD scaffold complex; sulfide acceptor

NA

Part of SUF BCD scaffold complex; ATPase activity

2 5.57

Plastid/ membrane

N

Pb Pf

Gisselberg et al. (2013), Haussig et al. (2014)

Part of SUF BCD scaffold complex; ATPase activity

2 5.29

Plastid/ membrane

N

Pb

Haussig et al. (2014)

SUFE

SUFS activator and sulfide “transferase”; part TGME49_277010 of SUF ES complex

2 3.31

Plastid/ soluble

N

Pb Pf

Charan et al. (2014), Gisselberg et al. (2013), Haussig et al. (2014)

SUFS

Cysteine desulfurase; generates sulfide from LTGME49_216170 cysteine

2 3.95

Plastid/ soluble

N

Pb Pf

Charan et al. (2014), Gisselberg et al. (2013), Haussig et al. (2014)

CPN60

Chaperonin; help in ISC transfer?

2 3.32

Plastid/ soluble

N

Tg Pf

Agrawal et al. (2009), Sato and Wilson (2005)

Chaperonin; help in ISC transfer?

2 4.02

Plastid/ soluble

N

Pf

Sato and Wilson (2005)

Plant-type FNR; redox system together with FD TGME49_298990 for electron transfer

2 3.03

Extracellular/ soluble

Y

Tg

Striepen et al. (2000)

FD

2 4.35

Plastid/ membrane

N

Tg Pf

Kimata-Ariga et al. (2007), Seeber et al. (2005)

BOLA-like

0.1

Plastid/ membrane

N

?

GRX14-like

Glutaredoxin-like transfer protein for ISC to apoTGME49_268730 proteins

2 2.13

Plastid/ soluble

N

?

HCF101-like

2 3.24

Cytoplasm/ soluble

N

?

SUFA HesB TGME49_297925 SUFB TogoCp26-t26_1 SUFC TGME49_225800 SUFD TGME49_273445

TGME49_240600 CPN20 TGME49_273960 FNR

TGME49_215070

Plant-type FD; electron transfer protein

Targeting/facilitating ISC insertion; GRX14TGME49_239320 interacting

TGME49_318590 a

Transfer protein for ISC to apo-proteins

According to Sidik et al. (2016). The more negative the value, the more this gene is considered essential under the experimental conditions tested, with genes shown to be important for tachyzoite proliferation having fitness scores between 22 and 26.89. Predictions were performed at the following servers: SignalP 5.0, http://www.cbs.dtu.dk/services/SignalP/; DeepLoc-1.0, http://www.cbs.dtu.dk/services/ DeepLoc-1.0/. N, No signal peptide; Y, predicted signal peptide. c Experimental confirmation of apicoplast localization in Tg, T. gondii; Pf, P. falciparum; Pb, P. berghei; ?, undetermined. FD, Ferredoxin; FNR, ferredoxin NADP 1 reductase. b

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11. The apicoplast and mitochondrion of Toxoplasma gondii

The exception is NFU-api for which genetic mutants were previously reported to be viable in both T. gondii (Sheiner et al., 2011) and P. berghei (Haussig et al., 2013). Its nonessentiality is in accordance with a potential redundancy of NFU-api and SUFA as shown in P. berghei (Haussig et al., 2013, 2014). While sequences similar to the plant plastid ISC transfer/targeting proteins HCF101, and GRXS14/16 and BOLA (Przybyla-Toscano et al., 2018) can be identified in the genomes of several Apicomplexa (Table 11.2), their localization and thus potential role in apicoplast ISC synthesis awaits characterization. ACP, a key protein in the type II FAS pathway, was one of the first NEAT proteins shown to be present in the apicoplast (Waller et al., 1998), implicating this organelle as a location for fatty acid synthesis. Fatty acids are of great importance for a variety of cellular functions, with their role as precursors for lipid synthesis being amongst the most important. The type II pathway, typical of plants, algae, and many bacteria, is mediated by a series of enzymes which have been identified in all the sequenced apicomplexan genomes except those of the piroplasms Babesia sp. and Theileria sp. (Table 11.1). Acetyl-CoA is a required precursor for the FAS II pathway and is generated from glycolysis-derived pyruvate by the action of the large multienzyme complex pyruvate dehydrogenase (PDH) (Fig. 11.11). To be active, one of its subunits (PDH-E2) must be posttranslationally modified by LA, a dithiol-fatty acid. LA synthesis requires two enzymes (LIPA and LIPB) that are localized to the apicoplast, as well as its substrate octanoyl-ACP, itself a product of FAS II. Thus this pathway indirectly requires its own product (Seeber and SoldatiFavre, 2010). A detailed description of FAS II and LA biochemistry is given in Chapter 8, Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake. Heme biosynthesis is another organellar process in eukaryotes, occurring in the

mitochondrion and cytosol of the human host. In contrast, in T. gondii and P. falciparum, heme biosynthesis is accomplished through metabolic cooperation between the apicoplast, the cytosol, and the mitochondrion (Fig. 11.11). This pathway is described in detail in Chapter 10, Metabolic networks and metabolomics. The enzymes required for the de novo synthesis of heme are encoded in the genomes of P. falciparum and Coccidia, while they are absent from the piroplasms (Table 11.1).

11.3 The mitochondrion 11.3.1 Appearance and ultrastructure The mitochondrion of T. gondii is essential for growth and survival of the disease-causing tachyzoite stage (Sections 11.3.4
11.3.6) and is a validated and effective drug target in both the tachyzoite and bradyzoite stages of the parasite life cycle (Section 11.3.7). Each parasite cell contains a single mitochondrion which is bounded by an outer and an inner membrane (Figs. 11.1 and 11.13). The inner membrane invaginates to form tubular cristae (Fig. 11.1B). The mitochondrion is sometimes found in close apposition to the apicoplast (Nishi et al., 2008; Ovciarikova et al., 2017), perhaps reflective of interrelated biochemical pathways that occur in these organelles (Section 11.3.6). In intracellular T. gondii the mitochondrion most commonly adopts a circular, “lasso-like” structure that loops around the periphery of the cell (Fig. 11.13) just beneath the IMC (Ovciarikova et al., 2017). This peripheral localization may result from as-yet undefined physical associations of the mitochondrion with the IMC. Time-lapse imaging reveals that the T. gondii mitochondrion is a highly dynamic organelle in intracellular parasites, with frequent branching and fusion events (Ovciarikova et al., 2017). None of the proteins known to mediate mitochondrial fusion events

Toxoplasma Gondii

11.3 The mitochondrion

529

FIGURE 11.13 Morphological appearance of the mitochondrion of Toxoplasma gondii. Phase contrast (A) and immunofluorescence image (B) of intracellular T. gondii tachyzoites in an eight-cell vacuole. In (B) mitochondria are labeled with an antibody against the outer membrane protein TgTOM40. Each parasite contains a single mitochondrion. Most mitochondria exhibit a “lasso” shape, with occasional branches emerging from nodes on the mitochondrion. Source: Image provided by GvD.

in other organisms are conserved in T. gondii, and the importance of this process for its mitochondrial biology remains unknown. Time-lapse imaging studies also reveal that the mitochondrion divides and segregates into daughter parasites late in the cell cycle of tachyzoites, in a process that involves the dynamin-related protein DRPC (Melatti et al., 2019). Upon egress of tachyzoites from their host cells, the mitochondrion undergoes rapid and profound morphological changes, with the lasso-shaped organelles differentiating to form “sperm-like” or collapsed structures. The organelle’s structure can also undergo dramatic changes in response to nutrient starvation or oxidative stress, fragmenting to form multiple mitochondria, ultimately leading to parasite death (Charvat and Arrizabalaga, 2016; Ghosh et al., 2012). This mitochondrial fragmentation has been linked to autophagy, a process by which cells consume their own cytoplasm, including organelles such as mitochondria. The reasons and mechanisms for mitochondrial fragmentation upon nutrient starvation and oxidative stress remain unclear.

11.3.2 Evolution Mitochondria arose early in eukaryotic evolution, most likely through an endosymbiotic association between a proteobacterium and an Asgard-like archaeal or proto-eukaryotic

cell (Eme et al., 2017). Most extant eukaryotic lineages retain a mitochondrion or a mitochondrion-related organelle. The mitochondrion-containing Last Eukaryotic Common Ancestor (LECA) existed B1.5 billion years ago, and within B300 million years after its appearance all the major extant lineages of eukaryotes evolved (Eme et al., 2017). As indicated by their presence in diverse lineages of eukaryotes, including apicomplexans, many of the canonical features we associate with mitochondria were present in LECA. These include metabolic and energy-producing pathways such as the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC), and biosynthetic pathways such as those for ISC, coenzyme Q, and heme biosynthesis (Seeber et al., 2008). Many housekeeping functions of the mitochondrion, including the machinery for transcription and translation of the mitochondrial genome and protein import apparatuses, were also present in LECA and are found in apicomplexans such as T. gondii today. Although the mitochondrion of T. gondii is in many respects canonical, emerging evidence suggests it has numerous novel or divergent features (Jacot et al., 2016; Seidi et al., 2018). A recent proteomic analysis of the mitochondrion of T. gondii identified B400 proteins, of which B50% lacked clear homologs in wellstudied organisms such as animals, fungi, and plants (Seidi et al., 2018). Notably, of those

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11. The apicoplast and mitochondrion of Toxoplasma gondii

putative mitochondrial proteins restricted to apicomplexans, B75% are predicted by genome-wide screens to be important for growth of tachyzoites (Seidi et al., 2018; Sidik et al., 2016). A particular challenge is to elucidate the functions of these important, divergent apicomplexan mitochondrial proteins. The last five years have seen considerable progress in defining the unique or unusual features of T. gondii mitochondrial biology, which will be covered in more detail later in this chapter. Many of the divergent mitochondrial proteins were present in the common ancestor of apicomplexans and their nearest, free-living relatives, the dinoflagellates, and chromerids (Danne et al., 2013; Jacot et al., 2016; Seidi et al., 2018). Together with the apicomplexans, they comprise a eukaryotic lineage known as the Myzozoa (Cavalier-Smith, 2018). The features of mitochondrial biology shared by and unique to myzozoans indicate that these features arose independently of the major lifestyle changes that occurred later in these lineages, including the evolution of parasitism in apicomplexans (Danne et al., 2013).

11.3.3 Replication and expression of the mitochondrial genome Mitochondria arose from a proteobacterium, and most mitochondria and related organelles retain a bacterially derived genome (Gray, 2012). Apicomplexan mitochondrial genomes, first identified in Plasmodium spp. in the late 1980s (reviewed in Feagin, 1994), are amongst the smallest mitochondrial genomes known. They are linear B6
8 kb molecules with a very limited gene content, encoding only three protein-coding genes and fragmented ribosomal RNAs (Fig. 11.14). In contrast to Plasmodium and numerous other apicomplexans, the sequence of the T. gondii mitochondrial genome has been elusive. Multiple partial copies of the mitochondrial genome of T. gondii

FIGURE 11.14 Comparisons of apicomplexan mitochondrial genomes. Schematic maps of the mitochondrial genomes of T. gondii, P. falciparum, and T. parva are shown with genes above or below the line depending on direction of transcription (left to right above the line). Protein-coding genes are indicated by their names; fragments of large and small subunit rRNAs are shown in light gray and dark gray boxes, respectively. Open boxes indicate small transcripts with characteristics similar to the rRNA fragments; these may correspond to less-conserved regions of rRNA. The P. falciparum mitochondrial genome has been most thoroughly studied, with fewer rRNA fragments identified for T. parva and none identified as yet for T. gondii.

have migrated into the nuclear genome of the parasite, complicating efforts to establish its sequence. Nevertheless, it is likely that the T. gondii mitochondrial genome contains many of the same features as those of other apicomplexans. Apicomplexan mitochondrial genomes have gone to extremes in reduction of coding capacity. Like the apicoplast genomes, they are conserved in gene content but not in organization or topology. The three protein-coding genes in the B6
8 kb mitochondrial genomes contrast with 13 in the 16 kb mammalian mitochondrial genomes (reviewed in Feagin, 2000). However, those three are hallmark mitochondrial genes: cytochrome b (COB), and cytochrome oxidase subunits I (COX1) and III (COX3). Apicomplexan mitochondrial genomes also contain short sequences similar to highly

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conserved portions of bacterial large and SSU rRNAs (reviewed in Feagin, 2000; see also Feagin et al., 2012). Unexpectedly, these sequences are not contiguous but are fragmented and scattered out of order across the mitochondrial genome. Fragmented rRNAs have been identified in a number of organisms but those encoded by apicomplexan mitochondrial genomes are by far the smallest, and their cumulative size is far less than conventional rRNAs. The P. falciparum mitochondrial rRNA genes and corresponding transcripts have been studied in greatest detail (Feagin et al., 2012). Thirty-four small RNAs, ranging in size from 23 to 190 nt, have been mapped to the P. falciparum mitochondrial genome. Of these, 27 specify regions of large and SSU rRNA. They encode highly conserved and functionally important parts of conventional rRNAs, with expected base-pairing, long-distance interactions, and sequence elements known to be associated with ribosomal function. When mapped to a three-dimensional model of the ribosome, they cluster at the interface between the large and small ribosomal subunits (Feagin et al., 2012). While their function in mitochondrial protein synthesis has not been formally demonstrated, all evidence suggests that these tiny transcripts comprise functional rRNAs which, when assembled into mitochondrial ribosomes, translate the three mitochondrial mRNAs. Similarly, the three proteincoding genes of apicomplexan mitochondrial genomes are the only ones present in the mitochondrial genome of the chromerid V. brassicaformis (Flegontov et al., 2015), indicating that the severe gene reduction in the mitochondrial genome evolved early in the myzozoan lineage. Few studies have addressed the transcription of mitochondrial genes in T. gondii. However, the COB transcript has been examined due to its predicted role in drug resistance. The COB gene encodes cytochrome b, a

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component of the cytochrome bc1 complex of the ETC, which is the site of action of atovaquone, endochin-like quinolones (ELQs), and several other drugs that target apicomplexan mitochondria (Section 11.3.7). Sequencing of cob cDNA from drug-resistant parasites led to the identification of numerous mutations that likely mediate resistance to atovaquone and ELQs (Alday et al., 2017; McFadden et al., 2000). While transcription from the T. gondii mitochondrial genome remains minimally investigated, some predictions may reasonably be made. It is common for mitochondrial genomes to be polycistronically transcribed, with the precursor RNA then cleaved to produce individual RNAs. It is likely that mitochondrial transcription in T. gondii will have similar characteristics as those described in other Apicomplexa (reviewed in Feagin, 2000; Feagin et al., 2012). Several lines of evidence indicate that translation of mitochondrial mRNA transcripts occurs and that this process is essential for T. gondii growth and viability. Numerous proteins required for mitochondrial translation, including ribosomal proteins and elongation factors, localize to the mitochondrion (Lacombe et al., 2019; Pino et al., 2010; Seidi et al., 2018). Regulated knockdown of a nuclear-encoded mitochondrial ribosomal protein impairs parasite growth and leads to defects in cytochrome c oxidase activity, likely because of reduced translation of COX1 and COX3 mRNAs from the mitochondrial genome (Lacombe et al., 2019). The mitochondrial genome of apicomplexans lacks tRNAs, which must therefore be encoded on the nuclear genome, and imported into the mitochondrion for protein synthesis to occur. Indeed, several tRNAs were shown to be imported into the T. gondii mitochondrion (Esseiva et al., 2004). To be functional, these tRNAs must be aminoacylated by tRNA synthetases, raising the question of whether they are aminoacylated before or after import into mitochondria. The dearth

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of tRNA synthetases in the mitochondrial proteome of T. gondii (Seidi et al., 2018) and the presence of tRNA synthetases for specific amino acids only in the cytosol and apicoplast (Pino et al., 2010) suggest that tRNAs are imported into the mitochondrion fully charged with their corresponding amino acids. A challenge with this scenario is that once the amino acid is transferred to the growing polypeptide, the tRNA cannot be recharged within the organelle. How tRNAs are recycled, or whether mitochondria must continually import aminoacylated tRNAs is an important outstanding question. Another curious feature is that, unlike mitochondria of other organisms, protein synthesis apparently does not start with an N-formylated methionine, as both the methionyl-tRNA formyltransferase and the peptide deformylase appear to be present only in the apicoplast (Pino et al., 2010). Mitochondrial protein synthesis is bacterial in nature and may therefore be a target of bacterial translation inhibitors such as doxycycline, azithromycin, and clindamycin. Nevertheless, available evidence suggests that the apicoplast is the major target of these drugs in apicomplexans (see Section 11.2.8). Understanding the extent to which these inhibitors act on translation in T. gondii mitochondria is a priority in the field and may be aided by the establishment of recent assays that measure the activity of mitochondrial ETC complexes that depend on mitochondrially encoded proteins (Lacombe et al., 2019; Seidi et al., 2018).

11.3.4 Protein trafficking to the mitochondrion All but three of the hundreds of proteins destined to function in the mitochondrion are nuclear-encoded and must traverse the membranes surrounding the organelle to reach their final destinations. In contrast to secondary

plastids (as mentioned in Section 11.2.7), entry into organelles derived by primary endosymbiosis—both mitochondria and plastids—typically does not involve the secretory system. Rather, proteins are translated on free cytosolic ribosomes, or on ribosomes associated with the organelle’s outer membrane, and enter the organelles directly by virtue of translocation machinery in the outer and inner membranes of the organelle. Many proteins target to the mitochondrial matrix (i.e., the inner compartment of the organelle) courtesy of an N-terminal targeting signal. In T. gondii, the fusion of the N-terminal region of proteins such as heat shock protein 60 to a nonmitochondrially targeted protein is sufficient to direct that protein to the organelle (Toursel et al., 2000). Tools designed to detect N-terminal targeting signals from other eukaryotes predict the presence of such signals on some mitochondrial proteins in T. gondii (Huet et al., 2018; Seidi et al., 2018). Of the .400 T. gondii mitochondrial matrix proteins identified in a proteomic study, only B50% had strongly predicted N-terminal mitochondrial targeting signals using such programs (Seidi et al., 2018). This may be because other targeting signals exist or because existing bioinformatic tools were not trained or developed using T. gondii mitochondrial proteins in their datasets (Lacombe et al., 2019; Seidi et al., 2018). Mitochondrial proteins that target to the membranes or the intermembrane space of eukaryotes such as yeast have a range of targeting signals, some encoded at the N-terminus, others internally or at the C-terminus. Such diversity of targeting signals also exists in T. gondii. For example, several mitochondrial proteins contain C-terminal transmembrane domains that anchor the protein in the outer membrane of the organelle and which are sufficient to mediate this targeting process (Padgett et al., 2017). Several mitochondrial proteins have been identified that also localize to the apicoplast.

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Dual targeting to the plastid and mitochondrion is reasonably common in organisms with primary plastids, where N-terminal plastid and mitochondrial targeting sequences share some resemblance. The presence of signal peptides at the N-terminus of NEAT proteins would make dual targeting seem improbable. Pino et al. (2007), however, found that the targeting signal at the N-terminus of the superoxide dismutase SOD2 could mediate dual targeting to the apicoplast and mitochondrion. The SOD2 targeting signal appears to be encoded in both the N-terminal region and in the mature domain of the protein (Pino et al., 2007). In other instances, dual targeting is mediated by alternative start codons (Saito et al., 2008). The predicted presequence of pyruvate kinase II contains multiple methionines, and when the third methionine was mutated to alanine, the protein was found in the apicoplast but not the mitochondrion. Conversely, when the N-terminal ER signal sequence was deleted and translation started at the third methionine, the protein was routed to the mitochondrion. The thioredoxin-dependent peroxidase protein TPX1 presents a more complex instance of a protein with multiple intracellular destinations in T. gondii; it is alternatively spliced to yield two different 50 transcript ends (Pino et al., 2007). When tagged constructs of the splicing variants were stably expressed in T. gondii, the proteins encoded by the shorter transcript localized to the cytosol while those encoded by the longer transcript localized to the apicoplast and mitochondrion. Protein translocons in outer and inner membrane of the mitochondrion facilitate trafficking of nuclear-encoded proteins into the mitochondrion. These translocons are multisubunit protein complexes: a translocon of the outer mitochondrial membrane (TOM complex) facilitates transport across the outer membrane and a translocon of the inner mitochondrial membrane (TIM complex) facilitates protein transport across the inner membrane and into the

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matrix. Protein complexes also exist to facilitate the integration of proteins into the outer and inner membranes. All these protein translocons have homologs in T. gondii and core subunits of these complexes target to its mitochondrion (van Dooren et al., 2016). The best studied of these complexes is the TOM complex. Like in yeast, the parasite complex is B400 kDa in mass and includes a protein (TOM40) that likely serves as the protein channel (van Dooren et al., 2016). It also contains homologs of the accessory proteins TOM22 and TOM7, which are important for parasite growth and for import of proteins into the mitochondrion. The yeast TOM22 protein has multiple roles in protein import, including an N-terminal, cytosolic domain rich in acidic amino acids that functions as a receptor for the presequence of mitochondrially targeting proteins, and a region encoded by the transmembrane domain that is important for assembly and stability of the complex. By contrast, T. gondii TOM22 lacks the N-terminal receptor domain but retains its key role in TOM complex assembly. This is similar to the TOM22 homologs of other, distantly related eukaryotes such as Trichomonas vaginalis (Makki et al., 2019; Mani et al., 2017), suggesting that the core role of TOM22 is in TOM complex organization and assembly and that the TOM22 protein from yeast and related organisms probably acquired a second function as a receptor for presequence recognition. Understanding the composition of mitochondrial protein translocons provides valuable insights into the evolution of these protein complexes across the course of eukaryotic evolution. The presence of “core” subunits of each of the translocons in diverse eukaryotic phyla, including apicomplexans, indicates that the LECA already had the central constituents of these complexes (Makki et al., 2019; van Dooren et al., 2016). Other proteins were then acquired. Most notably, the TOM complex receptor proteins that first

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recognize presequence-containing proteins differ markedly between different eukaryotic phyla, indicating that these evolved subsequent to radiation that led to the major eukaryotic phyla (Makki et al., 2019). The receptors of the T. gondii TOM complex and other apicomplexans are unknown, and identifying these is of particular interest.

11.3.5 Oxidative phosphorylation and energy metabolism The mitochondrion is commonly described as the powerhouse of the cell. It is the site of the TCA cycle and the ETC, which act in concert to convert the products of glucose and amino acid catabolism to ATP. Bioinformatic analyses of the genomes of T. gondii and other apicomplexans identified genes encoding TCA cycle and ETC proteins (Seeber et al., 2008). However, it was apparent that both processes differ markedly between apicomplexans and the well-studied animal hosts they infect. Apicomplexans lack homologs of mitochondrial PDH, the protein complex that converts pyruvate to acetyl-CoA, thereby feeding the product of glycolysis into the TCA cycle (Seeber et al., 2008). Instead, apicomplexans have repurposed a branched-chain α-keto acid dehydrogenase (BCKDH) complex to catalyze the conversion of pyruvate to acetyl-CoA (Oppenheim et al., 2014) (see Chapter 10: Metabolic networks and metabolomics for details). Similarly, several enzymes of the TCA cycle, including malate dehydrogenase, Class II fumarate hydratase, and NAD1-dependent isocitrate dehydrogenase appear to be absent from the mitochondrion of these organisms (Seeber et al., 2008; van Dooren et al., 2006). Apicomplexans encode a malate:quinone oxidoreductase which, like malate dehydrogenase, catalyzes the conversion of malate to oxaloacetate but which feeds electrons directly to coenzyme Q of the ETC rather than reducing

NAD1 (Hartuti et al., 2018). Apicomplexans encode a Class I fumarate hydratase, which is structurally unrelated to the Class II enzyme found in animals, and an NADP1-dependent isocitrate dehydrogenase (Seeber et al., 2008). As observed elsewhere in the mitochondrial biology of apicomplexans (as shown previously and next), most of the divergent features of pyruvate metabolism and the TCA cycle (with the possible exception of the Class I fumarate hydratase) were already present in the common, free-living ancestor of myzozoans (Danne et al., 2013). Most TCA cycle enzymes are predicted to be important for tachyzoite proliferation, with the interesting exception of the malate:quinone oxidoreductase (which has a phenotype score of 20.79, with scores below 22 considered indicative of genes important for tachyzoite growth; Sidik et al., 2016). Knockout of subunits from the BCKDH complex leads to severe defects in parasite growth in vitro; however, parasites remain viable (Oppenheim et al., 2014). This suggests that pyruvate is not the only substrate that feeds into the TCA cycle. Indeed, numerous studies indicate that catabolism of the amino acid glutamine to α-ketoglutarate and subsequent metabolism through the TCA cycle are important for mitochondrial ATP generation in T. gondii (for details see Chapter 10: Metabolic networks and metabolomics). T. gondii parasites have a complete pathway for the degradation of branched-chain amino acids (BCAAs), including a downstream methylcitrate cycle that serves to detoxify propionyl-CoA derived from BCAA degradation, and some of the enzymes of this pathway localize to the mitochondrion (Limenitakis et al., 2013; see Chapter 10: Metabolic networks and metabolomics). T. gondii parasites also encode enzymes for β-oxidation of fatty acids, a process by which fatty acids are catabolized to form acetyl-CoA, and which occurs in the mitochondrion of some organisms. These enzymes are probably not expressed in tachyzoites, and

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their localizations have not been experimentally determined. The BCAA degradation pathway is not required for tachyzoite growth, and the genes encoding β-oxidation enzymes are also not predicted to be essential for growth in tachyzoites. Thus these processes do not appear to be important for energy generation in tachyzoites, although this may differ at other stages of the parasite life cycle. The ETC transfers electrons from a range of redox reactions through a series of protein complexes, ultimately donating them to O2. The transfer of electrons is coupled to the translocation of protons from inside to outside the inner mitochondrial membrane, resulting in the generation of a proton gradient across the inner membrane in actively respiring mitochondria. This proton gradient can be “harvested” by the ATP synthase complex, with the energy of protons moving down their concentration gradient coupled to ADP phosphorylation to generate ATP. Like the TCA cycle, the ETC of T. gondii has both conserved and unique features compared to the ETC of vertebrate hosts. The first step of the ETC is the transfer of electrons to coenzyme Q, an electron carrier integral to the inner mitochondrial membrane. T. gondii is thought to contain six dehydrogenases capable of donating electrons to the ETC. Malate:quinone oxidoreductase and succinate dehydrogenase, components of the TCA cycle, catalyze the oxidation of malate and succinate, respectively. The TCA cycle also generates NADH. In animal mitochondria, a large multisubunit protein complex called NADH dehydrogenase oxidizes NADH and transfers electrons to coenzyme Q. T. gondii lacks the multisubunit NADH dehydrogenase. Instead, the parasite genome encodes two single subunit NADH dehydrogenase enzymes (also known as type II NADH dehydrogenases) that can carry out this reaction (Lin et al., 2011). T. gondii mitochondria contain a FADdependent glycerol 3-phosphate dehydrogenase enzyme, which is thought to feed

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electrons from the oxidation of cytosolic glycerol 3-phosphate into the ETC. The importance of this enzyme for parasite metabolism and ETC function remains to be determined, although one study found that glycerol 3phosphate can contribute to electron transport and O2 consumption in the parasite mitochondrion (Vercesi et al., 1998). The sixth and final enzyme that can donate electrons to coenzyme Q is dihydroorotate dehydrogenase (DHODH). DHODH catalyzes the conversion of dihydroorotate to orotate in the de novo pyrimidine biosynthesis pathway and localizes to the mitochondrion of T. gondii (Hortua Triana et al., 2012). T. gondii parasites can both synthesize and scavenge pyrimidines, and it is possible to knockout other pyrimidine biosynthesis enzymes if parasites are supplemented with an exogenous source of the pyrimidine uracil (see Chapter 9: Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other apicomplexa). However, while strains of T. gondii encoding a catalytically dead DHODH enzyme can be generated when grown in excess uracil, it has not been possible to entirely delete the DHODH gene (Hortua Triana et al., 2016). This indicates that DHODH has a key role in pyrimidine biosynthesis and that it may have a second function that is independent of enzyme activity. Reduced coenzyme Q donates electrons to the cytochrome bc1 complex. As discussed below, the bc1 complex is a major drug target in apicomplexan parasites, including T. gondii. Inhibition of this complex leads to reduced O2 consumption, and ultimately parasite death (Doggett et al., 2012; Seidi et al., 2018; Vercesi et al., 1998). The bc1 complex donates electrons to cytochrome c, a mobile electron carrier in the intermembrane space of the parasite. T. gondii encodes two cytochrome c paralogs; experimental evidence predicts that one of these is important for growth of tachyzoites under standard in vitro conditions (Sidik et al., 2016).

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11. The apicoplast and mitochondrion of Toxoplasma gondii

As for most eukaryotes, the terminal oxidase of the ETC in T. gondii is cytochrome c oxidase. Electrons from cytochrome c are passed through several heme- and copper-containing centers, ultimately reducing O2. Although the function of the T. gondii COX complex in reducing O2 is similar to its hosts, a recent proteomic analysis of the complex revealed numerous novel or divergent features (Seidi et al., 2018). The T. gondii COX complex is B600 kDa and likely contains 16 distinct proteins as compared to the B200 kDa mammalian complex containing 13 proteins (Lacombe et al., 2019; Seidi et al., 2018). Some of these subunits, including the core COX1, COX2, and COX3 proteins, occur in both the apicomplexan and mammalian complexes. COX2 is a hydrophobic protein that is encoded on the mitochondrial genome of most eukaryotes. By contrast, COX2 from apicomplexans is composed of two polypeptides, both of which are encoded on the nuclear genome (Funes et al., 2002). This unusual “splitting” of the COX2 gene may have reduced the overall hydrophobicity of the COX2 protein, enabling its posttranslational import into the mitochondrion, thereby allowing the gene to be encoded on the nuclear genome. Other subunits of the T. gondii COX complex have diverged substantially from equivalent proteins in the mammalian complex, to the extent that they are no longer identifiable by sequence comparisons (Seidi et al., 2018). Knockdown of ApiCOX25, a divergent homolog of the mammalian COX6A protein, led to severe impairment of parasite growth and selective inhibition of mitochondrial O2 consumption, pointing to the key role for the COX complex in these processes (Seidi et al., 2018). The transport of electrons through the ETC results in the translocation of protons from inside to outside the inner mitochondrial membrane, generating a membrane potential. The T. gondii mitochondrion can be stained with fluorescent dyes which accumulate in

organelles with a high transmembrane potential; accumulation is impaired when parasites are treated with ETC inhibitors. These data indicate conservation of the central role of the ETC in generating this mitochondrial membrane potential. ATP synthase is a large, multisubunit complex on the mitochondrial inner membrane that, in other organisms, utilizes the proton gradient to synthesize ATP. ATP synthase consists of two domains: F0 and F1. The F0 domain acts as a proton channel, and proton movement along an electrochemical gradient rotates the domain. This provides energy for synthesis of ATP at the F1 domain. Analysis of the genomes of T. gondii and other mitochondrion-containing apicomplexans readily identified canonical subunits of the F1 domain, but F0 domain proteins were not apparent, leading to the hypothesis that the ATP synthase of apicomplexans lacked the rotary motor of this complex, and was therefore nonfunctional. Two recent proteomic analyses of ATP synthase from T. gondii identified B25 subunits of this complex, many of which appear to be restricted to apicomplexans and related organisms (Huet et al., 2018; Salunke et al., 2018). Amongst these 25 proteins were highly divergent candidates for subunits a and c of the F0 domain, which together form the rotary motor of the complex, and a candidate for subunit b, a key component of the so-called stator domain that links the F0 and F1 domains of the complex (Huet et al., 2018; Salunke et al., 2018). Knockdown of the subunit b homolog (called ICAP2 in T. gondii) leads to defects in parasite growth and ATP synthesis, pointing to a key role for ATP synthase in these processes in the parasite (Huet et al., 2018). Emerging evidence indicates that the ETC of T. gondii and other apicomplexans contains many novel or highly divergent proteins compared to their potential hosts. Phylogenetic analyses indicate that, like most of the novel traits of apicomplexan mitochondria, these proteins are shared with other members of the

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myzozoan lineage, to the exclusion of other eukaryotes (Huet et al., 2018; Salunke et al., 2018; Seidi et al., 2018). This points to the scenario that these novel features evolved after the common ancestor of myzozoans and ciliates (the basal lineage of the Alveolata) but before the evolution of parasitism in apicomplexans (Danne et al., 2013). What prompted the evolution of these divergent mitochondrial features remains a mystery.

11.3.6 Biosynthetic pathways in the mitochondrion The importance of the TCA cycle and oxidative phosphorylation is so great in many cells that other functions of the mitochondrion are sometimes overlooked (Fig. 11.11). Nevertheless, mitochondria have numerous biosynthetic pathways that are critical for cellular survival. The mitochondrion of T. gondii harbors enzymes necessary for the synthesis of ISC, heme (as mentioned previously), coenzyme Q, and DHODH, which performs a key step in pyrimidine synthesis (as mentioned previously and Chapter 9: Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other apicomplexa). These enzymes are present in tachyzoites (Seidi et al., 2018), pointing to the importance of these pathways for the disease-causing stage of the parasite. The importance of ISC has been discussed previously (see Section 11.2.9). Mitochondrial ISC synthesis not only serves that organelle with these cofactors but also provides them for the cytosol and nucleus (Braymer and Lill, 2017). Indeed, almost all mitochondria and related organelles house this pathway, suggesting it is a core mitochondrial function. Bioinformatic analyses have allowed in silico assembly of a mitochondrial ISC synthesis pathway in T. gondii (Seeber et al., 2008), which parallels the mitochondrial pathway found in

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yeast and many other eukaryotes. A homolog of the protein frataxin, which is implicated in iron transport and assembly of ISC, has been annotated in recent versions of the T. gondii genome (TGME49_262810). No experimental verification has been reported on any of these components with the exception of the localization of two ISC scaffold proteins, ISCU and ISCA, to mitochondria (Pino et al., 2007). The ETC depends heavily on coenzyme Q, and its biosynthesis is described in Chapters 8
10. It should be noted that modification of the coenzyme Q precursor 4-hydroxybenzoic acid by a specific prenyltransferase to yield coenzyme Q requires isoprenoids of different complexities and lengths, and the precursors for those, IMPP and DMAPP, are synthesized in the apicoplast (Fig. 11.11; see Section 11.2.9). In the mitochondrion, these are then condensed by the enzyme farnesyl pyrophosphate synthase into more complex isoprenoids (Ling et al., 2007). Most eukaryotes have a type-II like mitochondrial FAS pathway, which is involved in synthesis of the essential cofactor LA (see Section 11.2.9). T. gondii is devoid of this entire synthetic route and LA is scavenged from host cell mitochondria rather than being imported from the adjacent apicoplast where it is synthesized (Crawford et al., 2006). LA is required by three mitochondrial enzymes for their activity: the E2 subunit of the 2-oxoglutarate dehydrogenase of the TCA cycle, the E2 subunit of BCKDH (Fig. 11.11; see also Chapter 10: Metabolic networks and metabolomics), and the H-protein of the glycine cleavage complex, which generates a precursor required in pyrimidine biosynthesis and folate metabolism (see Chapter 9: Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other apicomplexa). Why there is no metabolic crosstalk between the otherwise highly connected organelles with respect to LA exchange is not known.

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11.3.7 The mitochondrion as a drug target The mitochondrion, and the ETC in particular, is a major drug target in apicomplexan parasites, with several drugs that target the ETC used in the treatment of apicomplexan infections (Alday and Doggett, 2017), and others in clinical development (Llanos-Cuentas et al., 2018; Nilsen et al., 2013). Most drugs that disrupt the mitochondrial ETC in these parasites, including atovaquone and related hydroxynapthoquinones, ELQs, and decoquinate (Doggett et al., 2012), target the cytochrome bc1 complex. One drug, 1-hydroxy-2-dodecyl-4(1H) quinolone, is a potent inhibitor of growth and ETC functions in T. gondii and may have multiple targets in the ETC, including the single subunit NADH dehydrogenases, DHODH, and the bc1 complex (Hegewald et al., 2013). The triazolopyrimidine DSM1, and derivatives thereof, are potent and selective inhibitors of DHODH in T. gondii (Hortua Triana et al., 2016). The best-studied ETC inhibitor is atovaquone. Atovaquone treatment is effective against T. gondii infections in humans and is recommended for use in immunocompromised patients who cannot tolerate other anti
T. gondii drugs, as well as for the treatment of ocular toxoplasmosis (Dunay et al., 2018). Resistance to atovaquone can be generated in the laboratory upon chemically induced random DNA mutagenesis and some, but not all, of these mutations occur in the COB gene (McFadden et al., 2000). COB encodes cytochrome b, one of the core subunits of the bc1 complex. The function of the complex requires docking of coenzyme Q at two sites on the COB protein, termed the Qo and Qi sites. At the Qo site, reduced coenzyme Q donates one electron to an ISC of the complex and a second electron to a heme moiety on COB. The first electron ultimately reduces cytochrome c, while the second is donated to oxidized coenzyme Q bound to the Qi site. Notably, mutations that confer

atovaquone resistance in T. gondii map to the Qo site of COB (McFadden et al., 2000) and structural studies of the yeast bc1 complex indicate that atovaquone binds to this site (Birth et al., 2014). These data indicate that atovaquone is a Qo site inhibitor of the bc1 complex. By contrast, mutations in T. gondii that confer resistance to ELQ-316, a member of the ELQ class of bc1 complex inhibitors, reside in the Qi site (Alday et al., 2017). Other ELQ-family inhibitors, such as ELQ-400, are proposed to target both the Qi and Qo sites, making these particularly potent inhibitors of parasite growth (McConnell et al., 2018). The latent bradyzoite stage of T. gondii that develops after the initial acute infection is not cleared by the host immune system, so T. gondii parasites persist for the lifetime of an infected individual (Sullivan and Jeffers, 2012). This is a particular issue for individuals who subsequently become immunocompromised and are susceptible to tachyzoite reactivation. Inhibitors that target and eliminate the latent stage of the parasite are, therefore, highly desirable. The transition of active tachyzoites to encysted bradyzoites is stimulated upon changes in certain environmental conditions in vitro, such as changes in pH, alteration in nutrient accessibility, change in O2 tension, and exposure to compounds that limit or block mitochondrial function (Jeffers et al., 2018). Notably, tachyzoites transition to bradyzoites in vitro following exposure to atovaquone and other mitochondrial inhibitors, suggesting bradyzoites may be less reliant on mitochondrial respiration than intracellular tachyzoites. Nevertheless, treatment with bc1 complex inhibitors such as hydroxynapthoquinones and ELQs lowers the burden of tissue cysts in the brains of mice latently infected with T. gondii (Doggett et al., 2012), suggesting that the ETC of bradyzoites is important for their survival and that it is a useful target for treating chronically infected individuals. Mitochondria are grossly understudied in latent stages of the parasite, and the development of approaches to

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References

examine the mitochondrial ETC and other aspects of mitochondrial biology in bradyzoites should be a future priority.

11.4 Conclusion The endosymbiotic organelles of T. gondii and other apicomplexans have proven much more exciting than imagined years ago. From a multimembraned organelle of unknown significance, we now have a plastid with pathways that are essential for T. gondii’s intracellular survival, yielding targets for new drugs. The same is true for the mitochondrion, initially somewhat overlooked because of the novelty of the apicoplast. Its fragmented rRNAs provide a look at a minimal ribosome, and it has emerged as an important drug target. Much research on both organelles has previously focused on P. falciparum, for good reasons: the organelle genomes were first identified in P. falciparum, its nuclear genome sequence was completed years earlier than any other apicomplexan, and the magnitude of morbidity and mortality it causes is dramatically greater than for T. gondii. But now, with many tools in place, there has been a rapid expansion of knowledge about the apicoplast and mitochondrion of T. gondii, with implications for treatment and control for diseases caused by numerous apicomplexans. Numerous questions remain, however, and studies of apicoplast and mitochondrial functions are expected to generate further illuminating insights.

Acknowledgments We apologize to colleagues whose relevant work we were unable to cite due to reference restrictions. We are grateful to Drs. Isabelle Coppens, Amy DeRocher, Damien Jacot, and Michael Laue for images. We thank Dr. Michael Gray for advice on the evolution of endosymbiosis. Support for work in the authors’ labs was provided by NIH grants R01 AI50506 to MP and by the Robert Koch-Institute to FS.

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References Agrawal, S., Chung, D.W., Ponts, N., van Dooren, G.G., Prudhomme, J., Brooks, C.F., et al., 2013. An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins. PLoS Pathog. 9, e1003426. Agrawal, S., van Dooren, G.G., Beatty, W.L., Striepen, B., 2009. Genetic evidence that an endosymbiont-derived ERAD system functions in import of apicoplast proteins. J. Biol. Chem. 284, 33683
33691. Alday, P.H., Doggett, J.S., 2017. Drugs in development for toxoplasmosis: advances, challenges, and current status. Drug Des. Dev. Ther. 11, 273
293. Alday, P.H., Bruzual, I., Nilsen, A., Pou, S., Winter, R., Ben Mamoun, C., et al., 2017. Genetic evidence for cytochrome b Qi site inhibition by 4(1H)-quinolone-3diarylethers and antimycin in Toxoplasma gondii. Antimicrob. Agents Chemother. 61, e01866
01816. Amberg-Johnson, K., Yeh, E., 2019. Host cell metabolism contributes to delayed-death kinetics of apicoplast inhibitors in Toxoplasma gondii. Antimicrob. Agents Chemother. 63, e01646
01618. Amberg-Johnson, K., Hari, S.B., Ganesan, S.M., Lorenzi, H. A., Sauer, R.T., Niles, J.C., et al., 2017. Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens. eLife 6, e29865. Andenmatten, N., Egarter, S., Jackson, A.J., Jullien, N., Herman, J.P., Meissner, M., 2013. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods 10, 125
127. Archibald, J.M., 2015. Genomic perspectives on the birth and spread of plastids. Proc. Natl. Acad. Sci. U.S.A. 112, 10147
10153. Arisue, N., Hashimoto, T., 2015. Phylogeny and evolution of apicoplasts and apicomplexan parasites. Parasitol. Int. 64, 254
259. Bahl, A., Davis, P.H., Behnke, M., Dzierszinski, F., Jagalur, M., Chen, F., et al., 2010. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii. BMC Genomics 11, 603. Bansal, P., Tripathi, A., Thakur, V., Mohmmed, A., Sharma, P., 2017. Autophagy-related protein ATG18 regulates apicoplast biogenesis in apicomplexan parasites. mBio 8, e01468
01417. Baumeister, S., Wiesner, J., Reichenberg, A., Hintz, M., Bietz, S., Harb, O.S., et al., 2011. Fosmidomycin uptake into Plasmodium and Babesia-infected erythrocytes is facilitated by parasite-induced new permeability pathways. PLoS One 6, e19334. Beckers, C.J.M., Roos, D.S., Donald, R.G., Luft, B.J., Schwab, J.C., Cao, Y., et al., 1995. Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma

Toxoplasma Gondii

540

11. The apicoplast and mitochondrion of Toxoplasma gondii

gondii. Implications for the target of macrolide antibiotics. J. Clin. Invest. 95, 367
376. Biddau, M., Bouchut, A., Major, J., Saveria, T., Tottey, J., Oka, O., et al., 2018. Two essential thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii. PLoS Pathog. 14, e1006836. Birth, D., Kao, W.C., Hunte, C., 2014. Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action. Nat. Commun. 5, 4029. Blume, M., Seeber, F., 2018. Metabolic interactions between Toxoplasma gondii and its host. F1000Res. 7, 1719. Boucher, M.J., Ghosh, S., Zhang, L., Lal, A., Jang, S.W., Ju, A., et al., 2018. Integrative proteomics and bioinformatic prediction enable a high-confidence apicoplast proteome in malaria parasites. PLoS Biol. 16, e2005895. Bouchut, A., Geiger, J.A., DeRocher, A.E., Parsons, M., 2014. Vesicles bearing Toxoplasma apicoplast membrane proteins persist following loss of the relict plastid or Golgi body disruption. PLoS One 9, e112096. Braymer, J.J., Lill, R., 2017. Iron-sulfur cluster biogenesis and trafficking in mitochondria. J. Biol. Chem. 292, 12754
12763. Brooks, C.F., Johnsen, H., van Dooren, G.G., Muthalagi, M., Lin, S.S., Bohne, W., et al., 2010. The Toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival. Cell Host Microbe 7, 62
73. Camps, M., Arrizabalaga, G., Boothroyd, J., 2002. An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii. Mol. Microbiol. 43, 1309
1318. Cavalier-Smith, T., 2018. Kingdom Chromista and its eight phyla: a new synthesis emphasising periplastid protein targeting, cytoskeletal and periplastid evolution, and ancient divergences. Protoplasma 255, 297
357. Charan, M., Singh, N., Kumar, B., Srivastava, K., Siddiqi, M.I., Habib, S., 2014. Sulfur mobilization for Fe-S cluster assembly by the essential SUF pathway in the Plasmodium falciparum apicoplast and its inhibition. Antimicrob. Agents Chemother. 58, 3389
3398. Charvat, R.A., Arrizabalaga, G., 2016. Oxidative stress generated during monensin treatment contributes to altered Toxoplasma gondii mitochondrial function. Sci. Rep. 6, 22997. Chaubey, S., Kumar, A., Singh, D., Habib, S., 2005. The apicoplast of Plasmodium falciparum is translationally active. Mol. Microbiol. 56, 81
89. Chaudhari, R., Dey, V., Narayan, A., Sharma, S., Patankar, S., 2017. Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum. PeerJ 5, e3128. Chotewutmontri, P., Holbrook, K., Bruce, B.D., 2017. Plastid protein targeting: preprotein recognition and translocation. Int. Rev. Cell. Mol. Biol. 330, 227
294.

Cilingir, G., Broschat, S.L., Lau, A.O., 2012. ApicoAP: the first computational model for identifying apicoplasttargeted proteins in multiple species of Apicomplexa. PLoS One 7, e36598. Cilingir, G., Lau, A.O., Broschat, S.L., 2013. ApicoAMP: the first computational model for identifying apicoplasttargeted transmembrane proteins in Apicomplexa. J. Microbiol. Methods 95, 313
319. Cinar, H.N., Qvarnstrom, Y., Wei-Pridgeon, Y., Li, W., Nascimento, F.S., Arrowood, M.J., et al., 2016. Comparative sequence analysis of Cyclospora cayetanensis apicoplast genomes originating from diverse geographical regions. Parasites Vectors 9, 611. Crawford, M.J., Thomsen-Zieger, N., Ray, M., Schachtner, J., Roos, D.S., Seeber, F., 2006. Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast. EMBO J. 25, 3214
3222. Daher, W., Morlon-Guyot, J., Sheiner, L., Lentini, G., Berry, L., Tawk, L., et al., 2015. Lipid kinases are essential for apicoplast homeostasis in Toxoplasma gondii. Cell. Microbiol. 17, 559
578. Dahl, E.L., Rosenthal, P.J., 2007. Multiple antibiotics exert delayed effects against the Plasmodium falciparum apicoplast. Antimicrob. Agents Chemother. 51, 3485
3490. Danne, J.C., Gornik, S.G., Macrae, J.I., McConville, M.J., Waller, R.F., 2013. Alveolate mitochondrial metabolic evolution: dinoflagellates force reassessment of the role of parasitism as a driver of change in apicomplexans. Mol. Biol. Evol. 30, 123
139. DeRocher, A., Hagen, C.B., Froehlich, J.E., Feagin, J.E., Parsons, M., 2000. Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system. J. Cell. Sci. 113, 3969
3977. DeRocher, A., Gilbert, B., Feagin, J.E., Parsons, M., 2005. Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting. J. Cell. Sci. 118, 565
574. DeRocher, A.E., Coppens, I., Karnataki, A., Gilbert, L.A., Rome, M.E., Feagin, J.E., et al., 2008. A thioredoxin family protein of the apicoplast periphery identifies abundant candidate transport vesicles in Toxoplasma gondii. Eukaryot. Cell 7, 1518
1529. DeRocher, A.E., Karnataki, A., Vaney, P., Parsons, M., 2012. Apicoplast targeting of a T. gondii transmembrane protein requires a cytosolic tyrosine-based motif. Traffic 13, 694
704. Doggett, J.S., Nilsen, A., Forquer, I., Wegmann, K.W., Jones-Brando, L., Yolken, R.H., et al., 2012. Endochinlike quinolones are highly efficacious against acute and latent experimental toxoplasmosis. Proc. Natl. Acad. Sci. U.S.A. 109, 15936
15941. Dorrell, R.G., Gile, G., McCallum, G., Meheust, R., Bapteste, E.P., Klinger, C.M., et al., 2017. Chimeric

Toxoplasma Gondii

References

origins of ochrophytes and haptophytes revealed through an ancient plastid proteome. eLife 6, e23717. Dunay, I.R., Gajurel, K., Dhakal, R., Liesenfeld, O., Montoya, J.G., 2018. Treatment of toxoplasmosis: historical perspective, animal models, and current clinical practice. Clin. Microbiol. Rev. 31, e00057
00017. Dzierszinski, F., Nishi, M., Ouko, L., Roos, D.S., 2004. Dynamics of Toxoplasma gondii differentiation. Eukaryot. Cell 3, 992
1003. Eme, L., Spang, A., Lombard, J., Stairs, C.W., Ettema, T.J. G., 2017. Archaea and the origin of eukaryotes. Nat. Rev. Microbiol. 15, 711
723. Esseiva, A.C., Naguleswaran, A., Hemphill, A., Schneider, A., 2004. Mitochondrial tRNA import in Toxoplasma gondii. J. Biol. Chem. 279, 42363
42368. Feagin, J.E., 1994. The extrachromosomal DNAs of apicomplexan parasites. Annu. Rev. Microbiol. 48, 81
104. Feagin, J.E., 2000. Mitochondrial genome diversity in parasites. Int. J. Parasitol. 30, 371
390. Feagin, J.E., Drew, M.E., 1995. Plasmodium falciparum: alterations in organelle transcript abundance during the erythrocytic cycle. Exp. Parasitol. 80, 430
440. Feagin, J.E., Harrell, M.I., Lee, J.C., Coe, K.J., Sands, B.H., Cannone, J.J., et al., 2012. The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum. PLoS One 7, e38320. Fellows, J.D., Cipriano, M.J., Agrawal, S., Striepen, B., 2017. A plastid protein that evolved from ubiquitin and is required for apicoplast protein import in Toxoplasma gondii. mBio 8, e00950. Ferguson, D.J., Henriquez, F.L., Kirisits, M.J., Muench, S.P., Prigge, S.T., Rice, D.W., et al., 2005. Maternal inheritance and stage-specific variation of the apicoplast in Toxoplasma gondii during development in the intermediate and definitive host. Eukaryot. Cell 4, 814
826. Fichera, M.E., Bhopale, M.K., Roos, D.S., 1995. In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii. Antimicrob. Agents Chemother. 39, 1530
1537. Fichera, M.E., Roos, D.S., 1997. A plastid organelle as a drug target in apicomplexan parasites. Nature 390, 407
409. Flegontov, P., Michalek, J., Janouskovec, J., Lai, D.H., Jirku, M., Hajduskova, E., et al., 2015. Divergent mitochondrial respiratory chains in phototrophic relatives of apicomplexan parasites. Mol. Biol. Evol. 32, 1115
1131. Foth, B.J., Ralph, S.A., Tonkin, C.J., Struck, N.S., Fraunholz, M., Roos, D.S., et al., 2003. Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science 299, 705
708. Frenal, K., Jacot, D., Hammoudi, P.M., Graindorge, A., Maco, B., Soldati-Favre, D., 2017. Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat. Commun. 8, 15710.

541

Funes, S., Davidson, E., Reyes-Prieto, A., Magallon, S., Herion, P., King, M.P., et al., 2002. A green algal apicoplast ancestor. Science 298, 2155. Ghosh, D., Walton, J.L., Roepe, P.D., Sinai, A.P., 2012. Autophagy is a cell death mechanism in Toxoplasma gondii. Cell. Microbiol. 14, 589
607. Gibbs, S.P., 1981. The chloroplast endoplasmic reticulum: structure, function, and evolutionary signficance. Int. Rev. Cytol. 72, 49
99. Gisselberg, J.E., Dellibovi-Ragheb, T.A., Matthews, K.A., Bosch, G., Prigge, S.T., 2013. The Suf iron-sulfur cluster synthesis pathway is required for apicoplast maintenance in malaria parasites. PLoS Pathog. 9, e1003655. Glaser, S., van Dooren, G.G., Agrawal, S., Brooks, C.F., McFadden, G.I., Striepen, B., et al., 2012. Tic22 is an essential chaperone required for protein import into the apicoplast. J. Biol. Chem. 287, 39505
39512. Goodman, C.D., McFadden, G.I., 2014. Ycf93 (Orf105), a small apicoplast-encoded membrane protein in the relict plastid of the malaria parasite Plasmodium falciparum that is conserved in Apicomplexa. PLoS One 9, e91178. Gray, M.W., 2012. Mitochondrial evolution. Cold Spring Harb. Perspect. Biol. 4, a011403. Gray, M.W., 2017. Lynn Margulis and the endosymbiont hypothesis: 50 years later. Mol. Biol. Cell 28, 1285
1287. Gray, M.W., Lang, B.F., Burger, G., 2004. Mitochondria of protists. Annu. Rev. Genet. 38, 477
524. Hadariova, L., Vesteg, M., Hampl, V., Krajcovic, J., 2018. Reductive evolution of chloroplasts in nonphotosynthetic plants, algae and protists. Curr. Genet. 64, 365
387. Harb, O.S., Chatterjee, B., Fraunholz, M.J., Crawford, M.J., Nishi, M., Roos, D.S., 2004. Multiple functionally redundant signals mediate targeting to the apicoplast in the apicomplexan parasite Toxoplasma gondii. Eukaryot. Cell 3, 663
674. Hartuti, E.D., Inaoka, D.K., Komatsuya, K., Miyazaki, Y., Miller, R.J., Xinying, W., et al., 2018. Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target. Biochim. Biophys. Acta Bioenerg. 1859, 191
200. Haussig, J.M., Matuschewski, K., Kooij, T.W.A., 2013. Experimental genetics of Plasmodium berghei NFU in the apicoplast iron-sulfur cluster biogenesis pathway. PLoS One 8, e67269. Haussig, J.M., Matuschewski, K., Kooij, T.W.A., 2014. Identification of vital and dispensable sulfur utilization factors in the Plasmodium apicoplast. PLoS One 9, e89718. He, C.Y., Shaw, M.K., Pletcher, C.H., Striepen, B., Tilney, L. G., Roos, D.S., 2001. A plastid segregation defect in the protozoan parasite Toxoplasma gondii. EMBO J. 20, 330
339. Hegewald, J., Gross, U., Bohne, W., 2013. Identification of dihydroorotate dehydrogenase as a relevant drug target

Toxoplasma Gondii

542

11. The apicoplast and mitochondrion of Toxoplasma gondii

for 1-hydroxyquinolones in Toxoplasma gondii. Mol. Biochem. Parasitol. 190, 6
15. Heiny, S.R., Pautz, S., Recker, M., Przyborski, J.M., 2014. Protein traffic to the Plasmodium falciparum apicoplast: evidence for a sorting branch point at the Golgi. Traffic 15, 1290
1304. Heredero-Bermejo, I., Varberg, J.M., Charvat, R., Jacobs, K., Garbuz, T., Sullivan Jr., W.J., et al., 2019. TgDrpC, an atypical dynamin-related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division. Mol. Microbiol. 111, 46
64. Holstein, S.A., Hohl, R.J., 2004. Isoprenoids: remarkable diversity of form and function. Lipids 39, 293
309. Hortua Triana, M.A., Huynh, M.H., Garavito, M.F., Fox, B. A., Bzik, D.J., Carruthers, V.B., et al., 2012. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii. Mol. Biochem. Parasitol. 184, 71
81. Hortua Triana, M.A., Cajiao Herrera, D., Zimmermann, B. H., Fox, B.A., Bzik, D.J., 2016. Pyrimidine pathwaydependent and -independent functions of the Toxoplasma gondii mitochondrial dihydroorotate dehydrogenase. Infect. Immun. 84, 2974
2981. Howe, C.J., Purton, S., 2007. The little genome of apicomplexan plastids: its raison d’etre and a possible explanation for the ‘delayed death’ phenomenon. Protist 158, 121
133. Huet, D., Rajendran, E., van Dooren, G.G., Lourido, S., 2018. Identification of cryptic subunits from an apicomplexan ATP synthase. eLife 7, e38097. Imlay, L., Odom, A.R., 2014. Isoprenoid metabolism in apicomplexan parasites. Curr. Clin. Microbiol. Rep. 1, 37
50. Jacot, D., Daher, W., Soldati-Favre, D., 2013. Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance. EMBO J. 32, 1702
1716. Jacot, D., Waller, R.F., Soldati-Favre, D., Macpherson, D.A., Macrae, J.I., 2016. Apicomplexan energy metabolism: carbon source promiscuity and the quiescence hyperbole. Trends Parasitol. 32, 56
70. Janouskovec, J., Horak, A., Obornik, M., Lukeˇs, J., Keeling, P.J., 2010. A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids. Proc. Natl. Acad. Sci. U.S.A. 107, 10949
10954. Jeffers, V., Tampaki, Z., Kim, K., Sullivan, W.J., 2018. A latent ability to persist: differentiation in Toxoplasma gondii. Cell. Mol. Life Sci. 4, 1
19. Kadian, K., Gupta, Y., Singh, H.V., Kempaiah, P., Rawat, M., 2018. Apicoplast metabolism: parasite’s achilles’ heel. Curr. Top. Med. Chem. 18, 1987
1997. Karnataki, A., DeRocher, A., Coppens, I., Nash, C., Feagin, J.E., Parsons, M., 2007a. Cell cycle-regulated vesicular

trafficking of Toxoplasma APT1, a protein localized to multiple apicoplast membranes. Mol. Microbiol. 63, 1653
1668. Karnataki, A., DeRocher, A.E., Coppens, I., Feagin, J.E., Parsons, M., 2007b. A membrane protease is targeted to the relict plastid of Toxoplasma via an internal signal sequence. Traffic 8, 1543
1553. Kimata-Ariga, Y., Kurisu, G., Kusunoki, M., Aoki, S., Sato, D., Kobayashi, T., et al., 2007. Cloning and characterization of ferredoxin and ferredoxin-NADP 1 reductase from human malaria parasite. J. Biochem. 141, 421
428. Ko¨hler, S., Delwiche, C.F., Denny, P.W., Tilney, L.G., Webster, P., Wilson, R.J., et al., 1997. A plastid of probable green algal origin in Apicomplexan parasites. Science 275, 1485
1489. Lacombe, A., Maclean, A.E., Ovciarikova, J., Tottey, J., Mu¨hleip, A., Fernandes, P., Sheiner, L., 2019. Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation. Mol. Microbiol 112, 1235
1252. Lemgruber, L., Kudryashev, M., Dekiwadia, C., Riglar, D. T., Baum, J., Stahlberg, H., et al., 2013. Cryo-electron tomography reveals four-membrane architecture of the Plasmodium apicoplast. Malaria J. 12, 25. Leveque, M.F., Berry, L., Cipriano, M.J., Nguyen, H.M., Striepen, B., Besteiro, S., 2015. Autophagy-related protein ATG8 has a noncanonical function for apicoplast inheritance in Toxoplasma gondii. mBio 6, e01446
01415. Li, H.M., Teng, Y.S., 2013. Transit peptide design and plastid import regulation. Trends Plant. Sci. 18, 360
366. Lim, L., Sayers, C.P., Goodman, C.D., McFadden, G.I., 2016. Targeting of a transporter to the outer apicoplast membrane in the human malaria parasite Plasmodium falciparum. PLoS One 11, e0159603. Limenitakis, J., Oppenheim, R.D., Creek, D.J., Foth, B.J., Barrett, M.P., Soldati-Favre, D., 2013. The 2methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii. Mol. Microbiol. 87, 894
908. Lin, S.S., Gross, U., Bohne, W., 2011. Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth. Mol. Microbiol. 82, 209
221. Lin, T.Y., Nagano, S., Gardiner Heddle, J., 2015. Functional analyses of the Toxoplasma gondii DNA gyrase holoenzyme: a janus topoisomerase with supercoiling and decatenation abilities. Sci. Rep. 5, 14491. Ling, Y., Li, Z.H., Miranda, K., Oldfield, E., Moreno, S.N., 2007. The farnesyl-diphosphate/geranylgeranyl-diphosphate synthase of Toxoplasma gondii is a bifunctional

Toxoplasma Gondii

References

enzyme and a molecular target of bisphosphonates. J. Biol. Chem. 282, 30804
30816. Liu, S., Wang, L., Zheng, H., Xu, Z., Roellig, D.M., Li, N., et al., 2016. Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. BMC Genomics 17, 316. Llanos-Cuentas, A., Casapia, M., Chuquiyauri, R., Hinojosa, J.C., Kerr, N., Rosario, M., et al., 2018. Antimalarial activity of single-dose DSM265, a novel plasmodium dihydroorotate dehydrogenase inhibitor, in patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria infection: a proof-ofconcept, open-label, phase 2a study. Lancet Infect. Dis. 18, 874
883. Lorestani, A., Sheiner, L., Yang, K., Robertson, S.D., Sahoo, N., Brooks, C.F., et al., 2010. A Toxoplasma MORN1 null mutant undergoes repeated divisions but is defective in basal assembly, apicoplast division and cytokinesis. PLoS One 5, e12302. Makki, A., Rada, P., Zarsky, V., Kereiche, S., Kovacik, L., Novotny, M., et al., 2019. Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis. PLoS Biol. 17, e3000098. Mallo, N., Fellows, J., Johnson, C., Sheiner, L., 2018. Protein import into the endosymbiotic organelles of apicomplexan parasites. Genes (Basel) 9, E412. Mani, J., Rout, S., Desy, S., Schneider, A., 2017. Mitochondrial protein import—functional analysis of the highly diverged Tom22 orthologue of Trypanosoma brucei. Sci. Rep. 7, 40738. McConnell, E.V., Bruzual, I., Pou, S., Winter, R., Dodean, R. A., Smilkstein, M.J., et al., 2018. Targeted structureactivity analysis of endochin-like quinolones reveals potent Qi and Qo site inhibitors of Toxoplasma gondii and Plasmodium falciparum cytochrome bc1 and identifies ELQ-400 as a remarkably effective compound against acute experimental toxoplasmosis. ACS Infect. Dis. 4, 1574
1584. McFadden, D.C., Tomavo, S., Berry, E.A., Boothroyd, J.C., 2000. Characterization of cytochrome b from Toxoplasma gondii and Qo domain mutations as a mechanism of atovaquone-resistance. Mol. Biochem. Parasitol. 108, 1
12. McFadden, G.I., 2017. The cryptomonad nucleomorph. Protoplasma 254, 1903
1907. McFadden, G.I., Reith, M.E., Munholland, J., LangUnnasch, N., 1996. Plastid in human parasites. Nature 381, 482. McFadden, G.I., Yeh, E., 2017. The apicoplast: now you see it, now you don’t. Int. J. Parasitol. 47, 137
144. Melatti, C., Pieperhoff, M., Lemgruber, L., Pohl, E., Sheiner, L., Meissner, M., 2019. A unique dynamin-related

543

protein is essential for mitochondrial fission in Toxoplasma gondii. PLoS Pathog. 15, e1007512. Milton, M.E., Nelson, S.W., 2016. Replication and maintenance of the Plasmodium falciparum apicoplast genome. Mol. Biochem. Parasitol. 208, 56
64. Mukhopadhyay, A., Chen, C.Y., Doerig, C., Henriquez, F. L., Roberts, C.W., Barrett, M.P., 2009. The Toxoplasma gondii plastid replication and repair enzyme complex, PREX. Parasitology 136, 747
755. Mullin, K.A., Lim, L., Ralph, S.A., Spurck, T.P., Handman, E., McFadden, G.I., 2006. Membrane transporters in the relict plastid of malaria parasites. Proc. Natl. Acad. Sci. U.S.A. 103, 9572
9577. Nair, S.C., Brooks, C.F., Goodman, C.D., Strurm, A., McFadden, G.I., Sundriyal, S., et al., 2011. Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii. J. Exp. Med. 208, 1547
1559. Nilsen, A., Lacrue, A.N., White, K.L., Forquer, I.P., Cross, R.M., Marfurt, J., et al., 2013. Quinolone-3-diarylethers: a new class of antimalarial drug. Sci. Transl. Med. 5, 177ra37. Nisbet, R.E., McKenzie, J.L., 2016. Transcription of the apicoplast genome. Mol. Biochem. Parasitol. 210, 5
9. Nisbet, R.E.R., Kurniawan, D.P., Bowers, H.D., Howe, C.J., 2016. Transcripts in the Plasmodium apicoplast undergo cleavage at tRNAs and editing, and include antisense sequences. Protist 167, 377
388. Nishi, M., Hu, K., Murray, J.M., Roos, D.S., 2008. Organellar dynamics during the cell cycle of Toxoplasma gondii. J. Cell. Sci. 121, 1559
1568. Oppenheim, R.D., Creek, D.J., Macrae, J.I., Modrzynska, K. K., Pino, P., Limenitakis, J., et al., 2014. BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei. PLoS Pathog. 10, e1004263. Ovciarikova, J., Lemgruber, L., Stilger, K.L., Sullivan, W.J., Sheiner, L., 2017. Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii. Sci. Rep. 7, 42746. Padgett, L.R., Arrizabalaga, G., Sullivan Jr., W.J., 2017. Targeting of tail-anchored membrane proteins to subcellular organelles in Toxoplasma gondii. Traffic 18, 149
158. Pala, Z.R., Saxena, V., Saggu, G.S., Garg, S., 2018. Recent advances in the [Fe-S] cluster biogenesis (SUF) pathway functional in the apicoplast of Plasmodium. Trends Parasitol. 34, 800
809. Patron, N.J., Waller, R.F., 2007. Transit peptide diversity and divergence: a global analysis of plastid targeting signals. Bioessays 29, 1048
1058. Perard, J., Ollagnier de Choudens, S., 2018. Iron-sulfur clusters biogenesis by the SUF machinery: close to the

Toxoplasma Gondii

544

11. The apicoplast and mitochondrion of Toxoplasma gondii

molecular mechanism understanding. J. Biol. Inorg. Chem. 23, 581
596. Pham, J.S., Sakaguchi, R., Yeoh, L.M., De Silva, N.S., McFadden, G.I., Hou, Y.M., et al., 2014. A dual-targeted aminoacyl-tRNA synthetase in Plasmodium falciparum charges cytosolic and apicoplast tRNACys. Biochem. J. 458, 513
523. Pino, P., Foth, B.J., Kwok, L.Y., Sheiner, L., Schepers, R., Soldati, T., et al., 2007. Dual targeting of antioxidant and metabolic enzymes to the mitochondrion and the apicoplast of Toxoplasma gondii. PLoS Pathog. 3, e115. Pino, P., Aeby, E., Foth, B.J., Sheiner, L., Soldati, T., Schneider, A., et al., 2010. Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNAMet formylation in Apicomplexa. Mol. Microbiol. 76, 706
718. Przybyla-Toscano, J., Roland, M., Gaymard, F., Couturier, J., Rouhier, N., 2018. Roles and maturation of ironsulfur proteins in plastids. J. Biol. Inorg. Chem. 23, 545
566. Reiff, S.B., Vaishnava, S., Striepen, B., 2012. The HU protein is important for apicoplast genome maintenance and inheritance in Toxoplasma gondii. Eukaryot. Cell 11, 905
915. Saito, T., Nishi, M., Lim, M.I., Wu, B., Maeda, T., Hashimoto, H., et al., 2008. A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion. J. Biol. Chem. 283, 14041
14052. Salunke, R., Mourier, T., Banerjee, M., Pain, A., Shanmugam, D., 2018. Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii. PLoS Biol. 16, e2006128. Sato, S., Wilson, R.J., 2005. Organelle-specific cochaperonins in apicomplexan parasites. Mol. Biochem. Parasitol. 141, 133
143. Seeber, F., Soldati-Favre, D., 2010. Metabolic pathways in the apicoplast of Apicomplexa. Int. Rev. Cell. Mol. Biol. 281, 161
228. Seeber, F., Aliverti, A., Zanetti, G., 2005. The plant-type ferredoxin-NADP 1 reductase/ferredoxin redox system as a possible drug target against apicomplexan human parasites. Curr. Pharm. Des. 11, 3159
3172. Seeber, F., Limenitakis, J., Soldati-Favre, D., 2008. Apicomplexan mitochondrial metabolism: a story of gains, losses and retentions. Trends Parasitol. 24, 468
478. Seidi, A., Muellner-Wong, L.S., Rajendran, E., Tjhin, E.T., Dagley, L.F., Aw, V.Y., et al., 2018. Elucidating the mitochondrial proteome of Toxoplasma gondii reveals the presence of a divergent cytochrome c oxidase. eLife 7, e38131.

Sharma, D., Soni, R., Rai, P., Sharma, B., Bhatt, T.K., 2018. Relict plastidic metabolic process as a potential therapeutic target. Drug Discov. Today 23, 134
140. Sheiner, L., Demerly, J.L., Poulsen, N., Beatty, W.L., Lucas, O., Behnke, M.S., et al., 2011. A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor. PLoS Pathog. 7, e1002392. Sheiner, L., Fellows, J.D., Ovciarikova, J., Brooks, C.F., Agrawal, S., Holmes, Z.C., et al., 2015. Toxoplasma gondii Toc75 functions in import of stromal but not peripheral apicoplast proteins. Traffic 16, 1254
1269. Siddall, M.E., 1992. Hohlzylinders. Parasitol. Today 8, 90
91. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166, 1423
1435. Sieber, K.B., Bromley, R.E., Dunning Hotopp, J.C., 2017. Lateral gene transfer between prokaryotes and eukaryotes. Exp. Cell Res. 358, 421
426. Smith, D.R., Keeling, P.J., 2015. Mitochondrial and plastid genome architecture: reoccurring themes, but significant differences at the extremes. Proc. Natl. Acad. Sci. U.S.A. 112, 10177
10184. Sommer, M.S., Gould, S.B., Lehmann, P., Gruber, A., Przyborski, J.M., Maier, U.G., 2007. Der1-mediated preprotein import into the periplastid compartment of chromalveolates? Mol. Biol. Evol. 24, 918
928. Stewart, R.J., Ferguson, D.J., Whitehead, L., Bradin, C.H., Wu, H.J., Tonkin, C.J., 2016. Phosphorylation of αSNAP is required for secretory organelle biogenesis in Toxoplasma gondii. Traffic 17, 102
116. Striepen, B., Crawford, M.J., Shaw, M.K., Tilney, L.G., Seeber, F., Roos, D.S., 2000. The plastid of Toxoplasma gondii is divided by association with the centrosomes. J. Cell. Biol. 151, 1423
1434. Sullivan Jr., W.J., Jeffers, V., 2012. Mechanisms of Toxoplasma gondii persistence and latency. FEMS Microbiol. Rev. 36, 717
733. Tawk, L., Dubremetz, J.F., Montcourrier, P., Chicanne, G., Merezegue, F., Richard, V., et al., 2011. Phosphatidylinositol 3-monophosphate is involved in Toxoplasma apicoplast biogenesis. PLoS Pathog. 7, e1001286. Tomova, C., Humbel, B.M., Geerts, W.J., Entzeroth, R., Holthuis, J.C., Verkleij, A.J., 2009. Membrane contact sites between apicoplast and ER in Toxoplasma gondii revealed by electron tomography. Traffic 10, 1471
1480. Tonkin, C.J., Roos, D.S., McFadden, G.I., 2006. N-terminal positively charged amino acids, but not their exact position, are important for apicoplast transit peptide fidelity

Toxoplasma Gondii

References

in Toxoplasma gondii. Mol. Biochem. Parasitol. 150, 192
200. Tosetti, N., Dos Santos Pacheco, N., Soldati-Favre, D., Jacot, D., 2019. Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii. eLife 8, e42669. Toursel, C., Dzierszinski, F., Bernigaud, A., Mortuaire, M., Tomavo, S., 2000. Molecular cloning, organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii. Mol. Biochem. Parasitol. 111, 319
332. van Dooren, G.G., Hapuarachchi, S.V., 2017. The dark side of the chloroplast: biogenesis, metabolism and membrane biology of the apicoplast. Adv. Bot. Res. 84, 145
185. van Dooren, G.G., Su, V., D’Ombrain, M.C., McFadden, G. I., 2002. Processing of an apicoplast leader sequence in Plasmodium falciparum and the identification of a putative leader cleavage enzyme. J. Biol. Chem. 277, 23612
23619. van Dooren, G.G., Stimmler, L.M., McFadden, G.I., 2006. Metabolic maps and functions of the Plasmodium mitochondrion. FEMS Microbiol. Rev. 30, 596
630. van Dooren, G.G., Tomova, C., Agrawal, S., Humbel, B.M., Striepen, B., 2008. Toxoplasma gondii Tic20 is essential for apicoplast protein import. Proc. Natl. Acad. Sci. U.S. A. 105, 13574
13579. van Dooren, G.G., Reiff, S.B., Tomova, C., Meissner, M., Humbel, B.M., Striepen, B., 2009. A novel dynaminrelated protein has been recruited for apicoplast fission in Toxoplasma gondii. Curr. Biol. 19, 267
276. van Dooren, G.G., Yeoh, L.M., Striepen, B., McFadden, G.I., 2016. The import of proteins into the mitochondrion of Toxoplasma gondii. J. Biol. Chem. 291, 19335
19350. Vercesi, A.E., Rodrigues, C.O., Uyemura, S.A., Zhong, L., Moreno, S.N., 1998. Respiration and oxidative

545

phosphorylation in the apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 273, 31040
31047. Walczak, M., Ganesan, S.M., Niles, J.C., Yeh, E., 2018. ATG8 is essential specifically for an autophagyindependent function in apicoplast biogenesis in bloodstage malaria parasites. mBio 9, e02021
02017. Waller, R.F., Keeling, P.J., Donald, R.G., Striepen, B., Handman, E., Lang-Unnasch, N., et al., 1998. Nuclearencoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S. A. 95, 12352
12357. Williamson, D.H., Denny, P.W., Moore, P.W., Sato, S., McCready, S., Wilson, R.J., 2001. The in vivo conformation of the plastid DNA of Toxoplasma gondii: implications for replication. J. Mol. Biol. 306, 159
168. Williamson, D.H., Preiser, P.R., Moore, P.W., McCready, S., Strath, M., Wilson, R.J., 2002. The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms. Mol. Microbiol. 45, 533
542. Wilson, R.J.M., Denny, P.W., Preiser, P.R., Rangachari, K., Roberts, K., Roy, A., et al., 1996. Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum. J. Mol. Biol. 261, 155
172. Woo, Y.H., Ansari, H., Otto, T.D., Klinger, C.M., Kolisko, M., Michalek, J., et al., 2015. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. eLife 4, e06974. Yeh, E., DeRisi, J.L., 2011. Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum. PLoS Biol. 9, e1001138. Zuegge, J., Ralph, S., Schmuker, M., McFadden, G.I., Schneider, G., 2001. Deciphering apicoplast targeting signals—feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins. Gene 280, 19
26.

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C H A P T E R

12 Calcium storage and homeostasis in Toxoplasma gondii Douglas A. Pace1, Silvia N.J. Moreno2 and Sebastian Lourido3,4 1

Department of Biological Sciences, California State University Long Beach, Long Beach, CA, United States 2Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, United States 3Whitehead Institute for Biomedical Research, Cambridge, MA, United States 4Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States

12.1 Introduction Calcium ions (Ca21) are universal signaling molecules that participate in many cellular functions in all eukaryotes. Fluctuations in the cytosolic Ca21 concentration are highly regulated. An increase in cytoplasmic Ca21 is immediately followed by a regulatory phase that brings the ion concentrations back to normal basal levels to avoid the cellular damage caused by high cytosolic Ca21 concentrations. Perturbations of these homeostatic processes cause many prominent diseases in humans, such as heart failure or neuronal death. In Toxoplasma gondii, Ca21 plays a critical role in several vital functions including host cell invasion, motility, differentiation, and egress. The

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00012-8

cytosolic concentration of Ca21 is maintained at B70 nM—likely the result of the concerted operation of transporters at the plasma membrane and several intracellular stores. The endoplasmic reticulum (ER), the plant-like vacuole (PLV) [or vacuolar compartment (VAC)], and the acidocalcisomes have been identified as major Ca21 stores. Other potential calcium storage organelles include the Golgi and the mitochondrion. Although many of the molecular players involved in the control and regulation of T. gondii intracellular calcium are unknown, we will summarize the information available about the specialized systems for uptake and release of Ca21 in T. gondii and their consequences on parasite biology. We will also analyze the available methods to study Ca21 regulation in T. gondii.

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12. Calcium storage and homeostasis in Toxoplasma gondii

12.2 Fluorescent methods to study calcium in Toxoplasma Methods for intracellular Ca21 measurements have vastly advanced over the last 5 years. A number of new probes (chemical and genetic) have been created (Table 12.1), each with unique attributes lending to their use for different biological questions. Several research groups have contributed to a remarkable expansion of genetic Ca21 probes, many conveniently available from Addgene (addgene. com). Interestingly, the use of the chemical probe Fura-2 still remains the gold-standard for intracellular Ca21 quantification, largely due to its ability to provide quantitative Ca21 flux measurements (i.e., nM changes) (Moreno and Zhong, 1996; Pace et al., 2014). Fura-2, as well as other highly sensitive fluorescent probes used to study Ca21, was developed by Roger Tsien (Grynkiewicz et al., 1985; Tsien, 1989). There are other methodologies for measuring intracellular Ca21 [Ca21]i, such as 45 Ca21 and microelectrodes; however, the most widely used methods for Toxoplasma rely on fluorescent probes, both chemical and genetic.

12.2.1 Probes for measuring calcium in Toxoplasma gondii One of the most accessible techniques to measure cytosolic Ca21 fluctuations is the use of chemical fluorescent probes that exhibit significant changes in spectral properties upon Ca21 binding. These spectral shifts can be monitored and quantified in real time. In general, these probes possess a modular design in which there is a calcium-binding site—similar in structure to ethylene glycol-bis(β-aminoethyl ether)-N,N,N0 , N0 -tetraacetic acid (EGTA)—combined with a fluorescence indicator. There has been a rapid evolution of fluorescent probes over the past two decades, and there is now a wide range of commercially available probes for a variety of

purposes (Lock et al., 2015). It is important to keep in mind that there is no single probe that will be appropriate for all types of experiments. The selection of a fluorescent probe depends on a number of factors, including the wavelengths to be used, cell permeability, cellular compartment of interest, regulatory timescale of interest, and concentration range of Ca21 to be measured. The work of Tsien et al. resulted in the first generation of highly sensitive fluorescent probes that could be used to measure changes in cellular Ca21 concentrations (Tsien, 1989). Fluorescent probes have very low background light emission due to their wavelength specificity and exhibit a dramatic change in fluorescence upon calcium binding. Perhaps the most important development in fluorescent probes was the addition of an acetoxymethyl (AM) ester group to the indicators. This modification allows fluorescent probes to freely diffuse through the plasma membrane due to the masking of charged portions by the AM group (Bruton et al., 2012). Upon diffusing through the plasma membrane, the AM group is cleaved off by cytosolic esterases, effectively trapping the probe in the cytosol (Tsien, 1983). A significant issue when using AM probes is their propensity to become “compartmentalized” within organelles—the probes can diffuse into organelles before being deesterified. In addition, the cell impermeant version can become enriched in certain organelles or expelled from the cytosol through organicanion channels (Tsien, 1989). To overcome these issues, it is recommended to shorten loading times and to use the minimum effective concentration of fluorescent probe; this has the additional benefit of decreasing interference with calcium signaling due to the buffering effects of the probe binding free Ca21. The use of organic-anion channel blockers is also recommended to inhibit the translocation of the probes to organellar compartments (Tsien, 1989). The organic-anion channel blocker probenecid has been shown to be specific and

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TABLE 12.1

Ca21 indicators, characteristics, and their potential use for Toxoplasma gondii calcium measurements.

Indicator

Kd

Excitation/emissiona,* Notes

References

Fura-2

145 nM

Ex 5 380/340

Ratiometric

Moreno and Zhong (1996), Carruthers et al. (1999b), Vieira and Moreno (2000), Pace et al. (2014)

Indo-1

230 nM

Ratiometric

Pingret et al. (1996), Stommel et al. (1997)

Ratiometric. Lower affinity

1

Ratiometric. New probe from AAT bioquest more compatible with commons filter sets

1

Nonratiometric

1 ,11

Nonratiometric

1

Nonratiometric

Lovett and Sibley (2003), Wetzel et al. (2004)

Enhanced brightness. Easier loading

11 1 , Lock et al. (2015)

Enhanced brightness and sensitivity. Consistent loading and localization

11 1 , Lock et al. (2015)

Improved intracellular retention

11 1 , Lock et al. (2015)

Low sensitivity. Ideal for intracellular stores

1111

Low sensitivity. Ideal for intracellular stores

1111

Em 5 510 Ex 5 338 Em 5 485/405 Fura-FF

5.5 μM

Fura-8

260 nM

Ex 5 380/360 Em 5 505 Ex 5 415/354 Em 5 530 nm

Calcium Green-1

190 nM

Rhod-2

570 nM

Ex 5 497 Em 5 516 Ex 5 552 Em 5 581

Fluo-4

345 nM

Fluo-8

389 nM

Ex 5 494 Em 5 516 Ex 5 494 Em 5 517

Cal520

320 nM

Ex 5 492 Em 5 514

Calbryte520 1.2 μM

Ex 5 4 92 Em 5 514

Mag-Fura-2 25 μM

Ex 5 378/336 Em 5 503

Mag-Fluo4

22 μM

Ex 5 494 Em 5 516

a

Information for excitation and emission was taken from the AAT bioquest website: https://www.aatbio.com.

* Em: emission wavelength, Ex: excitation wavelength. Dual wavelengths for ratiometric probes are listed with regards to calcium-binding state (unbound/bound). 1 No published studies in Toxoplasma using these indicators. We have tested the discontinued Fura-5F and it loads fine and it is good to look at changes in cytosolic Ca2 1 with strong ionophores like ionomycin (unpublished). 11 Published study with Trypanosoma brucei (Huang et al., 2013). We have used Calcium Green-5N successfully in our lab on T. gondii using permeabilization strategies (unpublished results). 11 1 Superior performance to Fluo3 and Fluo4 for imaging and plate reader measurements. They are compared in Lock et al. (2015). 1111 Both of these indicators work for Toxoplasma measurements of organellar Ca2 1 (Li et al., unpublished).

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effective in reducing the sequestration of probe into organelles or leakage out of the cell (Di Virgilio et al., 1988). Calcium probes can be categorized as either nonratiometric (single wavelength) or ratiometric. Nonratiometric probes exhibit an increase in fluorescence intensity upon calcium-binding with negligible spectral shift. Calcium binding by ratiometric probes shifts their emission or excitation spectra, such that an intensity ratio can be calculated from the fluorescence emission at either of two excitation wavelengths (e.g., Fura-2-AM), or at two emission wavelengths following excitation with a single wavelength (Indo-1-AM). The ratio is calculated at wavelengths for which the difference of fluorescence between bound and free indicator is maximum. A widely used ratiometric probe is Fura-2 which can be excited at 340 or 380 nm while monitoring the emission at 510 nm. When Fura-2 is unbound to Ca21, its maximum fluorescence occurs when excited at 380 nm; however, the Ca21 bound state of Fura-2 has a maximum fluorescence at 340 nm. The primary advantage of ratiometric probes is that factors such as differences in cell density, cell volume, loading variability, photobleaching, and differences in path-length can be internally corrected (Bright et al., 1987). Given the batch-to-batch variability in cytosolic probe concentrations encountered when loading T. gondii tachyzoites with Fura-2-AM, the internal correction is useful when comparing different samples. Importantly, following calibration, the proportion of bound to unbound probe can be converted into relevant units of concentration (Grynkiewicz et al., 1985). Both nonratiometric and ratiometric probes can be purchased through commercial vendors (Thermo Fisher, AAT Bioquest, and other companies) and are available as cell-impermeable (non-AM ester) and cell-permeable (AM ester) versions (Tsien, 1981). Cell-impermeable versions of calcium probes are typically used when measuring

calcium in semipermeabilized cells (e.g., digitonin treatment) or when calcium needs to be monitored in the extracellular environment. Table 12.1 shows a list of chemical calcium probes that have been or could be used for T. gondii research and their relevant references. For a more complete comparison of available calcium probes, see Takahashi et al. (1999). The dissociation constant (Kd) of calcium probes (whether nonratiometric or ratiometric) should be similar to the concentration of calcium in the system being measured. Because the cytosolic calcium of T. gondii is B50100 nM (Moreno and Zhong, 1996), Fura-2, with a Kd of 140 nM, represents a good choice. However, dramatic calcium responses, such as those evoked by the calcium ionophores ionomycin or A23187, can result in cytosolic calcium concentrations in excess of 1 μM, well beyond the saturation point of the probe. High-affinity calcium probes such as Fura-2 also suffer from slow release times of bound calcium, an important consideration if calcium transients (i.e., waves and spikes) need to be studied. An option is to use probes such as Fura-5F (Kd of 400 nM), which is able to quantify higher concentrations of calcium and has the temporal resolution necessary to capture calcium transients. The ideal fluorescent indicator for studying Ca21 signals would have low basal fluorescence and a large change in fluorescence intensity in response to small changes of Ca21. In addition, it should have fast Ca21 binding and dissociation rates so they can be used to study transient changes in Ca21. A variety of new chemical indicators especially suitable for imaging have been created, and the majority of them have not been tested in Toxoplasma. A recent study compared the performance of these probes in human cells (Lock et al., 2015). In this regard, there are many probes available that have visible emission/excitation wavelengths in a variety of colors, making them useful for dual labeling studies (see AAT Bioquest https://www.aatbio.com/).

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12.2 Fluorescent methods to study calcium in Toxoplasma

Ratiometric probes, on the other hand, require specialized setups for rapid switching of the two wavelengths in use as well as a way of quantifying fluorescent signal. Ratiometric probes are most useful in plate-reader formats or large fluorometers using cuvettes and cell suspensions (e.g., Hitachi F-7000 or 7100). In this format the lower fluorescent signals are easily quantified and calibration is straightforward, resulting in real-time analysis of calcium regulatory events. From a biological standpoint, measurements using probes such as Fura-2 can be problematic due to their excitation wavelengths, which are in the UV range and can cause cellular damage. While longer wavelength probes such as Fluo-4 avoid this issue, there are still instances when visible wavelength excitation probes result in high background fluorescence (Mbatia and Burdette, 2012). It is always advisable to check for potential cytotoxicity when testing new probes and find optimal loading and washing conditions. For example, we tested the long wavelength (visible), nonratiometric probe calcium crimson. Although this probe should avoid the pitfalls of near-UV cytotoxicity, we found that it was toxic to parasites and therefore not useful for observing real-time calcium regulation. Fura-2-AM loaded cells have been used to study intracellular calcium stores in T. gondii (Moreno and Zhong, 1996) and Ca21 influx (Pace et al., 2014). In addition, Indo-1 was used to analyze the distribution of calcium in mammalian cells infected with T. gondii (Pingret et al., 1996) and to assess the effects of dithiols on such infected cells (Stommel et al., 2001). By contrast, Fluo-4 has been used to detect Ca21 changes induced by motility or host cell attachment, as its emission wavelength and intensity are more compatible with live video microscopy (Lovett and Sibley, 2003). For T. gondii the general protocol in our laboratories is to load with 5 μM of the ratiometric fluorescent probe, Fura-2-AM. We load the

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parasites for 26 minutes at 26 C and do not add probenecid (Pace et al., 2014). In addition, we keep the parasite final suspension on ice until their use for fluorescent measurements as this helps slow down the compartmentalization of the probe through organic-anion channels.

12.2.2 Ca21 buffers EGTA is highly selective for binding Ca21 over Mg21, and because of this, it is the most commonly used Ca21 buffer. However, the Ca21-binding activity of EGTA is pH dependent. When used at physiological pH, EGTA exists primarily as a protonated species (H2EGTA22) (highest pKas 8.90 and 9.52), releasing two H1 as a result of Ca21 binding and making the reaction pH dependent. A drop in pH from 7.2 to 7.1 changes the Kd(Ca21) of EGTA by 1.6-fold. Tsien developed an analog of EGTA in which the methylene links between oxygen and nitrogen atoms were replaced with benzene rings to yield a compound called 1,2-bis(o-aminophenoxy)ethaneN,N,N0 ,N0 -tetraacetic acid (BAPTA) (Tsien, 1980). This compound is considerably less pH sensitive than EGTA at physiological pH values since its highest pKas are 5.47 and 6.36 (Tsien, 1980). This characteristic makes BAPTA a less troublesome Ca21 buffer, although it can also display side effects. BAPTA has a potent microtubule depolymerizing effect and decreases the ATP pool of the cells (Saoudi et al., 2004). To study the correlation of a biological process with changes in intracellular calcium concentration ([Ca21]i), it is useful to be able to block the change in [Ca21]i with a calcium chelator. An easy approach for buffering intracellular Ca21 is by loading them with BAPTAAM, the ester form of BAPTA (Vieira and Moreno, 2000). Cells can be loaded with AM esters of BAPTA and a calcium indicator simultaneously since the same conditions can

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12. Calcium storage and homeostasis in Toxoplasma gondii

be used (Kao, 1994). Once cells are loaded with BAPTA-AM, it is important to show that it chelates intracellular Ca21, and this is shown by simultaneous loading with Fura-2-AM and performing measurements of intracellular Ca21. Addition of ionophores and inhibitors should result in dampened Ca21 responses (Vieira and Moreno, 2000). This BAPTA buffering method has been widely used to understand the role of calcium in microneme secretion by T. gondii (Carruthers et al., 1999a), conoid extrusion (Mondragon and Frixione, 1996), gliding motility (Wetzel et al., 2004), and invasion (Vieira and Moreno, 2000). Another option is to use EGTA-AM, the AM ester derivative of EGTA, which can be passively loaded into cells to generate intracellular EGTA. The slower on-rate of EGTA relative to BAPTA reduces its ability to inhibit Ca21 diffusion in cells (Dargan and Parker, 2003). The ionophores Br-A23187 and ionomycin form lipid-soluble complexes with divalent metal cations and increase the permeability of biological membranes to Ca21. There are significant differences in the properties of both ionophores. The ability of Ca21 transport by both ionophores is pH dependent, and this pH dependence differs (Liu and Hermann, 1978). Transport of Ca21 by Br-A23187 is best at pH 7.5, whereas Ca21 transport by ionomycin does not reach a maximum until pH 9.5. In addition, ionomycin has better selectivity for Ca21 over Mg21 whereas Br-A23187 shows no preference for one cation over the other (Liu and Hermann, 1978). Both ionophores are inefficient in mediating Ca21 transport at low Ca21 concentrations. Br-A23187 should be used instead of A23187 for experiments involving fluorescence since A23187 is fluorescent. Fig. 12.1 shows a typical tracing with Fura 2-loaded T. gondii tachyzoites in suspension in a buffer containing 1 mM EGTA. Under these conditions, fluorescent changes reflect Ca21 movements in the cytosol resulting from leakage/release from intracellular calcium stores.

FIGURE 12.1 Intracellular Ca21 response to TG and ionomycin (IO). Cytosolic Ca21 changes of Fura-2AMloaded tachyzoites upon addition of TG and IO, both at 1 μM. The effect of ionomycin is noticeably larger than the effect of TG. Parasites were loaded with Fura-2/AM as described in Pace et al. (2014). A 50 μL aliquot of tachyzoite suspension was diluted into a 2.5 mL of a reaction buffer in a cuvette placed in a Hitachi F-7000 spectrofluorometer. Excitation was at 340 and 380 nm, and emission was at 510 nm. The Fura-2 fluorescence relationship to Ca21 concentration was calibrated from the ratio of 340/380-nm fluorescence values after subtraction of the background fluorescence of the cells at 340 and 380 nm as described by Grynkiewicz et al. AM, Acetoxymethyl; IO, ionomycin; TG, thapsigargin. Source: Reproduced from Figure 2C of Pace, D. A., Mcknight, C.A., Liu, J., Jimenez, V., Moreno, S.N., 2014. Calcium entry in Toxoplasma gondii and its enhancing effect of invasion-linked traits. J. Biol. Chem. 289, 1963719647.

Addition of 1 μM thapsigargin (TG) results in cytosolic increase of Ca21 likely due to efflux from the ER. TG is a known inhibitor of the SERCA Ca21 ATPase, a Ca21 pump that localizes to the ER of Toxoplasma (Nagamune et al., 2007), and that continuously pumps Ca21 into the ER and its inhibition results in unsupervised efflux of Ca21 into the cytosol. Addition of ionomycin shows a large increase in intracellular calcium indicating that calcium is released from an intracellular compartment with neutral pH into the cytosol (likely the ER).

12.2.3 Genetic indicators Genetically encoded calcium indicators (GECIs) are powerful tools for the study of

Toxoplasma Gondii

12.2 Fluorescent methods to study calcium in Toxoplasma

Ca21 signaling, and in the last decade, they have significantly increased their performance to match those of organic chemical probes (McCombs and Palmer, 2008; Tian et al., 2009, 2012, Zhao et al., 2011; Akerboom et al., 2013). GECIs enable noninvasive imaging of defined cells and compartments, while loading cells with chemical probes may result in organellar compartmentalization and leakage from the cytosol, and/or toxic side effects (as previously discussed). GECIs can be targeted to various subcellular compartments and combined with high-resolution microscopy, making it possible to selectively monitor the dynamics of Ca21 with unprecedented spatial resolution. Stateof-the-art GECIs include the single-wavelength intensiometric (the intensity of the fluorescence increases proportionally to Ca21 increase) GCaMP’s, which are based on circularly permuted green fluorescent protein (cpGFP), calmodulin (CaM), and the Ca21/CaM-binding “M13” peptide (M13pep) and Fo¨rster resonance energy transfer (FRET)-based indicators, which are ratiometric and require dual-channel recording (Suzuki et al., 2016; Tian et al., 2012). Singlewavelength intensiometric GECIs have a larger dynamic range and thus tend to be preferred over FRET-based indicators. In mammalian cells a large number of probes have been produced for a variety of uses, including targeting to the ER, nucleus, mitochondria, Golgi, and endosome/peroxisomes (Suzuki et al., 2016). The GCaMP6 series are highly sensitive probes with three variants available that differ in their kinetics (Chen et al., 2013) (Table 12.2). GCaMP6s has the slowest kinetics, GCaMP6m is faster while maintaining a high response, and GCaMP6f is the fastest variant (Chen et al., 2013). The specific characteristics of these indicators have been recently reviewed (Deo and Lavis, 2018). Multicolor GECIs are also available such as the red-shifted variants jRGECO1a, jRCaMP1a/b, RCaMP, R-GECO, OGECO, and CAR-GECO and the blue-shifted variants such as B-GECO and GEM-GECO

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(Zhao et al., 2011; Akerboom et al., 2013). Red indicators are of particular interest because red fluorescence is absorbed much less than green fluorescence, especially in mammalian cells, and the recently developed jGECO1a has favorable kinetics to match GCaMP6s, giving red fluorophores a competitive advantage for in vivo imaging (Dana et al., 2016). GECIs combined with the genetic tractability of Toxoplasma have impacted the field of calcium signaling in these parasites, and we can now study Ca21 signaling during each step of the lytic cycle—egress, extracellular motility, invasion, and replication. In Toxoplasma, Ca21 signaling results in the stimulation of gliding motility, microneme secretion, conoid extrusion, invasion, and egress. Many studies of Ca21 signaling during the lytic cycle of T. gondii were done indirectly by loading extracellular parasites with fluorescent dyes to follow Ca21 changes during their gliding motility; through using Ca21 ionophores and other exogenous agents to elevate Ca21 in extracellular parasites and stimulate conoid extrusion or microneme secretion; or using intracellular or extracellular Ca21 chelators to prevent host cell invasion or egress. The potential use of GECIs across a wide diversity of applications is still beginning to emerge, and some pioneer studies have utilized GECIs in high-throughput screens of compounds that disrupt Ca21 signaling in both mammalian cells and parasites (Bassett and Monteith, 2017; Sidik et al., 2016). Recent studies with T. gondii tachyzoites expressing GECIs in the cytosol characterized Ca21 oscillations patterns during motility, and a direct correlation between distance traveled and amplitude of the oscillations was found (Borges-Pereira et al., 2015; Stewart et al., 2017). These findings indicated that Ca21 influx channels and reuptake mechanisms are highly active in gliding parasites and necessary for effective lytic cycle event (Lovett and Sibley, 2003; Borges-Pereira et al., 2015; Stewart et al., 2017). Efficient gliding required Ca21 oscillations, as

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TABLE 12.2 Genetically encoded Ca21 indicators and their use in Toxoplasma.a GECIs

Kd for Ca21

GCaMP3

542 nM

Dynamic range (Fmax/Fmin) References 13.5

Comments

Tian et al. (2012)

Improved dynamic range over first generation

Borges-Pereira et al. (2015) GCaMP5G

460 nM

32.7

Akerboom et al. (2012)

Improved dynamic range over GCaMP3

Sidik et al. (2016) R-GECO

482 nM

16.0

Zhao et al. (2011)

First generation red GECI

Borges-Pereira et al. (2015) B-GECO

164 nM

7

Zhao et al. (2011)

First generation blue GECI

Borges-Pereira et al. (2015) GCaMP6f

375 nM

51.8

Chen et al. (2013)

Fast on/off kinetics

Borges-Pereira et al. (2015) Brown et al. (2016) GCaMP6s

144 nM

63.2

Chen et al. (2013)

Slow on/off kinetics

Stewart et al. (2017) Kuchipudi et al. (2016) LAR-GECO1

24 μM

10

Wu et al. (2014)

Low affinity, endoplasmic reticulum targeted

LARGECO1.2

12 μM

8.7

Wu et al. (2014)

Low affinity, Mitochondria targeted

jRGECO1a

148 nM

11.6

Dana et al. (2016)

Second generation variant of R-GECO

jRCaMP1a

214 nM

3.2

Dana et al. (2016)

Second generation red GECI

jRCaMP1b

712 nM

7.2

Dana et al. (2016)

Second generation red GECI

a This table was reproduced from Vella et al. (2019). GECI, Genetically encoded calcium indicator.

opposed to simply prolonged, increased cytosolic Ca21 concentrations (Wetzel et al., 2004). It is possible that Ca21 oscillations enable the parasite to traverse longer distances over a longer period of time, via coordinating the release of micronemes only when necessary instead of constitutively (Borges-Pereira et al., 2015; Stewart et al., 2017). It was known for several years that Ca21 ionophores could trigger egress (Endo et al., 1982), but the use of cytoplasmic GECIs

showed conclusively that there was a cytosolic Ca21 increase right before egress (BorgesPereira et al., 2015). The use of these probes has vastly impacted the studies of Ca21 in intracellular parasites (Sidik et al., 2016; Kuchipudi et al., 2016; Stewart et al., 2017). Initiation of parasite motility results in mechanical pressure and final rupture of the host cell allowing the release of the parasites. The presence of extracellular Ca21 also affected the rate of egress that was blocked with the

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12.3 Regulation of [Ca2 1 ]i in Toxoplasma gondii

Ca21-channel blocker nifedipine Pereira et al., 2015). Despite all advances in Ca21 biology, the trigger ates Ca21 and subsequent parasite still a mystery.

(Borgesof these that initiegress is

12.3 Regulation of [Ca21]i in Toxoplasma gondii Discovery of Ca21 regulation strategies and their respective molecular mechanisms in Toxoplasma has benefited from the extensive literature in mammalian Ca21 regulation. Searches for homologous elements have identified some commonalities between Toxoplasma and the cells it parasitizes. However, the evolutionary distance between apicomplexan parasites and its mammalian host has also resulted in the identification of novel genes and characterization of regulatory pathways that have yet to be attributed to a specific set of genes in Toxoplasma. These represent novel therapeutic drug targets that may not overlap with host cell biological functions. The [Ca21]i in tachyzoites is about 70 6 6 nM when measured in the absence of extracellular Ca21 (with the Ca21 chelator EGTA added to the medium) and 100 6 9 nM in the presence of 1 mM extracellular Ca21, as detected in Fura-2loaded cells (Moreno and Zhong, 1996). These concentrations are in the range observed in many studies with eukaryotic cells (Grynkiewicz et al., 1985).

12.3.1 Ca21 transport across the plasma membrane As previously defined, the critical role of intracellular Ca21 stores in initiating parasite motility, adhesion, invasion, and egress has been well established (Wetzel et al., 2004; Carruthers et al., 1999b; Mondragon and Frixione, 1996; Arrizabalaga and Boothroyd, 2004; Lovett and Sibley, 2003). Whereas studies

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using the cell-permeable calcium chelator, BAPTA-AM, clearly demonstrated the immediate use of intracellularly stored Ca21 to initiate invasion-linked events, they could not address the relevance of the extracellular reservoir of Ca21 available to the parasite. This reservoir represents a near-infinite source of Ca21 and has a strong electrochemical gradient favoring its entry into the parasite. This extracellular source of Ca21 does provide a mechanism for enhancing invasion-linked traits and likely plays a long-term role in keeping intracellular stores fully loaded (Pace et al., 2014). Specifically, extracellular Ca21 increased motility (number of parasites and length traveled by each parasite), microneme secretion, conoid extrusion (number of elevated conoids and duration of extension), and invasion rate. While the addition of extracellular Ca21 was sufficient to increase these metrics, the pretreatment with an agent, such as the SERCA inhibitor, TG, resulted in a significant elevation of these responses. Real-time measurements of intracellular Ca21 concentration (using Fura 2-AM, as defined in this chapter) provided a mechanistic explanation for these observations. TG treatment resulted in an influx of Ca21 to the cytosol from the parasite ER. This resulted in an increased influx of Ca21 from the extracellular environment. As this effect of store emptying on extracellular Ca21 influx could not be reciprocated using the Ca21 analogs of manganese or barium in conjunction with TG, the phenomenon of store-operated calcium entry (SOCE) was eliminated as a possible pathway. In addition, the genetic components of the SOCE pathway, STIM and ORAI, are absent from the T. gondii genome (Prole and Taylor, 2011). It appears that the enhanced entry of Ca21 from the extracellular environment is a function of increased intracellular Ca21 concentration. Given that the influx of Ca21, as well as the enhancement of invasion-linked events, was nifedipine-sensitive (Pace et al., 2014), the entry

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pathway is likely through a voltage-gated calcium channel (VGCC). While the T. gondii genome encodes for two proteins with similarity to VGCCs (Nagamune and Sibley, 2006), their expression and importance awaits confirmation and characterization, respectively. It is likely that the large electrochemical gradient favoring Ca21 entry, coupled with the parasite’s need to keep intracellular stores full, makes the extracellular reservoir critical to the long-term invasion/egress capacity of the parasite as it repeats the lytic cycle dynamics during acute infection. The development of GECIs in T. gondii tachyzoites has allowed for greater observational power of Ca21 dynamics during the lytic cycle (Borges-Pereira et al., 2015; Sidik et al., 2016). Importantly, GECIs have been very useful for understanding Ca21 oscillations, simultaneously, in intracellular parasites, and the host cells where they reside. Such research has further established the importance of extracellular Ca21 in supporting the requisite lytic cycle dynamics of T. gondii. Research where both host cells and parasites were expressing color-specific GECIs determined that, upon ionomycin treatment, the availability of extracellular calcium resulted in greater parasite calcium spikes and faster egress time relative to controls where no extracellular Ca21 was provided. This enhancement of egress with extracellular calcium was negated with the use of nifedipine. Examination of extracellular tachyzoites, also expressing GCaMP6f, likewise confirmed the enhancement of parasite invasion in a Ca21 replete environment. Parasites in a physiological concentration of Ca21 (2 mM) exhibited more Ca21 spikes and invasion events relative to parasites in a calcium deplete extracellular environment (Borges-Pereira et al., 2015). Collectively, these results indicate that the extracellular environment provides enhancement of lytic cycle dynamics. A plausible model regarding the role of the extracellularly available Ca21 is that it can serve as an

immediate enhancer of motility and invasion for extracellular parasites and allows rapid replenishment of intracellular stores that are subsequently mobilized for host cell egress (Fig. 12.2) (Pace et al., 2014). Elucidation of the putative L-type calcium channel(s) that serves as the mechanism of calcium influx to the parasite may provide an important avenue for therapeutic treatment of infection.

12.3.2 Calcium storage 12.3.2.1 Endoplasmic reticulum The largest store of Ca21 in cells is usually found in the ER, with the concentration reaching millimolar levels. In order to concentrate Ca21 ions, the ER utilizes a SERCA-Ca21ATPase, a transmembrane P-type ATPase, that couples ATP hydrolysis to the transport of ions across biological membranes and against a concentration gradient (Fig. 12.2, homeostasis). SERCA pumps can be inhibited by various inhibitors including the very potent and highly specific TG, a sesquiterpene lactone derived from the plant Thapsia garganica (Sagara and Inesi, 1991; Thastrup et al., 1990). Inhibition of SERCA results in cytosolic Ca21 increase due to leakage from the ER through an unknown pathway. Ca21 leakage is the passive Ca21 efflux from the ER that is thought to prevent ER Ca21 overload and thus allow cytosolic Ca21 signaling (Clapham, 2007). Evidence for the presence of a SERCA-type Ca21-ATPase in T. gondii was first provided by experiments using Fura-2loaded tachyzoites in which TG was shown to increase [Ca21]i in tachyzoites (Moreno and Zhong, 1996) due to Ca21 efflux from the ER. Molecular evidence for the presence of a SERCA-type Ca21-ATPase in T. gondii has also been reported (Nagamune et al., 2007). The gene encoding this pump was able to complement yeast deficient in Ca21 pumps providing evidence of its function as a Ca21 pump, and the encoded protein has an

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12.3 Regulation of [Ca2 1 ]i in Toxoplasma gondii

557

FIGURE 12.2 Ca21 homeostasis and signaling in Toxoplasma gondii Schematic representation of the distribution of Ca21 in a Toxoplasma gondii tachyzoite. Ca21 entry is likely through a Ca21 channel. Once inside the cells, Ca21 can be pumped out to the extracellular milieu, primarily by the action of a PMCA. In addition, Ca21 can become sequestered by the ER by the action of the SERCA Ca21-ATPase, sequestered by the AC or the PLV by the action of a Ca21-ATPase (TgA1). An increase of cytosolic Ca21 during a signaling event could be the result of Ca21 influx or could be due to the release from the ER through an uncharacterized channel by the action of IP3 (generated from PIP2 by a PLC) or cADPribose (generated from NAD1 by an ADP ribosyl cyclase). Downstream to Ca21, the ion may bind a diversity of CBPs. CDPKs are stimulated by Ca21 and active CDPKs can phosphorylate protein substrates that are involved in gliding motility and microneme secretion, which is also stimulated by DOC2. AC, Acidocalcisome; CBP, calcium-binding protein; CDPK, calcium-dependent protein kinase; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PMCA, plasma membrane Ca21-ATPase; PLV, plant-like vacuole. Source: Reproduced from Figure 1 of Lourido, S., Moreno, S.N., 2015. The calcium signaling toolkit of the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell Calcium 57, 186193, with permission (Lourido and Moreno, 2015).

apparent molecular mass of 120 kDa. Inhibitors of the pump, such as TG (Thastrup et al., 1990), and artemisinin (Eckstein-Ludwig et al., 2003) were able to stimulate microneme secretion, a process that relies on elevated [Ca21]i. The steady-state concentration of ER luminal Ca21 is thought to be controlled by the presence of leak channels in the membrane of the ER of mammalian cells (Guerrero-Hernandez et al., 2010). Several types of membrane proteins have been proposed to be involved in the ER calcium leak pathway, Bcl-2, pannexin 1,

presenilins, and TRPC1. An active ER Ca21 release channel, termed transmembrane and coiled-coil domains 1 (TMCO1) or Ca21 loadactivated Ca21 channel, has been shown to assemble as a homotetramer upon ER Ca21 overload (Wang et al., 2016b). A small portion of TMCO1 exists as homotetramer even under normal levels of ER Ca21, which could also cause passive leak of Ca21 from the ER. The Toxoplasma genome contains ortholog genes for a presenilin (TGME49_204040) and two TRP channels (TGME49_247370 and

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12. Calcium storage and homeostasis in Toxoplasma gondii

TGME49_310560) (Prole and Taylor, 2011). These Toxoplasma molecules have not been characterized except for a recent study using high-affinity tags, which localized TGME49_247370 to the ER (Hortua Triana et al., 2018). The genome of Toxoplasma shows evidence for the presence of a TMCO1 ortholog (TGME49_310870), predicted to have a signal peptide at its N terminus. This Toxoplasma gene has not been characterized. In vertebrate cells binding of a ligand to a surface receptor such as a G proteincoupled receptor (GPCR) may result in the stimulation of the phosphoinositide-signaling pathway via the activation of a phosphatidylinositol phospholipase C (PI-PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Binding of IP3 to an IP3 receptor usually present in the membrane of the ER will result in its opening allowing Ca21 to be released into the cytosol (Berridge, 2009). A diversity of cell signaling pathways can be generated through this mechanism (Berridge, 2009). Cyclic ADP ribose (cADPR) is also able to release intracellular Ca21 acting through ryanodine receptors usually located in the ER (Lee, 2011). The T. gondii PI-PLC (TgPIPLC) was cloned and expressed in Escherichia coli and its enzymatic characteristics were investigated (Fang et al., 2006). Characterization of conditional mutants of the TgPIPLC gene showed its essentiality for parasite survival and its participation in a vast and currently incompletely understood signaling cascade (Bullen et al., 2016). It is not known which is the target of the IP3 generated by TgPIPLC because there is no genomic support for the presence of IP3 receptors in any apicomplexan parasite (Garcia et al., 2017). However, IP3 stimulates Ca21 release from isolated microsomes of T. gondii and cADPR does as well, presumably by stimulating a ryanodinetype receptor (Chini et al., 2005). Ca21 release by IP3 is inhibited by the IP3 receptor inhibitor

xestospongin C (Chini et al., 2005), which also inhibited secretion of micronemes, secretory organelles involved in host invasion (Lovett et al., 2002). Treatment with ethanol increased IP3 and [Ca21]i, and this pathway was sensitive to inhibitors of IP3R channels. Evidence for the presence of cADPR cyclase and hydrolase activities, the two enzymes that control cADPR levels was found in Toxoplasma (Chini et al., 2005). 12.3.2.2 Mitochondria Mammalian mitochondria possess a high capacity to sequester Ca21, although under physiologic conditions total mitochondrial Ca21 levels and free Ca21 parallel cytosolic Ca21. Transfer of Ca21 from the ER to the mitochondria, which occurs through membrane contact sites, is important for the regulation of the cell bioenergetics since Ca21 is known to provide reducing equivalents to support oxidative phosphorylation (Cardenas et al., 2010) through activation of three intramitochondrial dehydrogenases (Denton and McCormack, 1990; Hajnoczky et al., 1995) and the ATP synthase (Balaban, 2009). The inner mitochondrial membrane possesses a uniport carrier for Ca21, which allows the electrogenic entry of the cation driven by the electrochemical gradient generated by respiration or ATP hydrolysis. Calcium efflux, on the other hand, takes place by a different pathway, which appears to catalyze the electroneutral exchange of internal calcium with external sodium or protons. The role of the mitochondrion in Ca21 homeostasis and signaling is an untapped research area in Toxoplasma. Genomic evidence does not support the presence of any of the uniporter subunits in Toxoplasma or any apicomplexan parasite. Unlike mammalian mitochondria, where intracellular Ca21 regulates the activity of several dehydrogenases, no such Ca21-regulated dehydrogenases have been reported in T. gondii.

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12.3 Regulation of [Ca2 1 ]i in Toxoplasma gondii

12.3.2.3 Acidocalcisomes T. gondii tachyzoites possess a significant amount of Ca21 stored in an acidic compartment termed acidocalcisomes, that were first described in Trypanosoma brucei (Vercesi et al., 1994) and Trypanosoma cruzi (Docampo et al., 2005). These organelles are characterized by their acidic nature, high density [both in mass and by electron microscopy (EM)], and high content of pyrophosphate, polyphosphate (polyP), calcium, magnesium, and other elements (Docampo et al., 2005; Rodrigues et al., 2002). Acidocalcisomes are similar to the volutin or metachromatic granules first described almost a hundred years ago (Kunze, 1907) in Coccidia, and detected in 1966 in T. gondii for their ability to stain red when treated with toluidine blue (metachromasia) (Mira Gutierrez and Del Rey Calero, 1966). They were also named “black granules” (Bonhomme et al., 1993) and studies on Leishmania (Besteiro et al., 2008) and T. brucei (Huang et al., 2011) have suggested that acidocalcisomes are lysosome-related organelles. A large number of acidocalcisomes can be seen in EM images of whole cells directly dried on Formvar-coated grids (Luo et al., 2001). The advantage of this type of preparation is the observation of the whole parasite without the addition of fixatives or other chemicals used in the routine procedures for transmission EM. This significantly reduces the extraction of material from the acidocalcisomes, allowing the observation of the organelle in its “native” state (Luo et al., 2001). X-ray microanalysis revealed considerable amounts of oxygen, sodium, magnesium, phosphorus, chlorine, potassium, calcium, and zinc concentrated in these compartments (Scott et al., 1997; Rodrigues et al., 1999; Miranda et al., 2000, 2004; LeFurgey et al., 2001). Ca21 uptake by T. gondii acidocalcisomes is likely due to the activity of a Ca21-ATPase (TgA1) closely related to the family of plasma membrane calcium ATPases (Fig. 12.2,

559

homeostasis) (Luo et al., 2001). Ca21 transport activity was observed in enriched acidocalcisome fractions, which was insensitive to TG and sensitive to bafilomycin A1 (Rohloff et al., 2011). Mutants deficient in TgA1 were shown to have altered cytosolic Ca21 levels, deficient invasion of host cells and decreased virulence in vivo (Luo et al., 2005). The acidification of acidocalcisomes is due to the activity of two proton pumps: the T. gondii V-H1-PPase (TgVP1) (Drozdowicz et al., 2003) and a V-H1-ATPase (Rodrigues et al., 2002; Stasic et al., 2019). The TgVP1 gene (TgME49_248670) was cloned and a truncated version (without the N terminus) could be functionally expressed in yeast (Drozdowicz et al., 2003). TgVP1 belongs to the K1-stimulated group of V-H1-PPases (type I), and its biochemical characterization was done using T. gondii purified fractions enriched in the enzyme (Miranda et al., 2010; Rohloff et al., 2011). The activity of the enzyme has been successfully used as a marker for acidocalcisome purification (Rohloff et al., 2011). TgVP1 also localizes to the PLV/VAC (see next). Importantly, genetic knockout of the TgVP1 gene resulted in a significant reduction in parasite virulence (Liu et al., 2014). This observation is probably related to the localization of TgVP1 to not only acidocalcisomes but also the PLV/VAC compartment (see below for description of organelle). The V-H1ATPase was first identified in T. gondii by its sensitivity to bafilomycin A1, a specific inhibitor of this proton pump when used at low concentrations (Bowman et al., 1988). In experiments using intact tachyzoites loaded with the fluorescent Ca21 indicator Fura-2, bafilomycin A1 was able to release Ca21 from an intracellular compartment of T. gondii (Moreno and Zhong, 1996). The V-H1-ATPase was also shown to play a role in intracellular pH homeostasis (Moreno et al., 1998). Experimental evidence, utilizing spectrophotometric measurements with acridine orange, supports the presence of Na1- and

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12. Calcium storage and homeostasis in Toxoplasma gondii

Ca21-proton exchange mechanisms in acidocalcisomes, as well as in the PLV (Rohloff et al., 2011). Subsequent analysis of an annotated Ca1/H1 exchanger (TgME49_207910) demonstrated it was nonessential in T. gondii tachyzoites and exhibited low expression during the intracellular stage (undetectable by immunofluorescent staining) (Guttery et al., 2013). A number of genes have been identified in the genome of T. gondii that could potentially encode additional acidocalcisome transporters, for example, a putative phosphate transporter (TgGT1_240210), two putative chloride channels (TgGT1_26550 and TgGT1_290330), a neutral and basic amino acid transporter (TgGT1_314340), a Zn21 transporter (TgGT1_251630), and Na1/H1 exchangers (TGME49_299060 and TGME49_305180). All acidocalcisomes described so far have been found to have high levels of phosphorus in the form of inorganic pyrophosphate (PPi) and polyphosphate (poly P) (Rodrigues et al., 2000; Moreno et al., 2001). PPi is a byproduct of many biosynthetic reactions (synthesis of nucleic acids, coenzymes, proteins, activation of fatty acids, and isoprenoid synthesis), and its hydrolysis by inorganic pyrophosphatases makes these reactions thermodynamically favorable. The storage of phosphate as poly P reduces the osmotic effect of large pools of this important compound. Short- and long-chain poly P levels also rapidly decreased upon exposure of tachyzoites to agents that mobilize Ca21 such as calcium ionophores (ionomycin), alkalinizing agents (NH4Cl), or inhibitors of the V-H1-ATPase (bafilomycin A1) (Rodrigues et al., 2002). This would suggest a role for poly P in the adaptation of the parasites to environmental stress. 12.3.2.4 Plant-like vacuole/vacuolar compartment The PLV, also named VAC, is a lysosomelike organelle in the tachyzoite stage of T.

gondii visible by light microscopy and with broad functional similarity to the plant vacuole (Miranda et al., 2010; Parussini et al., 2010). This organelle expresses a plant-like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, several cathepsin proteases (TgCPL, TgCPB, others), an aquaporin (TgAQP1), a Na1/H1 exchanger (Francia et al., 2011), a zinc transporter (TgZnT) that appears to also localize to acidocalcisomes (Chasen et al., 2019), a chloroquine-resistance transporter (TgCRT) (Warring et al., 2014; Thornton et al., 2019) as well as Ca21/H1 exchange activity supporting its similarity to the plant vacuole. It is a dynamic structure that undergoes significant changes as the extracellular tachyzoite invades a host cell and begins replication within the parasitophorous vacuole (Parussini et al., 2010). In the extracellular tachyzoite stage the PLV occupies a large portion of the parasite and is multivesicular (Miranda et al., 2010). Physiological experiments in intact cells loaded with the Ca21 indicator Fura-2-AM showed release of Ca21 from an intracellular store stimulated by glycyl-Lphenylalanine-naphthylamide (GPN), which was different from other Ca21 stores such as the ER and acidocalcisomes. GPN is specifically hydrolyzed in the lysosomes of a variety of different cell types by a cathepsin C protease and had been used before to demonstrate the presence of Ca21 in lysosome-like compartments (Haller et al., 1996; Srinivas et al., 2002). This Ca21 release has also been implicated in enhancing calcium entry through the plasma membrane (described in Section 12.3.1). In addition, proton transport measurements in enriched fractions showed Ca21 exchange activity after pyrophosphate-driven acidification. Proteomic data and immunofluorescence assays showed the localization of TgA1 to this organelle (Moreno et al., unpublished observations) (Fig. 12.2, homeostasis). The localization of both TgVP1 and TgA1 to acidic organelles, acidocalcisomes, and PLV supports the

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12.4 Transducing Ca2 1 signals

hypothesis that both organelles interact or that the PLV has a role in the biogenesis of the acidocalcisome. The colocalization of TgAQP1 to both PLV and to acidocalcisomes also supports this hypothesis. The importance of the acidification of the PLV was directly assessed by knocking out TgVP1 (Liu et al., 2014) and one of the subunits of the V-H1-ATPase (TgVha1) (Stasic et al., 2019). While TgVP1 was dispensable (likely because of the overlap with the vacuolarATPase pump), its ablation resulted in reduced virulence in mice. Further analysis demonstrated significant reductions in parasite attachment, invasion, microneme secretion, and osmoregulation. Analysis of cathepsin L (CPL) maturation provided evidence that this PLVlocalized pump is utilized, along with the vacuolar-ATPase pump, in protein processing at the PLV/VAC compartment. Related to these proton-gradient establishing pumps, biochemical experiments of enriched PLV fractions have shown the presence of several proton exchange mechanisms, one of which is a calciumproton exchanger (Ca21/H1 exchanger). Immunofluorescence analysis also shows localization of a Na1/H1 exchanger (Francia et al., 2011). The significance of the PLV as a Ca21 store is still being characterized, but due to its large size and dynamic behavior, it seems likely that the PLV contributes directly to many of the Ca21-related behaviors that have been documented for T. gondii. A recent study characterized the function of the V-H1-ATPase (Stasic et al., 2019), a multisubunit protein complex that couples the hydrolysis of ATP to the pumping of protons across membranes. The Toxoplasma V-H1-ATPase complex localizes to the plasma membrane and to the PLV and characterization of conditional mutants of the a1 subunit (TgVha1) highlighted its function at both locations. The pump protected parasites against ionic stress and also was important for the maturation of secretory proteins (destined for the micronemes and

561

rhoptries) and their maturases in endosomal-like compartments during their traffic to their corresponding organelles (Stasic et al., 2019).

12.4 Transducing Ca21 signals 12.4.1 Calcium-binding proteins Calcium-binding proteins (CBPs) are a heterogenous group of proteins defined by their ability to bind calcium quickly and reversibly. These proteins have been extensively studied in mammals and plants (Yanez et al., 2012; Zeng et al., 2015). Given the importance of calcium signaling pathways, CBPs have many different functions and can be found in any given cellular location (Faas and Mody, 2014). CBPs primarily function as intracellular calcium buffers or as calcium-responsive signaling molecules. Most CBPs bind calcium by way of a highly conserved motif called the EF-hand domain. The EF-hand domain (e.g., InterPro ID: IPR011992) is composed of a 12-residue loop region that is flanked by 12-residue alpha helices. Each loop region of an EF-hand domain binds one Ca21 (Lewit-Bentley and Rety, 2000). In many CBPs the binding of Ca21 to the EF-hand domain results in a conformational change in the overall protein structure resulting in a change of activity state for the protein (i.e., becoming activated or deactivated). Most CBPs have EF-hand domains that occur in pairs or some other product of two and display cooperative Ca21 binding (LewitBentley and Rety, 2000). While the structural similarities and sequence conservation of EFhand domains are relatively high, their Ca21 affinity and function can be fine-tuned. This functional diversity allows CBPs to be both spatially and temporally responsive to calcium oscillations in an appropriate range for the signaling pathway under consideration (Bhattacharya et al., 2004). CaMs, calcineurins, troponin C, and parvalbumin are examples of

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canonical CBPs that are well studied and known to play essential roles in mammalian cells. Analysis of the ME49 strain of T. gondii reveals 69 genes that contain at least one EFhand domain (as defined by a search using the InterPro ID: IPR011992 on ToxoDB.org, release 43), including the family of calcium-dependent protein kinases (CDPKs), discussed in the next section. Orthologs of many well-known CBPs in mammalian cells (e.g., CaMs) can be found in T. gondii. Due to the known role of calciummediated signaling in virulence-linked traits such as motility, invasion, and egress, several CBPs have been characterized in T. gondii. However, this represents only a fraction of the CBPs that are predicted by in silico analysis, and their characterization is limited to the fastreplicating tachyzoite stage. The following is a brief summary of CBPs that have been characterized and their role in the tachyzoite lytic cycle. Members of the CaM family are major regulators of eukaryotic signal transduction. Once bound to calcium, CaM is activated and interacts with other critical signaling elements such as kinases and phosphatases (Chin and Means, 2000). In some cases autophosphorylation can render these kinases CaM-independent for a period of time following activation, providing a form of molecular memory that enables the integration of calcium spikes. Structurally, CaMs have two globular head regions, each with a pair of EF-hand domains, separated by a flexible linker region. This large and diverse family of proteins can be divided into archetypal CaMs, CaM-like proteins (CMLs), and centrins, also known as caltractins. The genome of T. gondii is predicted to contain only one CaM and several CMLs (Nagamune and Sibley, 2006). T. gondii CaM, TgCAM, has been shown to bind calcium in vitro (Seeber et al., 1999) and is localized to the apical region of the tachyzoite (Pezzella-D’Alessandro et al., 2001). Interestingly, a more recent study has

determined its localization more specifically to the conoid where it interacts with calcineurin (Paul et al., 2015). CMLs are loosely defined as having at least four EF-hand domains (but typically more), sequence similarity to CaM that is generally less than 50%, and, like CaM, a lack of additional functional domains (Zeng et al., 2015). Three CMLs have received recent attention due to their role in parasite motility and invasion. CAM1, CAM2, and CAM3 are located in the conoid and interact with the apical motor protein (Hu et al., 2006; Long et al., 2017a,b; Graindorge et al., 2016). Both CaM1 and CaM2 are individually dispensable; however, double knockouts are not viable. CaM3 is essential and acute depletion of the protein using an auxin-induced degradation strategy resulted in severe growth defects (Long et al., 2017b). Like the apical motor system driven by MyoH, the Myosin A (MyoA)-driven glideosome that is responsible for translocation of adhesins along the majority of the parasite membrane also relies on CMLs for its function. As myosin light chains are evolutionarily related to CaMs and contain EF-hand domains, recent studies have focused on the molecular mechanisms of calcium-dependent gliding motility in T. gondii. The term essential light chain has been used to distinguish some myosin light chains, although they are not strictly essential and in some cases are functionally indistinguishable from regulatory light chains. Essential light chains 1 and 2 (ELC1 and ELC2) both bind calcium and interact directly with MyoA. ELC1/ELC2 are predicted to increase the structural integrity of the lever arm of MyoA, thereby optimizing force transduction by the glideosome (Powell et al., 2017; Williams et al., 2015). While ELC1 and ELC2 appear to be redundant, at least one is required for the glideosome to be functional (Williams et al., 2015). It will be important to continue characterizing other CMLs and their role in the T. gondii lytic cycle.

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Centrins are CBPs typically found in eukaryotic centrosomes, which play critical roles in the cell cycle and generation of microtubules. Centrins typically contain four EF-hand domains and have a molecular weight of approximately 20 kDa. Three centrins have been described in T. gondii (Hu et al., 2006). TgCEN1 and TgCEN3 are predominantly located in centrosomes, while TgCEN2 has a more expansive localization. In addition to the parasite centrosome, TgCEN2 localizes to the apical region (preconoidal rings), the basal complex, and to 56 peripheral annuli that are located at the interface of the apical cap and parasite membrane cortex. TgCEN2 appears to be essential, since knockdown of TgCEN2 resulted in severe replication defects and diminished microneme secretion and invasion (Leung et al., 2019). Many cellular enzymes have dedicated mechanisms of calcium regulation. One notable example is calcineurin, which acts as a heterodimer composed of a catalytic subunit (CnA) with serine/threonine protein phosphatase activity and a regulatory subunit (CnB) that binds to Ca21 via four EF-hand domains (Rusnak and Mertz, 2000). Calcineurin is highly conserved and serves as an important player in calcium-dependent signal transduction in a wide range of eukaryotes. In T. gondii, both subunits are located throughout the cytosol during intracellular replication. However, upon entering the extracellular stage both subunits are enriched in the apical region and colocalize with CaM at the conoid (Paul et al., 2015). Conditional knockdown of CnA resulted in loss of host cell attachment, a result that mirrored the effect of conditional knockdown of CnB in the malaria parasite, Plasmodium falciparum (Paul et al., 2015). Two other CBPs were recently described, both containing transmembrane domains (Chang et al., 2019). TGGT1_216620 has a predicted molecular weight of 412 kDa, making it much larger than other CBPs, and 7

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transmembrane domains. This CBP was localized to the plasma membrane of the tachyzoite, and its four EF-hand domains are predicted to face the extracellular environment. Ablation of this gene resulted in smaller plaque sizes and reduced extracellular calcium influx. TGGT1_280480 has a predicted weight of 34.4 kDa, 2 EF-hand domains and a COPI domain within one of the two transmembrane domains. It was localized to the rhoptries. Knockout of this CBP resulted in smaller plaque sizes but no dysregulation of intracellular calcium (Chang et al., 2019). Recently, the divergent stator of the apicomplexan ATP synthase complex (ICAP2) was characterized (Huet et al., 2018) and predicted to contain a calcium-binding domain in its N terminus. This feature is absent from all other characterized stators, and its function in the mitochondrial ATP synthase of apicomplexans remains unknown. However, these results highlight that the evolution of calcium regulation is a dynamic process that can intersect with a variety of cellular pathways. While EF-hand domains are the most prevalent calcium-binding motifs, other conserved structures can bind calcium. Another notable group of CBPs is characterized by the presence of C2 domains (InterPro ID: IPR000008), which bind to lipid bilayers in a calcium-dependent manner and were originally characterized in calcium-dependent isoforms of protein kinase C (PKC). C2 domains are found in several mammalian proteins that mediate calcium-regulated vesicular fusion including synaptotagmin, Munc13, and ferlins, as well as other proteins with regulated membrane localization and activity such as Perforin-1 (Nalefski and Falke, 1996). C2 domain-containing proteins therefore participate in a wide range of cellular functions including membrane trafficking, protein phosphorylation, lipid second messenger systems (e.g., phosphoinositide-specific phospholipase C), and activation of GTPases (Nalefski and Falke, 1996). Analysis of the T. gondii ME49

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genome reveals 12 predicted proteins that contain one or more C2 domains (as defined by a search using the InterPro ID: IPR000008 on ToxoDB, release 43). A notable example is DOC2.1 that is thought to participate in recruiting machinery for vesicular fusion. Conditional knockdown of DOC2.1 in T. gondii resulted in a microneme secretion defect, impacting both parasite invasion and egress (Farrell et al., 2012). Most proteins bearing calcium-binding domains in T. gondii remain to be studied, and further research into CBPs will continue to be a rewarding avenue by which to understand the lytic cycle of T. gondii. However, the presence of EF-hands and C2 domains does not guarantee the involvement of a given protein in calciumregulated processes, and biochemical analyses should accompany the study of any proteins suspected of calcium binding. Domains structurally related to EF hands but lacking the ability to bind calcium are common in a variety of proteins, including some myosin light chains (Grabarek, 2006). By contrast, the affinity of some EF hands for calcium suggests that they would be found in their bound state even at basal calcium concentrations and are therefore unlikely to respond to the fluctuations characteristic of signaling. It will also be valuable to understand the role of CBPs in other stages of the life cycle (e.g., bradyzoites) so as to arrive at a fuller appreciation of their functions.

12.4.2 Calcium-dependent protein kinases and their function CDPKs are a group of kinases that are overrepresented in the genomes of some protists and plants but are altogether absent from animals. Canonical CDPKs have an intrinsic ability to transduce Ca21 signals based on the presence of EF hands on the same polypeptide as the serine/threonine protein kinase domain. Their confirmed importance in a number of biological processes has made apicomplexan

CDPKs the focus of intense study to understand their roles in calcium-regulated processes and develop inhibitors that may target apicomplexan parasites without harming their animal hosts. In animals, kinases respond to calcium by association with CaM (CaMKs) or regulated attachment to membranes through C2 domains (PKC). There is no evidence for the presence of either CaMKs or PKC in T. gondii, which instead has an expanded group of protein kinases bearing EF hands, the CDPKs. The T. gondii genome encodes 14 protein kinases with EF-hands (ToxoDB, release 43), of which six have the canonical domain architecture with four EF-hands in close proximity to the C terminus of the kinase domain (CDPK1, CDPK2, CDPK2A, CDPK2B, CDPK3, and CDPK5). Noncanonical CDPKs have variable numbers of EF-hands, sometimes N-terminal to the kinase domain and often displaying mutations or truncations that make them unlikely to participate in calcium binding. Several CDPKs have additional domains that likely contribute to the functions of the kinase, which include a starch-binding domain in CDPK2 and pleckstrin-homology (PH) domains in CDPK7 and CDPK7A. The starch-binding domain of CDPK2 mediates a physical interaction with amylose and was shown to contribute to the regulation of amylopectin levels in complementation studies (Uboldi et al., 2015). Activity of the PH domain from CDPK7 was also confirmed by assessing the binding of phosphoinositides by the recombinantly expressed domain, although its function in the context of the full-length protein remains to be determined (Morlon-Guyot et al., 2014). The function of CDPKs can further be modified through acylation by influencing the subcellular localization of the kinases. Several canonical and noncanonical CDPKs have predicted myristoylation and palmitoylation sites (Billker et al., 2009; Long et al., 2016). Dual modification mediates stable membrane association,

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whereas myristoylation alone tends to be reversible and, in the case of CDPK1 and CDPK7, generates a diffuse, punctate pattern of localization restricted to the cytosol (Long et al., 2016). The functional consequences of CDPK3 acylation have been studied in detail. CDPK3 is N-terminally myristoylated and palmitoylated, and both modifications are required for its correct localization to the plasma membrane (Lourido et al., 2012; Garrison et al., 2012; McCoy et al., 2012). Mutants deficient in dual acylation were unable to complement CDPK3defficient parasites, unless overexpressed (McCoy et al., 2012). This suggests that acylation is not entirely required for kinase activity but likely modifies efficient targeting to the relevant subcellular compartment. The direct relationship between calcium binding and kinase activity has only been established for selected canonical CDPKs, with particular emphasis on T. gondii CDPK1 and CDPK3 for which full-length structures are available (Wernimont et al., 2010; Ojo et al., 2010; Ingram et al., 2015). In these kinases a long alpha helix occludes the catalytic site in a manner analogous to the inhibited state of mammalian CaMKII. This inhibitory alpha helix comprises the junction domain and part of the first EF-hand. From studies of the homolog of CDPK3 in P. falciparum, it was predicted that calcium binding is highly cooperative, with the N-terminal lobe of the CaM-like fold playing a dominant role in calcium binding and the structural changes that accompany it (Zhao et al., 1994). Upon calcium binding the EF hands reorganize around the inhibitory alpha helix causing a dramatic reorganization that relocates the entire regulatory domain contralateral to the catalytic site of the protein (Wernimont et al., 2010). Up to this point the mechanism of activation resembles that of the derepression of CaMKII; however, the kinase domain of CDPK1 is intrinsically inactive, adopting an “αC-out” conformation that characterizes the inactive state of many protein

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kinases (Ingram et al., 2015; Meharena et al., 2013). Activation of the kinase domain therefore requires intramolecular allosteric activation, involving a latching mechanism that links the N-terminal lobe of the kinase domain with the calcium-bound regulatory domain. Mutation of a single phenylalanine in the Nterminal extension that mediates this interaction in CDPK1 leads to a near complete loss of kinase activity, emphasizing the importance of allosteric activation for at least some CDPKs (Ingram et al., 2015). As a result of these dramatic rearrangements, the studied CDPKs are exquisitely calcium sensitive exhibiting greater than 1000fold changes in activity when exposed to lowmicromolar concentrations of free calcium (Wernimont et al., 2010; Ingram et al., 2015; Donald et al., 2006). Unlike mammalian CaMKII, the activation of CDPKs appears to be fully reversible, following calcium chelation. Phosphorylation of CaMKII in the junction region has been shown to render the kinase calcium independent—a property important for the integration of calcium spikes and certain types of cellular memory (Lisman et al., 2002). No such calcium-independent state has been reported for CDPKs, but different phases of activation may exist in the cellular context, mediated by the effect of other parasite proteins, such as the interaction of CDPK1 with DJ-1 (Child et al., 2017). Further studies will also need to determine the relationship between calcium binding and kinase activity for other CDPKs, particularly those with noncanonical domain architectures. CDPKs participate in a variety of cellular pathways ranging from calcium-regulated processes such as gliding motility and microneme secretion to steps of cell division where the role of calcium is unclear. T. gondii CDPK1 has been implicated in the regulation of microneme protein secretion, which is additionally modulated by the CBP DOC2.1, discussed earlier (Lourido et al., 2010). The precise mechanism

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through which CDPK1 regulates microneme exocytosis remains to be elucidated. Toward this goal the unusually broad ATP-binding pocket of CDPK1 enabled researchers to use bulky ATP analogs, bearing a gamma thiophosphate, to label and capture direct substrates of the kinase in lysates (Lourido et al., 2013). Critically, a specific glycine at the base of the expanded ATP-binding pocket can be mutated to methionine, preserving kinase activity but abrogating binding of the bioorthogonal substrate, thereby providing an adequate control for the studies. These efforts identified a number of CDPK1 substrates including DrpB, which could be confirmed in vivo and has itself been implicated in the biogenesis of micronemes (Breinich et al., 2009). Because gliding motility and invasion are both dependent on microneme proteins, it is unclear whether the effect of CDPK1 on these processes is direct or indirect; however, it is likely that these kinases coordinate several pathways and will therefore have several relevant targets. T. gondii CDPK3 has also been implicated in the regulation of microneme exocytosis specifically in the context of ionophore-stimulated egress (Garrison et al., 2012; McCoy et al., 2012; Lourido et al., 2012). The high K1 concentrations experienced by intracellular parasites repress microneme secretion and egress (Moudy et al., 2001). It has been suggested that under such intracellular conditions, CDPK3 becomes necessary, but other pathways compensate for its loss under extracellular conditions (McCoy et al., 2012). Consistent with that notion, CDPK3 inhibition affected microneme secretion in extracellular parasites in a manner dependent on the agonist used, whereas inhibition of CDPK1 had a uniform effect (Lourido et al., 2012). The same study also reported that stimulation of the protein kinase G (PKG) pathway could overcome the inhibition of CDPK3 during egress. Similar epistasis has been observed between the homologs of CDPK1 and

PKG in Plasmodium berghei (Fang et al., 2018). Adding to the complexity, a forward genetic screen also identified a suppressor of egress (SCE1) whose loss could compensate for the absence of CDPK3 during ionophore-stimulated egress (McCoy et al., 2017). SCE1 was also found to be phosphorylated by CDPK3 and mutations that abrogate phosphorylation reduced egress, although knocking out SCE1 had no effect on otherwise normal parasites. Based on the multiple suggestions of compensation by other signaling pathways, it is not surprising that CDPK3 knockouts are viable, although mutations in CDPK3 have been associated with decreased virulence in mice and lower numbers of chronic-stage tissue cysts (Treeck et al., 2014). Kinase signaling networks are highly plastic, and the roles of individual kinases and their dispensability should always be appreciated in the context in which they are observed, since examining the entire life cycle of the parasite is usually not feasible. Beyond microneme secretion, CDPK3 has been associated with other physiological changes during egress and the activation of the motor complex that powers gliding motility. Consistent with these functions, a study comparing the phosphoproteome of wild-type and CDPK3-deficient parasites identified 156 phosphosites that differed in abundance between the two strains, which included peptides from a P-type ATPase and several known components of the gliding motility machinery (Garrison et al., 2012). These phosphoproteomics changes were correlated with higher basal-calcium concentrations observed in CDPK3-deficient parasites, which more detailed analyses suggest might be the aggregate effect of differences in the rate of recovery from calcium spikes (Stewart et al., 2017). A similar slow recovery was observed upon CDPK1 inhibition and was independent from the permeabilization of the parasitophorous vacuole by PLP1. The motor complex is extensively phosphorylated, with several sites exhibiting calcium dependency

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(Nebl et al., 2011; Treeck et al., 2014). However, detailed analyses of GAP45 and MLC1 point to much of the phosphorylation being superfluous or highly redundant (Jacot et al., 2014). By contrast, analysis of MyoA has identified sites that modulate the activity of the motor. In a textbook example of phosphomodulation, mutation of a cluster of serines in the neck region of MyoA (S20, S21, and S29) to alanines delayed egress, whereas mutation to phosphomimetic aspartate residues restored the phenotype (Tang et al., 2014). Pairs of phosphomimetic mutations in the neck region (S20/21D) or the motor region (S743/744D) of MyoA were later shown to overcome the loss of CDPK3 in ionophore-stimulated egress (Gaji et al., 2015). Structural and biophysical analysis of the motor indicates that these regulatory regions play different roles in the modulation of MyoA, with phosphorylation of the neck region enhancing myosin activity (as in S20/21D) and phosphorylation of the motor region decreasing its stability (as in S743D) (Powell et al., 2018). How the latter destabilization contributes to CDPK3 independence remains an open question for future studies. More broadly, the diverse pathways associated with CDPK1 and CDPK3 reflect their coordination of events across the series of cellular pathways that mediates motility from the secretion of adhesins at the apical end to the modulation of the motors that translocate them, and the quenching of the calcium signals that triggered the process in the first place. Consistent with this notion, a recent study has implicated CDPK1 in the initiation of the actin flows that mediate motility (Tosetti et al., 2019). Dissecting the phenotypes associated with specific CDPKs has uncovered new interactions between calcium signaling and previously unrelated cellular pathways. Most notably, T. gondii CDPK2 has been shown to play an important role in regulating the balance between starch biosynthesis and degradation (Uboldi et al., 2015). CDPK2 knockout parasites aberrantly accumulated starch granules, a

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phenotype exacerbated by conversion in the chronic bradyzoite stages and ultimately leading to a complete abrogation of cyst formation. Quantitative analysis of the proteome of CDPK2-defficient parasites revealed reduced abundance and phosphorylation of many enzymes involved in starch synthesis and utilization. Among the modulated enzymes, direct targeting of pyruvate phosphate dikinase by CDPK2 could be confirmed in lysates. Modulation of such enzymes is consistent with the localization of CDPK2 to starch granules discussed previously (Uboldi et al., 2015). Moreover, a complete block of calcium signaling using BAPTA-AM increased starch accumulation in a manner similar to the loss of CDPK2. The calcium concentration needed to activate CDPK2 will need to be measured to determine whether the kinase is active at the basal concentrations found within intracellular parasites and to fully understand the accumulation of starch observed in the CDPK2 knockout in that stage of the lytic cycle. A similar question remains for the activity of the noncanonical CDPK7, which was found to be involved in endodyogeny (Morlon-Guyot et al., 2014). Loss of CDPK7 was associated with mislocalization of organelles and general defects related to daughter cell budding. The function of most other CDPKs has not been studied in detail. Two studies used the increased efficiency of genetic manipulation afforded by CRISPR/ Cas9 to investigate the function of other noncanonical CDPKs, finding that most of them have no consequence for the tachyzoite cycle (Wang et al., 2016a; Long et al., 2016). One of the studies did identify an intermediate defect in plaque formation for parasites lacking CDPK6 (Long et al., 2016). Knocking out additional noncanonical CDPKs in the CDPK6-deficient background did not exacerbate the phenotype, and epistatic interactions were not identified for any of the kinase pairs examined. In P. berghei, studies involving sexual stages have identified the functions of several CDPKs that

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are dispensable for the asexual stages, raising the possibility that analogous studies in T. gondii will uncover the function of additional CDPKs. The importance of CDPKs for the T. gondii lytic cycle, their conservation among apicomplexans and their absence from mammalian genomes, makes these kinases attractive targets for the development of antiparasitic compounds. To this end, particular focus has been given to T. gondii CDPK1 and its orthologs, following the realization that their unusual ATPbinding pockets can be specifically targeted through stearic complementarity. Among all T. gondii protein kinases, CDPK1 is unique in encoding a glycine at the gatekeeper position, a residue that limits the depth of the ATPbinding pocket and is occupied by a bulky hydrophobic residue in most kinases. This feature allowed researchers to inhibit CDPK1 using repurposed bioorthogonal kinase inhibitors (sometimes called bumped kinase inhibitors; BKIs), originally designed to target kinases engineered to have similarly small gatekeeper residues (Ojo et al., 2010; Lourido et al., 2010; Sugi et al., 2010). The specificity of these compounds was demonstrated by expressing a CDPK1 allele bearing a methionine gatekeeper, which fully complemented the loss of the wild-type kinase, yet rendered parasites resistant to the bioorthogonal inhibitors during invasion, egress, and microneme secretion—phenotypes also linked to CDPK1 through genetic inactivation. It should be noted that other small residues (alanine, serine, or threonine) can sometimes accommodate BKIs, and other compounds that rely on extended ATP-binding pockets. The PKG inhibitors, Compound 1 and Compound 2, have been shown to have secondary activity against CDPK1 (Donald et al., 2006). For both kinases, bulky gatekeeper substitutions can dramatically decrease the sensitivity of the enzymes to the inhibitors. Like the PKG inhibitors, BKIs have also been shown to have secondary effects on other kinases (Sugi et al., 2010). The

promising effects of BKIs against CDPK1 have motivated further development to improve their pharmacokinetics and potency (reviewed in Cardew et al., 2018). Other modalities of CDPK inhibition may be possible through the design of peptides that mimic the inhibitory alpha helix or occlude the interactions for allosteric activation (Bansal et al., 2013; Ingram et al., 2015). Although more challenging to implement, these strategies may be able to target several CDPKs at once, reducing the likelihood of emergent resistance. As studies of the function of CDPKs continue, research into their inhibition will provide important tool compounds for their study and promising new therapeutic avenues.

12.5 Conclusion The field of Toxoplasma Ca21 signaling has advanced significantly since the last edition of this chapter. A large part of these advances in Toxoplasma can be credited to new genetic tools such as CRISPR-Cas9 for characterization of genes, the expression of genetically encoded Ca21 indicators, the use of biotinligase fusion proteins to identify proximal and interacting proteins with common subcellular localizations, and the adaptation of tools to transcriptionally or translationally regulate expression, enabling studies of more immediate phenotypic differences without the risk of compensatory changes. The results of these technical advances have expanded our understanding of the link between Ca21 signaling and critical facets of parasite biology (i.e., gliding motility, microneme secretion, host cell invasion, and egress). Importantly, they have also allowed for the discovery of new genes and Ca21 regulatory pathways. Ca21 signaling is controlled by the uptake and release of ions from different cellular compartments, and there are important differences between the processes that control Ca21

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References

homeostasis in T. gondii compared to other eukaryotic cells, providing opportunities for finding novel drug targets. Studying Ca21 signaling in Toxoplasma has also informed the basic mechanisms that regulate how Toxoplasma, and related pathogens, cause disease. Years after Endo (Endo et al., 1982) showed that ionophores trigger egress, the assumption that Ca21 oscillations occur prior to egress from host cells has been validated by observations of genetically encoded Ca21 sensors (GECIs). GECIs allow specific detection of Ca21 changes in live intracellular parasites and continued elaboration of calcium probes with different affinities and subcellular localizations will further enrich our understanding of T. gondii signaling. Questions remain about the cross talk between intracellular and extracellular Ca21 sources, and how they combine to deliver the threshold needed for increase in motility and egress. In recent years, substantial evidence of cross talk has emerged between Ca21 and other secondary messengers such as cGMP, cAMP, and phosphatidic acid (Fig. 12.2, activation). What connects the various signaling pathways and the order in which they are naturally elicited remains unclear. Major gaps also exist in understanding how homeostasis is reestablished following stimulation. Further research into CBPs and CDPKs will help clarify many of the gaps in our understanding of these critical signaling pathways. It will be the goal of future research to elucidate the full spectrum of interactions between Ca21 signaling and other cellular pathways and integrate this information into a comprehensive model of the dynamic behaviors of apicomplexan parasites.

Acknowledgments Work in our laboratories was funded by the US National Institutes of Health to SNJM (AI128356) and DAP (1SC3GM121223), and the NIH Director’s Early Independence Award (1DP5OD017892) and a grant from the Mathers Foundation to SL.

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References Akerboom, J., Chen, T.W., Wardill, T.J., Tian, L., Marvin, J. S., Mutlu, S., et al., 2012. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 1381913840. Akerboom, J., Carreras Calderon, N., Tian, L., Wabnig, S., Prigge, M., Tolo, J., et al., 2013. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2. Arrizabalaga, G., Boothroyd, J.C., 2004. Role of calcium during Toxoplasma gondii invasion and egress. Int. J. Parasitol. 34, 361368. Balaban, R.S., 2009. The role of Ca(2 1 ) signaling in the coordination of mitochondrial ATP production with cardiac work. Biochim. Biophys. Acta 1787, 13341341. Bansal, A., Singh, S., More, K.R., Hans, D., Nangalia, K., Yogavel, M., et al., 2013. Characterization of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) and its role in microneme secretion during erythrocyte invasion. J. Biol. Chem. 288, 15901602. Bassett, J.J., Monteith, G.R., 2017. Genetically encoded calcium indicators as probes to assess the role of calcium channels in disease and for high-throughput drug discovery. Adv. Pharmacol. 79, 141171. Berridge, M.J., 2009. Inositol trisphosphate and calcium signalling mechanisms. Biochim. Biophys. Acta 1793, 933940. Besteiro, S., Tonn, D., Tetley, L., Coombs, G.H., Mottram, J. C., 2008. The AP3 adaptor is involved in the transport of membrane proteins to acidocalcisomes of Leishmania. J. Cell. Sci. 121, 561570. Bhattacharya, S., Bunick, C.G., Chazin, W.J., 2004. Target selectivity in EF-hand calcium binding proteins. Biochim. Biophys. Acta 1742, 6979. Billker, O., Lourido, S., Sibley, L.D., 2009. Calciumdependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5, 612622. Bonhomme, A., Pingret, L., Bonhomme, P., Michel, J., Balossier, G., Lhotel, M., et al., 1993. Subcellular calcium localization in Toxoplasma gondii by electron microscopy and by X-ray and electron energy loss spectroscopies. Microsc. Res. Tech. 25, 276285. Borges-Pereira, L., Budu, A., Mcknight, C.A., Moore, C.A., Vella, S.A., Hortua Triana, M.A., et al., 2015. Calcium signaling throughout the Toxoplasma gondii lytic cycle: a study using genetically encoded calcium indicators. J. Biol. Chem. 290, 2691426926. Bowman, E.J., Siebers, A., Altendorf, K., 1988. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. U.S.A. 85, 79727976.

Toxoplasma Gondii

570

12. Calcium storage and homeostasis in Toxoplasma gondii

Breinich, M.S., Ferguson, D.J., Foth, B.J., Van Dooren, G.G., Lebrun, M., Quon, D.V., et al., 2009. A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii. Curr. Biol. 19, 277286. Bright, G.R., Fisher, G.W., Rogowska, J., Taylor, D.L., 1987. Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH. J. Cell Biol. 104, 10191033. Brown, K.M., Lourido, S., Sibley, L.D., 2016. Serum albumin stimulates protein kinase G-dependent microneme secretion in Toxoplasma gondii. J. Biol. Chem. 291, 95549565. Bruton, J.D., Cheng, A.J., Westerblad, H., 2012. Methods to detect Ca(2 1 ) in living cells. Adv. Exp. Med. Biol. 740, 2743. Bullen, H.E., Jia, Y., Yamaryo-Botte, Y., Bisio, H., Zhang, O., Jemelin, N.K., et al., 2016. Phosphatidic acidmediated signaling regulates microneme secretion in Toxoplasma. Cell Host Microbe 19, 349360. Cardenas, C., Miller, R.A., Smith, I., Bui, T., Molgo, J., Muller, M., et al., 2010. Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca21 transfer to mitochondria. Cell 142, 270283. Cardew, E.M., Verlinde, C., Pohl, E., 2018. The calciumdependent protein kinase 1 from Toxoplasma gondii as target for structure-based drug design. Parasitology 145, 210218. Carruthers, V.B., Giddings, O.K., Sibley, L.D., 1999a. Secretion of micronemal proteins is associated with toxoplasma invasion of host cells. Cell. Microbiol. 1, 225235. Carruthers, V.B., Moreno, S.N., Sibley, L.D., 1999b. Ethanol and acetaldehyde elevate intracellular [Ca2 1 ] and stimulate microneme discharge in Toxoplasma gondii. Biochem. J. 342 (Pt 2), 379386. Chang, L., Dykes, E.J., Li, J., Moreno, S.N.J., Hortua Triana, M.A., 2019. Characterization of two EF-hand domaincontaining proteins from Toxoplasma gondii. J. Eukaryot. Microbiol. 66, 343353. Chasen, N.M., Stasic, A.J., Asady, B., Coppens, I., Moreno, S.N.J., 2019. The vacuolar zinc transporter TgZnT protects Toxoplasma gondii from zinc toxicity. mSphere 4, e00086-19. Chen, T.W., Wardill, T.J., Sun, Y., Pulver, S.R., Renninger, S.L., Baohan, A., et al., 2013. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295300. Child, M.A., Garland, M., Foe, I., Madzelan, P., Treeck, M., Van Der Linden, W.A., et al., 2017. Toxoplasma DJ-1 regulates organelle secretion by a direct interaction with calcium-dependent protein kinase 1. mBio 8, e02189-16. Chin, D., Means, A.R., 2000. Calmodulin: a prototypical calcium sensor. Trends Cell Biol. 10, 322328.

Chini, E.N., Nagamune, K., Wetzel, D.M., Sibley, L.D., 2005. Evidence that the cADPR signalling pathway controls calcium-mediated microneme secretion in Toxoplasma gondii. Biochem. J. 389, 269277. Clapham, D.E., 2007. Calcium signaling. Cell 131, 10471058. Dana, H., Mohar, B., Sun, Y., Narayan, S., Gordus, A., Hasseman, J.P., et al., 2016. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727. Dargan, S.L., Parker, I., 2003. Buffer kinetics shape the spatiotemporal patterns of IP3-evoked Ca21 signals. J. Physiol. 553, 775788. Denton, R.M., McCormack, J.G., 1990. Ca21 as a second messenger within mitochondria of the heart and other tissues. Annu. Rev. Physiol. 52, 451466. Deo, C., Lavis, L.D., 2018. Synthetic and genetically encoded fluorescent neural activity indicators. Curr. Opin. Neurobiol. 50, 101108. Di Virgilio, F., Steinberg, T.H., Swanson, J.A., Silverstein, S. C., 1988. Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions. J. Immunol. 140, 915920. Docampo, R., De Souza, W., Miranda, K., Rohloff, P., Moreno, S.N., 2005. Acidocalcisomes  conserved from bacteria to man. Nat. Rev. Microbiol. 3, 251261. Donald, R.G., Zhong, T., Wiersma, H., Nare, B., Yao, D., Lee, A., et al., 2006. Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase. Mol. Biochem. Parasitol. 149, 8698. Drozdowicz, Y.M., Shaw, M., Nishi, M., Striepen, B., Liwinski, H.A., Roos, D.S., et al., 2003. Isolation and characterization of TgVP1, a type I vacuolar H1-translocating pyrophosphatase from Toxoplasma gondii. The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor. J. Biol. Chem. 278, 10751085. Eckstein-Ludwig, U., Webb, R.J., Van Goethem, I.D., East, J.M., Lee, A.G., Kimura, M., et al., 2003. Artemisinins target the SERCA of Plasmodium falciparum. Nature 424, 957961. Endo, T., Sethi, K.K., Piekarski, G., 1982. Toxoplasma gondii: calcium ionophore A23187-mediated exit of trophozoites from infected murine macrophages. Exp. Parasitol. 53, 179188. Faas, G.C., Mody, I., 2014. Measuring the Ca(2)(1)-binding kinetics of proteins. Cold Spring Harb. Protoc. 2014, 691693. Fang, J., Marchesini, N., Moreno, S.N.J., 2006. A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol. Biochem. J. 394, 417.

Toxoplasma Gondii

References

Fang, H., Gomes, A.R., Klages, N., Pino, P., Maco, B., Walker, E.M., et al., 2018. Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites. Nat. Commun. 9, 4248. Farrell, A., Thirugnanam, S., Lorestani, A., Dvorin, J.D., Eidell, K.P., Ferguson, D.J., et al., 2012. A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis. Science 335, 218221. Francia, M.E., Wicher, S., Pace, D.A., Sullivan, J., Moreno, S.N., Arrizabalaga, G., 2011. A Toxoplasma gondii protein with homology to intracellular type Na(1)/H(1) exchangers is important for osmoregulation and invasion. Exp. Cell Res. 317, 13821396. Gaji, R.Y., Johnson, D.E., Treeck, M., Wang, M., Hudmon, A., Arrizabalaga, G., 2015. Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid initiation of motility during Toxoplasma gondii egress. PLoS Pathog. 11, e1005268. Garcia, C.R.S., Alves, E., Pereira, P.H.S., Bartlett, P.J., Thomas, A.P., Mikoshiba, K., et al., 2017. InsP3 signaling in apicomplexan parasites. Curr. Top. Med. Chem. 17, 21582165. Garrison, E., Treeck, M., Ehret, E., Butz, H., Garbuz, T., Oswald, B.P., et al., 2012. A Forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma. PLoS Pathog. 8, e1003049. Grabarek, Z., 2006. Structural basis for diversity of the EFhand calcium-binding proteins. J. Mol. Biol. 359, 509525. Graindorge, A., Frenal, K., Jacot, D., Salamun, J., Marq, J.B., Soldati-Favre, D., 2016. The conoid associated motor MyoH is indispensable for Toxoplasma gondii entry and exit from host cells. PLoS Pathog. 12, e1005388. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 34403450. Guerrero-Hernandez, A., Dagnino-Acosta, A., Verkhratsky, A., 2010. An intelligent sarco-endoplasmic reticulum Ca21 store: release and leak channels have differential access to a concealed Ca21 pool. Cell Calcium 48, 143149. Guttery, D.S., Pittman, J.K., Frenal, K., Poulin, B., Mcfarlane, L.R., Slavic, K., et al., 2013. The Plasmodium berghei Ca(2 1 )/H(1) exchanger, PbCAX, is essential for tolerance to environmental Ca(2 1 ) during sexual development. PLoS Pathog. 9, e1003191. Hajnoczky, G., Robb-Gaspers, L.D., Seitz, M.B., Thomas, A. P., 1995. Decoding of cytosolic calcium oscillations in the mitochondria. Cell 82, 415424. Haller, T., Volkl, H., Deetjen, P., Dietl, P., 1996. The lysosomal Ca21 pool in MDCK cells can be released by ins (1,4,5)P3-dependent hormones or thapsigargin but does

571

not activate store-operated Ca21 entry. Biochem. J. 319 (Pt 3), 909912. Hortua Triana, M.A., Marquez-Nogueras, K.M., Chang, L., Stasic, A.J., Li, C., Spiegel, K.A., et al., 2018. Tagging of weakly expressed Toxoplasma gondii calcium-related genes with high-affinity tags. J. Eukaryot. Microbiol. 65, 709721. Hu, K., Johnson, J., Florens, L., Fraunholz, M., Suravajjala, S., Dilullo, C., et al., 2006. Cytoskeletal components of an invasion machine—the apical complex of Toxoplasma gondii. PLoS Pathog. 2, e13. Huang, G., Fang, J., Sant’anna, C., Li, Z.H., Wellems, D.L., Rohloff, P., et al., 2011. Adaptor protein-3 (AP-3) complex mediates the biogenesis of acidocalcisomes and is essential for growth and virulence of Trypanosoma brucei. J. Biol. Chem. 286, 3661936630. Huang, G., Bartlett, P.J., Thomas, A.P., Moreno, S.N., Docampo, R., 2013. Acidocalcisomes of Trypanosoma brucei have an inositol 1,4,5-trisphosphate receptor that is required for growth and infectivity. Proc. Natl. Acad. Sci. U.S.A. 110, 18871892. Huet, D., Rajendran, E., Van Dooren, G.G., Lourido, S., 2018. Identification of cryptic subunits from an apicomplexan ATP synthase. eLife 7, e38097. Ingram, J.R., Knockenhauer, K.E., Markus, B.M., Mandelbaum, J., Ramek, A., Shan, Y., et al., 2015. Allosteric activation of apicomplexan calciumdependent protein kinases. Proc. Natl. Acad. Sci. U.S.A. 112, E4975E4984. Jacot, D., Frenal, K., Marq, J.B., Sharma, P., Soldati-Favre, D., 2014. Assessment of phosphorylation in Toxoplasma glideosome assembly and function. Cell. Microbiol. 16, 15181532. Kao, J.P., 1994. Practical aspects of measuring [Ca21] with fluorescent indicators. Methods Cell Biol. 40, 155181. Kuchipudi, A., Arroyo-Olarte, R.D., Hoffmann, F., Brinkmann, V., Gupta, N., 2016. Optogenetic monitoring identifies phosphatidylthreonine-regulated calcium homeostasis in Toxoplasma gondii. Microb. Cell 3, 215223. Kunze, W., 1907. Uber Ocheobius herpobdellae schuberg et kunze. Arch. Protistenk. 9, 383390. Lee, H.C., 2011. Cyclic ADP-ribose and NAADP: fraternal twin messengers for calcium signaling. Sci. China Life Sci. 54, 699711. LeFurgey, A., Ingram, P., Blum, J.J., 2001. Compartmental responses to acute osmotic stress in Leishmania major result in rapid loss of Na1 and Cl. Comp. Biochem. Physiol., A. Mol. Integr. Physiol. 128, 385394. Leung, J.M., Liu, J., Wetzel, L.A., Hu, K., 2019. Centrin2 from the human parasite Toxoplasma gondii is required for its invasion and intracellular replication. J. Cell. Sci. 132 (13). Available from: https://doi.org/10.1242/ jcs.228791.

Toxoplasma Gondii

572

12. Calcium storage and homeostasis in Toxoplasma gondii

Lewit-Bentley, A., Rety, S., 2000. EF-hand calcium-binding proteins. Curr. Opin. Struct. Biol. 10, 637643. Lisman, J., Schulman, H., Cline, H., 2002. The molecular basis of CaMKII function in synaptic and behavioural memory. Nat. Rev. Neurosci. 3, 175190. Liu, C., Hermann, T.E., 1978. Characterization of ionomycin as a calcium ionophore. J. Biol. Chem. 253, 58925894. Liu, J., Pace, D., Dou, Z., King, T.P., Guidot, D., Li, Z.H., et al., 2014. A vacuolar-H(1)-pyrophosphatase (TgVP1) is required for microneme secretion, host cell invasion, and extracellular survival of Toxoplasma gondii. Mol. Microbiol. 93, 698712. Lock, J.T., Parker, I., Smith, I.F., 2015. A comparison of fluorescent Ca(2)(1) indicators for imaging local Ca(2)(1) signals in cultured cells. Cell Calcium 58, 638648. Long, S., Wang, Q., Sibley, L.D., 2016. Analysis of noncanonical calcium-dependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9. Infect. Immun. 84, 12621273. Long, S., Anthony, B., Drewry, L.L., Sibley, L.D., 2017a. A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii. Nat. Commun. 8, 2236. Long, S., Brown, K.M., Drewry, L.L., Anthony, B., Phan, I. Q.H., Sibley, L.D., 2017b. Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog. 13, e1006379. Lourido, S., Moreno, S.N., 2015. The calcium signaling toolkit of the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell Calcium 57, 186193. Lourido, S., Shuman, J., Zhang, C., Shokat, K.M., Hui, R., Sibley, L.D., 2010. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465, 359362. Lourido, S., Tang, K., Sibley, L.D., 2012. Distinct signalling pathways control Toxoplasma egress and host-cell invasion. EMBO J. 34, 4524. Lourido, S., Jeschke, G.R., Turk, B.E., Sibley, L.D., 2013. Exploiting the unique ATP-binding pocket of toxoplasma calcium-dependent protein kinase 1 to identify its substrates. ACS Chem. Biol. 8, 11551162. Lovett, J.L., Sibley, L.D., 2003. Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells. J. Cell. Sci. 116, 30093016. Lovett, J.L., Marchesini, N., Moreno, S.N., Sibley, L.D., 2002. Toxoplasma gondii microneme secretion involves intracellular Ca(2 1 ) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores. J. Biol. Chem. 277, 2587025876. Luo, S., Vieira, M., Graves, J., Zhong, L., Moreno, S.N., 2001. A plasma membrane-type Ca(2 1 )-ATPase colocalizes with a vacuolar H(1)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii. EMBO J. 20, 5564.

Luo, S., Ruiz, F.A., Moreno, S.N., 2005. The acidocalcisome Ca21-ATPase (TgA1) of Toxoplasma gondii is required for polyphosphate storage, intracellular calcium homeostasis and virulence. Mol. Microbiol. 55, 10341045. Mbatia, H.W., Burdette, S.C., 2012. Photochemical tools for studying metal ion signaling and homeostasis. Biochemistry 51, 72127224. McCombs, J.E., Palmer, A.E., 2008. Measuring calcium dynamics in living cells with genetically encodable calcium indicators. Methods 46, 152159. Mccoy, J.M., Whitehead, L., Van Dooren, G.G., Tonkin, C.J., 2012. TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells. PLoS Pathog. 8, e1003066. McCoy, J.M., Stewart, R.J., Uboldi, A.D., Li, D., Schroder, J., Scott, N.E., et al., 2017. A forward genetic screen identifies a negative regulator of rapid Ca(2 1 )-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii. J. Biol. Chem. 292, 76627674. Meharena, H.S., Chang, P., Keshwani, M.M., Oruganty, K., Nene, A.K., Kannan, N., et al., 2013. Deciphering the structural basis of eukaryotic protein kinase regulation. PLoS Biol. 11, e1001680. Mira Gutierrez, J., Del Rey Calero, J., 1966. Volutina in Toxoplasma gondii. Med. Trop. (Madr.) 42, 2029. Miranda, K., Benchimol, M., Docampo, R., De Souza, W., 2000. The fine structure of acidocalcisomes in Trypanosoma cruzi. Parasitol. Res. 86, 373384. Miranda, K., Rodrigues, C.O., Hentchel, J., Vercesi, A., Plattner, H., De Souza, W., et al., 2004. Acidocalcisomes of Phytomonas francai possess distinct morphological characteristics and contain iron. Microsc. Microanal. 10, 647655. Miranda, K., Pace, D.A., Cintron, R., Rodrigues, J.C., Fang, J., Smith, A., et al., 2010. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Mol. Microbiol. 76, 13581375. Mondragon, R., Frixione, E., 1996. Ca(2 1 )-dependence of conoid extrusion in Toxoplasma gondii tachyzoites. J. Eukaryot. Microbiol. 43, 120127. Moreno, S.N., Zhong, L., 1996. Acidocalcisomes in Toxoplasma gondii tachyzoites. Biochem. J. 313 (Pt 2), 655659. Moreno, S.N., Zhong, L., Lu, H.G., Souza, W.D., Benchimol, M., 1998. Vacuolar-type H1-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites. Biochem. J. 330 (Pt 2), 853860. Moreno, B., Bailey, B.N., Luo, S., Martin, M.B., Kuhlenschmidt, M., Moreno, S.N., et al., 2001. 31)P NMR of apicomplexans and the effects of risedronate on Cryptosporidium parvum growth. Biochem. Biophys. Res. Commun. 284, 632637.

Toxoplasma Gondii

References

Morlon-Guyot, J., Berry, L., Chen, C.T., Gubbels, M.J., Lebrun, M., Daher, W., 2014. The Toxoplasma gondii calcium-dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival. Cell. Microbiol. 16, 95114. Moudy, R., Manning, T.J., Beckers, C.J., 2001. The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii. J. Biol. Chem. 276, 4149241501. Nagamune, K., Sibley, L.D., 2006. Comparative genomic and phylogenetic analyses of calcium ATPases and calcium-regulated proteins in the Apicomplexa. Mol. Biol. Evol. 23, 16131627. Nagamune, K., Beatty, W.L., Sibley, L.D., 2007. Artemisinin induces calcium-dependent protein secretion in the protozoan parasite Toxoplasma gondii. Eukaryot. Cell 6, 21472156. Nalefski, E.A., Falke, J.J., 1996. The C2 domain calciumbinding motif: structural and functional diversity. Protein Sci. 5, 23752390. Nebl, T., Prieto, J.H., Kapp, E., Smith, B.J., Williams, M.J., Yates 3rd, J.R., et al., 2011. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex. PLoS Pathog. 7, e1002222. Ojo, K.K., Larson, E.T., Keyloun, K.R., Castaneda, L.J., Derocher, A.E., Inampudi, K.K., et al., 2010. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors. Nat. Struct. Mol. Biol. 17, 602607. Pace, D.A., Mcknight, C.A., Liu, J., Jimenez, V., Moreno, S. N., 2014. Calcium entry in Toxoplasma gondii and its enhancing effect of invasion-linked traits. J. Biol. Chem. 289, 1963719647. Parussini, F., Coppens, I., Shah, P.P., Diamond, S.L., Carruthers, V.B., 2010. Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii. Mol. Microbiol. 76, 13401357. Paul, A.S., Saha, S., Engelberg, K., Jiang, R.H., Coleman, B. I., Kosber, A.L., et al., 2015. Parasite calcineurin regulates host cell recognition and attachment by apicomplexans. Cell Host Microbe 18, 4960. Pezzella-D’Alessandro, N., Le Moal, H., Bonhomme, A., Valere, A., Klein, C., Gomez-Marin, J., et al., 2001. Calmodulin distribution and the actomyosin cytoskeleton in Toxoplasma gondii. J. Histochem. Cytochem. 49, 445454. Pingret, L., Millot, J.M., Sharonov, S., Bonhomme, A., Manfait, M., Pinon, J.M., 1996. Relationship between intracellular free calcium concentrations and the intracellular development of Toxoplasma gondii. J. Histochem. Cytochem. 44, 11231129.

573

Powell, C.J., Jenkins, M.L., Parker, M.L., Ramaswamy, R., Kelsen, A., Warshaw, D.M., et al., 2017. Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex. J. Biol. Chem. 292, 1946919477. Powell, C.J., Ramaswamy, R., Kelsen, A., Hamelin, D.J., Warshaw, D.M., Bosch, J., et al., 2018. Structural and mechanistic insights into the function of the unconventional class XIV myosin MyoA from Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 115, E10548E10555. Prole, D.L., Taylor, C.W., 2011. Identification of intracellular and plasma membrane calcium channel homologues in pathogenic parasites. PLoS One 6, e26218. Rodrigues, C.O., Scott, D.A., Docampo, R., 1999. Presence of a vacuolar H1-pyrophosphatase in promastigotes of Leishmania donovani and its localization to a different compartment from the vacuolar H1-ATPase. Biochem. J. 340 (Pt 3), 759766. Rodrigues, C.O., Scott, D.A., Bailey, B.N., De Souza, W., Benchimol, M., Moreno, B., et al., 2000. Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets. Biochem. J. 349 (Pt 3), 737745. Rodrigues, C.O., Ruiz, F.A., Rohloff, P., Scott, D.A., Moreno, S.N., 2002. Characterization of isolated acidocalcisomes from Toxoplasma gondii tachyzoites reveals a novel pool of hydrolyzable polyphosphate. J. Biol. Chem. 277, 4865048656. Rohloff, P., Miranda, K., Rodrigues, J.C., Fang, J., Galizzi, M., Plattner, H., et al., 2011. Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii. PLoS One 6, e18390. Rusnak, F., Mertz, P., 2000. Calcineurin: form and function. Physiol. Rev. 80, 14831521. Sagara, Y., Inesi, G., 1991. Inhibition of the sarcoplasmic reticulum Ca21 transport ATPase by thapsigargin at subnanomolar concentrations. J. Biol. Chem. 266, 1350313506. Saoudi, Y., Rousseau, B., Doussiere, J., Charrasse, S., Gauthier-Rouviere, C., Morin, N., et al., 2004. Calciumindependent cytoskeleton disassembly induced by BAPTA. Eur. J. Biochem. 271, 32553264. Scott, D.A., Docampo, R., Dvorak, J.A., Shi, S., Leapman, R. D., 1997. In situ compositional analysis of acidocalcisomes in Trypanosoma cruzi. J. Biol. Chem. 272, 2802028029. Seeber, F., Beuerle, B., Schmidt, H.H., 1999. Cloning and functional expression of the calmodulin gene from Toxoplasma gondii. Mol. Biochem. Parasitol. 99, 295299. Sidik, S.M., Hortua Triana, M.A., Paul, A.S., El Bakkouri, M., Hackett, C.G., Tran, F., et al., 2016. Using a genetically encoded sensor to identify inhibitors of Toxoplasma gondii Ca21 signaling. J. Biol. Chem. 291, 95669580.

Toxoplasma Gondii

574

12. Calcium storage and homeostasis in Toxoplasma gondii

Srinivas, S.P., Ong, A., Goon, L., Bonanno, J.A., 2002. Lysosomal Ca(2 1 ) stores in bovine corneal endothelium. Invest. Ophtalmol. Vis. Sci. 43, 23412350. Stasic, A.J., Chasen, N.M., Dykes, E.J., Vella, S.A., Asady, B., Starai, V.J., et al., 2019. The Toxoplasma vacuolar H (1)-ATPase regulates intracellular pH and impacts the maturation of essential secretory proteins. Cell Rep. 27, 21322146.e7. Stewart, R.J., Whitehead, L., Nijagal, B., Sleebs, B.E., Lessene, G., Mcconville, M.J., et al., 2017. Analysis of Ca (2)(1) mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules. Cell. Microbiol. 19, e12685. Stommel, E.W., Ely, K.H., Schwartzman, J.D., Kasper, L.H., 1997. Toxoplasma gondii: dithiol-induced Ca21 flux causes egress of parasites from the parasitophorous vacuole. Exp. Parasitol. 87, 8897. Stommel, E.W., Cho, E., Steide, J.A., Seguin, R., Barchowsky, A., Schwartzman, J.D., et al., 2001. Identification and role of thiols in Toxoplasma gondii egress. Exp. Biol. Med. (Maywood) 226, 229236. Sugi, T., Kato, K., Kobayashi, K., Watanabe, S., Kurokawa, H., Gong, H., et al., 2010. Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step. Eukaryot. Cell 9, 667670. Suzuki, J., Kanemaru, K., Iino, M., 2016. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophys. J. 111, 11191131. Takahashi, A., Camacho, P., Lechleiter, J.D., Herman, B., 1999. Measurement of intracellular calcium. Physiol. Rev. 79, 10891125. Tang, Q., Andenmatten, N., Hortua Triana, M.A., Deng, B., Meissner, M., Moreno, S.N., et al., 2014. Calciumdependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii. Mol. Biol. Cell 25, 25792591. Thastrup, O., Cullen, P.J., Drobak, B.K., Hanley, M.R., Dawson, A.P., 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca21 stores by specific inhibition of the endoplasmic reticulum Ca2(1)-ATPase. Proc. Natl. Acad. Sci. U.S.A. 87, 24662470. Thornton, L.B., Teehan, P., Floyd, K., Cochrane, C., Bergmann, A., Riegel, B., et al., 2019. An ortholog of Plasmodium falciparum chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion. PLoS Pathog. 15, e1007775. Tian, L., Hires, S.A., Mao, T., Huber, D., Chiappe, M.E., Chalasani, S.H., et al., 2009. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875881.

Tian, L., Hires, S.A., Looger, L.L., 2012. Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harb. Protoc. 2012, 647656. Tosetti, N., Dos Santos Pacheco, N., Soldati-Favre, D., Jacot, D., 2019. Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii. eLife 8, e42669. Treeck, M., Sanders, J.L., Gaji, R.Y., Lafavers, K.A., Child, M.A., Arrizabalaga, G., et al., 2014. The calciumdependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis. PLoS Pathog. 10, e1004197. Tsien, R.Y., 1980. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19, 23962404. Tsien, R.Y., 1981. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290, 527528. Tsien, R.Y., 1983. Intracellular measurements of ion activities. Annu. Rev. Biophys. Bioeng. 12, 91116. Tsien, R.Y., 1989. Fluorescent indicators of ion concentrations. Methods Cell Biol. 30, 127156. Uboldi, A.D., Mccoy, J.M., Blume, M., Gerlic, M., Ferguson, D.J., Dagley, L.F., et al., 2015. Regulation of starch stores by a Ca(2 1 )-dependent protein kinase is essential for viable cyst development in Toxoplasma gondii. Cell Host Microbe 18, 670681. Vella, S.A., Calixto, A., Asady, B., Li, Z.H., Moreno, S.N., 2019. Genetic indicators for calcium signaling studies in Toxoplasma gondii. Method. Mol. Biol. 2071, 18720. Vercesi, A.E., Moreno, S.N., Docampo, R., 1994. Ca21/H1 exchange in acidic vacuoles of Trypanosoma brucei. Biochem. J. 304 (Pt 1), 227233. Vieira, M.C., Moreno, S.N., 2000. Mobilization of intracellular calcium upon attachment of Toxoplasma gondii tachyzoites to human fibroblasts is required for invasion. Mol. Biochem. Parasitol. 106, 157162. Wang, J.L., Huang, S.Y., Li, T.T., Chen, K., Ning, H.R., Zhu, X.Q., 2016a. Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system. Parasitol. Res. 115, 697702. Wang, Q.C., Zheng, Q., Tan, H., Zhang, B., Li, X., Yang, Y., et al., 2016b. TMCO1 is an ER Ca(2 1 ) load-activated Ca(2 1 ) channel. Cell 165, 14541466. Warring, S.D., Dou, Z., Carruthers, V.B., Mcfadden, G.I., Van Dooren, G.G., 2014. Characterization of the chloroquine resistance transporter homologue in Toxoplasma gondii. Eukaryot. Cell 13, 13601370.

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References

Wernimont, A.K., Artz, J.D., Finerty Jr., P., Lin, Y.H., Amani, M., Allali-Hassani, A., et al., 2010. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. Nat. Struct. Mol. Biol. 17, 596601. Wetzel, D.M., Chen, L.A., Ruiz, F.A., Moreno, S.N., Sibley, L.D., 2004. Calcium-mediated protein secretion potentiates motility in Toxoplasma gondii. J. Cell. Sci. 117, 57395748. Williams, M.J., Alonso, H., Enciso, M., Egarter, S., Sheiner, L., Meissner, M., et al., 2015. Two essential light chains regulate the MyoA lever arm to promote Toxoplasma gliding motility. mBio 6, e00845-15. Wu, J., Prole, D.L., Shen, Y., Lin, Z., Gnanasekaran, A., Liu, Y., et al., 2014. Red fluorescent genetically encoded Ca21 indicators for use in mitochondria and endoplasmic reticulum. Biochem. J. 464, 1322.

575

Yanez, M., Gil-Longo, J., Campos-Toimil, M., 2012. Calcium binding proteins. Adv. Exp. Med. Biol. 740, 461482. Zeng, H., Xu, L., Singh, A., Wang, H., Du, L., Poovaiah, B. W., 2015. Involvement of calmodulin and calmodulinlike proteins in plant responses to abiotic stresses. Front. Plant Sci. 6, 600. Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R.M., Kappes, B., 1994. Calcium-binding properties of a calcium-dependent protein kinase from Plasmodium falciparum and the significance of individual calciumbinding sites for kinase activation. Biochemistry 33, 37143721. Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y.F., Nakano, M., et al., 2011. An expanded palette of genetically encoded Ca(2)(1) indicators. Science 333, 18881891.

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C H A P T E R

13 Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii Kevin M. Brown1, Christopher J. Tonkin2, Oliver Billker3 and L. David Sibley1 1

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, United States 2Division of Infectious Disease and Immune Defence, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 3Department of Molecular Biology, Norrland University Hospital, Umea˚, Sweden

13.1 Introduction Toxoplasma gondii is a ubiquitous parasite of animals, including humans, which causes infection due to the accidental ingestion of oocysts shed by cats or tissue cysts found in undercooked meat (Dubey, 2010). Although humans play almost no role in environmental transmission, they are commonly infected, and within its human host, the parasite undergoes development of the asexual stages, similar to other intermediate hosts (Montoya and Liesenfeld, 2004). Following the initial expansion of tachyzoites, which normally replicate rapidly and lyse their host cells, the parasite differentiates into a slow growing bradyzoite form that remains semidormant within tissue cysts (Knoll et al., 2014). During tachyzoite replication, the parasites reside within a membrane-bound vacuole called the parasitophorous vacuole (PV). This PV has

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00013-X

the unique properties of remaining segregated from the endosomal system, while also recruiting host mitochondria and endoplasmic reticulum. This protected compartment provides the niche for parasite replication, prior to egress. Bradyzoites also occupy a vacuole within their host cell, although they also elaborate a multilayered wall around the compartment, which remains intracellular (Ferguson, 1988). Unlike rapidly replicating tachyzoites, the bradyzoite stage replicates asynchronously, gradually leading to expansion of the cyst (Watts et al., 2015). It is thought that tissue cysts periodically rupture, releasing bradyzoites that can revert to rapid growth as tachyzoites, or give rise to daughter cysts, thus perpetuating the chronic infection. Despite a vigorous immune response, the host is unable to eliminate the infection, which remains semidormant yet capable of reactivating in the face of waning immunity.

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T. gondii is an obligate intracellular parasite, and it spends little time outside of its host cell. A common feature that defines the intracellular stages of T. gondii is that the parasite must control the decision to remain intracellular or to exit from one host cell, termed egress, and to seek a new host cell for invasion. Although bradyzoites remain within long lived cysts, the fate of tachyzoites is much more dynamic as they typically will exit from their host cell in 24
48 hours. As such, we know a great deal more about the invasion and egress pathways used by tachyzoites. The decision to egress and reinvade versus to remain within the host cell requires the coordinated action of systems that govern motility and protein secretion, two pathways that are tightly coupled to control these pathways. In this chapter, we review the cellular signaling pathways that control this critical decision point via the second messengers calcium and cyclic nucleotides, and two families of kinases that are regulated by these signals. Before summarizing the details of how these systems work in T. gondii, we briefly review the role of second messenger pathways in eukaryotic cells. Intracellular calcium is maintained at .10,000-fold lower concentrations in the cytosol than extracellular medium, and release of calcium from intracellular stores, or influx from the extracellular milieu, results in dramatic increases in cytoplasmic calcium (Berridge et al., 2003). Changes in intracellular calcium trigger many cellular pathways; hence, this second messenger is subjected to tight regulation in eukaryotic cells (Tsien, 1990). One common domain that binds calcium and regulates downstream partners is known as an EF hand, a motif first described in the structure of parvalbumin that contains a cluster of aspartic acid residues that bind calcium with high affinity. Calmodulin (CaM) is the canonical calcium
binding protein, and it contains four EF hand
containing domains (Kursula, 2014). Binding to calcium results in conformational

changes that can influence interactions and thereby the activity of many partner proteins that interact with CaM (Kursula, 2014). Related CaM-like domains function as myosin light chains and centrin, consequently, changes in intracellular calcium can regulate a wide range of cellular functions. Although T. gondii and Plasmodium falciparum respond to agonists of inositol triphosphate (IP3) and ryanodine receptors, such channels are not sufficiently conserved at the genome or protein level to be identified by homology and as such they have not yet been identified in apicomplexans (Garcia et al., 2017). A more in-depth analysis of calcium stores and regulation is found elsewhere in this volume (see Chapter 12: Calcium storage and homeostasis in Toxoplasma gondii). Cells also use cyclic nucleotides as second messengers, the cellular levels of which are not controlled by compartmentalization, but instead by the regulation of the enzymes that synthesize and degrade these. A 30 ,50 -cyclic nucleotide monophosphate (cNMP) is a signaling molecule that consists of a single-phosphate ribonucleotide with a 30 ,50 -phosphodiester bond between the phosphate and ribose groups (Beavo and Brunton, 2002). Subtle differences in the molecular structure of these nucleotides have enormous functional consequences by determining which effectors can recognize them, thus triggering specific signaling cascades. The cyclic monophosphate group functionally distinguishes cNMPs from NMPs, whereas the nucleobase, a purine (adenine or guanine) or pyrimidine (cytosine or uracil), distinguishes cNMPs from one another. Purine cNMPs (cAMP and cGMP) are widely conserved across the tree of life and are wellstudied, whereas pyrimidine cNMPs (cCMP and cUMP) are thought to have evolved more recently in mammals with little known about their respective functions (Seifert et al., 2015). Three groups of enzymes are directly involved in cNMP signaling (Beavo and Brunton, 2002).

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13.2 Motility

First, nucleotide cyclases synthesize cNMPs from nucleotide triphosphates. Once synthesized, cNMPs can then bind effector enzymes to activate or inhibit their function [e.g., cGMP activates protein kinase (PK) G (PKG)]. cNMPs are inactivated through cleavage of their 50 phosphodiester bond by a group of enzymes called phosphodiesterases (PDEs). PKs also control key signaling cascades, a process initiated by the transfer of the gamma phosphate from ATP to an acceptor hydroxyl residue typically on a Ser, Thr, or Tyr residue of the target, thereby altering interaction with downstream partners. Eukaryotic PKs share common structural features including globular N- and C-lobes linked by a hinge region that together comprise 12 conserved subdomains (Hanks and Hunter, 1995). The N-lobe contains a series of beta strands and a single alpha-helical region αC that interacts with the C-lobe. The Clobe comprises a series of alpha-helical regions, including a long F-helix, that defines the hydrophobic core. The pocket between the N- and C-lobes binds ATP, and together, they coordinate transfer the γ-phosphoryl group from ATP to the hydroxyl side chain residues in the target (Hanks and Hunter, 1995). Comparison of a large number of X-ray crystal structures reveals major conformational changes that accompany activation (Huse and Kuriyan, 2002). Although earlier studies emphasized differences in closed versus open conformations, more recent studies have defined two hydrophobic spines, referred to as the C, or catalytic spine, and the R, or regulatory spine, in controlling activation (Taylor et al., 2012a; Taylor and Kornev, 2011). In addition to their catalytic activity, kinases may impart signaling function by virtue of this bimolecular rearrangement and subsequent interaction with binding partners (Taylor et al., 2012b). This revised structural model explains many of the features of the activation mechanism of diverse kinases, including the allosteric interactions that allow pseudokinases to activate their partners (Shaw et al., 2014).

Although the human kinome contains more than 500 kinases, this number is reduced in apicomplexans, for example, T. gondii contains approximately 100 kinases (Lim et al., 2012). Apicomplexan parasites lack some normally conserved kinases such as PKC, although they contain members of the other major classes of PKs, including AGC (named for PKA, PKG and PKC), CMGC [named for cyclin dependent kinases, MAP kinases (MAPK), glycogen synthase kinase 3 and CDC-like kinases], CaM kinases (CaMK), and casein kinase 1 groups, as well as tyrosine kinases (TK)
like kinases (Miranda-Saavedra et al., 2012; Talevich et al., 2012). However, apicomplexans appear to lack conventional TK and have reduced numbers of MAPK family members (Miranda-Saavedra et al., 2012; Talevich et al., 2012). They also contain several expanded families, notably the FIKK kinases that are exported by P. falciparum into the infected red blood cell (Nunes et al., 2006; Schneider and Mercereau-Puijalon, 2005) and the rhoptry (ROP) kinase family (Peixoto et al., 2010; Talevich and Kannan, 2013), implicated in pathogenesis of T. gondii (Hunter and Sibley, 2012). Two of the families that have received the most attention in apicomplexans are calcium-dependent PKs (CDPKs), a group that is also found in plants, and cyclic nucleotide dependent kinases regulated by cGMP (PKG) and cAMP (PKA). Their respective roles in motility and cell invasion are considered in more detail next.

13.2 Motility T. gondii, such as other apicomplexan parasites, displays substrate-dependent gliding motility that is used for traversal across cellular barriers and to invade host cells (Sibley, 2004). Gliding motility can be observed on twodimensional substrates such as serum-coated glass where tachyzoites display several characteristic behaviors described based on their

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patterns as circular, helical gliding, and twirling (Ha˚kansson et al., 1999). Gliding is most commonly studied using immunofluorescence microscopy assays that rely on staining of surface membrane trails left on the substratum (Fig. 13.1). Gliding motility has also been studied based on time-lapse video microscopy, which allows more quantitative assessment of speed, directional changes, and time spent in various forms of motility (i.e., circular vs gliding). During circular gliding, the parasite remains parallel to the substrate and revolves in a counterclockwise circle that traces out the circumference dictated by the arc-shaped parasite. In contrast, during helical gliding the parasite rotates clockwise along its long axis,

rising above the substrate as it migrates forward. By spiraling along the long axis, it completes one forward step for each rotation and then reorients to initiate the next step. Twirling occurs when the parasite undergoes a similar helical twist but remains upright, tethered at its base to the substrate. Of these three behaviors, only helical gliding leads to progress across the substratum and results in cell invasion (Ha˚kansson et al., 1999; Morisaki et al., 1995). More recent studies have described tachyzoite motility in three-dimensional matrices, where parasites move in corkscrew-like trajectories, which resemble helical and twirling gliding patterns, albeit in a tissue-like environment (Leung et al., 2014).

FIGURE 13.1 Diagram of the intracellular lytic cycle of Toxoplasma gondii. (A) Gliding motility leads to deposits of surface membrane proteins on the substrate. Tachyzoites gliding on serum-coated glass, stained with antibodies to the surface protein SAG1 and secondary antibodies labeled with fluorescein isothiocyanate (FITC). Motility is actin-myosin dependent. (B) Upon attachment to host cells, the level of microneme proteins at the apical end of the parasite is greatly increased where it contains the host cell. Image stained for MIC2 using rabbit anti-MIC2 followed by secondary antibodies labeled with FITC. (C) During invasion, the parasite squeezes through a prominent constriction called the moving junction. The parasite image shown was labeled with an antibody to the surface protein SAG1 prior to detergent (red), then detergent permeabilized and stained with anti-SAG1 (green). The parasite shown is caught mid-way in invasion with the prominent constriction at the moving junction visible. (D) Vacuole rupture is initiated by release of the pore-forming protein TgPLP1. Active egress requires microneme secretion and actin-myosin based motility. Phase-contrast image showing newly emerged tachyzoites that are refractile. Source: (B) Image adapted from Carruthers, V.B., Sibley, L.D., 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol. Microbiol. 31, 421
428. The cartoon model at the bottom was provided by S. Lourido.

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13.2 Motility

The composition of the cytoskeleton is considered in more detail elsewhere in this volume (see Chapter 16: The Toxoplasma cytoskeleton: structures, proteins, and processes); here we briefly summarize the key components that are important for motility. Gliding motility is dependent on an actin
myosin motor complex that is anchored beneath the plasma membrane in T. gondii. Conventional actin is encoded by a single copy gene ACT1 that exhibits high homology to actin in other eukaryotes (Dobrowolski et al., 1997). Inhibition of parasite motility and host cell invasion by treatment with cytochalasin D, coupled with resistant mutants in host and parasite actins, indicate that ACT1 in T. gondii is largely responsible for the force needed for host cell invasion, while host cell actin plays a minor role in the process (Dobrowolski and Sibley, 1996). However, host actin is seen to polymerize at the site of invasion where it may serve a supporting role by providing an anchor point for traction (Gonzalez et al., 2009). During host cell invasion, a complex of parasite proteins that is secreted from the rhoptry necks (so-called RONs) is inserted into the host cell plasma membrane, thus providing a receptor (RON2) for recognition by AMA1, which is a transmembrane protein released from micronemes (Besteiro et al., 2011; Tonkin et al., 2011). Recent studies demonstrate that this RON complex also includes several parasite proteins that are involved in anchoring to the host cytoskeleton (Guerin et al., 2017). The unique ability to insert its own receptor into the host membrane and then bind to it with high affinity using a coevolved ligand, coupled with self-propulsion, provides an extremely efficient system for host cell invasion by T. gondii. Despite exhibiting a high degree of conservation with conventional actins, actin encoded by ACT1 in T. gondii exists largely as a globular protein sequestered with actin binding proteins such as profilin (Skillman et al., 2012) and actin-depolymerizing factor (Mehta and Sibley, 2010). This behavior contrasts sharply with

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conventional actin in mammalian systems, where a large fraction is assembled into stable filaments. Unlike the stable, long filaments seen in mammalian cells, actin filaments in T. gondii are short and relatively unstable (Sahoo et al., 2006). The difference in steady-state assembly of T. gondii actin filaments stems from intrinsically different polymerization kinetics (Skillman et al., 2013). Conventional actins show a strongly cooperative assembly where initial nucleation is rate limiting, followed by rapid elongation to form stable filaments. In contrast, T. gondii actin encoded by ACT1 polymerizes by an isodesmic mechanism, where gradual assembly of multimers is driven by concentration (Skillman et al., 2013). The prediction from the isodesmic assembly mechanism is that stable filaments will only form at high local concentration, and they should disassemble rapidly as concentrations drop due to sequestration of actin monomers. The features closely mirror what is seen for the behavior of actin in vivo. In part, these differences in kinetics can be explained by structural differences that impart instability in T. gondii actin by affecting cross bridges along and across the actin filament (Skillman et al., 2011). Conventional actin has a conserved hydrogen bond that forms between adjacent monomers across the filament, while substitute of a Ser200 to Gly in T. gondii ACT1 ablates this interaction (Skillman et al., 2011). Furthermore, T. gondii actin contains a charge residue Lys270 in place of a conserved hydrophobic residue (typically Met) that is important in stabilizing conventional filamentous actin (Skillman et al., 2011). Importantly, actin in P. falciparum shows cooperative assembly, although short unstable filaments also predominate in this system, again likely due to weak lateral interactions (Kumpula et al., 2017). The unusual kinetics of T. gondii actin imply that filaments are polymerized only when and where it is needed for motility. Actin filaments have been notoriously difficult to visualize in parasites, but one of the few examples where

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they are detected is under the membrane of gliding parasites, as captured using freeze-etch electron microscopy (Sahoo et al., 2006). These actin filaments are short, 50
100 nm in length (Sahoo et al., 2006), and are unbranched, consistent with an absence of Arp2/3 in apicomplexans (Gordon and Sibley, 2005). Given the intrinsic instability of T. gondii actin filaments, and absence of Arp2/3 for nucleation, formins likely play a critical role in assembly of actin in T. gondii. Consistent with this model, recent data implicate a family of three formins (FRM) as the major mediators of actin polymerization (Tosetti et al., 2019). FRM2 is involved in apicoplast inheritance, while FRM3 is restricted to the basal pole and involved in interconnections of daughter cells (Tosetti et al., 2019). In contrast, FRM1 is found in the conoid where it initiates F-actin polymerization needed for gliding motility (Tosetti et al., 2019). A combination of protein degradation of FRM1 together with visualizing actin using F-actin chromobody staining indicates that FRM1 nucleates actin at the apical end of the cell and that this polymerized actin then flows rearward in a process that depends on MyoH (Tosetti et al., 2019). Actin flux is further controlled by PKG, CDPK1, and apical complex lysine methyltransferase (AKMT) (Tosetti et al., 2019), which are described further below. Myosins in apicomplexans are also unusual, and they are encoded by a Class XIV family (Foth et al., 2006). In T. gondii, MyoA, which is localized beneath the plasma member, controls gliding motility (Meissner et al., 2002). MyoA exists in complex called the glideosome, which includes several proteins (i.e., GAP40, GAP45, GAP50) that anchor the motor to the inner membrane complex (IMC) (Frenal et al., 2010; Opitz and Soldati, 2002). MyoA is distributed along the length of the parasite, except for the apical and basal poles. In contrast, MyoH is found in the conoid where it plays a crucial role in initiating gliding motility by functioning at the apical cap (Graindorge et al., 2016) and MyoC, functions at the basal end (Delbac et al., 2001). Deletion of

MyoH renders parasites unable to progress past the point of apical attachment, suggesting this motor protein is important in initiating invasion (Graindorge et al., 2016). Similar to other myosins, MyoA is regulated by small CaM-like proteins that function as light chains to regulate motor function (Bookwalter et al., 2014; Williams et al., 2015). Collectively, the concerted action of MyoH and MyoA are thought to function in concert with filamentous actin that forms beneath the plasma membrane to power gliding motility. Myosins provide the force for moving along actin filaments, and the translocations of adhesive complexes (described further below) drives forward motion. The model for how gliding motility is controlled predicts that actin and myosin should be essential in T. gondii. Indeed, previous studies using regulated knockdown of MyoA (Meissner et al., 2002) or chemical inhibition of ACT1 (Dobrowolski and Sibley, 1996) support this conclusion. However, recent studies have questioned their absolute essentiality for invasion after showing it was possible to delete the genes for MyoA and ACT1 using more efficient approaches (Andenmatten et al., 2012; Egarter et al., 2014). However, loss of either gene results in a profound decrease in motility and invasion, suggesting that these proteins play a critical role in motility and cell invasion and any potential backup pathway is highly inefficient. An alternative explanation for the ability to delete these otherwise essential genes is that other myosins may compensate for loss of MyoA (Frenal et al., 2014), or residual protein expression from ACT1 may be sufficient for functionality (Drewry and Sibley, 2015), thus accounting for residual invasion that is observed.

13.3 Regulated secretion of micronemes Secretion of apically located micronemes and release of adhesins that they contain, is also essential for gliding motility in T. gondii and

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13.3 Regulated secretion of micronemes

related apicomplexans. Micronemes are secreted at the extreme apical end of the cell after traveling through the lumen of the conoid to dock at the plasma membrane where they fuse and release their contents (Carruthers et al., 1999b). Contact with the host appears to be a trigger for release, as there is a prominent accumulation of micronemal proteins at the site of initial attachment (Fig. 13.1). Secretion also requires a protein called DOC2.1 that contains two C2 domains that bind calcium (Farrell et al., 2012). DOC2.1 was identified in a temperaturesensitive genetic screen, and although its precise function is not known, it likely mediates membrane fusion, based on similarity to other C2-containing proteins. The production of phosphatidic acid (PA) in the plasma membrane is also thought to be important for release of micronemes. The action of diacylglycerol (DAG) kinase generates PA in the membrane and this lipid is recognized by a microneme membrane protein to facilitate association of these organelles with the plasma membrane (Bullen et al., 2016). Once micronemes fuse to the plasma membrane, they transfer their membrane components to the surface and release soluble proteins to the extracellular space. Micronemes contain numerous adhesive proteins that are predicted to adopt a transmembrane configuration in the plasma membrane. Thus positioned, they present extracellular domains that engage receptors and molecules found in the extracellular matrix and on host cell membranes (Dubois and Soldati-Favre, 2019). Many adhesins also have short cytoplasmic tails that are on the inside of the parasite membrane and are associated to the motor complex by a connecting protein called the glideosome associated connector (Jacot et al., 2016), which provides a necessary link between attachment and force transduction. It has therefore been proposed that the rearward translocation of microneme proteins engaged with host receptors by the actomyosin motor provides the mechanism for

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forward movement thus enabling gliding motility and cell invasion (Sibley, 2010). Following translocation, surface MIC proteins must be clipped, to release the attachment point. Rhomboid proteases, a group of intramembrane enzymes, have been shown to be important for MIC protein surface shedding and allowing tachyzoites to release from their substrate (Brossier et al., 2005; Dowse et al., 2005). The constant treadmilling of adhesins assures that the parasite attaches to the host cell with the apical end and that as the adhesin is translocated reward, the parasite moves forward across the substrate (Buguliskis et al., 2010). The importance of this directional translocation was revealed by mutants in the surface adhesin MIC2 that were inefficiently processed, leading to enhanced attachment to host cells in orientations that did not lead to productive entry (Brossier et al., 2003). Although the process of secretion, translocation and release is energetically costly, it also assures that proteins, which might be recognized by neutralizing host antibodies, do not remain on the surface for very long. Following intracellular replication, the parasite again initiates motility to actively egress from the host cell (Fig. 13.1). Signals that trigger natural egress are less well understood, but a decrease in pH, drop in [K1], and accumulation of vacuolar PA have been implicated in activating motility and triggering exit from the vacuole (Roiko et al., 2014; Bisio et al., 2019; Yang et al., 2019). Microneme secretion is important for releasing a perforin-like protein (TgPLP1) that helps to disrupt the PV membrane and rupture the host cell (Kafsack et al., 2009) (Fig. 13.1). Recent structural data implicate a protruding hydrophobic loop in the C terminal domain of TgPLP1 that inserts into the bilayer of the target membrane (Guerra et al., 2018). Active motility is also required for egress and this process is blocked by disruption of MyoH (Graindorge et al., 2016) or MyoA (Gaji et al., 2015). Egress also requires

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AKMT, which is important for activating motility although it is not required for microneme secretion (Heaslip et al., 2011). This finding suggests that posttranslational modification of proteins by methylation of substrates is important in regulating motility. Although parasites remain immotile during replication, they are always primed for egress as pharmacological elevations of calcium, PA, or cGMP, or disruption of host membranes, rapidly and efficiently stimulate egress of replicating parasites in vitro.

13.4 Release of intracellular calcium as a regulatory cascade Similar to other eukaryotic cells, intracellular calcium is maintained at low levels in the cytosol of T. gondii tachyzoites, and this second messenger is tightly regulated by a system that governs homeostasis (Moreno et al., 2011). Because calcium is such a potent second messenger, cells typically sequester it away in storage organelles to prevent unwanted or prolonged activation of signaling cascades. The compartments that store calcium within the cell, and various mechanisms to regulate cytosolic levels have been described previously (Lourido and Moreno, 2015). Here, we will briefly review the regulation of elevated intracellular calcium that coordinates protein section and motility in T. gondii tachyzoites. During gliding motility, intracellular calcium levels oscillate, as shown using a calcium sensitive probe called Fluo-4 (Lovett and Sibley, 2003) or more recently genetically encoded biological sensor called GCaMP (Stewart et al., 2017). Parasites undergoing active gliding are observed to undergo oscillations in calcium, while inactive cells are much less likely to show these patterns. The oscillations take place in the absence of extracellular calcium, suggesting that there are repeated rounds of calcium release and reuptake from

intracellular stores. Intriguingly, calcium levels are elevated during gliding and during initial attachment to host cells, but they are rapidly dampened once the parasite invades (Lovett and Sibley, 2003; Uboldi et al., 2018). In studies monitoring natural egress using calcium sensitive protein reporters, calcium levels also become elevated in the parasite prior to egress (Borges-Pereira et al., 2015). Pharmacological studies implicate intracellular calcium release channels in regulating elevated calcium and triggering microneme release. Artificial elevation of intracellular calcium using ionophores leads to secretion of micronemes, as detected by western blot for release of processed proteins (Carruthers et al., 2000), and more recently by release of luciferase fusion reporters (Brown et al., 2016). Consistent with this role, chelation of intracellular calcium using BAPTA-AM, a cell permeant analog of EGTA, blocks microneme secretion (Carruthers and Sibley, 1999). Similar to mammalian cells, ethanol and acetaldehyde stimulate calcium increases and stimulate microneme secretion in T. gondii (Carruthers et al., 1999a). Intracellular calcium is sufficient to control these events, since secretion can be triggered in the absence of extracellular calcium. Although there are multiple stores of calcium in the cell, the endoplasmic reticulum is the most probable source of calcium release leading to a rise in cytosolic levels. Consistent with this role, T. gondii contains a conserved reuptake channel located in the endoplasmic reticulum known as SERCA (sarcoplasmic/endoplasmic reticulum calcium ATPase) (Nagamune et al., 2007), and studies with the inhibitor thapsigargin are consistent with its role in calcium reuptake (Carruthers et al., 1999a). In addition, tachyzoites of T. gondii respond to ethanol by producing IP3 and releasing intracellular calcium and antagonists of IP3 receptors such as xestospongin block this pathway (Lovett et al., 2002). Agonists of the related ryanodine channel such as caffeine and ryanodine also trigger calcium release and microneme

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secretion (Lovett et al., 2002). The action of ethanol in triggering IP3 production is consistent with activation of phospholipase C, which also generates the second messenger DAG. Despite these pharmacological findings, the genomes of apicomplexan parasites do not contain conserved IP3 or ryanodine calcium release channels (Garcia et al., 2017). Hence, although all the available evidence points to intracellular calcium stores in controlling microneme secretion, it is presently unclear how release of this critical signal is mediated in T. gondii.

13.5 Calcium-dependent protein kinases A range of proteins can be activated by a rise in intracellular calcium levels. Similar to other apicomplexans, T. gondii contains an expanded family of CDPKs, which are found in green alga, oomycetes, and higher plants but absent in animal cells (Billker et al., 2009)

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(Fig. 13.2). CDPKs are serine/threonine kinases that contain multiple calcium binding domains known as EF hands, a feature also found in CaM (Hui et al., 2015). Unlike CaM-regulated kinases (CaMK) in mammalian cells, where the separate kinase domain is regulated by recruitment of CaM, CDPKs contain the CaM-like domain linked to the kinase domain through a junctional domain that together is called the calcium activation domain (CAD) (Wernimont et al., 2010, 2011). It has been proposed that CDPKs arose by fusion of a kinase domain with CaM, and this arrangement subsequently diversified separately in plants and apicomplexans (Billker et al., 2009; Hui et al., 2015). Canonical CDPKs contain an N-terminal kinase domain, junction region, and four EF hands, although other arrangements, including N-terminal CaM-like domains are also seen (e.g., CDPK6, CDPK7) (Fig. 13.2). Several members contain N terminal extensions (e.g., CDPK2, CDPK5, and CDPK6) or variable

FIGURE 13.2 Phylogenetic tree of CDPKs from apicomplexan parasites. Models of domains depict the relative domain structure of major CDPKs. EF hands are regions that contain calmodulin like calcium binding domains. (EF) denotes domains with mutations in one or more of the acidic residues that chelate cations. PH, Pleckstrin homology domain. Purple domains indicate unique N-terminal extensions. Orthologs do not share the same numbering convention due to historical differences in naming. For example, CDPK1 in Toxoplasma gondii is homologous to CDPK4 in Plasmodium falciparum. CDPK, Calcium-dependent protein kinase. Source: Adapted from Billker, O., Lourido, S., Sibley, L.D., 2009. Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5, 612
622.

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number of EF hands (e.g., CDPK4) or additional domains such as a pleckstrin homology (PH) domain (e.g., CDPK7) (Fig. 13.2). Overall, 14 genes encoding CDPKs have been identified in T. gondii (Long et al., 2016), although only seven are present in Plasmodium and Cryptosporidium (Billker et al., 2009; Hui et al., 2015). The numbering of CDPK genes is not the same across these organisms as they were historically identified and named at different times. Moreover, even where orthologs have been studied across different genera, their functions are not always conserved. For example, CDPK1 in T. gondii is the most closely related to CDPK4 in Plasmodium spp. However, CDPK1 controls microneme secretion in T. gondii (see next), but CDPK4 regulates male gamete formation in Plasmodium berghei (Billker et al., 2004). Likewise, CDPK5 is essential for egress in P. falciparum (Dvorin et al., 2010), but dispensable in T. gondii, despite being the closest related gene. The amplification of CDPKs in T. gondii, including several pairs of paralogs, suggests that they control an expanded

repertoire of functions or that they are partially redundant. CDPK1 and CDPK3 in T. gondii share a similar motif preference for substrates that is reminiscent of CaMK, with the presence of basic residues (Lys, Arg) flanking the Ser/ Thr that is phosphorylated (Lourido et al., 2010, 2012). CDPK1 (cytosolic) and CDPK3 (membrane) differ in their localization due to differences in N-myristoylation and/or palmitoylation, and these different locations may in part control substrate accessibility (Lourido et al., 2010, 2012). CDPKs exhibit a number of unique structural features. When in the noncalcium bound state, the CAD faces the kinase active site, occluding access of ATP and substrate (Wernimont et al., 2010, 2011) (Fig. 13.3). In the inactive structure the autoinhibitory region forms a triad of interacting residues that holds the CAD in place and blocks substrate binding (Hui et al., 2015). Upon binding to calcium the CAD undergoes a dramatic reorganization, splitting several of the long helical regions and flipping to the back of the kinase domain, thus

FIGURE 13.3 Models of CDPK1 in the active versus inactive state based on X-ray crystal structures. (A) Model of the inactive CDPK1 structure from Toxoplasma gondii (PDB code 3KU2). In the inactive structure, the CAD (gray) occludes the kinase surface and blocks the ATP and substrate binding regions. Within the autoinhibitory domain (red box) key residues (shown in insert) block substrate binding by simulating a pseudosubstrate interaction. (B) In the active conformation of CDPK1 from T. gondii (PDB code 3HX4), the CAD has rearranged to the posterior surface of the kinase and undergone substantial reorganization of the CH1 (blue) and CH2) helices (green arrows). The green spheres represent bound calcium ions. CAD, calcium activation domain; CDPK, Calcium-dependent protein kinase. Source: Adapted from Wernimont, A.K., Artz, J.D., Finerty, P., Lin, Y., Amani, M., Allali-Hassani, A., et al., 2010. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. Nat. Struct. Mol. Biol. 17, 596
601.

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exposing both the substrate binding and ATP binding pockets (Wernimont et al., 2010, 2011) (Fig. 13.3). The displacement of the CAD involves rotation of approximately 130 degrees, which is the most dramatic example yet described for activation of a kinase (Hui et al., 2015). In the active conformation the kinase domain adopts the usual conformation of the αC helix being positioned close to the ATP binding pocket and the activation loop being positioned to interact with ATP (Wernimont et al., 2010, 2011). CDPK1 was the first member of this family to be functionally examined, and it turned out to be essential for calcium-stimulated microneme secretion (Lourido et al., 2010). Genetic suppression of CDPK1 results in failure to upregulate microneme secretion in responses to elevated calcium, thus blocking motility, cell invasion, and egress (Lourido et al., 2010). In contrast, CDPK1 plays no appreciable role in intracellular replication. These findings were confirmed using a chemical inhibitor that was specific for the ATP-binding pocket of CDPK1 (Lourido et al., 2010), as defined further below. The closely related PK CDPK3 is also required for egress (Garrison et al., 2012; McCoy et al., 2012), although only partially required for microneme secretion in response to certain agonists (Lourido et al., 2012) and more important in intracellular parasites than extracellular (McCoy et al., 2012). Phosphoproteomic studies that defined the substrates of CDPK3 implicate this kinase in the control of calcium, suggesting it lies upstream of CDPK1 (Treeck et al., 2014; McCoy et al., 2017). Consistent with this prediction, overexpression of CDPK1 can rescue the egress defects of Δcdpk3 mutants (Treeck et al., 2014). In addition, the function of CDPK3 in egress can be compensated for by activation of PKG (Lourido et al., 2012), albeit with slower kinetics (McCoy et al., 2017), as described further below. Collectively, these findings suggest that CDPK1 and CDPK3 have partially overlapping functions that control

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secretion of micronemes and activation of the motor complex during motility and invasion. As discussed further below, these kinases likely have other functions given their broad substrate profiles that have been revealed by phosphoproteomic studies. Functions have also been attributed to several other CDPKs based on genetic knock out or knock down strategies. Depletion of CDPK7 using the tetracycline repressible promoter results in aberrant cell division, while motility invasion and egress are not affected (MorlonGuyot et al., 2014). Closer analysis of the defect reveals that while DNA content was normal in Δcdpk7 mutants, daughter cells were disrupted in the formation of centrosome and kinetochores (Morlon-Guyot et al., 2014). CDPK7 also contains a PH domain, implicated in binding to phosphoinositides (Morlon-Guyot et al., 2014), although the role of this domain in the function of CDPK7 remains uncertain. In contrast, CDPK2 has a canonical structure of N terminal kinase domain followed by four EF hand CaM-like domains. Disruption of CDPK2 in tachyzoites has no effect on growth, yet during differentiation of bradyzoites, survival of Δcdpk2 mutants is profoundly affected (Uboldi et al., 2015). CDPK2 contains a carbohydrate binding module (i.e., CBM20), implicated in recruiting it to amylopectin storage sites (Uboldi et al., 2015). CDPK2 regulates the accumulation of amylopectin, a sugar polymer thought to play a role in carbohydrate storage in bradyzoites (Uboldi et al., 2015). Increased production and loss of degradation of amylopectin in Δcdpk2 mutants results in accumulation of large amylopectin inclusions that causes loss of cell viability (Uboldi et al., 2015). T. gondii also contains a large number of noncanonical CDPKs, which contain variable numbers of EF hands, many of which are only partially conserved (Long et al., 2016) (Fig. 13.2). The loss of conserved acidic residues within these degenerate EF hands suggest that some of these kinases may not be

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regulated by calcium. Given the dramatic reorganization seen in the structures of CDPK1, these features also suggest that some of these CDPKs may not be catalytically active. Inactive kinases, also referred to as pseudokinases, often serve important roles in substrate binding or allosteric activation of partners (Boudeau et al., 2006). Using a modified CRISPR/Cas9 approach that contained two guide RNAs designed to cleave at 50 and 30 ends of the gene, seven of these CDPK genes were deleted entirely from the genome of Type 1 and Type 2 parasites (Long et al., 2016). Surprisingly, the majority of these mutants showed minimal growth defects in vitro or, in the case of Type 2 strains, in a mouse model of chronic infection (Long et al., 2016). The sole exception to this finding was the Δcdpk6 mutant that showed a mild plaque defect in vitro and reduced tissue cysts level during chronic infection of mice (Long et al., 2016). The modest phenotypes for individual mutants, combined with expanded gene family, suggest these genes are partially redundant. However, sequential deletion of genes with CRISPR/Cas9, and recycling of a selectable marker that was removed using Cre recombinase and LoxP sites, also failed to identify striking phenotypes for double and even triple mutants (Long et al., 2016). Although these proteins appear to be nonessential in tachyzoite and bradyzoites stages, they may function at other stages in the life cycle. One of the unique features of CDPK1 is that it contains a small residue at the so-called gatekeeper residue. The majority of mammalian and parasite kinases contain a bulky hydrophobic residue at the gate keeper, which forms part of the R spine in the kinase structure (Hui et al., 2015). Surprisingly, CDPK1 does not require such a bulky hydrophobic residue and instead has Gly, the smallest possible side chain (Wernimont et al., 2010; Lourido et al., 2010) (Fig. 13.4). This residue is called the gatekeeper because it controls access of bulky ATP

analogs into the ATP-binding pocket. Previous studies have taken advantage of this feature to sensitize mammalian kinases to bulky ATP analogs, thereby allowing selective inhibition of the modified kinase in a background where all other kinases are resistant (Lopez et al., 2014). The natural Gly gatekeeper of CDPK1 allows the selective inhibition of this kinase, since all other kinases in T. gondii contain insensitive gatekeepers. This chemical genetic approach was used to confirm the role of CDPK1 in microneme secretion (Lourido et al., 2010). Moreover, mutation of the Gly gatekeeper to Met renders the CDPK1, and parasite, completely insensitive to a class of inhibitors called pyrazolopyrimidines (Fig. 13.4). The combination of these findings demonstrates that CDPK1 is the primary target of this class of pyrazolopyrimidines in T. gondii. The unique sensitivity to pyrazolopyrimidines has been used to advance CDPK1 as a candidate for development of new therapeutic interventions, as summarized previously (Hui et al., 2015). Lead analogs of this series are highly potent and selective for CDPK1 and have shown good efficacy in mouse models of toxoplasmosis, including a chronic model of reactivation (Rutaganira et al., 2017). The Gly gatekeeper also presents the opportunity to identify substrates of CDPK1, as a similar orthogonal strategy has been used to label kinase substrates using bulky analogs of ATPγS that form a thiol adduct that can be purified and potential substrates identified by mass spectrometry (Allen et al., 2005). This labeling strategy is selective for small (i.e., Gly) gatekeeper residues; hence, it can be used in semipermeable, whole cell preparations. Using this approach, several candidate substrates of CDPK1 were identified including a dynaminlike protein implicated in vesicular trafficking (Lourido et al., 2013). Methods for improved detection of CDPK1 substrates using more quantitative mass spectrometry approaches have recently been summarized (Rothenberg et al., 2016).

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FIGURE 13.4

Gatekeep residues of CDPKs are key to inhibitor sensitivity. (A) Alignment of subdomain V of CDPKs from Tg showing the gate keeper reside. CDPK1 is unique in containing a small residue at this position. Alignment generated by S. Lourido. (B) Structure of two pyrazolopyrimidine inhibitors described previously (Lourido et al., 2013). (C) Sensitivity of WT parasites versus lines that express modified CDPK1 enzymes to contain either a Gly gate keeper (cKO/ WT) or methionine gate keeper (cKO/G128M). (D) Conditional tetracycline repressible lines, referred to as cKO, were used to assess the sensitivity of parasite growth to inhibition by inhibitors after the addition of anhydrotetracycline. The replacement of the Gly residue with Met result in a .20-fold shift in sensitivity. CDPK, Calcium-dependent protein kinase; cKO, conditional knockouts; Tg, Toxoplasma gondii; WT, wild type. Source: Adapted from Lourido, S., Shuman, J., Zhang, C., Shokat, K.M., Hui, R., Sibley, L.D., 2010. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465, 359
362. Inhibition assay data and curve fitting graphs were provided by S. Lourido.

Other approaches for identifying the substrates of CDPKs have relied on phosphoproteome analysis in cells that express wild-type complement of kinases versus genetic knockouts or knockdowns of select genes. These studies are complicated by the difficulty in deciphering proximal from distal effects, since kinases may phosphorylate and activate or inactive other enzymes, including downstream kinases and phosphatases. An added challenge of these studies is the need for quantitative comparisons and a variety of methods exist for using internally labeled samples such as SILAC or postprocessing labeling such as iTRAQ. What has proved useful is methods to enrich

for particular substrates. For example, CDPK2 requires association with amylopectin through its CBM20 domain and thus substrates are predicted to share this property. Thus SILAC labeling followed by substrate enrichment over amylose resin proved success in identifying CDPK2-dependent phosphorylation events over a range of enzymes predicted to be involved in starch metabolism, including pyruvate phosphate dikinase, glycogen phosphorylase, and alpha glucan water dikinase (Uboldi et al., 2015). These findings are consistent with the phenotype of Δcdpk2 knockouts that accumulate large stores of amylopectin and do not form viable bradyzoites (Uboldi et al., 2015).

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Phosphoproteomics analysis was also was used to compare wild type and CDPK3 knockouts identified potential substrates, including several myosins and other proteins implicated in egress (Treeck et al., 2014). This study detected increased phosphorylation of CDPK1 substrates, and CDPK1 itself, in the Δcdpk3 mutants, consistent the ability of CDPK1 to compensate for CDPK3 when the former enzyme is overexpressed (Treeck et al., 2014). Finally, this study also detected potential targets of CDPK1 in pathways that regulate ion homeostasis, and metabolism (Treeck et al., 2014), suggesting a broader role than motility and egress phenotypes than have thus far been explored. An alternative approach was taken by fusing the permissive biotin ligase BirA to CDPK3, thus identifying the MyoA motor complex as in close proximity to this kinase (Gaji et al., 2015). This finding was validated by mutating the respective sites Ser21 and Ser274, which failed to fully complement a null mutant, confirming the importance these phosphorylation sites in controlling motor activity (Gaji et al., 2015). Generation of phosphomimetic mutations of TgMyoA also complemented the motility defects of the Δcdpk3 mutant, demonstrating that this is one of the major substrates of this kinase controlling motility (Gaji et al., 2015). It should be noted, however, that phenotypes of these phosphomimetic sites were mild suggesting that though important they are not essential for activation of MyoA and thus motility. This BioID proximity-based approach offers an alternative for identifying substrates, which could be especially valuable for kinases that traditionally do not bind tightly to their substrates.

13.6 Nucleotide cyclases and cyclic nucleotide phosphodiesterases In addition to cyclic ADP-ribose, which is implicated in controlling calcium during secretion (Chini et al., 2005), cAMP and cGMP are

the only cyclic nucleotides have been definitively observed in apicomplexans (Gould and de Koning, 2011). Enzymes called purine nucleotide cyclases [e.g., adenylate cyclase (AC) or guanylate cyclase (GC)] convert NTPs (ATP or GTP) into cNMPs by forming a 30 ,50 phosphodiester bond between phosphate and ribose, releasing pyrophosphate (PPi). There are six classes of ACs that are structurally unrelated but perform the same ATP to cAMP reaction. All six classes of ACs can be found in prokaryotes, but only Class III ACs are found in eukaryotes. Most Class III ACs consist of two catalytic domains (C1 and C2), where C1 is flanked by sets of six transmembrane domains. The C1 and C2 domains dimerize to form a catalytic unit which can be activated or inhibited in various ways such as G protein
coupled receptor (GPCR) signaling, elevated calcium, or photoactivation. In large part our understanding of these group of enzymes in apicomplexans comes from work in Plasmodium spp. Apicomplexans have both transmembrane and soluble ACs, but it is unclear how they are activated since they lack hormones, GPCRs and do respond to mammalian AC agonists such as forskolin (Baker and Kelly, 2004). GCs, which convert GTP into cGMP, can also be found in prokaryotes and eukaryotes. GCs have been classically divided into two types (Type 1 and Type 2). Type 1 GCs are single catalytic domain transmembrane receptors that dimerize to form a catalytic core and are activated by peptide hormones. Type 2 GCs are soluble and can be activated by nitric oxide or light. Interestingly, apicomplexan GCs do not possess prototypical Type 1 or Type 2 features, instead appear to have evolved from Class III ACs and acquired an unrelated P-Type ATPase-like domain (Linder et al., 1999). Once formed, cNMPs act locally through diffusion, and they are sensed by effector proteins (e.g., cNMP-dependent kinases), acting as a “second messenger” for the primary signal.

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Chemical or genetic inhibition of apicomplexan cGMP-dependent PKs is lethal, indicating that cGMP is an essential signaling molecule (Brown et al., 2016, 2017; Donald et al., 2002, 2006; Donald and Liberator, 2002; Gurnett et al., 2002; Wiersma et al., 2004; Baker and Deng, 2005). Accordingly, apicomplexan GCs are fully conserved and likely essential throughout the phylum (Bisio et al., 2019; Yang et al., 2019; Carucci et al., 2000; Moon et al., 2009; Brochet et al., 2014; Gao et al., 2018; Brown and Sibley, 2018; Hirai et al., 2006). Apicomplexan GCs have an unusual domain architecture consisting of an N-terminal P-type ATPase-like domain and two C-terminal GC domains (C1 and C2) where C1 is flanked by sets of six transmembrane domains. This “protist-type” ATPase-GC was first recognized in a 1999 study on GCs of free-living ciliates (Linder et al., 1999), then extended to other alveolates, including Apicomplexa (Baker and Kelly, 2004), Stramenopiles, and Rhizaria (Brown and Sibley, 2018), indicating it likely emerged from a gene fusion in a common ancestor of the SAR supergroup of protists. In addition to the unique P-type ATPase domain, the dual catalytic GC domains more closely resemble the Class III ACs than Type 1 or Type 2 GCs that contain a single catalytic GC domain and a single transmembrane domain at most. This implies that apicomplexan GCs likely evolved from a Class III AC and acquired a P-type ATPase domain. Most apicomplexans encode a single GC gene, except for the malarial parasites which encodes two: GCα and GCβ (Carucci et al., 2000). GCα is likely essential for asexual blood stages based on resistance to repeated deletion attempts (Kenthirapalan et al., 2016), whereas GCβ regulates ookinete motility and invasion in the mosquito midgut (Moon et al., 2009; Hirai et al., 2006). The role for GCβ in ookinete gliding was elucidated further in Plasmodium yoelii, a rodent malarial parasite. Gao et al. (2018) demonstrated that PyGCβ is expressed

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in the cytoplasm of gametocytes and zygotes but is polarized at a unique position in mature ookinetes behind the apical collar coined the “ookinete extrados site”. PyGCβ is anchored by PyISP1, an IMC component, and further stabilized by a Class IV P-Type ATPase (P4ATPase) cofactor called PyCDC50A (Gao et al., 2018). Coexpression of the two enzymatic domains separately rendered PyGCβ nonfunctional, providing strong genetic evidence that apicomplexan ATPase-GCs require linked enzymatic domains for proper function (Gao et al., 2018). Several recent studies of T. gondii GC (TgGC) provide further insights into their mode of activation, localization, interactions, and function (Bisio et al., 2019; Yang et al., 2019; Brown and Sibley, 2018). TgGC is a large, 22 transmembrane pass protein with a predicted mass of 477 kDa that accumulates at the apical cap region of the plasma membrane of T. gondii (Brown and Sibley, 2018; Jia et al., 2017) (Fig. 13.5). Conditional knockdown approaches demonstrated that TgGC is essential for tachyzoite cell-to-cell transmission by regulating egress, migration, and invasion (Bisio et al., 2019; Yang et al., 2019; Brown and Sibley, 2018). The essentiality of TgGC was also established in an in vivo model of toxoplasmosis, where parasites lacking TgGC were incapable of producing lethal infection in highly sensitive C57Bl/6 mice due to defects in proliferation, dissemination, and persistence (Brown and Sibley, 2018). Mechanistically, TgGC is essential for its ability to produce cGMP and in turn activate TgPKG, a cGMP-dependent kinase required for cell-to-cell transmission by regulating microneme secretion and motility (Brown et al., 2017; Brown and Sibley, 2018). Conditional loss of TgGC significantly lowers basal cGMP levels (Bisio et al., 2019; Yang et al., 2019), indicative of GC activity. Furthermore, the defects in microneme secretion caused by TgGC depletion, but not

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FIGURE 13.5 Nucleotide cyclases of Toxoplasma gondii. (A) Domain arrangement of four TgACs and single TgGC. (B) Alignment of purine binding motifs of selected nucleotide cyclases demonstrating conserved residues for binding ATP (adenylate cyclases) or GTP (guanylate cyclases). (C) Cartoon diagram depicting topology of TgGC. TgGC, T. gondii GC.

TgPKG depletion, can be reversed by supplementing parasites with a cell-permeable cGMP analog (PET-cGMP), placing TgGC upstream of TgPKG in the signaling pathway (Brown and Sibley, 2018). If the primary function of TgGC is to produce cGMP, the function of its N-terminal P-type ATPase-like domain is uncertain. This question was partially addressed through mutant analysis. Complementation of TgGC knockdown with a panel of mutant constructs indicated that TgGC requires both P-type ATPase and GC domains to be catalytically active and linked in order for proper localization and function, suggesting that the P-type ATPase domain may serve as regulatory domain for GC activity (Brown and Sibley, 2018). A similar requirement was observed for P. yoelii GCβ (Gao et al., 2018). In further support of this hypothesis, TgGC was shown to complex with an essential P4-ATPase cofactor called TgCDC50.1 (Bisio et al., 2019), homologous to the requirement of P. yoelii PyGCβ for PyCDC50A (Gao et al., 2018). Since P4ATPases are phospholipid flippases, it is possible that phospholipid flipping provides a

novel mode of apicomplexan GC activation. Although no bulk defects in phospholipid flipping were observed following loss of TgGC in extracellular parasites, intracellular parasites lacking TgGC were unable to respond to elevations in TgDGK2-generated PA in the PV, an intrinsic stimulator of egress (Bisio et al., 2019). In addition to intravacuolar PA accumulation, sudden drops in host cytosolic K1 or pH also trigger T. gondii egress by elevating cytosolic calcium levels in the parasite cytosol, activating TgCDPK1 and TgCDPK3 (Yang et al., 2019). Interestingly, the calcium response to depressions in K 1 or pH is also TgGC dependent (Yang et al., 2019). Therefore the P-type ATPase domain of TgGC may function by transporting or sensing lipids or ions, thereby controlling the activity of the GC domain through an unknown mechanism. In addition to TgCDC50.1, a second TgGC interactor called TgUGO, an essential transmembrane protein, was also shown to be necessary for TgGC localization (Bisio et al., 2019). Whether the TgGC-TgCDC50.1-TgUGO complex is anchored to an apical cap protein of the IMC remains to be seen.

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13.6.1 Adenylate cyclases There are four putative ACs in T. gondii— TgACα1, TgACα2, TgACα3, and TgACβ (Brown and Sibley, 2018) (Fig. 13.5A). The three genes of the TgACα group share a putative bifunctional domain architecture in which an N-terminal ion channel-like domain (pfam00520) precedes a single cyclase homology domain and presumably anchors it to a membrane. This domain architecture is typical of ACs in the subphylum alveolata (Muhia et al., 2003). This group of organisms lacks the heterotrimeric G proteins that often regulate membrane ACs in metazoan, and the cyclase activity may therefore be regulated by the unique ion channel-like domain which resembles a voltage-gated K 1 channel. This idea requires testing in T. gondii, but there is supporting data from Paramecium, where a homologous AC from the cilia copurifies with a voltage-gated K1 channel that links membrane hyperpolarization to cAMP production (Schultz et al., 1992). In T. gondii all three α type ACs can be readily disrupted individually in tachyzoites, and only the deletion of α2 and α3 produces a mild plaque phenotype (Brown and Sibley, 2018), pointing toward either functional complementation or roles at other life cycle stages. Evidence that apicomplexan zoites can dispense of ACs of the α type comes from P. falciparum, where there is only one ACα homolog. PfACα has enzymatically verified AC activity (Muhia et al., 2003). In P. berghei ACα was readily disrupted in the blood stages, and no change in phenotype was observed throughout blood and mosquito development except for an incomplete block of regulated apical protein exocytosis and hepatocyte invasion at the sporozoite stage (Ono et al., 2008). TgACβ lacks transmembrane domains and most closely resembles the bicarbonatesensitive soluble ACs found in pro- and eukaryotes (Kobayashi et al., 2004). In tachyzoites, TgACβ localizes to the cytosolic face of the

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rhoptry membrane, where it defines the proximal portion of the rhoptry neck, closest to the bulb (Mueller et al., 2016). TgACβ is recruited to its precise location by the armadillo repeats only protein (ARO), which is essential for the apical organization of rhoptries (Mueller et al., 2013; Beck et al., 2013). The localization of ARO in turn relies on palmitoylation by DHHC7, the only rhoptry-specific protein acyl transferase (Beck et al., 2013; Frenal et al., 2013). The stabilization of TgACβ requires the ARO-interacting protein, which may function as a bridge between ARO and the cyclase (Mueller et al., 2016). Evidence for AC activity of ACβ is only available from recombinantly expressed protein of the P. falciparum ortholog, which is thought to be essential in the asexual erythrocytic phase of the life cycle (Salazar et al., 2012). The deletion of ACβ in T. gondii tachyzoites has a severe plaque formation phenotype (Brown and Sibley, 2018). Future work will have to elucidate whether AC activity is required for ACβ to be recruited to the rhoptries and whether its specific localization is critical for the ability of one of the PKA catalytic subunits, PKAc1, to negatively regulate egress and motility (Jia et al., 2017). Whether combinatorial deletion of TgACs produces synthetic lethality is currently not known. It also remains to be seen whether TgAC mutants mimic the hypermotility and “restless invasion” phenotypes that explain the fitness defects seen with loss of TgPKAc1 (Uboldi et al., 2018; Jia et al., 2017). Therefore the primary function of TgACs may be to produce cAMP to activate TgPKAc1, by the release of the regulatory domain TgPKAr, to suppress motility during intracellular replication.

13.6.2 Phosphodiesterases Cyclic nucleotides are inactivated through cleavage of their 50 -phosphodiester bond by a group of enzymes called PDEs, thus providing

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another layer of control for the activity of cNMP-dependent kinases. Phylogenetic analysis of apicomplexan PDE catalytic domains reveals 10 natural clades that are yet to be named (Fig. 13.6). Only one clade is shared among all apicomplexans, three are specific to Coccidians (which include T. gondii), one is specific to Hematazoans (which includes the genus Plasmodium), one is shared between

Coccidians and Hematazoans, and four clades are shared between Coccidians and Gregarines (which include Cryptosporidium). Much of what we know about apicomplexan PDEs comes from studies on Plasmodium spp. Members of this genus contain only four putative PDEs, which all share a similar overall organization comprising of up to six predicted transmembrane domains in the N-terminal to central

FIGURE 13.6 Phylogenetic clades of apicomplexan phosphodiesterases. A bootstrap consensus tree of 101 apicomplexan PDEs (EuPathDB accessions) aligned catalytic domains was generated with MEGA6 (http://www.megasoftware.net/) using maximum likelihood, tested by Bootstrap method with 1000 replications, Jones-Thornton-Taylor model for amino acid substitutions, with nearest neighbor-interchange. The MEGA6 tree was redrawn as a radial cladogram with dendroscope (http://dendroscope.org/). PDE, phosphodiesterase.

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part of the protein and a PDE homology domain toward the C-terminal end (Wentzinger et al., 2008). Only the catalytic domain of PDEα has been successfully expressed in E. coli and was found to be specific for cGMP (Yuasa et al., 2005). In contrast, PDEβ precipitated from schizonts as an HAtagged protein was similarly active toward cGMP and cAMP (Flueck et al., 2019). For the remaining two enzymes, catalytic specificity can so far only be inferred from the phenotypes of knockout mutants. Although the PDEα gene is most highly expressed in the asexual blood stages, its disruption had no growth effect on P. falciparum in culture (Wentzinger et al., 2008). The role for this gene in sexual and mosquito stages remains to be investigated. PDEβ is most highly expressed and essential in asexual blood stages (Flueck et al., 2019; Bushell et al., 2017). A conditional mutant in which a catalytic PDEβ domain was inducibly deleted using a dimerizable Cre recombinase showed PDEβ to be important both for red blood cell invasion and subsequent growth (Flueck et al., 2019). Although PDEβ has dual specificity, only cAMP levels were increased in the mutant, and overactivation of the PKA catalytic domain therefore the most likely cause of the phenotype. In fact, PKA activity is prolonged in asexual blood stages after inducible disruption of PDEβ, and many phosphosites in these parasites resembled PKA phosphorylation motifs (Flueck et al., 2019). More work is required to define and explain the relative importance of PDEβ to regulate PKA- versus PKG-dependent downstream pathways. The PDEγ gene can be disrupted in P. yoelii without a marked effect on asexual blood stages and mosquito stage development, but gliding motility and hepatocyte invasion by sporozoites are strongly reduced (Lakshmanan et al., 2015). A much elevated cGMP level in mutant sporozoites provides indirect evidence that PDEγ may hydrolyze cGMP (Lakshmanan et al., 2015), which fits

595

with the role of PKG in sporozoite gliding and motility (Govindasamy et al., 2016). cAMP levels in the mutant were not investigated. Peak expression of PDEδ is in gametocytes, the sexual precursor stages, and disruption of this gene interferes with sexual development in both P. falciparum (Taylor et al., 2008) and P. berghei (Moon et al., 2009), although differences are observed in the precise stage where these phenotypes become evident. In contrast to Plasmodium spp., there are 18 putative cNMP PDEs found in the genome of T. gondii (Figs. 13.6 and 13.7), and little is known about their substrate specificity (cAMP, cGMP, or both), localization, essentiality, or function. The cGMP-PDE inhibitors Zaprinast and BIPPO stimulate microneme secretion and egress in T. gondii in a PKG-dependent fashion (Stewart et al., 2017; Brown et al., 2016; Sidik et al., 2016a), suggesting that T. gondii expresses one or more PDEs to regulate cGMP levels. There is also ample indirect evidence for the importance of both cGMP-PDEs and cAMP-PDEs based on the essentiality of TgPKG (Brown et al., 2017) and TgPKA (Uboldi et al., 2018; Jia et al., 2017), the two primary signaling kinases for cGMP and cAMP in T. gondii, respectively. To date, only TgPDE1 (TgGT1_202540) and TgPDE2 (TgGT1_293000) have been partially investigated. TgPDE1 and TgPDE2 localize to the parasite cytosol (Jia et al., 2017) and may be essential based on a genome-wide CRISPR disruption screen in tachyzoites (Sidik et al., 2016b). A recent RNAseq transcriptomic analysis of T. gondii strain CZ (Hehl et al., 2015) indicated that there are stage-independent and stage-dependent TgPDEs expressed in tachyzoites, bradyzoites, and merozoites, which provides a plausible explanation for why there are so many seemingly redundant PDEs in T. gondii. Further studies are needed to define the substrate specificity (cAMP, cGMP, or both), localization, interactions, essentiality, and precise function these PDEs.

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FIGURE 13.7 Cyclic nucleotide signaling in Toxoplasma gondii. Genes predicted to be responsible for cGMP signaling in T. gondii. Cyclic GMP drives parasite motility and transmission whereas cAMP blocks motility for parasite replication.

13.6.3 Cyclic GMP-dependent protein kinase(PKG) Apicomplexan parasites encode a single PKG gene with three cGMP binding domains within a regulatory domain (Gurnett et al., 2002; Baker and Deng, 2005). However, several genera of tissue-cyst forming coccidian parasites, including T. gondii, Hammondia, Neospora, and Eimeria, express two isoforms of the PKG protein by alternative translation initiation. N-Acylated TgPKG-I localizes to the plasma membrane, whereas TgPKG-II, lacking the N-acylated residues, is cytosolic (Donald et al., 2002; Donald and Liberator, 2002; Gurnett et al., 2002). Noncoccidian parasites, such as Plasmodium spp., have a single cytosolic isoform. Chemical genetic studies have demonstrated that PKG activity is required for the lytic lifecycles of all apicomplexan parasites (Brown et al., 2016; Donald et al., 2002, 2006;

Donald and Liberator, 2002; Gurnett et al., 2002; Baker and Deng, 2005; Diaz et al., 2006; Doerig, 2004; Taylor et al., 2010). These studies used a trisubstituted pyrrole inhibitor (compound 1) (Gurnett et al., 2002) or imidazopyridine inhibitor (compound 2) (Donald et al., 2006), in parasites expressing sensitive or resistant versions of PKG. Genetic evidence also exists for the essentiality of PKGs. Using a stage-specific FlpL/FRT recombination system in P. berghei (Buchholz et al., 1996; Combe et al., 2009), PKG was partially deleted in sporozoites causing a block in late liver stage development in a mouse model of infection (Falae et al., 2010). Phosphoproteomic studies in Plasmodium spp. have identified a variety of targets downstream of PKG, including PfCDPK1, components of the actin-myosin motor (Alam et al., 2015), and in enzymesmediating phosphoinositide metabolism (Brochet et al., 2014). These later studies

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suggest a mechanism for data placing PKG upstream of calcium release during schizont egress and merozoite invasion in P. falciparum, as well as gametocyte activation and ookinete gliding in P. berghei (Brochet et al., 2014). Genetic interaction studies in P. berghei using hypomorphic PKG alleles also demonstrate crosstalk between cyclic nucleotide and CDPKs (Fang et al., 2018). In T. gondii, the separate functions of the two PKG isoforms were analyzed using simultaneous conditional knockdown of both endogenous isoforms followed by mutant complementation (Brown et al., 2017). Conditional depletion of TgPKG-I and TgPKG-II blocked microneme secretion, paralyzing all motile functions, validating prior pharmacological inhibitor data. Complementation experiments showed that the plasma membrane
associated TgPKG-I isoform is necessary and sufficient for PKG-dependent processes, whereas cytosolic TgPKG-II is largely insufficient and dispensable (Brown et al., 2017). Mechanistically, the relevant substrates of TgPKG for microneme secretion have not yet been identified. However, several studies indicate that TgPKG is required for calcium upregulation (Yang et al., 2019; Stewart et al., 2017; Sidik et al., 2016a) which is required for microneme secretion and motility. However, TgPKG cannot be bypassed by artificial elevation of calcium (Brown et al., 2016, 2017; Brown and Sibley, 2018), indicating that TgPKG likely controls both an initial and final step in microneme secretion.

13.6.4 Cyclic AMP-dependent protein kinase (PKA) PKA is the only known kinase that directly responds to cAMP. This activation is achieved when cAMP binds to the regulatory subunit (PKAr) releasing it from the catalytic subunit (PKAc), allowing for activation. While extremely well studied in mammalian systems

597

the function of PKA in apicomplexan parasites remains rudimentary. As discussed previously in more detail, the role of PKA has been indirectly studied in P. falciparum by the functional removal of cAMP-specific PDEβ (Flueck et al., 2019). Inactivation of PDEβ shows no effect on schizont development or egress but a strong effect on invasion, suggesting that PfPKA also likely functions in a similar manner. Interestingly, PDEβ deletion in schizont stages results in a greater amount of phosphorylation of substrates that conform to a PKA-like consensus motif and include sites on MyoA, ACβ, CDPK1, and the cytoplasmic tail of AMA1 (Flueck et al., 2019). Indeed, phosphorylation of AMA1 Ser610 by PfPKA has previously been characterized and shown to play a very important role in invasion (Leykauf et al., 2010). It yet remains unknown what the function of this phosphorylation site is, nor any other PKA phosphorylation sites. Calcium and cAMP signaling also appear connected in P. falciparum, whereby loss of PfCDPK1 results in less phosphorylation of a site on PfPKAr (Kumar et al., 2017). What role this phosphorylation site plays in activation/repression of PfPKA and the true nature of communication between calcium and cAMP still remains largely unclear. The role that PKA plays in T. gondii is somewhat more advanced. While members of the Plasmodium genus have only one PKA ortholog, T. gondii has three. TgPKAc2 appears not to be expressed in the lytic tachyzoite stages, while TgPKAc3 negatively regulates bradyzoite differentiation (Sugi et al., 2016). Studies have investigated the function of TgPKAc1, the closest ortholog to the form of PKA found in Plasmodium spp. Jia et al. (2017) produced a conditional dominant negative mutant of TgPKAr1, by preventing its ability to interact with cAMP, thus not allowing for the activation of TgPKAc1. By doing so they showed that loss of PKA led to an early egress phenotype suggesting that cAMP signaling may negatively regulate exit from the host cell. Furthermore, this

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study also showed that loss of TgPKA also led to ‘restless’ invasion, whereby host cell became damaged upon exposure to these mutant parasites. This invasion phenotype was further fleshed-out in subsequent paper, showing that loss of PKA, using the tet-off system to downregulate expression leads to the egress of host cell shortly after invasion, which was mediated by PLP-1 dependent pathway. Furthermore, loss of PKAc prevents the rapid suppression of cytoplasmic calcium levels that normally occurs after invasion, which is presumably responsible for the rapid egress of this mutant parasite (Uboldi et al., 2018). Interestingly, these studies provide evidence that PKA could act to suppress cGMP, which acts upstream of calcium release (Uboldi et al., 2018; Jia et al., 2017). Initially, it was shown that PKA is required for phosphorylation of a specific residue on a PDE (Jia et al., 2017), and separately, it was shown that loss of PKA sensitized parasites to a PDE inhibitor, leading to a higher levels of cytosolic calcium at lower concentrations of BIPPO (Uboldi et al., 2018). This dichotomy further highlights the opposing roles of cAMP and cGMP in activation of motility in T. gondii.

13.7 Conclusion and future directions Studies over the past several decades have defined two important signaling pathways that control motility during the intracellular/extracellular transitions of apicomplexan parasites. The coordinated action of cyclic nucleotide and calcium-dependent kinases are used to control protein section, motility, cell invasion, and egress, and to downregulate this process to remain intracellular during replication. Although we now have a reasonable framework for how these pathways are activated and how they interact to achieve particular outcomes in tachyzoites, similar studies have not yet been done to investigate how these pathways operate in other life cycle stages such as bradyzoites

and sporozoites. In addition, there are still uncertainties about how these pathways function in tachyzoites. For example, although the available evidence suggests that PKG controls calcium release from intracellular stores, we lack specific information on how this release is achieved. Identifying additional substrates of PKG may help elucidate the trigger for calcium release. As well, the nature of calcium releases channels is completely unknown, given the lack of obvious homology with release channels in model systems. Studies in Plasmodium have revealed differences between species for the precise roles played by individual cyclase and PDE genes illustrating a degree of flexibility in how activation of cyclic nucleotide
dependent PK can be achieved. It will be interesting to look for similar interactions in Toxoplasma, especially given the expanded family of PDEs that occurs in this group. The signals that regulate activation of the cyclases are also unclear, although available evidence suggests that the GC in T. gondii may sense ions or lipids. Such signals are likely to differ between when parasites are intracellular versus extracellular, and establishing assays that can be used to define the binding of agonists and how these relay signals for activation will be key to unraveling their function in cells. Finally, although the evidence suggests that there is a rapid switch from activation of PKG to PKA upon invasion of cells, how this is achieved is still uncertain. Although we have excellent genetic tools to dissect these interactions, understanding them at a mechanistic level will also require biochemical and physiological studies to define the interactions among partners in these signaling networks and reconstitute them in vivo.

Acknowledgments The chapter is supported in part by grants from the NIH (AI118426 and AI034036). We are grateful to Sebastian Lourido for assistance with the figures and comments on the manuscript.

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References

References Alam, M.M., Solyakov, L., Bottrill, A.R., Flueck, C., Siddiqui, F.A., Singh, S., et al., 2015. Phosphoproteomics reveals malaria parasite protein kinase G as a signalling hub regulating egress and invasion. Nat. Commun. 6, 7285. PMID: 26149123. Allen, J.J., Lazerwith, S.E., Shokat, K.M., 2005. Bioorthogonal affinity purification of direct kinase substrates. J. Am. Chem. Soc. 127 (15), 5288
5289. PMID: 15826144. Andenmatten, N., Egarter, S., Jackson, A.J., Jullien, N., Herman, J.P., Meissner, M., 2012. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods. PMID: 23263690. Baker, D.A., Deng, W., 2005. Cyclic GMP-dependent protein kinases in protozoa. Front. Biosci. 10, 1229
1238. Baker, D.A., Kelly, J.M., 2004. Structure, function and evolution of microbial adenylyl and guanylyl cyclases. Mol. Microbiol. 52 (5), 1229
1242. PMID: 15165228. Beavo, J.A., Brunton, L.L., 2002. Cyclic nucleotide research
still expanding after half a century. Nat. Rev. Mol. Cell Biol. 3 (9), 710
718. PMID: 12209131. Beck, J.R., Fung, C., Straub, K.W., Coppens, I., Vashisht, A. A., Wohlschlegel, J.A., et al., 2013. A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress. PLoS Pathog. 9 (2), e1003162. PMID: 23408890; PMC3567180. Berridge, M.J., Bootman, M.D., Roderick, H.L., 2003. Calcium signaling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517
529. Besteiro, S., Dubremetz, J.F., Lebrun, M., 2011. The moving junction of apicomplexan parasites: a key structure for invasion. Cell. Microbiol. 13 (6), 797
805. PMID: 21535344. Billker, O., Tewari, R., Franke-Fayard, B., Brinkman, V., 2004. Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 117, 503
514. Billker, O., Lourido, S., Sibley, L.D., 2009. Calciumdependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5, 612
622. Bisio, H., Lunghi, M., Brochet, M., Soldati-Favre, D., 2019. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. Nat. Microbiol. 4, 420
428. PMID: 30742070. Bookwalter, C.S., Kelsen, A., Leung, J.M., Ward, G.E., Trybus, K.M., 2014. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite cochaperone, requires two light chains for fast motility. J. Biol. Chem. 289 (44), 30832
30841. PMID: 25231988; PMC4215259.

599

Borges-Pereira, L., Budu, A., McKnight, C.A., Moore, C.A., Vella, S.A., Hortua Triana, M.A., et al., 2015. Calcium signaling throughout the Toxoplasma gondii lytic cycle: a study using genetically encoded calcium indicators. J. Biol. Chem. 290 (45), 26914
26926. PMID: 26374900; PMC4646405. Boudeau, J., Miranda-Saavedra, D., Barton, G.J., Alessi, D. R., 2006. Emerging roles of pseudokinases. Trends Cell Biol. 16, 443
452. Brochet, M., Collins, M.O., Smith, T.K., Thompson, E., Sebastian, S., Volkmann, K., et al., 2014. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca21 signals at key decision points in the life cycle of malaria parasites. PLoS Biol. 12 (3), e1001806. PMID: 24594931; 3942320. Brossier, F., Jewett, T.J., Lovett, J.L., Sibley, L.D., 2003. Cterminal processing of the Toxoplasma protein MIC2 is essential for invasion into host cells. J. Biol. Chem. 278, 6229
6234. Brossier, F., Jewett, T.J., Sibley, L.D., Urban, S., 2005. A spatially-localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc. Natl. Acad. Sci. U.S.A. 102, 4146
4151. Brown, K.M., Sibley, L.D., 2018. Essential cGMP signaling in Toxoplasma is initiated by a hybrid P-type ATPaseguanylate cyclase. Cell Host Microbe 24 (6), 804
816. e806. PMID: 30449726; PMC6292738. Brown, K.M., Lourido, S., Sibley, L.D., 2016. Serum albumin stimulates protein kinase G-dependent microneme secretion in Toxoplasma gondii. J. Biol. Chem. 291 (18), 9554
9565. PMID: 26933037; PMC4850294. Brown, K.M., Long, S., Sibley, L.D., 2017. Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii. MBio 8 (3), pii: e00375-17, doi: 10.1128/ mBio.00375-17, PMID: 28465425; PMC5414004. Buchholz, F., Ringrose, L., Angrand, P.O., Rossi, F., Stewart, A.F., 1996. Different thermostabilities of FLP and Cre recombinases: implications for applied sitespecific recombination. Nucleic Acids Res. 24 (21), 4256
4262. PMID: 8932381; PMC146240. Buguliskis, J.S., Brossier, F., Shuman, J., Sibley, L.D., 2010. Rhomboid 4 (ROM4) affects the processing of surface adhesins and facilitates host cell invasion by Toxoplasma gondii. PLoS Pathog. 6 (4), e1000858. PMID: 20421941; 2858701. Bullen, H.E., Jia, Y., Yamaryo-Botte, Y., Bisio, H., Zhang, O., Jemelin, N.K., et al., 2016. Phosphatidic acidmediated signaling regulates microneme secretion in Toxoplasma. Cell Host Microbe 19 (3), 349
360. PMID: 26962945. Bushell, E., Gomes, A.R., Sanderson, T., Anar, B., Girling, G., Herd, C., et al., 2017. Functional profiling of a Plasmodium genome reveals an abundance of essential genes. Cell. 170 (2), 260
272. e8. PMID: 28708996.

Toxoplasma Gondii

600

13. Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii

Carruthers, V.B., Sibley, L.D., 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol. Microbiol. 31, 421
428. Carruthers, V.B., Moreno, S.N.J., Sibley, L.D., 1999a. Ethanol and acetaldehyde elevate intracellular [Ca21] calcium and stimulate microneme discharge in Toxoplasma gondii. Biochem. J. 342, 379
386. Carruthers, V.B., Giddings, O.K., Sibley, L.D., 1999b. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell. Microbiol. 1, 225
236. Carruthers, V.B., Ha˚kansson, S., Giddings, O.K., Sibley, L. D., 2000. Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment. Infect. Immun. 68 (7), 4005
4011. Carucci, D.J., Witney, A.A., Muhia, D.K., Warhurst, D.C., Schaap, P., Meima, M., et al., 2000. Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum. J. Biol. Chem. 275 (29), 22147
22156. PMID: 10747978. Chini, E.N., Nagamune, K., Wetzel, D.M., Sibley, L.D., 2005. Evidence that the cADPR signaling pathway controls calcium-mediated secretion in Toxoplasma gondii. Biochem J. 389, 269
277. Combe, A., Giovannini, D., Carvalho, T.G., Spath, S., Boisson, B., Loussert, C., et al., 2009. Clonal conditional mutagenesis in malaria parasites. Cell Host Microbe 5 (4), 386
396. PMID: 19380117. Delbac, F., Sanger, A., Neuhaus, E.M., Stratmann, R., Ajioka, J.W., Toursel, C., et al., 2001. Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division. J. Cell Biol. 155, 613
623. Diaz, C.A., Allocco, J., Powles, M.A., Yeung, L., Donald, R. G., Anderson, J.W., et al., 2006. Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): antiparasitic activity of a PKG inhibitor. Mol. Biochem. Parasitol. 146 (1), 78
88. PMID: 16325279. Dobrowolski, J.M., Sibley, L.D., 1996. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell. 84 (6), 933
939. Dobrowolski, J.M., Niesman, I.R., Sibley, L.D., 1997. Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1 and exists primarily in a globular form. Cell Motil. Cytoskel. 37 (3), 253
262. Doerig, C., 2004. Protein kinases as targets for anti-parasitic chemotherapy. Biochim. Biophys. Acta. 1697 (1
2), 155
168. PMID: 15023358. Donald, R.G.K., Liberator, P.A., 2002. Molecular characterization of a coccidian parasite cGMP dependent protein kinase. Mol. Biochem. Parasitol. 120, 165
175. Donald, R.G., Allocco, J.J., Singh, S.B., Nare, B., Salowe, S. P., Wiltsie, J., et al., 2002. Toxoplasma gondii cyclic GMP-

dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase. Eukaryot. Cell 1, 317
328. Donald, R.G., Zhong, T., Wiersma, H., Nare, B., Yao, D., Lee, A., et al., 2006. Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase. Mol. Biochem. Parasitol. 149 (1), 86
98. PMID: 16765465. Dowse, T.J., Pascall, J.C., Brown, K.D., Soldati, D., 2005. Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion. Int. J. Parasitol. 35, 747
756. Drewry, L.L., Sibley, L.D., 2015. Toxoplasma actin is required for efficient host cell invasion. MBio 6 (3), e00557. PMID: 26081631; PMC4471557. Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans. CRC Press, Boca Raton, FL, 313 p. Dubois, D.J., Soldati-Favre, D., 2019. Biogenesis and secretion of micronemes in Toxoplasma gondii. Cell. Microbiol. 21, e13018. PMID: 30791192. Dvorin, J.D., Martyn, D.C., Patel, S.D., Grimley, J.S., Collins, C.R., Hopp, C.S., et al., 2010. A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes. Science 328 (5980), 910
912. PMID: 20466936. Egarter, S., Andenmatten, N., Jackson, A.J., Whitelaw, J.A., Pall, G., Black, J.A., et al., 2014. The Toxoplasma ActoMyoA motor complex is important but not essential for gliding motility and host cell invasion. PLoS One 9 (3), e91819. PMID: 24632839; 3954763. Falae, A., Combe, A., Amaladoss, A., Carvalho, T., Menard, R., Bhanot, P., 2010. Role of Plasmodium berghei cGMPdependent protein kinase in late liver stage development. J. Biol. Chem. 285 (5), 3282
3288. PMID: 19940133; PMC2823412. Fang, H., Gomes, A.R., Klages, N., Pino, P., Maco, B., Walker, E.M., et al., 2018. Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites. Nat. Commun. 9 (1), 4248. PMID: 30315162; PMC6185908. Farrell, A., Thirugnanam, S., Lorestani, A., Dvorin, J.D., Eidell, K.P., Ferguson, D.J., et al., 2012. A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis. Science 335 (6065), 218
221. PMID: 22246776; 3354045. Ferguson, D.J.P., 1988. An immuno-electron microscopic study of the tissue cyst of Toxoplasma gondii in mouse brain. EUREM 3, 207
208. Flueck, C., Drought, L.G., Jones, A., Patel, A., Perrin, A.J., Walker, E.M., et al., 2019. Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion. PLoS Biol. 17 (2), e3000154. PMID: 30794532; PMC6402698.

Toxoplasma Gondii

References

Foth, B.J., Goedecke, M.C., Soldati, D., 2006. New insights into myosin evolution and classification. Proc. Natl. Acad. Sci. U.S.A. 103, 3681
3686. Frenal, K., Polonais, V., Marq, J.B., Stratmann, R., Limenitakis, J., Soldati-Favre, D., 2010. Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe 8 (4), 343
357. PMID: 20951968. Frenal, K., Tay, C.L., Mueller, C., Bushell, E.S., Jia, Y., Graindorge, A., et al., 2013. Global analysis of apicomplexan protein S-acyl transferases reveals an enzyme essential for invasion. Traffic 14 (8), 895
911. PMID: 23638681; 3813974. Frenal, K., Marq, J.B., Jacot, D., Polonais, V., Soldati-Favre, D., 2014. Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion. PLoS Pathog. 10 (10), e1004504. PMID: 25393004; PMC4231161. Gaji, R.Y., Johnson, D.E., Treeck, M., Wang, M., Hudmon, A., Arrizabalaga, G., 2015. Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid initiation of motility during Toxoplasma gondii egress. PLoS Pathog. 11 (11), e1005268. PMID: 26544049; PMC4636360. Gao, H., Yang, Z., Wang, X., Qian, P., Hong, R., Chen, X., et al., 2018. ISP1-anchored polarization of GCbeta/ CDC50A complex initiates malaria ookinete gliding motility. Curr. Biol. 28 (17), 2763
2776.e6. PMID: 30146157. Garcia, C.R., Alves, E., Pereira, P.H., Bartlett, P.J., Thomas, A.P., Mikoshiba, K., et al., 2017. InsP3 signaling in apicomplexan parasites. Curr. Top. Med. Chem. PMID: 28137231; PMC5490149. Garrison, E., Treeck, M., Ehret, E., Butz, H., Garbuz, T., Oswald, B.P., et al., 2012. A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma. PLoS Pathog. 8 (11), e1003049. PMID: 23209419; 3510250. Gonzalez, V., Combe, A., David, V., Malmquist, N.A., Delorme, V., Leroy, C., et al., 2009. Host cell entry by apicomplexa parasites requires actin polymerization in the host cell. Cell Host Microbe 5 (3), 259
272. PMID: 19286135. Gordon, J.L., Sibley, L.D., 2005. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites. BMC Genomics 6, e179. Gould, M.K., de Koning, H.P., 2011. Cyclic-nucleotide signalling in protozoa. FEMS Microbiol. Rev. 35 (3), 515
541. PMID: 21223322. Govindasamy, K., Jebiwott, S., Jaijyan, D.K., Davidow, A., Ojo, K.K., Van Voorhis, W.C., et al., 2016. Invasion of hepatocytes by Plasmodium sporozoites requires cGMPdependent protein kinase and calcium dependent protein kinase 4. Mol. Microbiol. 102 (2), 349
363. PMID: 27425827.

601

Graindorge, A., Frenal, K., Jacot, D., Salamun, J., Marq, J.B., Soldati-Favre, D., 2016. The conoid associated motor MyoH is indispensable for Toxoplasma gondii entry and exit from host cells. PLoS Pathog. 12 (1), e1005388. PMID: 26760042; PMC4711953. Guerin, A., Corrales, R.M., Parker, M.L., Lamarque, M.H., Jacot, D., El Hajj, H., et al., 2017. Efficient invasion by Toxoplasma depends on the subversion of host protein networks. Nat. Microbiol. 2 (10), 1358
1366. PMID: 28848228. Guerra, A.J., Zhang, O., Bahr, C.M.E., Huynh, M.H., DelProposto, J., Brown, W.C., et al., 2018. Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress. PLoS Pathog. 14 (12), e1007476. PMID: 30513119; PMC6294395. Gurnett, A.M., Liberator, P.A., Dulski, P.M., Salowe, S.P., Donald, R.G., Anderson, J.W., et al., 2002. Purification and molecular characterization of cGMP-dependent protein kinase from apicomplexan parasites. A novel chemotherapeutic target. J. Biol. Chem. 277, 15913
15922. Ha˚kansson, S., Morisaki, H., Heuser, J.E., Sibley, L.D., 1999. Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion. Mol. Biol. Cell. 10, 3539
3547. Hanks, S.K., Hunter, T., 1995. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576
596. Heaslip, A.T., Nishi, M., Stein, B., Hu, K., 2011. The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase. PLoS Pathog. 7 (9), e1002201. PMID: 21909263; 3164638. Hehl, A.B., Basso, W.U., Lippuner, C., Ramakrishnan, C., Okoniewski, M., Walker, R.A., et al., 2015. Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of nonoverlapping gene families to attach, invade, and replicate within feline enterocytes. BMC Genomics 16, 66. PMID: 25757795; PMC4340605. Hirai, M., Arai, M., Kawai, S., Matsuoka, H., 2006. PbGCbeta is essential for Plasmodium ookinete motility to invade midgut cell and for successful completion of parasite life cycle in mosquitoes. J. Biochem. 140 (5), 747
757. PMID: 17030505. Hui, R., El Bakkouri, M., Sibley, L.D., 2015. Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans. Trends Pharmacol. Sci. 36 (7), 452
460. PMID: 26002073; 4485940. Hunter, C.A., Sibley, L.D., 2012. Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat. Rev. Microbiol. 10, 766
778.

Toxoplasma Gondii

602

13. Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii

Huse, M., Kuriyan, J., 2002. The conformational plasticity of protein kinases. Cell 109, 275
282. Jacot, D., Tosetti, N., Pires, I., Stock, J., Graindorge, A., Hung, Y.F., et al., 2016. An apicomplexan actin-binding protein serves as a connector and lipid sensor to coordinate motility and invasion. Cell Host Microbe 20 (6), 731
743. PMID: 27978434. Jia, Y., Marq, J.B., Bisio, H., Jacot, D., Mueller, C., Yu, L., et al., 2017. Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii. EMBO J. 36 (21), 3250
3267. PMID: 29030485; PMC5666616. Kafsack, B.F., Pena, J.D., Coppens, I., Ravindran, S., Boothroyd, J.C., Carruthers, V.B., 2009. Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 323, 530
533. Kenthirapalan, S., Waters, A.P., Matuschewski, K., Kooij, T. W., 2016. Functional profiles of orphan membrane transporters in the life cycle of the malaria parasite. Nat. Commun. 7, 10519. PMID: 26796412; PMC4736113. Knoll, L.J., Tomita, T., Weiss, L.M., 2014. Bradyzoite development. In: Weiss, L.M., Kim, K. (Eds.), Toxoplasma gondii: The Model Apicomplexan: Perspectives and Methods, second ed. Academic Press, New York, pp. 521
551. Kobayashi, M., Buck, J., Levin, L.R., 2004. Conservation of functional domain structure in bicarbonate-regulated “soluble” adenylyl cyclases in bacteria and eukaryotes. Dev. Genes Evol. 214 (10), 503
509. PMID: 15322879; PMC3644946. Kumar, S., Kumar, M., Ekka, R., Dvorin, J.D., Paul, A.S., Madugundu, A.K., et al., 2017. PfCDPK1 mediated signaling in erythrocytic stages of Plasmodium falciparum. Nat. Commun. 8 (1), 63. PMID: 28680058; PMC5498596. Kumpula, E.P., Pires, I., Lasiwa, D., Piirainen, H., Bergmann, U., Vahokoski, J., et al., 2017. Apicomplexan actin polymerization depends on nucleation. Sci. Rep. 7 (1), 12137. PMID: 28939886; PMC5610305. Kursula, P., 2014. The many structural faces of calmodulin: a multitasking molecular jackknife. Amino Acids 46 (10), 2295
2304. PMID: 25005783. Lakshmanan, V., Fishbaugher, M.E., Morrison, B., Baldwin, M., Macarulay, M., Vaughan, A.M., et al., 2015. Cyclic GMP balance is critical for malaria parasite transmission from the mosquito to the mammalian host. MBio 6 (2), e02330. PMID: 25784701; PMC4453516. Leung, J.M., Rould, M.A., Konradt, C., Hunter, C.A., Ward, G.E., 2014. Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay. PLoS One 9 (1), e85763. PMID: 24489670; PMC3906025.

Leykauf, K., Treeck, M., Gilson, P.R., Nebl, T., Braulke, T., Cowman, A.F., et al., 2010. Protein kinase a dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite. PLoS Pathog. 6 (6), e1000941. PMID: 20532217; 2880582. Lim, D.C., Cooke, B.M., Doerig, C., Saeij, J.P., 2012. Toxoplasma and Plasmodium protein kinases: roles in invasion and host cell remodelling. Int J Parasitol. 42 (1), 21
32. PMID: 22154850; PMC3428259. Linder, J.U., Engel, P., Reimer, A., Kruger, T., Plattner, H., Schultz, A., et al., 1999. Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an Nterminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium. EMBO J. 18 (15), 4222
4232. PMID: 10428960; PMC1171498. Long, S., Wang, Q., Sibley, L.D., 2016. Analysis of noncanonical calcium-dependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9. Infect. Immun. 84 (5), 1262
1273. PMID: 26755159; 4862710. Lopez, M.S., Kliegman, J.I., Shokat, K.M., 2014. The logic and design of analog-sensitive kinases and their small molecule inhibitors. Methods Enzymol. 548, 189
213. PMID: 25399647. Lourido, S., Moreno, S.N., 2015. The calcium signaling toolkit of the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell Calcium. 57 (3), 186
193. PMID: 25605521; PMC4428288. Lourido, S., Shuman, J., Zhang, C., Shokat, K.M., Hui, R., Sibley, L.D., 2010. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465, 359
362. Lourido, S., Tang, K., Sibley, L.D., 2012. Distinct signalling pathways control Toxoplasma egress and host-cell invasion. EMBO J. 31, 4524
4534. Lourido, S., Jeschke, G.R., Turk, B.E., Sibley, L.D., 2013. Exploiting the unique ATP-binding pocket of Toxoplasma calcium-dependent protein kinase 1 to identify its substrates. ACS Chem. Biol. 8, 1155
1162. Lovett, J.L., Sibley, L.D., 2003. Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells. J. Cell Sci. 116, 3009
3016. Lovett, J.L., Marchesini, N., Moreno, S.N., Sibley, L.D., 2002. Toxoplasma gondii microneme secretion involves intracellular Ca21 release from IP3/ryanodine sensitive stores. J. Biol. Chem 277 (29), 25870
25876. PMID: 12011085. McCoy, J.M., Whitehead, L., van Dooren, G.G., Tonkin, C. J., 2012. TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells. PLoS Pathog. 8 (12), e1003066. PMID: 23226109; 3514314.

Toxoplasma Gondii

References

McCoy, J.M., Stewart, R.J., Uboldi, A.D., Li, D., Schroder, J., Scott, N.E., et al., 2017. A forward genetic screen identifies a negative regulator of rapid Ca(2 1 )-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii. J. Biol. Chem 292 (18), 7662
7674. PMID: 28258212; PMC5418062. Mehta, S., Sibley, L.D., 2010. Toxoplasma gondii actin depolymerizing factor acts primarily to sequester G-actin. J. Biol. Chem. 285, 6835
6847. Meissner, M., Schluter, D., Soldati, D., 2002. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837
840. Miranda-Saavedra, D., Gabaldon, T., Barton, G.J., Langsley, G., Doerig, C., 2012. The kinomes of apicomplexan parasites. Microbes Infect. 14 (10), 796
810. PMID: 22587893. Montoya, J.G., Liesenfeld, O., 2004. Toxoplasmosis. Lancet 363, 1965
1976. Moon, R.W., Taylor, C.J., Bex, C., Schepers, R., Goulding, D., Janse, C.J., et al., 2009. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite. PLoS Pathog. 5 (9), e1000599. PMID: 19779564; 2742896. Moreno, S.N., Ayong, L., Pace, D.A., 2011. Calcium storage and function in apicomplexan parasites. Essays Biochem. 51, 97
110. PMID: 22023444; 3488345. Morisaki, J.H., Heuser, J.E., Sibley, L.D., 1995. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J. Cell Sci. 108, 2457
2464. Morlon-Guyot, J., Berry, L., Chen, C.T., Gubbels, M.J., Lebrun, M., Daher, W., 2014. The Toxoplasma gondii calcium-dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival. Cell. Microbiol. 16 (1), 95
114. PMID: 24011186. Mueller, C., Klages, N., Jacot, D., Santos, J.M., Cabrera, A., Gilberger, T.W., et al., 2013. The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion. Cell Host Microbe 13 (3), 289
301. PMID: 23498954. Mueller, C., Samoo, A., Hammoudi, P.M., Klages, N., Kallio, J.P., Kursula, I., et al., 2016. Structural and functional dissection of Toxoplasma gondii armadillo repeats only protein. J. Cell Sci. 129 (5), 1031
1045. PMID: 26769898. Muhia, D.K., Swales, C.A., Eckstein-Ludwig, U., Saran, S., Polley, S.D., Kelly, J.M., et al., 2003. Multiple splice variants encode a novel adenylyl cyclase of possible plastid origin expressed in the sexual stage of the malaria parasite Plasmodium falciparum. J. Biol. Chem. 278 (24), 22014
22022. Nagamune, K., Beatty, W.L., Sibley, L.D., 2007. Artemisinin induces calcium-dependent secretion in Toxoplasma gondii. Eukaryot. Cell. 6, 2147
2156.

603

Nunes, M., Goldring, J.P.D., Doerig, C., Scherf, A., 2006. A novel protein kinase family in Plasmodium falciparum is differentially transcribed and secreted to various cellular compartments of the host cell. Mol. Microbiol. 63, 391
403. Ono, T., Cabrita-Santos, L., Leitao, R., Bettiol, E., Purcell, L. A., Diaz-Pulido, O., et al., 2008. Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection. PLoS Pathog. 4 (2), e1000008. PMID: 18389080. Opitz, C., Soldati, D., 2002. ‘The glideosome’: a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii. Mol. Microbiol. 45, 597
604. Peixoto, L., Chen, F., Harb, O.S., Davis, P.H., Beiting, D.P., Brownback, C.S., et al., 2010. Integrative genomics approaches highlight a family of parasite-specific kinases that regulate host responses. Cell Host Microbe 8, 208
218. Roiko, M.S., Svezhova, N., Carruthers, V.B., 2014. Acidification activates Toxoplasma gondii motility and egress by enhancing protein secretion and cytolytic activity. PLoS Pathog. 10 (11), e1004488. PMID: 25375818; PMC4223073. Rothenberg, D.A., Gordon, E.A., White, F.M., Lourido, S., 2016. Identification of direct kinase substrates using analogue-sensitive alleles. Methods Mol. Biol. 1355, 71
84. PMID: 26584919. Rutaganira, F.U., Barks, J., Dhason, M.S., Wang, Q., Lopez, M.S., Long, S., et al., 2017. Inhibition of calcium dependent protein kinase 1 (CDPK1) by pyrazolopyrimidine analogs decreases establishment and reoccurrence of central nervous system disease by Toxoplasma gondii. J. Med. Chem. 60, 9976
9989. PMID: 28933846; PMC5746462. Sahoo, N., Beatty, W.L., Heuser, J.E., Sept, D., Sibley, L.D., 2006. Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii. Mol. Biol. Cell. 17, 895
906. Salazar, E., Bank, E.M., Ramsey, N., Hess, K.C., Deitsch, K. W., Levin, L.R., et al., 2012. Characterization of Plasmodium falciparum adenylyl cyclase-beta and its role in erythrocytic stage parasites. PLoS One 7 (6), e39769. PMID: 22761895; PMC3383692. Schneider, A.G., Mercereau-Puijalon, O., 2005. A new Apicomplexa-specific protein kinase family: multiple members in Plasmodium falciparum, all with an export signature. BMC Genomics 6, 30. Schultz, J.E., Klumpp, S., Benz, R., Schurhoff-Goeters, W.J., Schmid, A., 1992. Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance. Science (New York, NY.) 255 (5044), 600
603. PMID: 1371017.

Toxoplasma Gondii

604

13. Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii

Seifert, R., Schneider, E.H., Bahre, H., 2015. From canonical to non-canonical cyclic nucleotides as second messengers: pharmacological implications. Pharmacol. Ther. 148, 154
184. PMID: 25527911. Shaw, A.S., Kornev, A.P., Hu, J., Ahuja, L.G., Taylor, S.S., 2014. Kinases and pseudokinases: lessons from RAF. Mol. Cell. Biol. 34 (9), 1538
1546. PMID: 24567368; 3993607. Sibley, L.D., 2004. Invasion strategies of intracellular parasites. Science 304, 248
253. Sibley, L.D., 2010. How apicomplexan parasites move in and out of cells. Curr. Opin. Biotechnol. 21, 592
598. Sidik, S.M., Hortua Triana, M.A., Paul, A.S., El Bakkouri, M., Hackett, C.G., Tran, F., et al., 2016a. Using a genetically encoded sensor to identify inhibitors of Toxoplasma gondii Ca2 1 signaling. J. Biol. Chem. 291 (18), 9566
9580. PMID: 26933036; PMC4850295. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016b. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166 (6), 1423
1435.e12. PMID: 27594426; PMC5017925. Skillman, K.M., Diraviyam, K., Khan, A., Tang, K., Sept, D., Sibley, L.D., 2011. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility of apicomplexan parasites. PLoS Pathog. 7, e1002280. Skillman, K.M., Daher, W., Ma, C., Soldati-Favre, D., Sibley, L.D., 2012. Toxoplasma gondii profilin acts primarily to sequester G-actin while formins initiate actin filament formation in vitro. Biochemistry 51, 2486
2495. Skillman, K.M., Ma, C.I., Fremont, D.H., Diraviyam, K., Cooper, J.A., Sept, D., et al., 2013. The unusual dynamics of parasite actin result from isodesmic polymerization. Nat. Commun. 4, 2285. Stewart, R.J., Whitehead, L., Nijagal, B., Sleebs, B.E., Lessene, G., McConville, M.J., et al., 2017. Analysis of Ca(2)(1) mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules. Cell. Microbiol. 19 (4), PMID: 27781359. Sugi, T., Ma, Y.F., Tomita, T., Murakoshi, F., Eaton, M.S., Yakubu, R., et al., 2016. Toxoplasma gondii cyclic AMPdependent protein kinase subunit 3 is involved in the switch from tachyzoite to bradyzoite development. MBio 7 (3), pii: e00755-16. doi: 10.1128/mBio.00755-16, PMID: 27247232; PMC4895117. Talevich, E., Kannan, N., 2013. Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors. BMC Evol. Biol. 13, 117. PMID: 23742205; 3682881. Talevich, E., Tobin, A.B., Kannan, N., Doerig, C., 2012. An evolutionary perspective on the kinome of malaria

parasites. Philos. Trans. R. Soc. London, B: Biol. Sci. 367 (1602), 2607
2618. PMID: 22889911; 3415840. Taylor, S.S., Kornev, A.P., 2011. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem. Sci. 36 (2), 65
77. PMID: 20971646; 3084033. Taylor, C.J., McRobert, L., Baker, D.A., 2008. Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis. Mol. Microbiol. 69 (1), 110
118. PMID: 18452584. Taylor, H.M., McRobert, L., Grainger, M., Sicard, A., Dluzewski, A.R., Hopp, C.S., et al., 2010. The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony. Eukaryot. Cell. 9 (1), 37
45. PMID: 19915077; PMC2805293. Taylor, S.S., Keshwani, M.M., Steichen, J.M., Kornev, A.P., 2012a. Evolution of the eukaryotic protein kinases as dynamic molecular switches. Philos. Trans. R. Soc. London, B: Biol. Sci. 367 (1602), 2517
2528. PMID: 22889904; 3415842. Taylor, S.S., Ilouz, R., Zhang, P., Kornev, A.P., 2012b. Assembly of allosteric macromolecular switches: lessons from PKA. Nat. Rev. Mol. Cell Biol. 13 (10), 646
658. PMID: 22992589; 3985763. Tonkin, M.L., Roques, M., Lamarque, M.H., Pugniere, M., Douguet, D., Crawford, J., et al., 2011. Host cell invasion by apicomplexan parasites: insights from the costructure of AMA1 with a RON2 peptide. Science 333 (6041), 463
467. PMID: 21778402. Tosetti, N., Dos Santos Pacheco, N., Soldati-Favre, D., Jacot, D., 2019. Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii. Elife. 8, pii: e42669. doi: 10.7554/ eLife.42669, PMID: 30753127; PMC6372287. Treeck, M., Sanders, J.L., Gaji, R.Y., LaFavers, K.A., Child, M.A., Arrizabalaga, G., et al., 2014. The calciumdependent protein kinase 3 of Toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis. PLoS Pathog. 10 (6), e1004197. PMID: 24945436. Tsien, R.W., 1990. Calcium channels, stores, and oscillations. Annu. Rev. Cell Biol. 6, 715
760. Uboldi, A.D., McCoy, J.M., Blume, M., Gerlic, M., Ferguson, D.J., Dagley, L.F., et al., 2015. Regulation of starch stores by a Ca(2 1 )-dependent protein kinase is essential for viable cyst development in Toxoplasma gondii. Cell Host Microbe 18 (6), 670
681. PMID: 26651943. Uboldi, A.D., Wilde, M.L., McRae, E.A., Stewart, R.J., Dagley, L.F., Yang, L., et al., 2018. Protein kinase A negatively regulates Ca2 1 signalling in Toxoplasma gondii. PLoS Biol. 16 (9), e2005642. PMID: 30208022; PMC6152992.

Toxoplasma Gondii

References

Watts, E., Zhao, Y., Dhara, A., Eller, B., Patwardhan, A., Sinai, A.P., 2015. Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. MBio 6 (5), e01155-15. PMID: 26350965; PMC4600105. Wentzinger, L., Bopp, S., Tenor, H., Klar, J., Brun, R., Beck, H.P., et al., 2008. Cyclic nucleotide-specific phosphodiesterases of Plasmodium falciparum: PfPDEalpha, a non-essential cGMP-specific PDE that is an integral membrane protein. Int. J. Parasitol. 38 (14), 1625
1637. PMID: 18590734. Wernimont, A.K., Artz, J.D., Finerty, P., Lin, Y., Amani, M., Allali-Hassani, A., et al., 2010. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. Nat. Struct. Mol. Biol. 17, 596
601. Wernimont, A.K., Amani, M., Qiu, W., Pizarro, J.C., Artz, J. D., Lin, Y.H., et al., 2011. Structures of parasitic CDPK domains point to a common mechanism of activation. Proteins 79 (3), 803
820. PMID: 21287613.

605

Wiersma, H.I., Galuska, S.E., Tomley, F.M., Sibley, L.D., Liberator, P.A., Donald, R.G.K., 2004. A role for coccidian cGMP-dependent protein kinase in motility and invasion. Int. J. Parasitol. 34, 369
380. Williams, M.J., Alonso, H., Enciso, M., Egarter, S., Sheiner, L., Meissner, M., et al., 2015. Two essential light chains regulate the MyoA lever arm to promote Toxoplasma gliding motility. MBio 6 (5), e00845
00815. PMID: 26374117; PMC4600101. Yang, L., Uboldi, A.D., Seizova, S., Wilde, M.L., Coffey, M. J., Katris, N.J., et al., 2019. An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca(2 1 ) signalling and motility in Toxoplasma gondii. J. Biol. Chem PMID: 30992368. Yuasa, K., Mi-Ichi, F., Kobayashi, T., Yamanouchi, M., Kotera, J., Kita, K., 2005. Omori K. PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum. Biochem. J. 392 (Pt 1), 221
229. PMID: 16038615; PMC1317681.

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C H A P T E R

14 Toxoplasma secretory proteins and their roles in parasite cell cycle and infection Maryse Lebrun1, Vern B. Carruthers2 and Marie-France Cesbron-Delauw3 1

UMR 5235 CNRS, University of Montpellier, Montpellier, France 2Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, United States 3UMR 5525 CNRS, University of Grenoble, Grenoble, France

14.1 Introduction Being an obligate intracellular parasite, Toxoplasma gondii has a limited capacity to survive outside of a cell during infection. Invading a new host cell is therefore crucial for survival and spreading of infection. Host cell invasion by T. gondii is an active, parasitedriven process (Morisaki et al., 1995). It leads to the formation of a new specialized compartment termed the parasitophorous vacuole (PV). The membrane of the vacuole—the PV membrane (PVM)—derives mostly from the host (Suss-Toby et al., 1996), does not acidify (Sibley et al., 1985), or fuse with endosomal compartments (Mordue et al., 1999b; Jones et al., 1972a), ensuring safe replication of the parasite in a vacuolar compartment separated from host compartments. Following replication, the parasite actively exits the host cell, a mechanism dependent upon its motility, and reinvades a new host cell. Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00014-1

The organelles of the apical complex— micronemes, rhoptries, and dense granules (DGs)—play their part successively in the processes of motility, invasion, and formation of the specialized vacuolar compartment by exocytosing their contents (Dubremetz et al., 1993; Carruthers and Sibley, 1997). Microneme proteins, followed by rhoptry proteins, are sequentially secreted during invasion (Carruthers and Sibley, 1997) and play distinct functions coordinated in time and space. The microneme proteins are redistributed to the parasite surface once secreted where they play diverse functions. They contribute to motility, attachment of the parasite to the host, and induce signaling in the host cell. Their release is also tied to the subsequent secretion of rhoptries (Fig. 14.1, part I). Rhoptry proteins, in contrast, are introduced into the host cell and contribute to two main functions. First, in synergy with microneme proteins, they participate in the penetration step, and second, they modify the host cell

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FIGURE 14.1 Secretory organelle protein functions. Schematic representation of the respective contributions of microneme (I), rhoptry (II), and dense granule proteins (III) to the Toxoplasma gondii
host cell interaction. Most functions are driven by single proteins with the most important exception being the AMA1-RONs cooperation in the moving junction process.

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14.2 Motility and invasion

response to disarm host immune defense (Fig. 14.1, part II). DG proteins are massively secreted into the PV lumen (Leriche and Dubremetz, 1991; Dubremetz et al., 1993; Carruthers and Sibley, 1997) at the end of the invasion process, after PV formation, and then later during the intracellular growth of the parasite. DG proteins are involved in the subsequent PV remodeling but are also key players together with rhoptry proteins in hijacking the host cell (Fig. 14.1, part III). After parasite intracellular replication proteins secreted from the DGs and the micronemes also contribute to parasite egress from infected cells. This chapter will describe how secretory proteins contribute to the intracellular life-style of T. gondii, focusing on their structural functions, that is, for invasion and biogenesis of PV, along with their roles in parasite egress. Some peripheral aspects such as secretion of the organelles are also described. Trafficking of secretory proteins and the biogenesis of secretory organelles is discussed briefly herein, but is more extensively covered in Chapter 15, Endomembrane trafficking pathways in Toxoplasma. The function of effectors of rhoptry and DG proteins will be described in Chapter 17, Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages.

14.2 Motility and invasion 14.2.1 Rapid and active processes unique to apicomplexan parasites 14.2.1.1 Motility Motility and invasion mechanisms are highly conserved in the Apicomplexa, meaning that results obtained with one organism can generally be extrapolated to other members of the phylum. Accordingly, in places we will describe T. gondii motility and invasion using supplemental findings obtained from other

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Apicomplexa. Almost all of our knowledge on T. gondii
host cell interactions are derived from in vitro studies using tachyzoites, the stage that is the most amenable to experimentation. Like other apicomplexan zoites, the invasive stages of T. gondii are motile. T. gondii uses motility not only to invade cells of its hosts but also to egress from these cells at the end of its lytic cycle. Motility and invasion are closely linked, and the molecular mechanisms driving these two phenomena are considered as closely related. Toxoplasma cells do not have specialized organelles for motility such as cilia or flagella, yet they are able to move along a substrate or a host cell surface by a gliding process, with anteroposterior polarity. This gliding motility, which is specific for Apicomplexa and highly conserved within the phylum, is defined by the absence of shape change, in contrast to amebae and others vertebrate cells that use crawling motility accompanied by the emission of pseudopods and lamellipods in the direction of motion. Apicomplexa gliding motility involves cell-surface contacts with substrates. This locomotion is exceptionally fast, reaching speeds up to 10 μm/s in vitro, that is, 10
50 times faster than the rates of motility of the majority of the other cells (keratinocytes, amoebas, etc.). Videomicroscopy of extracellular T. gondii tachyzoites on coated two-dimensional (2D) surfaces reveals that this gliding motility consists of a succession of several stereotyped behaviors: (1) circular gliding, which commences while the crescent-shaped parasite lies on its right side, from where it moves in a counter-clockwise manner; (2) upright twirling, which occurs when the parasite is attached to the substrate by its posterior end, producing clockwise spinning; and (3) helical rotation, which is a horizontal twirling movement resulting in forward displacement (Hakansson et al., 1999). In contrast to twirling motion, the helical and circular types of gliding depend on

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actin. Helical gliding is the only long-distance productive movement observed in vitro and is suggested to support host cell invasion (Hakansson et al., 1999). In a three-dimensional (3D) matrigel-based environment the motility is strikingly different, in that all parasites move in irregular corkscrew-like trajectories; and this behavior was shown to be dependent on the parasite’s crescent shape (Leung et al., 2014). It should be noted that Plasmodium sporozoites exhibit only circular gliding in vitro but also adopt corkscrew-like trajectories as they migrate through murine hepatocytes in vivo (Frevert et al., 2005). It is proposed that helical gliding could be the result of a parasite attempting to undergo 3D corkscrews constrained by the 2D surface (Leung et al., 2014). The directional and twisting nature of helical motility strongly suggests a connection between the driving system of the parasite and the helical organization of the subpellicular microtubules (see Chapter 2: The ultrastructure of Toxoplasma gondii). This connection could be established indirectly by the intramembrane particles (IMPs) present on the protoplasmic faces of the inner membrane complex (IMC) (see Chapter 2: The ultrastructure of Toxoplasma gondii). 14.2.1.2 Invasion Host cell invasion by T. gondii is fundamentally different from phagocytosis or endocytosis induced by intracellular pathogens such as viruses, bacteria, or Trypanosoma cruzi (Finlay and Cossart, 1997; Antoine et al., 1998; Sibley and Andrews, 2000). Invasion is essentially completed in less than 20 seconds and occurs with an apparent passivity of the host cell (i.e., without inducing host cell membrane ruffling, or tyrosine phosphorylation of host cell) (Morisaki et al., 1995). Entry mechanisms are largely driven by the parasite itself, although host cell may contribute to some extent in the invasion of T. gondii tachyzoite (Caldas et al., 2009; Gonzalez et al., 2009; Sweeney et al.,

2010; Bichet et al., 2016). Therefore Apicomplexa invasion is termed “active invasion” to contrast with “induced invasion” used during entry of bacteria. T. gondii shows two major peculiarities when compared to other members of the phylum. First, it has almost no host specificity, being able to invade all cell types, from mammalian to fish and even insect cells. Only plant protoplasts have proven refractory to invasion (Werk and Fischer, 1982). Second, contrasting with most other Apicomplexa that multiply by schizogony, T. gondii tachyzoites proliferate by endodyogeny in its intermediate hosts, bypassing the dedifferentiation stage that occurs in schizonts. Tachyzoites are thus invasive at any stage of their cell cycle, although they show lower invasiveness during mitosis and cytokinesis (Gaji et al., 2011a). This active invasion has been observed in many different cell types, including professional phagocytes where the parasite settles in a vacuole compartment that fails to fuse with lysosomes (Jones et al., 1972b; Jones and Hirsch, 1972) (see next). Contrasting with this active invasion, opsonized parasites are internalized by macrophages over a period of 2
4 minutes in a spacious vacuole, involving a series of profound changes of the host cell similar to those triggered when a bacterium is captured (Morisaki et al., 1995). The vacuole surrounding the parasite is then a traditional phagosome that quickly acquires the markers of endocytosis, fuses with endosomes/lysosomes, and rapidly acidifies (Sibley et al., 1985; Morisaki et al., 1995; Mordue and Sibley, 1997). The parasite is then destroyed by lysosomal enzymes. Opsonized internalization does not involve reorientation of the parasite to bring the apical end into contact with the host cell as observed for active invasion and does not involve secretion of apical proteins stored in secretory organelles (Morisaki et al., 1995). The fate of the parasite is therefore largely dependent on its ability to actively invade.

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14.2.1.3 Kinematic analysis of invasion process During helical gliding, Toxoplasma comes into direct contact with the host cell by its apical end. An apical attachment step is difficult to visualize, as invasion is a rapid process and both gliding locomotion and invasion appear continuous. However, there is some evidence of attachment with apical reorientation, since treatment of invading parasites with cytochalasin D (cytD), an actin-disrupting drug which blocks motility and invasion, does not affect attachment (Miller et al., 1979; Dobrowolski et al., 1997), suggesting that attachment and active invasion are uncoupled. The kinematic analysis of tachyzoites entry showed that the vast majority of invasion events start by an initial “impulse” of the parasite (pivoting movement) of a few microns per second speed (Bichet et al., 2014). This “impulse” might promote a correct positioning of the apex of the parasite prior to internalization and may relate to the clear apical reorientation of Plasmodium merozoites entering a red blood cell (Dvorak et al., 1975). The conoid, a thimble-shaped cytoskeletal structure at the extreme apex of the parasite, may also contribute to bringing the apex of the parasite in close proximity to the host cell surface (Schwartzman and Saffer, 1992). The conoid extends and retracts repeatedly as the parasite move along the host cell surface. A compound (inhibitor 6) identified in a small molecule screen selectively blocks conoid extrusion and parasite invasion, suggesting a role for conoid extrution in parasite entry (Carey et al., 2004b). However, conoid extrusion is not involved in motility since this same inhibitor has no effect on either parasite motility or microneme secretion. The projection of the conoid during motility or invasion is evident in Toxoplasma, Eimeria, and Sarcocystis, apicomplexan species that initiate infection through the gut. The absence of this organelle in Plasmodium sp. suggests

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that it may be an accessory apparatus used to penetrate particularly robust barriers such as the intestinal epithelium (Mondragon and Frixione, 1996). Following the movement that precedes the penetration of the parasite, an area of close contact between the cell and the apex of the parasite creates the “moving junction” (MJ) (also called tight junction) through which apicomplexan zoites propel themselves into the nascent PV (Aikawa et al., 1978). The MJ is a region of tight apposition but not fusion between parasite and host cell membranes, the latter being markedly thickened at this level (Aikawa et al., 1978). Freeze-fracture of invading tachyzoites (Chapter 2: The ultrastructure of Toxoplasma gondii) shows that the MJ contains rhomboidally arrayed particles and is identical in appearance to that formed by invading Plasmodium knowlesi merozoites (Aikawa et al., 1981). The MJ corresponds initially to a small area of contact between the parasite and host that turns into a circumferential ring around the parasite as invasion proceeds (see Chapter 2: The ultrastructure of Toxoplasma gondii). Although this interface has been called the “MJ,” it is actually anchored to the host cell cortex and remains stationery during invasion, while the parasite exercises a traction force and glides through this junction. However, in some cases, when the parasite encounters resistance or if the junction is not strongly anchored to the host cell cortex, the junction moves retrogradely with the host cell membrane along the parasite surface resulting eventually in a functional vacuole (Bichet et al., 2014). The MJ coincides generally but not systematically with a prominent constriction of the parasite at the site of penetration. This constriction may result from physical constraints imposed by the host cytoskeleton. The MJ then closes at the posterior end when entry is completed by fusion of host membrane behind the parasite (Aikawa et al., 1981; Lebrun et al., 2005). The parasite progresses forward into the

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nascent PV at an average speed of about 0.24 μm/s. Two changes in parasite body curvature are observed during invasion (Bichet et al., 2014). The first corresponds to a screwlike behavior, likely directed by the crescent shape of the parasite, while the second anticlockwise twisting motion, ending the entry process contributes to the fission process during PV separation (Pavlou et al., 2018). 14.2.1.4 Alternative routes of invasion Pioneering studies of invasion were mainly performed with the highly virulent type I RH tachyzoite. While the MJ-dependent invasion is the hallmark of invasion process of type I strains, in macrophages, type II avirulent strains use preferentially a noncanonical pathway called “phagosome to vacuole invasion (PTVI)” (Zhao et al., 2014). In this pathway the parasites are initially internalized through phagocytosis, and then actively invade through a MJ-process from within a phagosomal compartment to form eventually the classical specialized PV. This PTVI route might represent a Trojan horse strategy for phagocyte infection and may favor systemic dissemination and immune stimulation, leading to better control of avirulent strains and the establishment of chronic infection. Due to technical limitations, the invasion of the two transmissible stages—bradyzoites and sporozoites—are very poorly studied in Toxoplasma. The invasion of bradyzoites remains uncharacterized apart from one electron microscopy study that noted host projections are more prominent during bradyzoite entry and that the MJ is much less conspicuous or even absent (Sasono and Smith, 1998). It should be noted that this study compared RH tachyzoites with ME49 bradyzoites; thus it remains to be determined whether differences were due strain or stage-specific traits. Sporozoite entry in vitro appears also quite different from tachyzoite invasion. It is characterized by a large primary vacuole containing a single parasite and lacking molecular pores

(Speer et al., 1997; Tilley et al., 1997), contrasting with the tight-fitting PV formed by tachyzoites. The parasite does not replicate in this vacuole, and instead within 24 hours, it forms and exits into a secondary vacuole, where DG proteins are secreted (Tilley et al., 1997). The significance of this two-step invasion process is unknown but since the parasite replicates only within the secondary vacuole (Speer et al., 1995), specific secretory modification of the compartment is probably crucial for parasite intracellular survival and multiplication. Finally, while the important role of gliding for host cell invasion was experimentally verified for tachyzoites, the genetic depletion of gliding machinery uncovered the potential existence of alternative invasion mechanisms that account for residual invasion (Andenmatten et al., 2013; Egarter et al., 2014; Whitelaw et al., 2017).

14.2.2 Motility and invasion: central role of micronemes The first insight into understanding gliding motility and invasion by apicomplexan zoites came from observing relocalization of surfacebound molecules. Vanderberg (1974) described the circumsporozoite reaction whereby antibodies were capped on the trailing end of gliding Plasmodium berghei sporozoites. Then, Dubremetz and Ferreira (1978) showed that cationized ferritin bound the Eimeria sporozoite surface and was rapidly capped posteriorly. The velocity and susceptibility to cytD and low temperature of this capping phenomenon and of gliding motility were identical (Dubremetz and Ferreira, 1978; Russell and Sinden, 1981). Further studies eventually led to a model in which parasite gliding results from anteroposterior translocation of surface proteins interacting with cell surface or substrate receptors while being coupled to a submembranous actomyosin motor [see reviews (Sultan et al., 1997; Opitz and Soldati, 2002)]. Several studies have shown that these proteins were not resident at

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14.2 Motility and invasion

the parasite surface, but came from microneme exocytosis. Microneme exocytosis is an inducible process that has been first observed to occur at the apical attachment site between parasite and host cell before invasion. Inhibition of microneme exocytosis is correlated with inhibition of invasion (Carruthers and Sibley, 1999). After apical exocytosis, MIC proteins are translocated distally and released posteriorly as the parasite glides on a substrate or enters a cell (Asai et al., 1995; Carruthers and Sibley, 1997; Kappe et al., 1999; Garcia-Reguet et al., 2000; Opitz et al., 2002). This anteroposterior relocalization matches exactly the progression of the invasion. In addition, it is inhibited by cytD, suggesting a link between these proteins and the inner actin cytoskeleton. The molecular characterization of microneme proteins (MICs) has shown that adhesive motifs usually found in higher eukaryote proteins are present in these proteins (Tomley et al., 2001). A functional part played by these adhesins in host cell attachment, motility, invasion, and of a synergistic role of MICs in the infectious process (Huynh et al., 2003, 2014; Cerede et al., 2005; Mital et al., 2005; Huynh and Carruthers, 2006; Kessler et al., 2008; Friedrich et al., 2010a; Marchant et al., 2012; Sidik et al., 2016) has been demonstrated. In addition, the C-terminal cytosolic domain (CD) of transmembrane (TM) MICs play a critical role of in motility and invasion (Kappe et al., 1999; Jewett and Sibley, 2003; Sheiner et al., 2008; Lamarque et al., 2014). This domain, which in many cases is highly conserved in Apicomplexa, is proposed to serve as a linker between the extracellular domains of MICs interacting with host cell receptors and the submembranous actomyosin motor. In the molecular model of invasion that has emerged, secreted MICs serve as ligands recognizing both the host cell surface and, via adaptor proteins, the submembranous cytoskeletonassociated motor, called the glideosome (Fig. 14.2). The parasite myosin contractile

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system is fixed in place on the IMC, the actin filaments pull on MIC molecules allowing anterior-to-posterior translocation of MICs, the outermost membrane of the IMC providing a conveyor belt-like system. When a TM MIC protein is tethered to a fixed extracellular receptor, the parasite is propelled forward. Thus the parasite motion relative to host cells (invasion) or substrate (motility) is driven by rearward transport of actin filaments and their bound MICs relative to the parasite body. The flux of F-actin is initiated at the apical end via the conoidal formin 1 that nucleates and polymerizes actin (Tosetti et al., 2019). It depends on myosins and is crucial for motility, invasion and egress (Tosetti et al., 2019). Recent biophysical approaches (laser trap and optical trap) confirm that motile forces are indeed generated at the plasma membrane (Quadt et al., 2016; Stadler et al., 2017) and depend on actomyosin activity (Stadler et al., 2017). The posterior release of the interaction needed for efficient motion or invasion involves specific proteolytic activities. In this model, although experimental proof is still lacking, the MJ is thought to provide anchorage to the host cell while the parasite’s actin-myosin motor powers invasion. In this context, gliding machinery plays the central part in the invasion process and in egress from the host cells.

14.2.3 Moving junction formation: cooperative role between micronemes and rhoptries The molecular structure of the MJ has long been an enigma. MIC protein complexes associated with host cell receptors were believed to build up the junction, although initially no convincing data supported this hypothesis. Two independent studies in T. gondii led to the characterization of the first components of the MJ, an important step toward solving the molecular architecture of this structure central to

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FIGURE 14.2 Molecular components of the MJ during invasion. Model of the glideosome system at the MJ. As discussed in the text, TM MIC complexes are thought to bind host receptors and transmit the glideosome’s mechanical force by connecting through aldolase to F-actin, which is translocated posteriorly by the ATP driven myoA complex anchored into the IMC. IMP consisting of TM proteins may connect the IMC with subpellicular microtubules and IF-like structures on the cytosolic face of the IMC. Also shown is the AMA1-RON2/4/4L1/5/8 complex, which is proposed to form the MJ. RON2 is predicted to be an integral membrane protein anchored into the host plasma membrane (as depicted here). RON2 interacts directly with AMA1 exposed on the parasite surface, providing a bridge between the parasite and its host cell. AMA1 is also suggested as a bridging protein that physically connects the glideosome to other components of the MJ complex (via its ectodomain) and thus plays a critical role in the posterior translocation of the MJ complex during invasion; but this model awaits further demonstration. The other members of the RON complex (RON4/4L1/5/8) are tethered to RON2 and exposed to the cytosolic face of the host cell membrane, where they interact with host proteins to anchor the MJ to the host cell cytoskeleton. IMC, Inner membrane complex; IMP, intramembrane particles; IF, intermediate filament; MJ, moving junction.

apicomplexan invasion (Alexander et al., 2005; Lebrun et al., 2005). Among these, MJ proteins were hypothetical proteins restricted to Apicomplexa and derived from the anterior part of the rhoptries, known as rhoptry neck proteins (RONs), and the microneme protein apical membrane antigen 1 (AMA1). Further studies on the functional characterization of the AMA1RONs complex revealed that the MJ derives from collaboration between MICs and RONs (Fig. 14.1). These findings are detailed next.

14.3 Parasitophorous vacuole formation and maturation 14.3.1 Parasitophorous vacuole formation: role of rhoptries At the point of entry the force generated by the parasite induces the invagination of the host cell membrane that contributes to the membrane of the nascent vacuole. A real-time electrophysiological study of tachyzoite host

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14.3 Parasitophorous vacuole formation and maturation

cell entry elegantly demonstrated that the lipid bilayer of the vacuole derives predominantly from the host plasma membrane (Suss-Toby et al., 1996). However, the vacuole is also modified by the fusion of rhoptry-derived secretory vesicles (Hakansson et al., 2001). Rhoptry exocytosis is visualized at the beginning of invasion and rhoptry proteins (ROPs) are found associated with the PVM at early stages of biogenesis (Kimata and Tanabe, 1987; Carruthers and Sibley, 1997; Sinai and Joiner, 2001). The rhoptry contents sometimes appear as multilamellar vesicles both before and after exocytosis in T. gondii and in Plasmodium (Nichols et al., 1983; Bannister et al., 1986; Stewart et al., 1986). When invasion is interrupted with cytD, the intracellular accumulation of this membranelike material called evacuoles is particularly conspicuous even though the PV is not formed (Hakansson et al., 2001). How these vesicles assume a multilamellar structure is not known, but their morphology suggests they contain lipids organized in sheets or bilayers. Their lipid contents are strictly parasite derived (Hakansson et al., 2001), and, similar to the PVM, the evacuoles do not fuse with the endosomal compartments (Hakansson et al., 2001) (as stated previously). Subcellular fractionation of rhoptries has also shown an abundance of lipids in these organelles (Foussard et al., 1991) and especially an enrichment in cholesterol, but this latter was not confirmed in a subsequent study (Besteiro et al., 2008). Therefore rhoptry exocytosis serves to export proteins and lipids modifying the membrane of the developing PV. Some rhoptry proteins are also introduced into the host nucleus. Targeting to host nucleus and decorating the nascent PVM, ROPs proteins function to counteract host defenses (Fig. 14.1) (the role as effectors of ROP proteins is described in Chapter 17: Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages).

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While mostly of host nature, the early vacuole membrane appears devoid of intramembranous particles by freeze-fracture (Porchet-Hennere and Nicolas, 1983). A striking difference in the density of IMP between the P face of the host cell and P face of the PV is observed (Aikawa et al., 1981; Dubremetz et al., 1993), illustrating the diffusion barrier that excludes TM proteins at the MJ (Mordue et al., 1999a). This observation suggests that the MJdependent invasion process directly impacts the composition of the nascent PVM and selectively excludes some membrane proteins during invasion. Molecular insights into this exclusion process were studied by Mordue et al. (1999a) who first demonstrated that not all the host proteins are stripped, and secondly that sorting seems dependent on the anchoring to cytoskeleton. Host lipid
anchored proteins are retained associated with the PVM, while type I-single spanning TM proteins associated with cytoskeleton are excluded (Mordue et al., 1999a). However, the lipid composition of the host cell does not seem to influence the sorting because some raft associated proteins are retained, while other are excluded from the PVM (Charron and Sibley, 2004). How this exclusion operates is still unknown. While there is no experimental proof, RON proteins are ideally positioned to control the selective access of host proteins to PVM. The ability of the parasite to strip most of host proteins likely prevents lysosomal fusion and rapid killing of the parasite.

14.3.2 Maturation of the vacuole: a prominent role of dense granules Following entry into the host, the parasite is isolated from the cytoplasm of the host cell within a membrane-bound compartment, which separates it from nutrients present in the host cytosol and organelles. To access the

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pools of host-derived nutrients, the parasite transforms its vacuole into a dynamic structure. It also manipulates the host cell and hijacks organelles for its own benefit. Here we will succinctly present these profound modifications in order to introduce how ROPs and GRAs contribute to the maturation of the vacuole into a compartment suitable for survival and replication. 14.3.2.1 A complex network of tubules and vesicles Within 10
20 minutes postinvasion, the parasite builds up a complex network of tubules— the tubulo-vesicular network (TVN; also known as the intravacuolar network)—within the vacuole. These structures were initially estimated to be 40 6 60 nm in diameter (Sibley et al., 1995) and around 30 nm by superresolution electron microscopy (Magno et al., 2005a; de Souza and Attias, 2015). This network expands during replication by salvage of host lipids (Caffaro and Boothroyd, 2011; Nolan et al., 2017). Some of these tubules appear connected to the PVM (Sibley et al., 1986, 1995). The formation of a TVN strictly correlates with the intravacuolar release of DG proteins (GRAs) that accumulate into a posterior invaginated pocket of the parasite membrane formed 10
20 minutes postinvasion (Sibley et al., 1995) (Fig. 14.1, part III). Many proteins discharged from DGs segregate to either the TVN or PVM or both (Mercier et al., 2005), and some are critical players in the formation and architecture of TVN, as detailed next. The role of the TVN remains unclear but based on its spatial importance within the vacuole, current hypotheses and recent studies favor a role in metabolic exchange between the parasite and the host cell (Dou et al., 2014; Nolan et al., 2017). Ingestion of cytosolic host cell proteins during parasite replication is impeded in a GRA2 mutant (Dou et al., 2014), which also displays reduced scavenging of host lipid droplets (Nolan et al., 2017). Among

the other functions are a role in spatial organization of the parasites within the vacuole (Magno et al., 2005a) and a role in immunomodulation of the host response (Lopez et al., 2015). Some of the GRAs associated with TVN regulate access of parasite antigens to the MHC class I pathway, illustrating how the TVN influence crosstalk of the parasite with the host cell. In summary, the TVN will display two functions: it will act as an “umbilical cord” for feeding nutrients from the host and as a “trap” to reduce the exposure of immunogenic proteins to the host (Santi-Rocca and Blanchard, 2017). In addition to the TVN, the PV contains many fibril-containing vesicles of unknown function and actin filaments present inside membranous structures (de Souza and Attias, 2015; Periz et al., 2017). These membranous structures of 50 6 60-nm connect the PVM to the TVN and the parasites between them. These tubules control the synchronicity of parasite divisions and mediate the transfer of material between parasites (Frenal et al., 2017b; Periz et al., 2017). The PVM is not a “smooth” membrane. It remarkably shapes into outward-membranous tubules (Dubremetz et al., 1993) that extend into the host cytoplasm, increasing the PVM surface area for host cell interactions. These long membranous structures interconnect vacuoles formed in the same host cell, but also PVs located in neighboring cells (Dubremetz et al., 1993). Similar to the TVN, GRA proteins are also associated with these outward membranous structures and proposed to be exchanged between vacuoles via these structures (Rome et al., 2008). ROP proteins also decorate the outward-membranous tubules. In summary many membranous structures have been described. Whether these different structures are connected or separate, and whether they are formed with a similar kinetics during replication remain unknown. Both GRA proteins and the network persist during

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replication of the parasite within the PV. Later, when parasite stage conversion to bradyzoites takes place, the vacuolar structures redistribute and contribute to the formation of an intracellular cyst (Torpier et al., 1993; Ferguson, 2004). Hence, an essential function of some GRAs might be at the encystation stage for the construction and maintenance of the cyst wall. 14.3.2.2 Pore inside the parasitophorous vacuole membrane The parasite salvages essential nutrients from the host cell to multiply rapidly. Schwab et al. demonstrated that the PVM surrounding T. gondii permits free bidirectional diffusion of small charged and uncharged molecules (,1300 Da) between the host cell cytoplasm and the vacuolar space (Schwab et al., 1994). This finding is consistent with a pore of protein origin crossing the PVM. Since most of the integral membrane proteins from the host are excluded from the PVM (Mordue et al., 1999a), the pore has probably to be parasite derived. An elegant study by Gold et al. demonstrated that proteins discharged from DGs are directly responsible for the formation of a pore inside the PVM [(Gold et al., 2015) Fig. 14.1, part III; described later in Section 14.7.6.1]. 14.3.2.3 Attraction of host organelles and structures to the parasitophorous vacuole membrane Shortly after invasion, the limiting membrane of vacuoles associates with host mitochondria and rough endoplasmic reticulum (ER) (Sinai et al., 1997; Magno et al., 2005b). A molecular exchange between the host ER and the PV has been described and proposed to facilitate the presentation of parasite antigens at the host plasma membrane (Goldszmid et al., 2009). The PV attracts also host multivesicular bodies and lipid droplets (Nolan et al., 2017). The host lipid droplets are then internalized within the PV and eventually incorporate into both the parasite membranes and lipid

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droplets (Nolan et al., 2017). Mutant parasites impaired in TVN formation (GRA2 mutant) display diminished capacity for lipid uptake from host lipid droplets (LD) and this process is dependent upon a parasite TVN-localized phospholipase A2. This suggests a major contribution of the TVN and GRA proteins for host LD processing in the PV and lipid content release (Fig. 14.1, part III). The Golgi is also found associated with the PVM. Interestingly, it looks destabilized and sliced into ministacks that encircle the PV (Romano et al., 2013). The function of this fragmentation is unknown but might serve to intercept host Golgi-derived vesicles. Accordingly, many host vesicles containing proteins regulating intracellular trafficking like Rab GTPases are trapped within the PV. Among them are Rabassociated Golgi vesicles (Romano et al., 2013, 2017). There is also some evidence for Microtubule Organizing Center
PV association (Coppens et al., 2006; Walker et al., 2008; Wang et al., 2010), which might help the parasite to regulate the movement of host organelles and vesicles and attract them to the PV. In support of this hypothesis, the parasite also usurps the host microtubular network to create invagination of the PVM and reroute host endo-lysosomes to the PV (Coppens et al., 2006). The tubules mediating the host endolysosomal delivery system were named HOST for Host OrganelleSequestering Tubulo-structures. (Coppens et al., 2006). Trapped into the PV lumen these host endo-lysosome vesicles are proposed to allow the parasite to acquire a source of membrane lipids necessary for its replication. The mechanism of rerouting host organelles or vesicles to the PVM is unknown, except that the DG protein mitochondria association factor 1 (MAF1) directly contributes in the anchoring of host mitochondria to the PV membrane in type I strains (Pernas and Boothroyd, 2010) (Fig. 14.1, part III). Whether other parasite effectors are involved in host organelle interception is unknown, but likely.

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14.3.2.4 Targeting ROPs and GRAs to the PVM and host cell to neutralize host defense As mentioned above, many secreted parasite proteins are translocated to the PVM during the invasion process (ROPs) or during intracellular growth (GRAs), where they manipulate the host cell to the parasite’s benefit. Recent studies highlight that ROPs and GRAs targeted to the PVM also act in concert (Dunn et al., 2008; Alaganan et al., 2014). Finally, both GRAs and ROPs also relocate in the host cytosol or nucleus to hijack the host cell (Fig. 14.1). These emerging roles are discussed in Chapter 17, Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages.

et al., 2009). Proteins from the DGs also promote egress by acting on phospholipids to initiate signaling within the parasite (DGK2) (Bisio et al., 2019) and possibly help weaken the PVM (LCAT) (Pszenny et al., 2016; Schultz and Carruthers, 2018). Further, at least two DG proteins (GRA22 and GRA41) function to prevent premature egress from infected host cells (Okada et al., 2013; LaFavers et al., 2017). Parasites lacking rhoptry secretion egress normally (Beck et al., 2013; Mueller et al., 2013) suggesting that the contents of rhoptries do not contribute significantly to parasite exit. Thus micronemes promote egress by supporting gliding motility and rupture of the PVM, whereas DG proteins influence the process and timing of egress.

14.4 Egress

14.5 Micronemes

In addition to the aforementioned roles, it has emerged that proteins secreted from micronemes and DGs also play important roles in parasite egress following intracellular replication. Early studies showed that treatments that elevate calcium in infected host cells trigger egress (Pingret et al., 1996; Stommel et al., 1997). Although at the time it was thought that such treatments initiated egress by elevating host cytosolic Ca21, it is now appreciated that elevation of Ca21 within the parasite cytosol is critical because of its role in stimulating microneme secretion and activating the glideosome (Lourido and Moreno, 2015; Carruthers, 2019). Microneme contents contribute to egress in two distinct ways (Fig. 14.1, part III). First, as a source of adhesive proteins (e.g., MIC2) necessary for effective gliding motility, microneme proteins help translate the mechanical force needed to traverse membranes, organelles, and cytoskeletal structures of the host for successful exit (Frenal et al., 2017a; Gras et al., 2017). Second, micronemes deliver a poreforming protein (PLP1) that is critical for efficient liberation of the parasite from the PVM (Kafsack

14.5.1 Trafficking of MICs and the biogenesis of microneme subpopulations This section will briefly discuss the general pathway and mechanisms of MIC trafficking along with the biogenesis of micronemes including recently identified subpopulations. A more extensive description of this topic is provided in Chapter 15, Endomembrane trafficking pathways in Toxoplasma. A total of 50
100 micronemes (from Greek for “small threads”) populate the apical portion of invasive Toxoplasma zoites. Micronemes are often seen in an arc-like pattern by their association with the cytoplasmic face of the IMC in the apical region. This association is likely mediated by binding to subpellicular microtubules, as the destabilization of microtubules leads to spatial redistribution of the micronemes (Leung et al., 2017). Some micronemes are also found more centrally located and even in the mid-region of the parasite, implying that not all micronemes are microtubule associated.

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14.5 Micronemes

MIC proteins are synthesized on membranebound ribosomes in the ER where some are modified by N- and O-linked glycans, and at least one (SUB1) gains a glycosylphosphatidylinositol (GPI) anchor. About 40% of T. gondii MICs have at least one TM domain, which permits interaction of the CD with trafficking machinery in the cytosol. Migration of MICs from the ER to the Golgi is presumed to depend on COPII-coated vesicles via interaction with the CD of TM MICs. Several MICs that do not have TM domains hitchhike on TM MICs for trafficking through the endomembranous system (Reiss et al., 2001). It remains unknown how “soluble” MICs (e.g., MIC5 and MIC10) that do not have stable partners move through the ER and Golgi. A handoff from the COPII system to SORTLR probably occurs at the trans-Golgi network (TGN). SORTLR is an ortholog of VPS10/sortilin, which binds to and escorts proteins through the endolysosomal system (Sloves et al., 2012). It localizes to the parasite Golgi, TGN, and endosome-like compartments (ELCs). SORTLR binds both to AP-1 vesicular coat proteins and to several microneme and rhoptry proteins. It, together with the AP-1 complex (Venugopal et al., 2017), is crucial for the biogenesis of micronemes and rhoptries (Sloves et al., 2012). The discovery of SORTLR along with other studies including those that have colocalized various MICs and ROPs with markers of endocytic compartments have firmly established that MICs and ROPs traffic through the endocytic system prior to packaging in their respective organelles. Thus Toxoplasma has repurposed part of its endolyososmal system as a conduit for the trafficking and processing of microneme and rhoptry proteins. Study of proMICs, a subset of MICs that are synthesized as immature proteins containing a propeptide, has identified a second trafficking element in addition to SORTLR. Propeptides are typically N-terminal and are proteolytically cleaved by endosomal proteolytic maturases

619

(cathepsin protease L. and ASP3; Parussini et al., 2010; Dogga et al., 2017), which helps to lock at least one MIC protein complex (MIC2/ M2AP) into a stable complex (Harper et al., 2006; Huynh et al., 2015). MIC propeptides are necessary for proper trafficking to the micronemes (Binder and Kim, 2004; Harper et al., 2006; Brydges et al., 2007; El Hajj et al., 2008) and contain crucial residues near their Nterminus that function late in trafficking (Gaji et al., 2011b), possibly at the site of packaging of MICs into nascent micronemes. Precisely how MIC propeptides function in MIC trafficking remains to be determined, but could involve interaction with SORTLR or a separate sorting receptor functioning downstream of SORTLR. The studies described above support a model in which forward targeting signals from the CD of a TM MIC license it to escort soluble MICs through the ER to the Golgi where SORTLR then navigate the complexes from the Golgi through some portion of the endosomal system and to the micronemes, possible in conjunction with or prior to a MIC propeptide dependent step. After transporting its cargo to the micronemes, SORTLR returns to the Golgi to reload for another delivery cycle. The role of SORTLR in ROP trafficking and rhoptry biogenesis is discussed in greater detail in Section 14.6.1. To this date, only one study reports a role for a microneme protein in the morphology of micronemes (Hammoudi et al., 2018). In this study the authors identified transporter facilitator protein 1 (TFP1), phylogenetically related to TFPs, associated with micronemes and participating in the maturation of microneme organelles. Conditional abalation of TFP1 leads to a significant decrease in the number of micronemes and to a strong defect in their exocytosis. In the mutant the remaining micronemes are larger and adopt an ovoid morphology, suggesting a role of TFP1 in the transport of molecules necessary for organelle maintenance, maturation and proper exocytosis.

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

14.5.2 Microneme subpopulations Pioneering work by Kremer et al. (2013) revealed that, despite their ultrastructural homogeneity, not all micronemes are alike. This study showed that overexpression of wild-type or dominant negative Rab5A or Rab5C resulted in mislocalization of MIC3, MIC8, and MIC11. In contrast, localization of MIC2, M2AP, AMA1, and PLP1 was largely normal. Electron microscopy revealed that Rab5A or C overexpression strains were devoid of lateral micronemes but retained a subset of central micronemes clustered near the conoid. Superresolution STED microscopy additionally indicated the existence of distinct subsets of micronemes wherein MIC2 and M2AP show strong colocalization, but less colocalization was seen between M2AP and MIC3, AMA1 and MIC8, and also MIC3 and MIC8, despite the previous suggestion that MIC3 and MIC8 form a complex (Meissner et al., 2002). Similar to overexpression of Rab5A or Rab5C, subsequent work conditionally ablating expression of the Rab5 GEF Vps9 showed mistargeting of MIC3 without substantial mistargeting of M2AP (Sakura et al., 2016). In the absence of VpS11, a subunit of HOPS and CORVET tethering complexes, all lateral micronemes were lost while apical micronemes were still detected (Morlon-Guyot et al., 2015). Together, these studies suggest distinct trafficking mechanisms to at least two different subsets of micronemes that are spatially segregated.

purification (Donahue et al., 2000). Genome sequencing of T. gondii has facilitated the identification of additional MICs, either by similarity to MICs of other Apicomplexa [AMA1, MIC2, SUB1, MIC17, SPATR, (GPI-anchored microneme antigen GAMA)] (Wan et al., 1997; Hehl et al., 2000; Miller et al., 2001; Chen et al., 2008; Sohn et al., 2011; Huynh et al., 2014; Huynh and Carruthers, 2016), by searching for TM domains and CDs with homology to those of TRAP/MIC2 (MIC6, MIC7, MIC8, MIC9, and MIC12) (Meissner et al., 2002; Opitz et al., 2002) or by similarity to known Toxoplasma MICs (MIC13 and MIC16) (Friedrich et al., 2010a; Sheiner et al., 2010). Novel MICs were identified by cell fractionation and calciummodulating compounds that enhance or block microneme secretion, and N-terminal sequencing (MIC4, MIC5, MIC10, and MIC11) (Brydges et al., 2000; Brecht et al., 2001; Hoff et al., 2001; Harper et al., 2004b). Coprecipitation allowed the identification of M2AP (Rabenau et al., 2001). Highly sensitive and complementary technologies such as 2-DE/MS and LC/ESIMS
MS have added new candidates on the already long list of MICs previously identified (Zhou et al., 2005; Kawase et al., 2007). Although at least 28 microneme proteins have been identified to date, it is likely that additional MICs will be discovered through new screening methodologies.

14.5.3 Microneme proteins

14.5.3.1 MICs sharing homologies with structural domains of eukaryotic proteins involved in protein
protein or protein
carbohydrate interactions

T. gondii expresses a large, diverse array of MICs including TM and soluble proteins. Several approaches have been used to identify MICs. Before the genome sequencing era, MICs were discovered using monoclonal antibodies (MIC1, MIC2, and MIC3), followed by immunoscreening of cDNA libraries (Achbarou et al., 1991a; Fourmaux et al., 1996b; GarciaReguet et al., 2000) or immunoaffinity

The molecular characterization of MICs has revealed a striking conservation of structural domains known in higher eukaryotic cells (see Table 14.1). These domains are found on both soluble and TM MICs, in single or multiple copies, and in a variety of combinations so that every MIC protein is structurally unique. The repertoire of MICs in T. gondii constitutes a patchwork of proteins, some of them sharing

Toxoplasma Gondii

TABLE 14.1

Properties of Toxoplasma secretory proteins: microneme (MIC) proteins.

Location/Protein

Calculated MW (kDa)a

Microneme MIC1

49

Domains(no.) MAR(2), galectin-like domain(1)

Interacting partners

Mutant phenotypes

MIC4, MIC6 Nonessential protein

KO mutant is less invasive and less virulent in mice MIC2

83

A/I-domain(1), TSR(5), degenerate TSR (1), TM(1)

M2AP

Key protein

Function

Postsecretory trafficking

Adhesion, Secreted and posterior binding to capping sialylated oligosaccharides, folding, assembly of the MIC1/4/6 complex Escorter, adhesion

Secreted and posterior capping

C-terminal truncation mutant was not recoverableSubstitution with Eimeria MIC1 is viable but less invasiveConditional knockdown mutant showed markedly reduced gliding and invasion MIC3

MIC4

38 (90 dimer)

CBL(1), EGF(5)

63

Pan/Apple(6)

MIC8

Nonessential protein

Fourmaux et al. (1996a), Cerede et al. (2005), Saouros et al. (2005) Blumenschein et al. (2007), Sawmynaden et al. (2008), Garnett et al. (2009) Wan et al. (1997), Carruthers et al. (1999), Huynh et al. (2004), Jewett and Sibley (2004) Huynh and Carruthers (2006), Starnes et al. (2006), Tonkin et al. (2010)

Adhesion

Secreted and posterior capping

Garcia-Reguet et al. (2000), Cerede et al. (2002), Cerede et al. (2005)

Adhesion, binding to galactose

Secreted and posterior capping

Brecht et al. (2001), Reiss et al. (2001), Marchant et al. (2012)

KO mutant has reduced virulence in mice MIC1, MIC6 Nonessential protein

References

(Continued)

TABLE 14.1

(Continued)

Location/Protein

Calculated MW (kDa)a

MIC5

20

Domains(no.)

Interacting partners

Protease prodomain

MIC6

37

EGF(3), acidic

MIC7

36

EGF(5), TM(1)

MIC8

75

CBL(1), EGF (10), TM(1)

MIC9

32

EGF(3), TM(1)

MIC10

23

MIC11

22

Mutant phenotypes

Function

Postsecretory trafficking

References

Nonessential protein

Subtilisin protease inhibitor

Secreted and posterior capping

Brydges et al. (2000), Saouros et al. (2012)

KO mutant shows elevated SUB1 proteolysis of other secretory proteins MIC1, MIC4 Nonessential protein

Reiss et al. (2001)

Escorter

Meissner et al. (2002) MIC3

Meissner et al. (2002) Meissner et al. (2002) Secreted

Hoff et al. (2001)

Strong charge asymmetry

Secreted

Harper et al. (2004b)

MIC12 234 (TGME49_267680)

EGF(31), TSR(4), TM(1)

Secreted

Opitz et al. (2002)

MIC13 (MCP2)

MAR(3)

51

Adhesion, binding to sialylated oligosaccharides

Friedrich et al. (2010a, b), Fritz et al. (2012a)

MIC14 106 (TGME49_218310)

TSR(2), TM(1)

Unpublished

MIC15 320 (TGME49_247195)

TSR(3), TM(1)

Unpublished

MIC16 75 (TGME49_289630)

TSR(6), TM(1)

Unpublished

Pan/apple(4)

Chen et al. (2008), Sohn et al. (2011)

MIC17A, B, C

B39

AMA1

60

Pan/apple(2), TM(1)

RON2

Key protein

MJ organization, Secreted binding to RON2 Conditional knockdown Detected on both the mutant can attach, external and internal secrete rhoptries, but regions of invading cannot penetrateFails to parasites organize the MJ Visible at the MJ in conditional knockdown mutant

Secreted and associated with MJ in absence of AMA1

Donahue et al. (2000), Hehl et al. (2000), Alexander et al. (2005), Howell et al. (2005), Mital et al. (2005), Bargieri et al. (2013) Besteiro et al. (2009), Crawford et al. (2010), Lamarque et al. (2011), Santos et al. (2011), Tonkin et al. (2011), Tyler and Boothroyd (2011), Parussini et al. (2012), Lamarque et al. (2014)

AMA2

RON2

Nonessential protein in tachyzoites

SporoAMA1/ AMA3

SporoRON2 (RON2L2)

Nonessential protein in tachyzoites

Fritz et al. (2012a, 2012b), Poukchanski et al. (2013)

EGF

RON2L1

Nonessential protein in tachyzoite

Lamarque et al. (2014), Parker et al. (2016)

Galectin-like(1), coil(2)

MIC2

Nonessential protein

Sporozoitespecific microneme protein AMA4 Sporozoite microneme protein M2AP

35

KO mutant shows defects in MIC2 trafficking; deficient attachment and invasion; virulence defect

Secreted and posterior capping

Lamarque et al. (2014)

Rabenau et al. (2001), Huynh et al. (2003)

(Continued)

TABLE 14.1

(Continued)

Location/Protein

Calculated MW (kDa)a

Domains(no.)

SUB1

85

Subtilase, GPI

Interacting partners

Mutant phenotypes

Function

Postsecretory trafficking

References

Not essential protein

Proteolysis

Secreted and posterior capping

Miller et al. (2001), Binder and Kim (2004), Lagal et al. (2010)

KO mutant shows defects the surface processing of several secretory proteins; deficient in gliding motility and invasion; virulence defect ROM1

28

Rhomboid, TM (7)

Nonessential protein

Proteolysis

Brossier et al. (2005), Dowse et al. (2005)

KO mutant shows a fitness defect due to modest deficiencies in invasion and replication SPATR

58

EGF(1), TSR(2)

Brossier et al. (2008)

Nonessential protein

Secreted

Kawase et al. (2007), Huynh et al. (2014)

Secreted

Laliberte and Carruthers (2011)

KO is partially defective in invasion and virulence Toxolysin 4 (TLN4)

247

Metalloprotease (M16 family)

Nonessential protein

Proteolysis

Difficult to KO in RH but KO in Δku80 is viable GAMA

101

Nonessential protein KO is partially defective in attachment to host cells

Agrawal and Carruthers (unpublished) Adhesion

Secreted and posterior capping

Huynh and Carruthers (2016)

CLAMP

42

PLP1

128

MACPF

KO is defective in egress from host cells

Pore formation

TFP1

60

Major facilitator superfamily, TM(12)

Conditional knockdown mutant is impaired microneme biogenesis and leads to a comblock in exocytosis

TFP1 participates in the condensation of the microneme content

a

KO fails to secrete rhoptries and is defective in invasion

Secreted and posterior capping

Sidik et al. (2016)

Kafsack et al. (2009), Roiko and Carruthers (2013), Roiko et al. (2014), Guerra et al. (2018), Ni et al. (2018) TM protein of microneme membrane, with Cterminal part facing the cytosol

Hammoudi et al. (2018)

Based on the complete open reading frame including signal sequence or GPI anchor signal, if present. CBL, Chitin-binding-like domain; CCP, domain abundant in complement control proteins; CLAMP, claudin-like apicomplexan microneme protein; CobT, cobalamin biosynthesis protein; DUF1222, domain of unknown function; EGF, epidermal growth factor; GAMA, glycosylphosphatidylinositol-anchored microneme antigen; GPI, glycosylphosphatidylinositol; KO, knockout; MJ, moving junction; MAR, microneme adhesive repeat; MACPF, membrane attack complex perforin; TSR, thrombospondin type-1 repeat; TM, transmembrane.

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

orthologs in the other Apicomplexa. Several of these conserved structural domains are known in higher eukaryotes to be responsible for protein
protein or carbohydrate
protein interactions. The cell surface binding properties of MICs have been demonstrated (MIC1, MIC2, MIC3, and MIC4) (Fourmaux et al., 1996a; Carruthers and Sibley, 1999; Garcia-Reguet et al., 2000; Marchant et al., 2012), but the presence of such domains has not been systematically associated with an attachment function (e.g., MIC6, which does not directly bind host receptors) (Saouros et al., 2005). In this section, we will review the various structural domains found in MICs. 14.5.3.1.1 I- or A-domain:

MIC2 is the only characterized Toxoplasma protein known to possess this domain (Wan et al., 1997). It is a member of the TRAP family, highly conserved among the phylum (Robson et al., 1988; Clarke et al., 1990; Spano et al., 1998). The contributions of MIC2 to infection have been reported in two studies. Huynh et al. showed that conditional ablation of MIC2 expression markedly decreased parasite gliding motility and parasite attachment, resulting in diminished invasion and loss of virulence in a mouse model of acute toxoplasmosis (Huynh and Carruthers, 2006). By generating parasites completely lacking MIC2, Gras et al. (2017) confirmed the role of MIC2 in gliding motility and attachment and identified a previously unrecognized contribution to egress but did not detect a deficiency in parasite virulence in mice. The different parasite strain backgrounds and distinct inocula that were used in these studies might underlie the apparent discrepancy in virulence. MIC2 possesses a single integrin-like Adomain. This domain is present in the α-chain of some integrins, which are type 1 integral membrane proteins that promote cell
cell and cell
extracellular matrix (ECM) contacts (Pytela, 1988; Larson et al., 2009). A similar

domain is also found in various plasma proteins (e.g., von Willebrand factor), in soluble matrix proteins, or in proteins involved in cell
cell and cell
ECM matrix interactions during homeostasis, inflammation, or cell migration (Whittaker and Hynes, 2002). Adomains bind to various ligands including collagens, laminin, fibronectin, ICAM-1, and the complement product iC3b. A unique feature of these domains is that they possess a metal-iondependent adhesion site (MIDAS) motif composed of five noncontiguous amino acids (Lee et al., 1995). Similar to the A-domain of Plasmodium falciparum TRAP (McCormick et al., 1999), MIC2 A-domain binds selectively to heparin (Harper et al., 2004a), a ubiquitous sulfated proteoglycan found in the ECM. This binding is not dependent on the MIDAS site, a property that is also supported by the absence of a divalent metal in the structure of the MIDAS domain of MIC2 (Tonkin et al., 2010) and of binding to heparin in the absence of divalent cations for the P. falciparum TRAP-A domain (McCormick et al., 1999). These findings suggest spatial separation of the putative heparin-binding site from the MIDAS region, indicating that these distinct molecular surfaces may be involved in the recognition of multiple receptors (Akhouri et al., 2004). However, the functional importance of MIDAS metals remains debated because chelation of metals inhibits cell binding (Pihlajamaa et al., 2013), a result in agreement with an earlier mutagenesis experiment which demonstrated that an intact MIDAS of TRAP is necessary for malaria parasite infection (Matuschewski et al., 2002). An important question is the identity of the extracellular molecules displayed on host cells that can interact with A-domain. The TRAP Adomain of P. bergehi interacts with fetuin-A on hepatocyte membranes and this interaction enhances the parasite’s ability to invade hepatocytes (Jethwaney et al., 2005). TRAP binds also to saglin, a salivary gland
specific surface

Toxoplasma Gondii

14.5 Micronemes

protein (Ghosh et al., 2009). The TRAP domain-A directly interacts with saglin and this interaction may contribute to sporozoite invasion of the salivary gland. Finally, a recent study showed the contribution of the TRAP-A domain in binding to the human integrin αvβ3, an interaction that might be used for dermal migration of sporozoites (Dundas et al., 2018). In T. gondii, MIC2 binds to ICAM-1, and this interaction facilitates migration across polarized epithelial cells (Barragan et al., 2005). 14.5.3.1.2 Thrombospondin type 1 (TSR) repeat domain:

This domain is found in MIC2, MIC16, and SPATR (Wan et al., 1997; Opitz et al., 2002; Kawase et al., 2010; Sheiner et al., 2010), with one degenerated motif in each case. The TSR domain is present in many proteins from distantly related organisms. These proteins are usually involved in cell
cell and cell
matrix interactions (Lawler, 1986) in clotting or innate immunity (Haefliger et al., 1989). The TSR domain allows thrombospondin and properdin to bind sulfated sugars and especially glycosaminoglycans (Holt et al., 1990; Chen et al., 2000). It shows also a high affinity for heparin (Guo et al., 1992). There are no reports of an adhesive function for the TSR domain of MIC2. However, in Plasmodium, the TRAP TSR domain binds heparan sulfate proteoglycans on the hepatocyte surface in vivo and in vitro (Muller et al., 1993). The importance of the TRAP TSRheparan sulfate interaction in the host cell invasion by Plasmodium sporozoite was demonstrated by Matuschewski et al. (2002). Structural data on the TSP-1 of thrombospondin (Tan et al., 2002) suggests that the MIC2 TSR likely forms an extended “stalk” on which the A-domain sits, optimally positioned for interaction with host receptors. The TSR domain is also required for interaction of MIC2 with M2AP (Huynh et al., 2015). The sixth TSR of MIC2 is necessary and

627

sufficient for interaction with hydrophobic residues in a galectin-like binding pocket of M2AP. Interestingly the sixth TSR is divergent in sequence from the other TSRs, suggesting it evolved a specialized role for interaction with M2AP. Several of the MIC2 TSRs are C-mannosylated and O-fucosylated (Bandini et al., 2019; Khurana et al., 2019). Targeted deletion of an O-fucosyl transferase (POFUT2) or a fucose transporter (NST2) established that the MIC2 TSRs are the main, if not exclusive, O-fucosylated domains in the parasite. Whereas one study concluded that O-fucosylation of MIC2 TSRs is not important for MIC2 function (Khurana et al., 2019), a parallel study concluded that O-fucosylation of MIC2 TSRs is necessary for quantitative expression and trafficking of MIC2 to the micronemes, thereby influencing invasion and egress. The authors of these studies suggested that the discrepancies could be due to methodologic differences in how phenotypes were measured. MIC14 and MIC15 have not been characterized, but are annotated in Toxodb.org. MIC16 is a TM protein that displays 6 TSRs in its ectodomain and is conserved amongst coccidian parasites (Sheiner et al., 2010). Although the function of MIC16 remains to be determined, it appears to be shed from the parasite surface by intramembrane cleavage similar to MIC2 and AMA1. The CD of MIC16 lacks targeting information for the micronemes, implying that MIC16 interacts with another MIC protein(s) that guides it to the micronemes. Hoffman and colleagues implicated a P. falciparum surface protein called Secreted Protein with altered thrombospondin repeat (PfSPATR) in sporozoite invasion (Chattopadhyay et al., 2003). PfSPATR is expressed in merozoites, gametocytes, and sporozoites, making it an attractive vaccine candidate. Recombinant PfSPATR bound to HepG2 cells and an antibody to PfSPATR reduced sporozoites invasion, suggesting an

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

adhesive role for the protein. Similar findings were reported for P. knowlesi SPATR (Mahajan et al., 2005). More recently a Toxoplasma ortholog, TgSPATR, was identified in a proteomics analysis of secretory proteins (Kawase et al., 2007). Further characterization showed that TgSPATR is secreted from the micronemes and is on the surface of extracellular tachyzoites (Kawase et al., 2010). Like its Plasmodium counterpart, TgSPATR contains an N-terminal epidermal growth factor (EGF) domain and one (Kawase et al., 2007) or two C-terminal TSR repeats (Huynh et al., 2014). TgSPATR knockout (KO) parasites show normal gliding motility and egress but are moderately deficient in invasion and virulence. Genetic complementation Δspatr parasites with domain deletion mutants established that the TgSPATR EGF domain is required for correct trafficking to the micronemes and that each of the two TSR domains is necessary for TgSPATR function in parasite invasion. Together, these studies in Plasmodium and Toxoplasma suggest that the conserved SPATR family contributes measurably to infection by apicomplexan parasites. 14.5.3.1.3 Epidermal growth factor-like domain

EGF-LDs are found in a large variety of proteins, mainly of animal origin (growth factors, lipoprotein receptors, selectins, clotting factors, ECM proteins, etc.) and are frequently seen in tandem repeats with various degrees of conservation. The functional significance of these domains is not understood yet. A common feature of these repeated domains is that they are found in the extracellular portion of TM proteins or in secreted proteins engaged in protein interactions (Appella et al., 1988; Davis, 1990). EGF domains typically contain six cysteine residues that form disulfide bridges. The subdomain lengths between cysteines vary extensively. Some EGF-LDs have the capacity to bind calcium and are therefore termed calcium-binding EGF (cbEGF). cbEGF domains form a more rigid, protease resistant structure

upon binding calcium, as shown for the Eimeria protein EtMIC4 (Periz et al., 2005), a TM protein containing 31 EGF-like and 12 TSR domains (Tomley et al., 2001). This property may help form an elongated stalk-like structure to maximally project cbEGF-containing proteins from the cell surface. EGF-LDs are found in apicomplexan MICs and on resident surface proteins of Plasmodium. Five of the EGF containing proteins in T. gondii are TM (MIC6, 7, 8, 9 and 12) (Reiss et al., 2001; Meissner et al., 2002; Opitz et al., 2002) and two are soluble (MIC3 and TgSPATR) (GarciaReguet et al., 2000; Kawase et al., 2010). MIC3, 6, 8, and TgSPATR are expressed by tachyzoites and bradyzoites (Garcia-Reguet et al., 2000; Meissner et al., 2002), whereas MIC7 and MIC9 are predominantly expressed by bradyzoites (Meissner et al., 2002). A Toxoplasma ortholog of EtMIC4 exists in the ToxoDB database. This protein likely corresponds to T. gondii MIC12 since the deduced amino acid sequence is virtually identical to the partial sequence of MIC12 published by Opitz et al. (2002). At least two of the EGF-containing MICs contribute to tachyzoite cell invasion. MIC6 KO parasites show mislocalization of the carbohydrate binding adhesins MIC1 and MIC4, resulting in a B50% loss of cell invasive (Sawmynaden et al., 2008). Conditional ablation of MIC8 revealed an essential role in invasion characterized by a block in rhoptry secretion, but normal gliding motility and apical attachment (Kessler et al., 2008), suggest a potential role for MIC8 in signaling the discharge of rhoptries for parasite invasion (see next). EGF domains of MIC proteins play multiple nonadhesive roles in their cognate proteins. A function for EGF domains in assembly of MIC protein complexes was revealed upon showing that the second and third EGF domains of MIC6 associate with the MIC1 galectin-LD (GLD) (Sawmynaden et al., 2008). This association is fundamental for assembly of the

Toxoplasma Gondii

14.5 Micronemes

MIC6/MIC1/MIC4 complex. The structure of the MIC6
EGF2/MIC1
GLD interface revealed a novel interaction mode for an EGF domain involving an intimate hydrophobic surface. Multicopy assembly of this complex supported by the MIC6
EGF/MIC1
GLD interaction has been proposed to enhance multivalent exposure MIC1 on the parasite surface during invasion (Reiss et al., 2001). A second role for MIC-EGF domains in activation of host signaling was revealed using recombinant proteins and KO parasites (Muniz-Feliciano et al., 2013). This work showed that WT parasites, but not those lacking MIC3 or MIC6, activate an EGF receptor/ PI3P/Akt-signaling pathway that suppresses autophagy-mediated killing of the parasite in human cells. Recombinant MIC3 or MIC6, but not M2AP or MIC4, was sufficient to activate the signaling pathway and promote parasite survival. These findings suggest a role for EGF MIC proteins in immune evasion of host defenses. In a possible third role for MIC EGFs, yeast two-hybrid experiments identified interactions of the MIC3 EGF domains with two human proteins, namely, spermatogenesis-associated protein 3 (Spata3) and Dickkopf-related protein 2 (Dkk2) (Wang et al., 2015). Spata3 is a testisspecific protein of unknown function, whereas Dkk2 is a more broadly expressed secretory protein that antagonizes Wnt signaling. The biological significance of MIC3 interaction with Spata3 and Dkk2 awaits further investigation. Finally, MIC3 has been implicated as an activator of TNF-alpha production by mouse spenocytes and peritoneal macrophages (Qiu et al., 2016), although whether this activity is dependent upon its EGF domains remain to be determined. 14.5.3.1.4 Plasminogen, apple, nematode/apple module

The apple module contains a conserved core of three disulfide bridges. In some members of

629

the family, an additional disulfide bridge links the N- and C-termini of the domain. This later type is commonly seen in tandem repeats (Tordai et al., 1999) and mediates protein
protein or protein
carbohydrate interactions. It is found in the N-terminal domain of members of the plasminogen/hepatocyte growth factor family, in the plasma prekallikrein/coagulation factor XI family (Tordai et al., 1999), in various nematode proteins, and recently in apicomplexan MIC proteins (Brown et al., 2001). The apple domains of plasma prekallikrein are known to mediate its binding to high molecular weight kininogen (Herwald et al., 1996), and the apple domains of factor XI bind to factor XIIa, platelets, kininogen, factor IX, and heparin (Ho et al., 1998). This domain was described for the first time in MIC4 through sequence homology searches (Brecht et al., 2001). It is also found in the micronemes of two other Apicomplexa: Eimeria tenella MIC5, which contains 11 apple domains (Brown et al., 2000), and the Sarcocystis muris lectin, which contains two of these domains (Klein et al., 1998). Brecht et al. (2001) suggested that MIC4, which contains six apple domains, could be an adhesin. However, subsequent studies have shown that MIC4 does not directly bind host cells and that one of its partner proteins, MIC1, is responsible for receptor recognition (Lourenco et al., 2001, Saouros et al., 2005). More recently the fifth apple domain of MIC4 was shown to bind specifically to galactose terminating oligosaccharides (Marchant et al., 2012). The authors suggested that the proteolytic release of the fifth and sixth apple domains from the parasite surface could mimic the function of galectins involved in innate immunity, thus modulating the host immune response. The apple domain is a subfamily of the PAN (plasminogen, apple, nematode) module that has been detected in the AMA1 (Pizarro et al., 2005), a microneme protein found in all Plasmodium species and in at least six other

Toxoplasma Gondii

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Apicomplexa including T. gondii, Neospora caninum, Sarcocystis neurona, E. tenella, Babesia bovis, and Theileria. The structural characterization of the AMA1 ectodomain from P. falciparum (Bai et al., 2005), Plasmodium vivax (Pizarro et al., 2005), T. gondi (Crawford et al., 2010), N. caninum, and Babesia divergens (Tonkin et al., 2012) has revealed a three domain architecture, originally proposed based on the disulfide bonding pattern (Hodder et al., 1996). Domains I (DI) and II (DII) are structurally homologous whereas domain III (DIII) is highly divergent. The seminal study of Pizarro et al. (2005) established that DI and DII of P. vivax AMA1 adopted a PAN motif. Intriguingly, however, the DI and DII of TgAMA1, BdAMA1, and NcAMA1 are not recognized as PAN modulecontaining proteins (Crawford et al., 2010; Tonkin et al., 2012), as was identified for Pf/ PvAMA1. The membrane proximal DIII displays the most structural divergence of the three ectoplasmic AMA1 domains. In TgAMA1, NcAMA1, and PvAMA1, it adopts the structurally ultrastable cystine knot motif that probably stabilizes and orients the ectodomain. In contrast, BdAMA1 DIII is noticeably less compact, forming a more extended layer across the base of DI and DII. The DIII has been shown to function as an adhesin for Plasmodium attachment to erythrocytes (Kato et al., 2005), based on its expression on CHO cells being sufficient to bind to the Kx membrane protein on trypsin-treated erythrocytes. Erythrocyte binding activity was also conferred by a construct comprising domains I and II of Plasmodium yoelii AMA1 (Fraser et al., 2001). However, other investigations failed to demonstrate binding of PfAMA1 (shed from cultured supernatent, and encompassing most of the PfAMA1 ectodomain) to host cells (Howell et al., 2001). Of particular interest is a conserved apical hydrophobic groove in the AMA1 DI, which is surrounded by a series of polymorphic loops from domain I and by an extended

nonpolymorphic DII loop. This structural feature, conserved across the AMA1 molecules of all apicomplexans examined, is crucial for the invasion. Known to be the target for invasioninhibitory monoclonal antibodies (Pizarro et al., 2005; Coley et al., 2006, 2007; Collins et al., 2009) and a peptide identified from a phage-display library (Richard et al., 2010), this trough has been recently shown to bind the rhoptry neck protein RON2 (Tonkin et al., 2011; Vulliez-Le Normand et al., 2012), a component of a multiprotein complex that is crucial for host cell invasion (Alexander et al., 2006; Besteiro et al., 2009; Lamarque et al., 2011; Tyler and Boothroyd, 2011). These and other recent studies discerning the role of the AMA1 ectodomain during invasion are described in greater detail next. Proteomic analysis of secretory products in T. gondii revealed at least three additional proteins containing PAN/apple domains (Zhou et al., 2005). The same series of paralogous genes were identified in an in silico screen for putative secretory proteins (Chen et al., 2008). The N. caninum ortholog of one member of this family was recently identified by a monoclonal antibody that stains the micronemes, providing strong evidence of its localization to these organelles (Sohn et al., 2011). These proteins were termed NcMIC17A, NcMIC17B, and NcMIC17C, according to their order on the chromosome. NcMIC17B was the family member identified as being expressed in N. caninum, which is consistent with transcriptomic data indicating robust expression of NcMIC17B, but little or no expression of NcMIC17A or NcMIC17C. Interestingly, based on proteomic and transcriptomic data in Toxodb.org, it appears that TgMIC17C is the principally expressed member of the family in T. gondii. Whether this genus-specific expression pattern contributes to the distinct biology of N. caninum and T. gondii awaits further study. A novel PAN/apple domain-containing protein has been recently described (Gong et al.,

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2012). It is composed of 10 putative PAN/ apple domains and displays affinity for chondroitin sulfate. It localizes to the apical pole of the parasite, with partial colocalization with micronemes.

been termed MIC8-like1 (Kessler et al., 2008) or MIC8.2 (Sheiner et al., 2010). The CD of this TM protein is functionally interchangeable with the CD of MIC8 (Kessler et al., 2008; Sheiner et al., 2010).

14.5.3.1.5 The chitin-binding-like domain

14.5.3.1.6 Galectin-like domain

MIC3 and MIC8 contain in their N-terminal region a domain homologous to one found in chitin-binding proteins (“hevein domain” or “chitin-binding” motif), followed by several EGF-LDs (Garcia-Reguet et al., 2000; Meissner et al., 2002). This chitin-binding-LD (CBL) is also found in an E. tenella protein (ETH_00017540, Toxodb.org) possessing a signal peptide and two EGF-LDs. The chitinbinding domain is a well conserved 30
40 AA stretch found in plants and fungi (Wright et al., 1991). It binds specifically to N-acetyl glucosamine oligosacharides and is involved in the cross-linking of chitin subunits. One of the best-known plant lectins is the wheat germ agglutinin (WGA), a homodimeric protein. Each subunit contains four repeats of the CBL domain (Wright and Kellogg, 1996), which features eight conserved cysteine residues implicated in four disulfide bridges that are responsible for structural conformation. A conserved serine residue forms a hydrogen bond with the nonreduced end of chitin polymers and stabilizes the interaction. This serine residue is followed by an aromatic AA pocket responsible for sugar binding (Wright et al., 1991). The CBL domain has been shown to be responsible for cell
surface interaction in MIC3 (Cerede et al., 2002) and in MIC8 (O. Ce´re`de and M. Lebrun, unpublished). This domain could allow binding to a large array of cell types (Cerede et al., 2002). Similar to WGA, the binding ability of MIC3 involves aromatic amino acids (Cerede et al., 2005). The CBL domain is also found in three additional ORFs of the T. gondii genome (TgME49_086740, TgME49_044180, and TgME49_038220). TgME49_086740 has also

A GLD is present in the C-terminal part of MIC1 (Saouros et al., 2005) and in the Nterminal part of M2AP (Huynh et al., 2015). Galectins are a unique family of soluble carbohydrate binding proteins (Barondes et al., 1994). The carbohydrate recognition involves critical AAs in the central region of the sixstranded beta-sheet and comprises an array of hydrophilic residues and a key aromatic sidechain (Seetharaman et al., 1998; Umemoto et al., 2003). These positions are not conserved in the equivalent locations within MIC1 and M2AP. Instead, these proteins share a hydrophobic environment in this region, reminiscent of protein
protein interface (Saouros et al., 2005; Huynh et al., 2015). Carbohydrate binding experiments with MIC1 showed no detectable binding except for 100 mM lactose which substantiates an altered binding motif (Saouros et al., 2005). The GLD of MIC1 is involved in the interaction with MIC6, assisting the correct folding of MIC6 and stabilizing the C-terminal fragment encompassing the third EGF and the acidic region (Saouros et al., 2005). This allows the MIC1/4/6 complex to exit the ER (Reiss et al., 2001). Similarly, the M2AP GLD binds to MIC2 via hydrophobic residues in the carbohydrate-binding region. Together, these studies provide two welldocumented examples of Toxoplasma repurposing a GLD for protein
protein interactions underlying assemble of MIC protein complexes. 14.5.3.1.7 Microneme adhesive repeat domain

The N-terminal region of MIC1 was initially thought to contain degenerate TSR domains (Fourmaux et al., 1996a). However, the crystal

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structure of this region revealed that it consists of a new fold termed the microneme adhesive repeat (MAR) (Blumenschein et al., 2007). MIC1 has two MAR domains that bind specifically to sialylated oligosaccharides in a novelbinding mode involving a key threonine in the binding pocket rather than conventional basic amino acids (arginine or lysine). Additional studies revealed that this binding site is found in several parasite, bacterial, and viral proteins (Garnett et al., 2009). The tandem MAR domains bind to branched sialylated oligosaccharides. Active site mutation of either domain abolishes binding to host cells, confirming the importance of its multivalent binding interaction. The significance of parasite recognizing sialic acids as receptors is underscored by the B30%
90% diminished invasion of sialic acid deficient host cells (Monteiro et al., 1998; Blumenschein et al., 2007) and the B50% reduced invasion by MIC1 KO parasites (Cerede et al., 2005). Three additional MAR containing proteins, initially termed MCP2, MCP3, and MCP4, are encoded in the Toxoplasma genome (Friedrich et al., 2010a). MCP2, now termed MIC13, is poorly expressed in tachyzoites but is abundantly expressed in bradyzoites and sporozoites (Fritz et al., 2012a). Like MIC1, MIC13 binds sialylated oligosaccharides, but with some unique preferences that suggest a role for parasite binding to sialylated receptors in the gut. Interestingly, a similar situation appears to exist for the Eimeria MAR protein EtMIC4, which preferentially recognizes the non
Nglycolylated sialylated oligosaccharides that are expressed in the gut of its chicken host (Lai et al., 2011). Consistent with its low or null expression in tachyzoites, a recent study showed that MIC13 does not contribute to acute infection (Ye et al., 2019). However, the same study suggested that targeted deletion of MIC13 resulted in fewer bradyzoites per cysts in vitro. Whether this indicates an unusual role for MIC13 in parasite replication or is a

consequence of MIC13 contributing to bradyzoite invasion awaits further study, as does defining its role in bradyzoite and sporozoite initiation of infection in the gut. MCP3 and MCP4 do not have the key active site threonine, a finding that is consistent with the absence of detectable host cell binding activity (Friedrich et al., 2010a). The subcellular location of MCP3 has not been determined, but it appears to be moderately expressed in tachyzoites and highly expressed in bradyzoites. MCP4 is mainly expressed in bradyzoites where it is a component of the cyst lumen and wall where it might play a structural role (Buchholz et al., 2011). 14.5.3.2 Other MICs Although many MICs contain structural motifs suggestive of a role in protein
protein interactions and parasite attachment, a growing subset of microneme proteins (MICs) do not possess obvious adhesive features, suggesting they may play alternative functions (MIC5, MIC10, MIC11). MIC5 encodes a previously identified immunodominant antigen called H4 (Johnson and Illana, 1991; Brydges et al., 2000). Although MIC5 possesses a consensus sequence characteristic of members of the parvulin family of peptidyl
prolyl cis
trans isomerases (PPIases), no PPIase activity was detected with recombinant MIC5 (Brydges et al., 2006). Targeted deletion of MIC5 resulted in hyperproteolysis of MICs on the parasite surface, suggesting that MIC5 regulates protease activity. Recent NMR analysis of MIC5 revealed that it mimics the structure of the prodomain of subtilisin proteases (Saouros et al., 2012). Like many proteases, subtilisins contain an N-terminal prodomain that suppresses activity by occluding the active site. Consistent with it mimicking this feature, MIC5 inhibits the activity of the micronemal protease SUB1 via a flexible C-terminal peptide. The flexible peptide contains conserved amino acids that are thought to mediate interactions with the

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SUB1 active site. Together, the findings suggest that MIC5 regulates SUB1 activity on the parasite surface to preclude over processing of specific substrates or inappropriate processing of other surface or secretory proteins. MIC10 is an 18 kDa protein lacking a putative TM domain. Although MIC10 is secreted during invasion it does not remain associated with the parasite membrane after microneme discharge like other MICs and does not bind to host cells (Hoff et al., 2001). The role of MIC10 during infection remains to be determined. MIC11 is also a small soluble protein of unknown function that displays a pronounced charge asymmetry with an acidic N-terminal region (pI 4.3) and a basic C-terminal region (pI 8.2) (Harper et al., 2004b), reminiscent of the rhoptry protein ROP1 (Ossorio et al., 1992). It is hypothesized that ROP1 and MIC11 proteins act as molecular organizers within rhoptries and micronemes, respectively, using their charge asymmetry to form an organized scaffold based on homotypic ionic interactions (Hoff et al., 2001). Perforin-like protein 1, which was discovered in a proteomics screen of secretory products (Zhou et al., 2005), contains a membrane attack complex (MAC)/perforin (MACPF) domain implicated in membrane disruption. The MACPF domain is the main functional entity allowing the immune effector proteins perforin and terminal components of the MAC to form large oligomeric ring-like pores in target membranes. Consistent with a similar membranolytic function, PLP1 expression is necessary for the formation of large membrane lesions in the PV membrane during induced parasite egress (Kafsack et al., 2009). Further support for a role in egress came from showing that PLP1 deficient parasites show a significant delay in escape from the cell, with some parasites failing to leave host cells altogether (Kafsack et al., 2009). PLP1 null parasites are profoundly attenuated in a mouse model of acute toxoplasmosis, revealing this secreted

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microneme protein as a key virulence factor (Kafsack et al., 2009). These findings also highlight the importance of rapid egress for progression of lethal infection by a type I parasite strain, and they established that micronemes contribute to parasite egress in addition to invasion. T. gondii GAMA was identified in a bioinformatics screen for apical secretory proteins that are conserved between T. gondii and Plasmodium (Huynh and Carruthers, 2016). Initial studies of PfGAMA using recombinant protein and neutralizing antibodies suggested a role in merozoite attachment to erythrocytes (Hinds et al., 2009). Consistent with such a role, targeted deletion of Toxoplasma GAMA resulted in reduced parasite attachment and invasion without affecting gliding motility or egress. Since GAMA lacks recognizable adhesive domains, precisely how it contributes to attachment remains unknown. Nevertheless, since GAMA is found in all apicomplexans except Cryptosporidium, these findings suggest a conserved, albeit nonessential, role for GAMA in host cell recognition by many apicomplexan parasites. If GAMA directly participates in parasite attachment then its cognate receptor is probably also expressed on the surface of many cell types including nucleated cells and erythrocytes. Claudin-like apicomplexan microneme protein (CLAMP) was initially identified in a genome-wide CRISPR-Cas9 screen as an indispensible conserved apicomplexan protein (Sidik et al., 2016). CLAMP resembles the tight junction protein claudin with 4 TM domains and a proline rich cytosolic C-terminal domain. CLAMP is secreted from the micronemes onto the parasite surface during apical attachment to host cells and it redistributes to the posterior end along with the MJ during parasite entry. Tachyzoites conditionally lacking CLAMP show normal gliding motility and attachment to host cells, but they fail to discharge rhoptries, thus resulting in a marked invasion

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defect. Blood stage P. falciparum parasites conditionally lacking PfCLAMP are rapidly lost from in vitro cultures. Collectively, these studies indicate a crucial role for CLAMP in cell invasion by apicomplexan parasites at a step upstream of rhoptry secretion. At least three proteases are found in micronemes. SUB1 is a subtilisin-type serine protease that is recognized by an anti
Plasmodium SUB1 antibody, suggesting that the two subtilases share areas of conserved antigenic structure (Miller et al., 2001). SUB1 trims other MIC proteins on the parasite surface during invasion and this appears to modulate their adhesive activity. Another serine protease belonging to the rhomboid family has been identified in micronemes, named ROM1 (Brossier et al., 2005). Conditional disruption of ROM1 resulted in moderately reduced invasion and intracellular replication (Brossier et al., 2008), thus conferring a parasite fitness defect. TLN4 is a metalloproteinase of the insulinase family that was recently identified in the micronemes (Laliberte and Carruthers, 2011). Although the substrates for ROM1 and TLN4 are not known, these proteases are likely to play a role in MIC processing before or after secretion of the organelles. 14.5.3.3 MICs assemble in complexes Another characteristic feature of Toxoplasma MICs is to assemble into protein complexes that work in concert. The first demonstration of the occurrence of such a complex was obtained by MIC6 gene deletion (Reiss et al., 2001). Indeed, the latter led to MIC1 and MIC4 missorting to the PV instead of microneme. In addition, artificial expression of MIC6 at the parasite plasma membrane induced the same localization for MIC1 and MIC4. Coimmunoprecipitation assay confirmed the association of the 3 proteins into a stable complex (Reiss et al., 2001). Then, the MIC2/M2AP complex was also found (Rabenau et al., 2001). In this case, a 450 kDa hexameric complex was found to form in the ER, to be

targeted in microneme and to persist on the surface during invasion (Rabenau et al., 2001; Jewett and Sibley, 2004). Last, the existence of a MIC3/MIC8 complex has been suggested by the expression of MIC3 on the surface of the parasite expressing artificially a GPI anchored version of MIC8 (Meissner et al., 2002). The density of MICs in complexes may serve two functions. They are likely to be important for proper trafficking in the secretory pathway. A role in quality control has been shown for MIC1 (Reiss et al., 2001; Saouros et al., 2005). Indeed, soluble MIC1 and MIC4 require interaction with the membrane-bound protein MIC6 to ensure proper targeting to the micronemes. As mentioned previously, the galectin domain of MIC1 binds MIC6 and ensures proper folding of MIC6 to pass the ER quality control. Then, MIC6 ensures targeting of the complex to micronemes. A similar role has been postulated for the soluble protein M2AP (Huynh et al., 2003, 2004). It stabilizes the complex formed with MIC2 and influences the correct targeting to micronemes. In the absence of M2AP, MIC2 appears to trimerize correctly but does not target efficiently to the micronemes and as a consequence undergoes degradation. Interestingly though, unlike the other confirmed MIC complexes MIC3 and MIC8 are not interdependent for microneme targeting since targeted disruption of either protein does not alter the localization of the other protein (Cerede et al., 2005; Kessler et al., 2008). A second function of MICs complexes may be in cooperative binding to enhance cell recognition and facilitate cell entry. Indeed, oligomerization is often critical for enhancing affinity, and, in addition, to cluster receptors and others components within complexes to induce signaling. Multimerization of MIC2 has been shown to increase the number of interactions with host cell receptors, thereby forming a multivalent adhesive protein (Harper et al., 2004a). MIC3 is a homodimer and this oligomerization is required for host cell binding

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(Cerede et al., 2002). The MIC3 homodimer contains 2 CBL domains and the TM protein MIC8 possesses one, the complex being therefore expressing 3 CBL domains. CBL domain oligomerization has been shown to increase its avidity (Cerede et al., 2002). CBL domain oligomerization in the MIC3/MIC8 complex would therefore be used to increase the affinity with the host cell surface. MIC1 has affinity for host cell surfaces when dimerized (Fourmaux et al., 1996a) and evidence of MIC1 as a trimer has also been reported (Marchant et al., 2012). Mature MIC6 has two binding sites for MIC1 and each copy of MIC1 appears to be associated with one copy of MIC4 (Sawmynaden et al., 2008; Marchant et al., 2012). Accordingly, each molecule of MIC6 can theoretically support the display of six copies of MIC1 and six copies of MIC4 on the parasite surface during invasion. To summarize, all microneme proteins described so far as showing affinity for host cells work as oligomers and this increased valency likely supports a robust interface for apical attachment to host cells prior to and during cell invasion. 14.5.3.4 Cytosolic domain of transmembrane MICs The CDs of several TM MICs is highly conserved in Apicomplexa. Replacing the CD of TRAP with that of TgMIC2 in P. berghei sporozoites resulted in normal motility, invasive capability, and infectivity in vivo (Kappe et al., 1999), underscoring the functional conservation of the invasive mechanism in Apicomplexa. Although this domain was initially reported to bind aldolase as an adaptor protein connected to actin and the parasite glideosome (Kappe et al., 1999; Jewett and Sibley, 2003; Sheiner et al., 2010), more recently it was shown that aldolase does not function in this role (Shen and Sibley, 2014). Subsequently, a conserved Armadillo repeat protein called the glideosomeassociated connector (GAC) was shown to link the CD of MIC2 with actin (Jacot et al., 2016)

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(see Chapter 16: The Toxoplasma Cytoskeleton: structures, proteins, and processes). An interaction between GAC and MIC2 was established by showing that accumulation of MIC2 on the parasite surface in ROM4 deficient parasites resulted in the deposition of MIC2 and GAC in membranous trails left behind by gliding parasites. An interaction was also directly established using recombinant proteins. Conditional ablation of GAC severely impaired gliding motility, invasion, and egress. Although it remains to be determined if GAC interacts with other TM MICs, the striking phenotypes and its conservation among the Apicomplexa suggest that GAC is an integral and essential connector between TM invasion proteins and the glideosome.

14.5.4 Microneme secretion Microneme secretion is a regulated by an intricate signaling pathway involving fluxes in cyclic nucleotides, phospholipids, and calcium within the parasite (Dubois and Soldati-Favre, 2019). Chapter 12, Calcium storage and homeostasis in Toxoplasma gondii, and Chapter 13, Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii, provide in-depth coverage of calcium and signaling networks, respectively. Here, we will briefly discuss studies related to the docking and fusion of micronemes with the apical membrane. Precisely, where and how micronemes discharge their contents remain poorly understood. Upon stimulation of microneme secretion with the calcium ionophore A23187, possible fusion of a microneme with the socalled anterior vesicle, an enigmatic 50 nm spherical structure immediately underlies the parasite apical tip (Carruthers and Sibley, 1999). This together with multiple reports of microneme proteins occupying the apical surface upon parasite attachment to host cells support a model of microneme discharge via

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fusion at the extreme apical tip of the parasite. After A23187 induced discharge, several small lucent vesicles were observed along the two microtubules that hang from the apical tip through the conoid (Carruthers and Sibley, 1999). Although the identity of these lucent vesicles remains to be determined, they could indicate partial reabsorption of empty micronemes after discharge. Alternatively, fusion with the apical tip could incorporate the microneme membrane lipids into the parasite plasma membrane, thus increasing membrane surface area at the apical end. Such a scheme has been incorporated into a “fountain-flow” model wherein delivery of new lipids at the anterior end drives rearward flow of lipids and surface proteins for subsequent internalization via endocytosis (Gras et al., 2019). Upon identifying a role for phosphatidic acid (PA) in microneme discharge, Bullen et al. (2016) searched for parasite proteins containing a pleckstrin homology (PH) domain as candidates for binding PA. Among seven PH domain proteins in T. gondii, only one protein termed acylated PH (APH) was also conserved in other apicomplexans including Plasmodium. APH is associated with the cytosolic face of micronemes via lipidation with myristate and palmitate. Lipid binding experiment showed that APH preferentially recognizes PA and that is PH domain is sufficient to confer binding. Conditional ablation of APH resulted in severe attenuation of microneme secretion together with defects in plaque formation, invasion, and egress. Together, these findings suggest a role for APH in binding PA to bring the micronemes into close apposition with the apical membrane for subsequent fusion and discharge. Mutational profiling has identified DOC2.1, a protein conserved in Apicomplexa and involved in calcium-dependent secretion of micronemes (Farrell et al., 2012). DOC2.1 contains a tandem C2 domain that is known to bind Ca21 and to be involved in Ca21-

mediated exocytosis (e.g., neurotransmitter release), facilitating membrane fusion of secretory vesicles with the plasma membrane (Friedrich et al., 2010b; Groffen et al., 2010). Thus DOC2.1 is proposed to facilitate Ca21dependent fusion of micronemes with the apical membrane, possible working in concert with a yet-to-be SNARE complex.

14.5.5 Postsecretory traffic of MICs 14.5.5.1 Parasite surface exposition and posterior capping of MICs As the parasite penetrates the host cell, most MICs are excluded from entering the vacuole and are progressively capped behind the MJ, remaining confined to the portion of the parasite that still protrudes out of the host cell (Carruthers and Sibley, 1997; Carruthers et al., 1999; Garcia-Reguet et al., 2000). As a consequence of MIC protein capping, binding to a fixed substrate would lead to forward locomotion, and binding to cell-surface receptors would lead to penetration into the cell. The backward capping of T. gondii MICs is an actin-dependent process, implying that either actin polymerization or actin filaments are required (Jensen and Edgar, 1976; Ryning and Remington, 1978; Miller et al., 1979; Russell and Sinden, 1981). The current view is that the actomyosin motor, located beneath the plasma membrane of the parasite, interacts indirectly with the cytoplasmic tail of the TM MICs including MIC2 and AMA1 and translocates them toward the posterior end of the parasite (Sibley, 2003; Keeley and Soldati, 2004; Soldati and Meissner, 2004; Sheiner et al., 2010). Thus as an immobilized myosin walks along the actin filament, the MICs
cell receptor complexes are capped backwards and the parasite propels itself on the substrate or into the host cell. Substantial evidence of this capping model has been obtained by reverse genetic

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approaches in Plasmodium. Deletion of the TRAP C-terminus abolishes gliding motility and cell invasion (Kappe et al., 1999). Directional invasion and helical gliding motility are sustained by connection of the actomyosin motor with the IMC. This additional membrane layer is supported by microtubules that provide the pitch for the spiral gliding action and serves as the “tramline” for the capping reaction. The myosin is indeed anchored in the IMC of the pellicle and generates the mechanochemical force for translocating the actin filaments (Gaskins et al., 2004). The different players of the invasion apparatus are called glideosome and described in Chapter 16, The Toxoplasma Cytoskeleton: structures, proteins, and processes. 14.5.5.2 Proteolytic cleavages during invasion MIC proteins are extensively processed on the parasite surface. These postexocytosis processing events likely regulate adhesion and facilitate dissociation of the parasite
host interaction at the end of the invasion process. Postexocytosis proteolysis is conferred by several distinct protease activities that were initially termed microneme protein protease 1, 2, and 3 (MMP1, MMP2, and MMP3) (Carruthers et al., 2000; Zhou et al., 2004). As detailed next, ROM4 and ROM5 are responsible for MMP1 activity, which sheds TM MIC proteins from the parasite surface by intramembrane cleavage near the C-terminus. SUB1 conveys MMP2 and possibly MMP3 activity on the parasite surface, resulting in the trimming of terminal peptides from MICs. The postexocytosis C-terminal cleavage of MIC2, MIC6, MIC8, MIC12, and AMA1 by MMP1 sheds these TM MICs from the parasite surface (Carruthers et al., 2000; Donahue et al., 2000; Reiss et al., 2001; Meissner et al., 2002; Opitz et al., 2002). Genetic and biochemical evidence shows that C-terminal cleavage occurs within the TM domain by regulated

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intramembrane proteolysis (Opitz et al., 2002; Zhou et al., 2004; Howell et al., 2005). The intramembrane cleavage site is conserved between TM MICs of Toxoplasma and other Apicomplexa (Dowse et al., 2005). These cleavage sites resemble the recognition sequence for rhomboid-like proteases, whose activity is sensitive to the serine protease inhibitor 3,4dichloroisocoumarin (DIC) (Urban and Freeman, 2003). DIC inhibits intramembrane cleavage of TM MICs (Howell et al., 2005) and reduces host cell invasion by T. gondii (Conseil et al., 1999), consistent with the participation of a rhomboid-like protease. Rhomboids are multipass membrane serine proteases that cleave within the TM of their substrate. First evidence that a parasite rhomboid may cleave TM MICs came from a study by Urban and Freeman (2003) showing that Rhomboid-1 from Drosophila and RHBDL2 from humans cleave chimeric proteins containing the TM domain of MIC2, MIC6, or MIC12 (Urban and Freeman, 2003). Rhomboid-like genes are present in the genome of all apicomplexan parasites currently sequenced. T. gondii contains six rhomboid genes (ROM1-6) (Dowse and Soldati, 2005). ROM2 and ROM3 are mainly expressed in sporozoites, ruling out their role as MMP1 activities in all stages of the parasite (Brossier et al., 2005). MMP1 activity is predicted to be constitutive on the parasite surface (Opitz et al., 2002). ROM1 localizes to micronemes, and ROM4 and ROM5 are expressed at the plasma membrane (Brossier et al., 2005; Dowse et al., 2005). Whereas ROM4 is evenly distributed along the parasite surface, ROM5 localizes at the periphery of the parasite with a prominent accumulation at the posterior pole of the parasite (Brossier et al., 2005; Rugarabamu et al., 2015). Definitive evidence of ROM4’s involvement in intramembrane cleavage of MIC2 came from conditional and complete ablation of ROM4 expression (Buguliskis et al., 2010; Shen et al., 2014; Rugarabamu et al., 2015). ROM4

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deficient parasites fail to shed MIC2 into the culture supernatant, resulting in the accumulation of MIC2 on the parasite surface. Mutant parasites showed increased adhesion to host cells, but reduced invasion thereof and defective gliding motility, supporting the model that intramembrane shedding of MIC2 is important, but not essential, for parasite active entry into host cells. Shedding of AMA1 was markedly reduced, but not eliminated in ROM4 deficient parasites (Rugarabamu et al., 2015). Whereas disruption of ROM5 alone had no effect on shedding of MIC2 or AMA1, deletion of ROM4 and ROM5 eliminated residual shedding of AMA1 and other AMA1 paralogs (Rugarabamu et al., 2015). Despite being in the micronemes, ROM1 plays no role in shedding of any of the MICs examined. Parasites lacking ROM4 and ROM5 exhibit decreased invasion because they stick to host cells via their posterior end and have difficulty reorienting for apically directed invasion. However, those that are able to reorient appear to invade normally. Altogether, these findings indicate MMP1 activity is mainly conferred by ROM4 and supplemented by ROM5 for AMA1 and AMA1 paralogs. The work further suggests that intramembrane cleavage of TM MICs is not necessary to complete the invasion process including pinching off of the PVM, which presumably results in vesicular releases of uncleaved TM MICs in ROM4/5 double KO parasites. Thus the main role of intramembrane cleavage is to promote an apical to posterior gradient of adhesive MICs on the parasite surface, thereby enhancing apical attachment and avoiding detrimental posterior accumulation of such proteins. Intramembrane cleavage of AMA1 was initially suggested to play a role in commencing parasite replication after invasion based on overexpression of a catalytically inactive mutant of ROM4 (Santos et al., 2011). However, this conclusion has been disputed by subsequent work with ROM KOs described

above together with the absence of a replication defect in parasites lacking AMA1 (Bargieri et al., 2013) or those expressing a mutant of AMA1 that is refractory to intramembrane cleavage (Parussini et al., 2012). Thus intramembrane cleavage of AMA1 by ROM4 is not necessary to begin parasite replication, as initially reported. It remains unclear how expression of a catalytically inactive mutant of ROM4 impairs commencement of parasite cell division. MMP2 trims off a short N-terminal extension upstream of the A-domain from MIC2 and it cleaves the M2AP C-terminal domain at several sites (Zhou et al., 2004). This protease also processes MIC4 at its N-terminus and Cterminus, releasing the last two apple domains (Brecht et al., 2001; Zhou et al., 2004). MMP2 activity is markedly enhanced by treatment with cytD, which blocks the capping of MICs toward the posterior end of the parasite. This finding was interpreted as evidence that MMP2 is a resident apical surface protein or microneme protein since cytD restricts MICs to the apical surface, thereby facilitating processing (Carruthers et al., 2000; Zhou et al., 2004). Based on partial inhibition by chymostatin and PMSF and nearly complete inhibition by ALLN and ALLM, MMP2 was predicted to be a serine or cysteine protease. The likely identity of MMP2 was revealed upon finding that SUB1 deficient parasites failed to process any of the MMP2 substrates including MIC2, M2AP, and MIC4 (Lagal et al., 2010). As mentioned earlier, SUB1 is a subtilisin-like protease that is secreted from the micronemes onto the parasite apical surface during invasion, placing it in the correct location for MMP2 activity. SUB1 activity is also sensitive to PMSF (Miller et al., 2001), which is consistent with the inhibitor profile of MMP2. Interestingly, ablation of SUB1 also extinguished MMP3 activity, which was identified previously by its ability to cleave the C-terminal most peptide of M2AP in an ALLN

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insensitive manner (Zhou et al., 2004). SUB1 cleavage of the C-terminal most M2AP peptide might occur rapidly upon secretion of both the protease and substrate, thus potentially explaining its refractivity to ALLN. The Nterminal processing of MIC2 now attributed to SUB1 has been shown to facilitate MIC2 interaction with ICAM-1, presumably by optimal exposure of the MIC2 A-domain (Barragan et al., 2005). SUB1 deficient parasites are moderately deficient in gliding motility and parasite attachment to host cells, consistent with a general role of surface trimming in the activation of MIC adhesins. The lack of surface trimming and activation of MIC adhesins also results in partial attenuation of virulence in mice, confirming the in vivo significance of this form of proteolysis (Lagal et al., 2010).

14.5.6 Why does Toxoplasma gondii exhibit this patchwork of MICs? The vast MIC repertoire of T. gondii MICs may be correlated with the broad host cell specificity in vitro and the spreading of infection in all organs in toxoplasmosis, contrasting with the high cell and organ specificity found in Plasmodium. Apicomplexa show variable cell specificity, particularly at different stages of infection: specificity may be related to the MIC repertoire, quite different from one genus to the other, and depend upon the stage in the life cycle. MIC gene deletion in T. gondii has shown that many MICs do not play critical role in vitro (M2AP, MIC1, MIC3, MIC4, MIC5, MIC6, SUB1, ROM1, PLP1, SPATR, GAMA, and TLN4). However, in vitro findings using a limited number of often nonphysiologically relevant cell types might not reflect the biological roles of MICs in vivo. Indeed, MIC3 gene disruption does not modify fibroblast invasion but induces a death delay in mice (Cerede et al., 2005). Consistent with this, MIC3 could

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be essential for invading specific cell types other than those that have been tested thus far in vitro. This hypothesis is supported by the demonstration that the binding ability of MIC3 to host cells is crucial for parasite virulence in vivo (Cerede et al., 2005). This suggests also that the importance of MICs might be better evaluated in vivo than in vitro. Indeed, motility, adhesion, and invasion, which are functions postulated for MIC proteins, are multifactorial phenomena, the expression of which is likely to differ in vivo from what occurs in the static environment found in a culture dish. Similar to rhoptry proteins (see next and Chapter 15: Endomembrane trafficking pathways in Toxoplasma) the role played by MICs (such as MIC3 and MIC6) in host cell signaling might be only highlighted in vivo. Deletion of the MIC1 gene results in defective targeting of both MIC4 and MIC6 in micronemes (Reiss et al., 2001) and mic1KO parasites are 50% impaired in invasion (Cerede et al., 2005). This defect in invasion can thus be assigned to the absence of the MIC1/4/6 complex, without further precision. As previously mentioned, only MIC1 binds host cell in vitro (Saouros et al., 2005). Therefore MIC6 anchors the two other proteins and interacts with the underlying motor, whereas MIC1 would establish specific interactions with host cell receptors necessary for host cell invasion. The disruption of M2AP gives also a partial phenotype. M2AP KO parasites were .80% impaired in host cell entry and show delayed death in mice (Huynh et al., 2003; Harper et al., 2006). In these parasites, MIC2 partially accumulates in the parasite ER/Golgi apparatus and is poorly secreted. This invasion defect is likely due to defective expression of MIC2/ M2AP complex. The importance of MIC protein diversity has been further stressed by simultaneous disruption of MICs (Cerede et al., 2005). Indeed, a spectacular decrease in virulence in vivo was

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observed by simultaneous disruption of the MIC1 and MIC3 genes (which corresponds in fact to a MIC1/3/4/6 functional KO). This result demonstrates that MICs have a synergistic effect on infection in vivo. Whereas individual deletion of several MICs results in moderate phenotypes, four MICs appear to be particularly important for parasite invasion. Each of these proteins likely plays a distinct role in entry. 14.5.6.1 MIC2: role in attachment and motility Conditional or complete ablation of MIC2 resulted in .80% impaired in invasion because of a defect in attachment (Huynh and Carruthers, 2006; Gras et al., 2017). Whether MIC2 is involved in the MJ formation has not been established, it has been shown to occupy the interface between the parasite and host membranes during invasion (Carruthers et al., 1999). In Plasmodium, the two adhesive motifs of TRAP have been suggested to be involved in internalization. Indeed, independent or simultaneous mutation in Plasmodium TRAP Adomain and TSP does not alter sporozoite motility but specifically decreases, or abolishes, in the case of simultaneous mutations, host cell invasion in vitro and in vivo (Matuschewski et al., 2002). Therefore gliding motility and host cell invasion likely involve distinct extracellular associations, perhaps with invasion requiring stronger binding to host receptors than what gliding motility needs. Nevertheless, despite the substantial invasion defect, MIC2 deficient parasites are viable and capable of entering cells, indicating MIC2 independent mechanisms of cell invasion. 14.5.6.2 MIC8 and claudin-like apicomplexan microneme protein: potential role in triggering rhoptry secretion Conditional ablation of MIC8 revealed key role in tachyzoite invasion (Kessler et al., 2008). Although MIC8 deficient parasites showed

normal egress, gliding motility, apical attachment, they failed to form a MJ for invasion of host cells. The arrest in invasion appears to correspond to a block in rhoptry secretion characterized by the absence of RON secretion at the MJ and ROP secretion into host cells in the form of evacuoles. The findings point toward a role for MIC8 in the assembly of the MJ and/or rhoptry discharge. The TM microneme protein CLAMP appears to play a similar role based on observing in normal gliding motility and apical attachment in CLAMP deficient parasites but a failure to secrete rhoptries, resulting in a severe defect in cell invasion. Whether MIC8 and/or CLAMP play structural or signaling roles remains to be defined. 14.5.6.3 AMA1 and AMA1 homologs: role in moving junction formation Like MIC2 and CLAMP, AMA1 is an integral membrane protein widely conserved in apicomplexan parasites. Considerable evidence points toward a role of AMA1 in invasion. The first demonstration of such a role came from the inhibitory effects of anti
AMA1 antibodies on invasion of P. knowlesi into erythrocytes (Thomas et al., 1984), then confirmed in other apicomplexan parasites with anti
Toxoplasma AMA1 antibodies (Hehl et al., 2000) or anti
Babesia AMA1 (Gaffar et al., 2004). Finally, anti
AMA1 antibodies inhibit sporozoite invasion, suggesting that the protein is also involved during invasion of hepatocytes (Silvie et al., 2004). AMA1 was proposed to be involved in the formation of a tight binding interface between merozoite and erythrocyte surfaces (Mitchell et al., 2004). Indeed, the initial random surface attachment of merozoites to red blood cells is not affected by the presence of inhibitory antibodies, but the normal apical reorientation of merozoites does not occur and the close junctional contact (6 nm) is absent. In this study the existence of membranous blebs inside some red blood cells suggested that some transient apical contacts

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occur in presence of anti-AMA1 and are sufficient to induce the secretion of the rhoptries, although insufficient to create the prolonged intimate contact between the red blood cell membrane and merozoite apex, which is required to allow completion of invasion. Consistent with this, Mital et al. (2005) generated a conditional knockdown of T. gondii AMA1 and showed that AMA1 is not involved in gliding motility, or in the initial step of attachment, or in microneme release but fails to attach intimately to host cells. Altogether, these data suggested a role for AMA1 in building the MJ. This has been then supported by recent studies, who showed that secreted AMA1 is associated at the parasite surface with a complex of secreted rhoptries neck proteins that relocalize at the MJ (Alexander et al., 2005; Besteiro et al., 2009; Lamarque et al., 2011; Tyler and Boothroyd, 2011). During invasion, AMA1 displays a surface localization with a high steady-state level (Howell et al., 2005). AMA-1 is present at the MJ but the majority of AMA1 is clear on both sides of this adhesion zone. But in knock-down parasites expressing low level of AMA1, this protein precisely colocalizes with RON4 at the MJ (Alexander et al., 2005). These studies suggest a model in which AMA1, with the cooperation of RONs, is involved in the formation of the MJ, being a major player in invasion (see more details in Section 14.6.4.3.1). Conditional ablation of Toxoplasma AMA1 reduced expression to undetectable levels of AMA1 and resulted in .80% impaired in invasion (Mital et al., 2005), showing a key role for AMA1 during invasion, but suggesting also compensatory mechanisms in the Toxoplasma. An universal and essential role of the AMA1-RON complex in MJ formation was then disputed by Giovannini et al. (2011), who showed that when AMA1 was reduced to undetectable level in P. berghei sporozoites, the entry of hepatocytes is not affected, while the parasites fail to invade erythrocytes. This

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illustrates that the invasion machinery components may be different at different stages of parasite life cycle. In contrast to PbAMA1, PbRON4 is required for sporozoite invasion of hepatocytes (Giovannini et al., 2011), which implied that AMA1 works independently of the RON proteins at the hepatic stage. They also reexamined the conditional Toxoplasma AMA1 and showed that (1) the ring staining of RON4 at the MJ appears normal in absence of any detectable AMA1; (2) in the absence of surface AMA1, tachyzoites failed to adhere throughout their length and instead bound only via their anterior portion; and (3) finally, the parasites invade at the same speed as wild-type parasite. The authors proposed that the AMA1-RON complex does not fulfill the force-transducing role of the MJ, and moreover that AMA1 is not involved in MJ formation per se but contributes to an independent step before MJ formation that helps the parasite to bind host cell surfaces through its entire length. More recently, a complete KO of Toxoplasma AMA1 was obtained, confirming a role for AMA1 in intimate attachment to host cells and for building the MJ (Lamarque et al., 2014). Interestingly, AMA1 null parasites showed upregulation of an orthologous AMA termed AMA2, present at the MJ, suggesting a compensatory mechanism. Double deletion of AMA1 and AMA2 further reduced, but still did not completely eliminate, invasion. Analysis of the double mutant showed upregulation of a RON2 ortholog, RON2L1, together with increased expression of yet another AMA ortholog, AMA4, with AMA4 and RON2L1 forming an additional complex (Parker et al., 2016). Collectively, the apparent requirement for elaborate compensation in the absence of AMA1 strongly supports a key role for AMA1 in tight binding of the parasite to host cells during formation of the MJ. In summary, T. gondii probably displays a patchwork of MICs ensure efficient invasion as

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an essential step in its life cycle. It achieves this by expressing accessory MICs that augment adhesion together with other MICs that play more of a central role in invasion at the steps of parasite attachment (MIC2), secretion of rhoptry proteins (MIC8 and CLAMP), and building of the MJ (AMA1).

channels (Lemgruber et al., 2011). Altogether, these features illustrate the complex organization of the rhoptry, which requires sophisticated processes of biogenesis coupled to rhoptry proteins targeting toward different subcompartments or subdomains.

14.6 Rhoptries

T. gondii possesses a highly polarized secretory system, which assembles de novo micronemes, rhoptries, and DGs. Rhoptries are formed during daughter cell assembly by budding of vesicles containing newly synthesized ROP and RON proteins from the ELC. The biogenesis of rhoptries involves the correct synchronization of cell division, rhoptry protein synthesis and packaging. Immature rhoptries (or prerhoptries) are spherical organelles located above the Golgi. Just prior to cytokinesis prerhoptries elongate to form mature rhoptries that eventually localize to the parasite apex (see Chapter 2: The ultrastructure of Toxoplasma gondii). Both rhoptries and prerhoptries are acidic compartments and were the only acidified organelles detected in the parasite in one study (Shaw et al., 1998), with the prerhoptries being more acidic than the mature compartment. Of note, during maturation of rhoptries, many ROPs/RONs are subjected to proteolytic processing. Early studies identified motifs within ROP proteins that are necessary for their trafficking to rhoptries (Hoppe and Joiner, 2000; Ngo et al., 2003). The authors proposed a mechanism involving a direct binding of adaptins with the tyrosine-based (YXXΦ) or dileucinebased (LL) motifs contained in the C-terminal end of ROP2 family proteins. These studies suggested that rhoptry targeting occurs along the endocytic pathway and is mediated by adaptins. This model of sorting was then questioned upon solving the structure of the ROP2 protein (Labesse et al., 2009; Qiu et al., 2009), which

14.6.1 Biogenesis of rhoptries— clustering and tethering to the apical end 14.6.1.1 Rhoptry: a complex organelle with subcompartments Rhoptries are present at a multiplicity of 6
12 per cell in T. gondii, while Plasmodium zoites contain only two. Rhoptries are clubshaped organelles with a bulbous base and an extended duct. They are grouped together at the apical end of the parasite but only one or two access the internal part of the conoid at the same time. The neck appears uniformly electron dense while the bulb may show a less homogenous electron-dense appearance (see Chapter 2: The ultrastructure of Toxoplasma gondii). There is a clear segregation of luminal rhoptry proteins between the neck and the bulb (Roger et al., 1988), although no membrane delimits these subcompartments. ROP are those proteins located in the bulb and RON the proteins associated with the neck of the rhoptry. A third subcompartment separating the rhoptry bulb and neck has been observed using freeze-fracture and quick-freeze/freezefracture/deep-etching (Lemgruber et al., 2011). This region of intermediate electron density is also defined by specific rhoptry proteins exposed on its cytoplasmic surface (Mueller et al., 2016). Freeze-fracture studies of the bulb surface also detected intramembranous particles (IMPs) organized in parallel rows around the bulb, along with pore-like structures, possibly reflecting the existence of pores or ion

14.6.1.2 Reshaping of the endosomal pathway for rhoptry biogenesis

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ruled out the previous structural model (Sinai and Joiner, 2001) implying that these proteins exposed the adaptin-binding motifs outside the rhoptry. The hydrophobic stretch considered to be the TM was found buried inside the crystal structure of ROP2, demonstrating that these proteins could not adopt a type 1 TM insertion, and that a direct interaction with adaptins was unlikely. In addition, others studies described correctly delivered to rhoptries in absence of a dileucine and YXXø motif (Bradley et al., 2004; Etheridge et al., 2014; El Hajj et al., 2007a). The first key study revealing an endolysosomal route for trafficking of rhoptries was the demonstration that sortilin plays a role in rhoptry and microneme biogenesis (Sloves et al., 2012). Sortilin, also known as VPS10 in yeast, is a cargo receptor that functions in mannose-6-phosphate independent sorting to the endolysosomal system. An endolysosomal route was then supported by conditional ablation of retromer-associated vacuolar sorting proteins Vsp9, Vps26, and Vsp35 (MorlonGuyot et al., 2015; Pieperhoff et al., 2015; Sakura et al., 2016; Sangare et al., 2016), which mediates retrograde transport from endosomes to the TGN (Kim et al., 2010), or the depletion of AP-clathrin adaptors AP1 (Venugopal et al., 2017), and finally proteins of the endosomal HOPS/CORVET complex (VPS11 and VPS8) (Morlon-Guyot et al., 2015; Morlon-Guyot et al., 2018b). Interfering with additional proteins involved in vesicular trafficking, such as the dynamin-related protein B (DrpB) (Breinich et al., 2009), clathrin heavy chain 1 (CHC1) (Pieperhoff et al., 2015), or the Rab GTPases Rab5A and Rab5C (Kremer et al., 2013) also results in both rhoptry and micronemes biogenesis defects. All these studies converged toward the demonstration that Toxoplasma has reshaped the endosomal pathway to build up not only the rhoptries but also micronemes. As discussed in Chapter 15, Endomembrane trafficking pathways in Toxoplasma, the emergent

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model suggests that SORTLR binds to rhoptry or microneme proteins in the Golgi lumen and recruits cytosolic cargo sorting proteins to form a sorting complex, which is presumably associated with vesicles that exit from the external Golgi cisternae. SORTLR then guides its cargo through to prerhoptries, or immature micronemes before releasing its payload. The “empty” cargo receptor then recruits components of the retromer complex for retrograde translocation and recycling to the Golgi to reload with new cargo. How SORTLR recognizes different cargo proteins from rhoptries and micronemes remains to be elucidated. One possible explanation is that the timing of expression dictates specificity. As rhoptries are formed prior to micronemes during daughter cell formation (Nishi et al., 2008), SORTLR might facilitate rhoptry biogenesis prior to turning its attention to packaging micronemes. This recycling model would allow the biogenesis of two distinct compartments using the same molecular machinery. Sorting within the Golgi might also occur via a clustering mechanism whereby proteins en route to a particular destination aggregate into distinct subdomains with only a subset of proteins forming direct contacts with the cargo receptor. In support of this, rhoptry proteins complexes exist in T. gondii (Besteiro et al., 2009; Lamarque et al., 2012) and complex formation is required for proper sorting to the rhoptries (Lamarque et al., 2012; Beck et al., 2014; Lamarque et al., 2014; Guerin et al., 2017a). 14.6.1.3 Rhoptry-targeting signals Most of the rhoptry proteins contain a signal peptide and are consequently targeted to the lumen of the organelles. In contrast, some rhoptry proteins devoid of a signal peptide have are associated with the cytosolic face of the rhoptry (Herm-Gotz et al., 2007; Cabrera et al., 2012; Beck et al., 2013; Mueller et al., 2016; Chasen et al., 2017) via N-terminal

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lipidation (Cabrera et al., 2012; Beck et al., 2013) or GPI anchoring (Chasen et al., 2017). Others contain multiple TM segments and are likely integral membrane proteins (Beck et al., 2013; Wang et al., 2016; Hammoudi et al., 2018). The formation of complexes might also account for targeting of proteins to the surface of rhoptries. For example, complexed to the armadillo repeats-only (ARO) protein, the ARO interacting protein (AIP) and adenylate cyclase β (ACβ) reach the surface of the organelle (Mueller et al., 2016). While ARO is distributed over the entire membrane (neck and bulb), its associated partners AIP and ACβ delineate the specific intermediate subcompartment separating the rhoptry bulb and neck, suggesting that other determinants are responsible for this peculiar location (Mueller et al., 2016). Luminal proteins usually partition to either bulb or neck. To the best of our knowledge only one, toxofilin, is described to be present in both compartments (Delorme-Walker et al., 2012). The mechanism by which luminal proteins are partitioned within a single membrane-bound organelle is not understood. Data from P. falciparum argues for the presence of a bulbretention signal, as RAP1 appears to possess a distinct signal that avoids relocalization of the protein from the bulb of the rhoptries to the neck (Richard et al., 2009). These findings are consistent with the observation that the prodomain of the T. gondii rhoptry bulb ROP1 protein directs trafficking of a reporter more prominently to the rhoptry neck, whereas full-length ROP1 is enriched in bulb (Bradley and Boothroyd, 2001). Mapping of the domains involved in targeting have also involved the propeptides of the rhoptry neck protein RON8 and bulb protein Toxolysin-1 as sorting signals (Straub et al., 2009; Hajagos et al., 2012). However, these sequences lack apparent similarities at the primary sequence level, precluding the identification of a region or a consensus sequence sufficient for rhoptry targeting.

14.6.1.4 Rhoptry morphogenesis and clustering to apical end The factors that contribute to the morphogenesis of the rhoptries during maturation of the organelles are poorly described. A transporter, TFP2, phylogenetically related to the Transporter Facilitator Proteins (TFPs) (Hammoudi et al., 2018), a carbonic anhydrase (CA)-related protein (CA-RP) (Chasen et al., 2017), and the rhoptry bulb protein ROP1 (Soldati et al., 1995), has been implicated in the morphological development of the organelle; however, mechanical insights are still missing. How the rhoptries are packed together and tethered to the parasite apex remained an enigma until two studies demonstrated the crucial role played by ARO in both steps. Upon conditional depletion of ARO, mature rhoptries are randomly dispersed within the parasite cytosol (Beck et al., 2013; Mueller et al., 2013). How ARO contributes to this processes is unclear, but it seems to be dependent on an actomyosin-based process (Jacot et al., 2013; Mueller et al., 2013), analogous to the actomyosin-dependent movement of organelles in yeast and melanocytes (Weisman, 2006; Hume and Seabra, 2011). Functional complementation of conditional KO mutant with deletion versions then revealed a role for ARO in holding rhopries in bundles (Mueller et al., 2016). ARO is conserved in apicomplexan parasites and likely plays similar role in P. falciparum, since complementation of Toxoplasma ARO mutant with its P. falciparum ortholog rescues the phenotype, highlighting that clustering and tethering are conserved processes within the phylum. A recent study however pointed also toward determinants specific to coccidian parasites (Morlon-Guyot et al., 2018a).

14.6.2 ROPs and RONs processing Most characterized T. gondii rhoptry proteins are proteolytically cleaved during transit

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in the secretory pathway. Three prodomain cleavage sites (in ROP1, SUB2, and ROP13) have been confirmed experimentally at a conserved SφXE site (φ is hydrophobic, X is any amino acid) (Bradley and Boothroyd, 1999; Bradley et al., 2002; Miller et al., 2003; Turetzky et al., 2010). This site has become a predictive tool for cleavage of ROPs/RONs without experimental confirmation (Carey et al., 2004a; El Hajj et al., 2007a; Besteiro et al., 2009; Turetzky et al., 2010). A degenerated site has been shown to be cleaved in Toxolysin-1 (SφXD) (Hajagos et al., 2012), allowing refinement of the predicted cleavage site to SφX(E/ D). The processings of ROPs and RONs are blocked by brefeldin A (Soldati et al., 1998; Besteiro et al., 2009). Once cleaved, the prodomains are likely degraded, as specific sera generated against the prodomains labeled only the prerhoptries and did not label the mature organelle in S phase parasites (Carey et al., 2004a; Besteiro et al., 2009; Turetzky et al., 2010; Hajagos et al., 2012). Altogether this indicates that cleavage should take place post-Golgi, but before maturation of rhoptries. The rhoptry resident serine protease SUB2 was initially proposed to be responsible of maturation of ROPs/RONs at SφXE/D site (Miller et al., 2003); however, disruption of the SUB2 gene eventually excluded the presumed role of SUB2 as maturase for ROPs/RONs (Dogga et al., 2017). Instead, the aspartyl protease 3 (ASP3) located mainly to the ELC has recently been demonstrated to act as a maturase for most RONs and ROPs (validated for ROP1, 4, 7, 13, 18, RON4, 2, 5, 11, and Toxolysin-1) (Dogga et al., 2017). ASP3 activity is optimal at pH 5.5
6.5, which is probably favored by the acidification of the pro-rhoptry lumen (Shaw et al., 1998). Comparing the Nterminome of wild-type and ASP3-depleted parasites, Dogga et al. (2017) then increased the repertoire of (direct or indirect) ASP3 substrates and identified four additional ROP proteins called TAILS5, 6, 7, and 8. This work also

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revealed that additional rhoptry maturases are present in the secretory pathway because the C-terminal cleavage site SXLkQ for Toxolysin1 (Hajagos et al., 2012) remains processed in absence of ASP3 (Dogga et al., 2017). The importance of these cleavages is largely unknown. It does not seem to play a critical role in trafficking for many ROPs/RONs (Bradley et al., 2002; Turetzky et al., 2010; Hajagos et al., 2012; Dogga et al., 2017), with two exception, RON11 and TAILS8 (TGME49_321650) which exhibit an altered distribution within the organelle in the absence of ASP3 (Dogga et al., 2017).

14.6.3 Secretion of rhoptries Rhoptry discharge occurs early during the invasion process. Electron microscopic observations show empty rhoptries as early as the initial apical contact creating the MJ. How many rhoptries are secreted during invasion is unknown, but not all rhoptries are discharged in tachyzoites. In contrast, in P. falciparum the RAP1 rhoptry signal disappears from the parasite just after invasion, indicating that this parasite exocytoses the contents of its two rhoptries (Riglar et al., 2011). Interestingly, electron micrograph shows the fusion between the neck of two rhoptries at the initial stage of invasion (Aikawa et al., 1978; Hanssen et al., 2013), implying a fusion machinery at this step. A single burst of rhoptry secretion occurs during the invasion process, as proteins associated with the MJ in T. gondii are found exclusively at the MJ and not in the vacuole. After the burst of rhoptry secretion during invasion, the secretion of micronemes is probably switched off, because the MICs proteins are neither found in the lumen of the vacuole nor associated with the intracellular part of the parasite. The secretion of rhoptries has long been seen as the necessary step in the invasion process, which remains true, but recent studies

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show that it may not be used exclusively for this purpose. Indeed, tachyzoites also inject rhoptry proteins into cells they do not productively invade (Koshy et al., 2012). While less rhoptry proteins are injected compared to productively invaded cells, the amounts introduced are nonetheless sufficient to produce physiologically relevant changes induced by rhoptry effectors (see next and Chapter 17: Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages). This nonproductive invasion mechanism also operates in vivo and is actually more common than productive invasion, especially in the mouse brain where Toxoplasma encysts and persists. While one cannot exclude the possibility that parasites invade but are cleared soon after invasion, this mechanism is proposed to substantially contribute to the pathogenesis of the disease. In contrast to micronemes, rhoptry secretion has been difficult to study because no triggers or inducers have yet been identified to facilitate biochemical approaches. It is proposed that rhoptry secretion depends on microneme proteins (MICs), their translocation onto the parasite surface, and their binding to host cell receptors, although this is largely uncharacterized. In Toxoplasma in the absence of the microneme proteins AMA1 (Mital et al., 2005) and MIC8 (Kessler et al., 2008), the ROP proteins discharge is reduced and these parasites display a major defect of invasion. Since AMA1 is involved in the formation of the MJ (see next), the decrease of rhoptry content export into the host cell might be the result of improper intimate attachment of the parasite to the host cell for an efficient injection. In the case of MIC8, the deletion of the cytoplasmic domain is sufficient to impair secretion of rhoptry proteins during invasion (Kessler et al., 2008). MIC8 possesses an extracellular lectin-binding domain (Cerede et al., 2002), suggesting that MIC8 might combine cell adhesion with

intracellular signaling functions. Altogether, these results indicated that the intimate attachment of the parasite to the host cell is a prerequisite to efficient rhoptry secretion and that interaction of MICs with a ligand probably triggers signal transduction pathways through their CDs. This was supported by a study in P. falciparum (Singh et al., 2010), which demonstrates that the interaction of EBA175 with glycophorin A, its receptor on erythrocytes, diminishes the elevated cytosolic calcium levels necessary for microneme secretion (as stated previously) and, importantly, triggers release of rhoptry proteins. These observations were also observed with EBA140-glycophorin C receptor engagement (Singh et al., 2010). A third microneme protein, CLAMP, is essential for invasion and involved in a cellular event following, egress, motility, and microneme secretion (Sidik et al., 2016), pointing for a potential role in rhoptry secretion for an additional MIC protein. Rhoptry secretion is minimally a two-step process. First, the rhoptry membrane docks and fuses with the apical plasma membrane of the parasite (exocytosis). This is followed by the transfer of content through the host cell membrane (export). The secretory material (ROPs/RONs and lipids) is not released outside the cell as in synaptic vesicles or densecore vesicles but is instead injected directly inside the host (Miller et al., 1979, Hakansson et al., 2001), which is a unique feature of this exocytosis process. The signals and molecular mechanisms defining how rhoptries fuse to the parasite membrane are almost entirely unknown. A first report suggested the contribution of a phospholipase (Ravindran et al., 2009). An inhibitor of phospholipase A2s called 4-bromophenacyl bromide has been shown to interfere with rhoptry secretion, while it did not affect microneme secretion. T. gondii encodes in its genome many predicted phospholipases that might be direct targets of this inhibitor.

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Second, a recent study highlighted the role played by an Apicomplexa conserved protein, Toxoplasma Ferlin 2 (FER2), for rhoptry secretion and invasion (Coleman et al., 2018). FER2 is part of ferlins, an ancient eukaryotic protein family typically functioning in membrane fusion, vesicle trafficking, and membrane repair (Lek et al., 2012). FER2 is present in defined cytoplasmic puncta suggestive of either inclusion bodies or membranous structure of unknown identity. It contains five potential C2 domains (lipid-calcium binding domains), two sharing features consistent with calcium binding. It is therefore tempting to speculate that FER2 is involved in a calciumtransducing signal. Additional studies are needed to determine if, similar to microneme secretion, the rhoptry secretion signaling pathways are calcium dependent. How rhoptry content is introduced into the host cell remains a mystery. Details of rhoptry structure during red cell invasion by Plasmodium show a dome-like structure of the red cell membrane where the tip of the rhoptry duct enters (Bannister and Mitchell, 1989). Concomitant with the intimate attachment of the apical tip of the parasite to the host cell and rhoptry secretion is a transient rise in the conductance of the host cell membrane (Suss-Toby et al., 1996). This has been interpreted as a transient break in the host membrane allowing rhoptry proteins to enter the host cell. Whether it corresponds to the 40 nm pore observed by freeze-fracture during the invasion process (Dubremetz, 2007), which seems to connect the rhoptry contents with the host cell cytoplasm, remains to be demonstrated.

14.6.4 Rhoptry proteins and functions Once secreted, rhoptry proteins associate with different compartments to perform a wide variety of roles: (1) some RONs are associated with the MJ for invasion; (2) many ROPs are

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associated with the PVM where they disarm immune defenses; (3) others are injected in the host cytosol to hijack the host during invasion; or (4) are targeted to the host cell nucleus to interplay with host cell signaling pathways by coopting transcription factors, principally those related to IFN-γ production. This section will describe the rhoptry contents and functions played by ROPs/RONs (see Table 14.2), with a focus on their structural role. The details of the functional insights of ROP effectors are described in Chapter 17, Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages. Identification of the major components of the rhoptries in T. gondii has been obtained initially by subcellular fractionation and generation of monoclonal antibodies (Leriche and Dubremetz, 1991; Ossorio et al., 1992; Saffer et al., 1992; Herion et al., 1993; Hajj et al., 2005; Lebrun et al., 2005; El Hajj et al., 2007a, 2007b; Lamarque et al., 2012) . Proteomics analysis of purified rhoptries has further allowed the characterization of 38 previously unidentified proteins of which some have been confirmed to be localized in the organelles (Bradley et al., 2005; Besteiro et al., 2009; Straub et al., 2009; Hajagos et al., 2012; Lentini et al., 2017). The mRNA expression timeline of rhoptry genes [maximum expression in S to M phase (Behnke et al., 2010)] was also used as a criteria to found new rhoptry proteins (Camejo et al., 2014; Kim et al., 2016). Others were discovered by affinity capture with a lectin (Wang et al., 2016), by in silico searches for homologs of known ROPs (Peixoto et al., 2010; Lamarque et al., 2014) or simply by studying proteins with particular domains (proteases, transporters, etc.) (Miller et al., 2001; Hajagos et al., 2012; Chasen et al., 2017; Hammoudi et al., 2018). More recently, a TAILS analysis to compare the N-terminomes of wild-type parasites and an ASP3 mutant (maturase of ROPs/RONs) extended the rhoptry proteome (Dogga et al., 2017).

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TABLE 14.2

Properties of Toxoplasma secretory proteins: rhoptry proteins.

Location/protein

Domains(#)a characteristics

Interacting partners

Mutant phenotypes

Function

Postsecretory trafficking

References

Rhoptry Rhoptry proteins associated with the cytosolic face of the rhoptry Rab11

Small GTPase

ARO

Armadillo-like repeats(2)

Rhoptry neck and bulb AIP

ARO and ACβ

Intermediate subcompartment separating the rhoptry bulb and neck ACβ Intermediate subcompartment separating the rhoptry bulb and neck CA-RP/CAH1

MyoF and AIP

Adenylate cyclase

CA-RP, GPI anchor

AIP

Conditional knockdown mutant

Vesicular trafficking?

Bradley et al. (2005)

Clustering and apical targeting of rhoptries

Cabrera et al. (2012), Beck et al. (2013), Mueller et al. (2013, 2016) Mueller et al. (2013, 2016)

Nonessential protein KO shows normal growth, invasion and virulence

Nonessential protein

Mueller et al. (2013, 2016)

KO shows normal growth, invasion and virulence

Nonessential protein.

Rhoptry morphology

Bradley et al. (2005), Mueller et al. (2016), Chasen et al. (2017)

Palmitoyl acyltransferase

Beck et al. (2013), Frenal et al. (2013)

Rhoptry morphology

Hammoudi et al. (2018)

The rhoptries appear fragmented. The mutant invades less efficiently and the virulence in mice is slightly attenuated

Integral membrane proteins DHHC7

DHHC zinc finger(1)

Key protein

TFP2

Transporter facilitator protein 2

Nonessential protein

Rhoptry bulb

Integral membrane protein of rhoptries

Length of the rhoptries in Δtfp2 parasites is increased

Rhoptry neck and bulb

Knockdown results in untethered rhoptries, failure of rhoptry secretion and loss of invasion

Transporter facilitator protein 3

Nonessential protein

Hammoudi et al. (2018)

Rhoptry bulb RON11

EF-hand(2)

Nonessential protein

Wang et al. (2016) Integral membrane protein of rhoptries

Sodium Hydrogen Exchanger

Nonessential protein

Strong charge asymmetry

Nonessential protein

Kinase domain

Nonessential proteins in vitro

TFP3

Rhoptry neck NHE2 Rhoptry bulb

KO has no discernable phenotype Ion homeostasis?

Karasov et al. (2005)

KO is defective in calciumdependent egress

Luminal proteins ROP1 Rhoptry bulb

ROP2a/ROP2b/ROP8

Rhoptry morphology

KO shows normal growth, invasion, and virulence, but abnormal rhoptry morphology

Gene duplication

Deletion of locus induces a moderately decreased cyst burden

ROPKs family

Associated with the PVM

Ossorio et al. (1992), Saffer et al. (1992), Soldati et al. (1995)

Associated with the PVM

Sadak et al. (1988), Beckers et al. (1994), Sinai and Joiner (2001), Fox et al. (2016)

Associated with the PVM and phosphorylated

Carey et al. (2004a,b), Hajj et al. (2005), Fox et al. (2016)

Associated with the PVM

Leriche and Dubremetz (1991), Bradley et al. (2005), El Hajj et al. (2007b), Reese et al. (2011), Behnke et al. (2012, 2015), Alaganan et al. (2014), Etheridge et al. (2014), Fox et al. (2016), El Hajj et al. (submitted)

Rhoptry bulb ROP4/ROP7

Kinase domain

Nonessential proteins in vitro

ROPKs family

Deletion of locus induces a moderately decreased of cyst burdens

Rhoptry bulb ROP5 Gene duplication Rhoptry bulb

Kinase domain

In complex with ROP17/ ROP18/ GRA7

Nonessential protein in vitro

Control INFγ immune response

ROKs family ROP6

Protease activity and host cell binding

ROP9 (P36)

Ahn et al. (2006) Reichmann et al. (2002)

Rhoptry bulb ROP10 Rhoptry bulb

In type I, nonessential protein in vitro and in vivo

Bradley et al. (2005), Wang et al. (2017a)

(Continued)

TABLE 14.2

(Continued)

Location/protein

Domains(#)a characteristics

ROP11

Kinase domain

Interacting partners

Mutant phenotypes

Function

Postsecretory trafficking

Bradley et al. (2005), Fox et al. (2016), Wang et al. (2017a)

In type I, nonessential protein in vitro and in vivo

ROKs family

References

Cyst burden not significantly modified in type II KO Bradley et al. (2005)

ROP12 Rhoptry bulb ROP13

In type I strain, not essential for growth in fibroblasts or for virulence in vivo

Rhoptry bulb ROP14

Host cell

Bradley et al. (2005), Turetzky et al. (2010)

Bradley et al. (2005)

DUF1222(1)

Rhoptry bulb ROP15

In type I, nonessential protein in vitro and in vivo

Rhoptry bulb ROP16

Kinase domain

Nonessential protein in vitro

Rhoptry bulb

Phosphorylates host proteins STAT3 and STAT6

ROKs family

Bradley et al. (2005), Wang et al. (2017a)

Control host immune response

Associated with the host cell nucleus

Bradley et al. (2005), Saeij et al. (2007), Fox et al. (2016)

Control host immune response

Associated with the PVM

Bradley et al. (2005), Alaganan et al. (2014), Etheridge et al. (2014), Fox et al. (2016)

Control host immune response

Associated with the PVM

Bradley et al. (2005), Saeij et al. (2006), Taylor et al. (2006), El Hajj et al. (2007a), Fentress et al. (2010), Alaganan et al. (2014), Etheridge et al. (2014), Behnke et al. (2015), Fox et al. (2016)

KO ROP16 in type II strain exhibits a significant increase in cyst burden ROP17

Kinase domain

Rhoptry bulb ROKs family ROP18

Kinase domain

Rhoptry bulb ROKs family

ROP20 Rhoptry bulb ROKs family

Kinase domain

In complex with ROP5, ROP18 and GRA7

Nonessential protein in vitro

In complex with ROP5, ROP17, and GRA7

Nonessential protein in vitro

Essential for chronic infection

Essential for chronic infection Phosphorylates host immunityrelated GTPases to prevent their accumulation on the PVM and thereby preserve the PV integrity

ROP23

Kinase domain

Cyst burden moderately decreased in type II KO

Fox et al. (2016)

Kinase domain

In type I, nonessential protein in vitro and in vivo

Fox et al. (2016), Wang et al. (2017a)

Rhoptry bulb ROKs family ROP24 Rhoptry bulb ROKs family ROP25

Cyst burden not significantly modified in type II KO Kinase domain

Cyst burden not significantly modified in type II KO

Fox et al. (2016)

Kinase domain

Cyst burden moderately decreased in type II KO

Fox et al. (2016)

Kinase domain

Cyst burden moderately decreased in type II KO

Fox et al. (2016)

Kinase domain

Nonessential

Jones et al. (2017)

Rhoptry bulb ROKs family ROP26 Rhoptry bulb ROKs family ROP28 Rhoptry bulb ROKs family ROP30

KO is normal for in vitro growth and bradyzoite differentiation ROP31

Kinase domain

Cyst burden not significantly modified in type II KO

Fox et al. (2016)

Kinase domain

In type I, nonessential protein in vitro and in vivo

Fox et al. (2016), Wang et al. (2017a)

Rhoptry bulb ROKs family ROP32 Rhoptry bulb ROKs family ROP36 Rhoptry bulb ROKs family

Cyst burden moderately decreased in type II KO Kinase domain

Essential for chronic infection In type I, nonessential protein in vitro and in vivo

Peixoto et al. (2010), Fox et al. (2016), Wang et al. (2017a)

(Continued)

TABLE 14.2

(Continued)

Location/protein

Domains(#)a characteristics

ROP37

Kinase domain

Rhoptry bulb

Interacting partners

Mutant phenotypes

Cyst burden moderately decreased in type II KO

ROP38, ROP29, ROP19 Kinase domain

Cyst burden moderately decreased in type II KO by deletion of ROP38, 29, 19

Rhoptry bulb

References

Control host immune response

Peixoto et al. (2010), Fox et al. (2016)

ROP38 downregulates host genes associated with MAPK signaling and the control of apoptosis and proliferation

ROKs family

ROP39

Postsecretory trafficking

Fox et al. (2016), Wang et al. (2017a)

In type I, nonessential protein in vitro and in vivo

ROKs family

Gene duplication

Function

Kinase domain

Essential for chronic infection

Fox et al. (2016)

Kinase domain

Cyst burden not significantly modified in type II KO

Fox et al. (2016)

Kinase domain

In type I, nonessential protein in vitro and in vivo

Fox et al. (2016), Wang et al. (2017a)

Rhoptry bulb ROKs family ROP40 Rhoptry bulb ROKs family ROP41/ROP42/ ROP44 Gene duplication Rhoptry bulb ROKs family ROP45

Cyst burdens were moderately decreased in type II KO Kinase domain

Cyst burden not significantly modified in type II KO

Fox et al. (2016)

Kinase domain

Cyst burden not significantly modified in type II KO

Fox et al. (2016)

Rhoptry bulb ROKs family ROP46 Rhoptry bulb ROKs family

ROP47

Kinase domain

In type I, nonessential protein in vitro and in vivo

Secreted and traffics to the host cell nucleus

Disruption of ROP47 gene in a type II strain does not impair parasite virulence during acute or chronic phases ROP48

In type I, nonessential protein in vitro and in vivo

Camejo et al. (2014), Wang et al. (2017a)

Camejo et al. (2014), Wang et al. (2017a)

In type II strain, not implicated in evading the INFγ response, or in acute or chronic infections. ROP49

Kinase domain

Talevich and Kannan (2013)

ROP50 ROP54

Talevich and Kannan (2013) Kinase domain

Modulates GBP2 loading onto parasite-containing vacuoles

Evasion of innate immunity

Associated with PVM

Kim et al. (2016)

Metalloprotease (M16 family)

Nonessential protein

Proteolysis

Associated with the PVM

Bradley et al. (2005), Hajagos et al. (2012), Dogga et al. (2017)

Subtilase

Initially described as essential rhoptry maturase, but recent results showed that SUB2 is a nonessential protein

Rhoptry bulb ROKs family TLN1, Toxolysin 1 Rhoptry bulb SUB2

Autocatalytic processing PPC2-hn

PP2C

Nonessential gene. PP2C KO in type I strain shows only a mild growth defect in vitro and no virulence defect in mice

DegP

Serine protease

Nonessential gene

Miller et al. (2003), Binder and Kim (2004), Dogga et al. (2017)

Targeted to nucleus

Gilbert et al. (2007)

Lentini et al. (2017)

Dispensable for the lytic cycle of both RH-type I and Pru-type II strains. Important for virulence of type II parasites BRP1

Nonessential gene

Rhoptry bulb (BRP1)

No phenotype in vitro and in vivo

Schwarz et al. (2005)

(Continued)

TABLE 14.2

(Continued)

Location/protein

Domains(#)a characteristics

Interacting partners

Toxofilin

Mutant phenotypes

Function

Postsecretory trafficking

References

Nonessential protein

Control host cortical actin cytoskeleton dynamics

Exported into cytosol of the host cell

Bradley et al. (2005), Delorme-Walker et al. (2012), Lodoen et al. (2010), Delorme-Walker et al. (2012)

Rhoptry neck and bulb RON1

Bradley et al. (2005)

CobT(1) CCP(1)

Rhoptry neck RON2

Interacts with AMA1

Rhoptry neck

In complex with RON2, RON4, RON5, RON4L1 and RON8

Conditional knockdown mutant can secret rhoptries, but fails to organize the MJ and is severely defective for invasion. RON4 and RON5 are mistargeted in the mutant and their expression reduced.

MJ formation and invasion

MJ localization, inserted in host membrane, exposing an extracellular domain that binds to AMA1

Bradley et al. (2005), Lebrun et al. (2005), Besteiro et al. (2009), Tonkin et al. (2011), Lamarque et al. (2014), Guerin et al. (2017b)

Interacts with host proteins CD2AP and CIN85 RON2L1 Sporozoite Rhoptry neck RON2L2/sporoRON2 Sporozoite-specific Rhoptry neck protein

Interacts with AMA4

Lamarque et al. (2014), Parker et al. (2016)

Interacts with SporoAMA1 (AMA3)

Invasion of sporozoites

Fritz et al. (2012b), Poukchanski et al. (2013)

RON3

Bradley et al. (2005)

Rhoptry neck RON4

In complex with RON2, RON5, RON4L1 and RON8

Rhoptry neck

Interacts with host proteins ALIX,

Mutant secret rhoptries, but is severely impair for invasion. RON2 and RON5 are mistargeted in the mutant and their expression reduced.

Invasion, MJ

MJ localization, exported into host cell membrane

Bradley et al. (2005), Lebrun et al. (2005), Guerin et al. (2017a), Guerin et al. (2017b), Wang et al. (2017b)

CD2AP and CIN85 RON4L1

Nonessential protein

In complex with RON2, RON4, RON5 and RON8

KO only displays a slight phenotype in vivo

RON5

In complex with RON2, RON4, RON4L1 and RON8

Conditional knockdown mutant has a strong invasion phenotype. RON2 and RON4 are mistargeted in the mutant and their expression reduced

Rhoptry neck

Interacts with host proteins CD2AP, CIN85 and TSG101

Rhoptry neck

Invasion, MJ

MJ localization, exported into host cell membrane

Fritz et al. (2012b), Guerin et al. (2017b)

MJ localization, exported into host cell membrane

Bradley et al. (2005), Lebrun et al. (2005), Besteiro et al. (2009), Straub et al. (2009), Beck et al. (2014), Guerin et al. (2017a), Guerin et al. (2017b)

RON6

Bradley (unpublished)

Rhoptry neck RON8

In complex with RON2, RON4, RON5, and RON4L1

Rhoptry neck

RON9 Rhoptry neck

RON10 Rhoptry neck

Sushi or (1), Ankirin (6)

Nonessential protein KO is defective in attachment, invasion and virulence

Interacts with RON10

Nonessential protein

Interacts with RON9

Nonessential protein

Invasion

MJ localization, exported into host cell membrane

Besteiro et al. (2009), Straub et al. (2009), Straub et al. (2011)

Lamarque et al. (2012)

KO has no discernable phenotype apart from mislocalization of RON10 Lamarque et al. (2012)

KO has no discernable phenotype apart from mislocalization of RON9

(Continued)

TABLE 14.2

(Continued)

Location/protein

Domains(#)a characteristics

Interacting partners

Mutant phenotypes

Function

Postsecretory trafficking

References Camejo et al. (2014)

RON12 Rhoptry neck

Update on proteins previously annotated as ROPs ROP21

Kinase domain

ROKs family Not associated with rhoptry

In type I, nonessential protein in vitro and in vivo

PV and cyst matrix

Peixoto et al. (2010), Fox et al. (2016), Jones et al. (2017), Wang et al. (2017a)

Cyst burden not significantly modified in type II KO KO is normal for in vitro growth and bradyzoite differentiation Combined Δrop21/Δrop17 KO led to a 50% reduction in cyst burden in vivo

ROP22

Kinase domain

Cyst burdens were moderately decreased in type II KO

Kinase domain

Cyst burden not significantly modified in type II KO

ROKs family

Peixoto et al. (2010), Fox et al. (2016)

Annotated as rhoptry bulb protein but experimentally invalidated ROP27 ROKs family

KO is normal for in vitro growth and bradyzoite differentiation

Annotated as rhoptry bulb protein but experimentally invalidated

Combined Δrop21/Δrop17 KO led to a 50% reduction in cyst burden in vivo

ROP33 ROKs family Annotated as rhoptry bulb protein but

Kinase domain

In type I, nonessential protein in vitro and in vivo Cyst burden not significantly modified in type II KO

PV and cyst matrix

Fox et al. (2016), Jones et al. (2017)

Fox et al. (2016), Wang et al. (2017a), Beraki et al. (2019)

presumably GRA protein homolog to WNG1 and WNG2 ROP34 (WNG2) ROKs family

Degenerated kinase

In type I, nonessential protein in vitro and in vivo

Kinase domain

In type I, nonessential protein in vitro and in vivo

PV lumen

Wang et al. (2017a), Beraki et al. (2019)

PV lumen

Wang et al. (2017a), Beraki et al. (2019)

Annotated as rhoptry bulb protein but experimentally assigned as a GRA protein ROP35 (WNG1) ROKs family

Unstable TVN in WNG1 KO

Annotated as rhoptry bulb protein but experimentally assigned as a GRA protein

Phosphorylates GRAs

Biogenesis of TVN

a

Based on the complete open-reading frame including signal sequence or GPI anchor signal, if present.

CBL, Chitin-binding-like-domain; CCP, domain abundant in complement control proteins; CobT, cobalamin biosynthesis protein; DUF1222, domain of unknown function; TSR, thrombospondin type-1 repeat; TM, transmembrane; ACβ, adenylate cyclase β; ARO, armadillo repeats-only; AIP, ARO interacting protein; BRP1, bradyzoite-specific rhoptry protein 1; CA-RP, carbonic anhydrase-related protein; INF, interferon; PV, parasitophorous vacuole; PVM, PV membrane; MJ, moving junction; KO, knockout; PP2C, phosphatase 2C; GPI, glycosylphosphatidylinositol.

658

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

To date, most of the proteins contained in the bulb are restricted to Toxoplasma [RON9 (Reichmann et al., 2002), RON14 (Bradley et al., 2005) being exceptions]. As they are exported to the host cell and function as effectors to modulate the host cell machinery, one can therefore suggest that these proteins have been subjected to evolutionary pressure and may have derived from some specialized property of these organisms, such as their ability to form tissues cysts or from their complex heteroxenous life cycle. In contrast, most of the proteins located in the rhoptry neck, RONs show homologies restricted to Apicomplexa (Bradley et al., 2005) and therefore have more generic functions across the phylum, such as MJ formation. 14.6.4.1 Rhoptry proteins associated with the cytosolic face of the rhoptry 14.6.4.1.1 Rab11a

Rab11a was found in the purified rhoptry proteome (Bradley et al., 2005). Rab11a is highly dynamic and can be also observed in an ELC and at the IMC in T. gondii (Agop-Nersesian et al., 2009). In higher eukaryotes, Rab11 belongs to the family of small GTPases involved in the regulation of vesicular traffic. It is usually localized to early endosomes, perinuclear recycling endosomes, as well as at the TGN. It is considered as controlling slow endosomal recycling, as well as traffic to the Golgi apparatus (Ullrich et al., 1996; Chen et al., 1998). Toxoplasma Rab11a, which does not contain a signal peptide, is likely associated with the cytoplasmic side of the rhoptry membrane through geranyl
geranyl modification of the two cysteines of the CCXX site (Bradley et al., 2005), a usual feature of Rab11. It was therefore proposed that Rab11a may act as a regulator of trafficking to the rhoptries (Bradley et al., 2005). Parasites expressing a Rab11a dominant negative mutant show a severe growth defect (Herm-Gotz et al., 2007). However, no defects in rhoptries are observed in this mutant. The

fate of other organelles, including micronemes, DGs, Golgi, apicoplast, and mitochondria, and nucleus are not changed; and the formation and early elongation of the IMC appeared normal (Agop-Nersesian et al., 2009). However, ablation of Rab11a function results in daughter parasites having an incompletely formed IMC, leading to a block at a late stage of division. In fact, Rab11a appears to regulate the assembly of the motor complex at the IMC, an essential step in parasite development. Why Rab11a is associated to rhoptries remains unknown. 14.6.4.1.2 ARO and its partners AIP and ACβ: apical targeting of rhoptries

A conserved apicomplexan protein termed ARO protein is localized to the cytosolic face of P. falciparum and T. gondii rhoptries (Cabrera et al., 2012). Although ARO does not possess a signal peptide for entering the secretory pathway it is targeted exclusively to the rhoptries by attachment to the cytosolic face of the membrane by N-terminal acyl-modifications. The acylation of ARO is dependent upon the acyltransferase DHHC7 that localizes to the rhoptries (Beck et al., 2013; Frenal et al., 2013). This palmitoylation event likely takes place late during the biogenesis of the rhoptries because the association of ARO to the organelle is only visible in mature rhoptries. Gene disruption experiments revealed a specific role for ARO in apical distribution of rhoptries (Mueller et al., 2013, 2016) (see Section 14.6.1). ARO mediates the surface localization of two soluble proteins named AIP and ACβ (Mueller et al., 2013, 2016), AIP binding being a prerequisite for the targeting of ACβ to the rhoptry. ARO covers the surface of both the rhoptry and the neck, while AIP and ACβ delineate the intermediate compartment. 14.6.4.1.3 Carbonic anhydrase
related protein

A CA-RP was initially identified in the proteome of rhoptries (Bradley et al., 2005) and then confirmed by Mueller et al. (2016) to be

Toxoplasma Gondii

14.6 Rhoptries

associated with the rhoptries. CA-RP shares structural similarities with CA isoforms but lacks enzymatic activity. Two of the three histidines of the Zn21 coordination domain are substituted in CA-RP and the recombinant protein lacks the activity (Chasen et al., 2017). Named initially CAH1 (Mueller et al., 2016) and later CA-RP (Chasen et al., 2017), it is posttranslationally modified at its C terminus with a GPI anchor that is required for its localization to rhoptries (Chasen et al., 2017). CA-RT is present in immature and mature rhoptries (Chasen et al., 2017) and exposed on the cytosolic face of the organelle (Mueller et al., 2016). CA-RT is not essential for the parasite, but in its absence, the morphology of the rhoptries is profoundly affected (Chasen et al., 2017). The rhoptries appear fragmented and not connected to a rhoptry neck structure, and then probably suboptimally functional. Consequently, the mutants invade less efficiently and the virulence in mice is slightly attenuated. 14.6.4.2 Integral membrane proteins 14.6.4.2.1 Acyltransferase DHHC7

DHHC7 is a S-acyl transferase associated with the rhoptries (Beck et al., 2013; Frenal et al., 2013). DHHC7 is present along the length of the entire rhoptry in both the bulbous body and duct-like neck. A conserved C-terminal region is required for its proper rhoptry targeting. DHHC7 is conserved in Apicomplexa (Frenal et al., 2013). It contains four TMDs and likely adopts a membrane topology that positions the DHHC catalytic domain in the cytosol where it would enable the palmitoylation of ARO and its anchoring to the cytosolic face of the rhoptries. Accordingly, a mutant for DHHC7 recapitulates the phenotype of an inducible knockdown of ARO, with rhoptries dispersed in the cytosol (Beck et al., 2013; Frenal et al., 2013). To date, ARO is the only known candidate substrate for DHHC7.

659

14.6.4.2.2 Transporters Na1/H1

exchanger: In searches of the Toxoplasma genome, three homologs of sodium-hydrogen exchangers have been found. These proteins catalyze Na1/H1 exchange and are involved in regulation of internal pH and cell volume. One of them, NHE2, with 12 predicted TMDs is associated with rhoptries (Karasov et al., 2005). The pH of rhoptries changes from acidic (estimated to be B3.5
5.5) for nascent rhoptries to more neutral (B5.0
7.0) in mature organelles (Shaw et al., 1998). It is therefore hypothesized that NHE2 may be involved in pH regulation during the course of rhoptry biogenesis and rhoptry protein processing. Disruption of NHE2 does not affect targeting of several rhoptry proteins, including ROP1 and ROP2/3/4, nor does it alter rate of growth or virulence in vivo (Karasov et al., 2005). Transporter facilitator proteins: TFP2 and TFP3:

The major facilitator superfamily (MFS) represents the largest group of transporters driving ions or solutes across membranes (Pao et al., 1998). Toxoplasma genome predicts more than 30 MFS members, two of them, TFP2 and TFP3 localize to the rhoptry bulbs (Hammoudi et al., 2018). TFP2 and TFP3 are polytopic membrane proteins, their C-terminal ends facing the cytosol. Both are dispensable for the parasite lytic cycle. While no phenotype was observed in absence of TFP3, the length of the rhoptries in Δtfp2 parasites is increased by about 40%, while the number, the abundance, the processing or the secretion of rhoptry contents is not affected. RON11: RON11 is a rhoptry neck protein that contains a calcium-binding domain (Beck et al., 2014; Wang et al., 2016). RON11 is a substrate for the maturase ASP3 (Dogga et al., 2017); it does not contain a signal sequence but has four predicted TMDs. This protein is then

Toxoplasma Gondii

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

likely inserted in the membrane of the rhoptry. It is highly conserved in apicomplexans, suggesting an important role in rhoptry-mediated functions. However, disruption of RON11 induces no effects on invasion or intracellular survival (Wang et al., 2016), a surprising result taking into account of the predicted essentiality of RON11 (fitness score of 23.25) (Sidik et al., 2016). It remains possible that another protein can compensate the loss of RON11. 14.6.4.3 Luminal rhoptry proteins 14.6.4.3.1 Rhoptry neck complex RON2/RON4/ RON5/RON8/RON4L1: role in moving junction formation and invasion

Independent studies, one investigating AMA1 associated proteins and the other searching for antigens recognized by an antibody that stained the MJ, isolated a complex of RON proteins (Alexander et al., 2005; Lebrun et al., 2005; Besteiro et al., 2009; Straub et al., 2009). This complex was composed of RON2, RON4, RON5, and RON8, conserved in other Apicomplexa, except RON8, which is specific to Coccidian parasites (Besteiro et al., 2009; Straub et al., 2009; Besteiro et al., 2011; Guerin et al., 2017b). Once secreted, all these proteins are associated with the MJ and involved in invasion (Straub et al., 2011; Beck et al., 2014; Lamarque et al., 2014; Guerin et al., 2017a; Wang et al., 2017b). Accordingly, Cryptosporidium, which remains extracytoplasmic, does not possess orthologs of the MJ RONs complex in its genome. A fifth member, RON4L1, was recently identified (Guerin et al., 2017b). RON4L1 displays some sequence similarity with RON4 and similarly to RON8 is a coccidian-specific protein. These proteins do not bear recognizable domains or motifs that could suggest a particular molecular interaction. They are subjected to proteolytic maturation (Besteiro et al., 2009; Straub et al., 2009; Beck et al., 2014; Guerin et al., 2017b) in the prerhoptry compartments, which is not a

prerequisite for their interaction, as immature proteins were found to be interacting in vitro (Besteiro et al., 2009). Bioinformatic analysis indicates that RON4, RON4L1, RON5, and RON8 are putative soluble proteins, while RON2 is predicted to have two TM domains. How the complex is organized and its stoichiometry are not yet known. The RON2/ RON4/ RON4L1/RON5/RON8 complex is secreted during invasion and then associated with the MJ during all the invasion process, giving the characteristic ring-shaped labeling by immunofluorescence. This complex is secreted into the host cell membrane, where RON2 is inserted as an integral membrane protein in the host cell (Lamarque et al., 2011), and RON4, RON4L1, RON5, and RON8, which do not contain TM domains, are exposed to the cytosolic face of the host membrane (Besteiro et al., 2009; Lamarque et al., 2011; Guerin et al., 2017b). The N-terminal part of RON2 is exposed inside the host cell and presumably maintains the rest of the complex at the host membrane, while the Nterminal part is outside and interacts with the microneme protein AMA1 (Besteiro et al., 2009; Lamarque et al., 2011), which is secreted just prior to rhoptry secretion at the tip of the parasite and exposed on the parasite surface. The discovery that RON2 binds directly to the microneme protein AMA1 allows us to propose a model where the parasite would be inserting its own receptor (RON2 and associated RON proteins) for the ligand AMA1 to form the close apposition of the host cell and parasite plasma membranes which constitutes the MJ. The AMA1
RON2 interaction has been detected by coimmunopurification in vitro. The functional relevance of this interaction in vivo has been further validated by showing that the invasion process is inhibited by incubating tachyzoites with a recombinant protein corresponding to a C-terminal portion of RON2 (Lamarque et al., 2011; Tyler and Boothroyd, 2011) and that a RON2 mutant selectively compromised in AMA1 binding is also defective for invasion (Lamarque et al., 2014).

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14.6 Rhoptries

Deletions and mutations in the RON2 ectodomain define the essential AMA1 binding domain of RON2 to a U-shaped polypeptide loop containing a pair of cysteines residues in a disulfide bridge (Tonkin et al., 2011). Cocrystallization of AMA1 with this peptide reveals that the RON2 peptide is deeply anchored within the hydrophobic grove of AMA1 to form an embedded structure, which likely withstands the mechanic forces that occur during the invasion process (Tonkin et al., 2011). These atomic details provide insight into the molecular basis of the intimate attachment and are consistent with the 6 nm distance between the host cell and parasite plasma membranes at the MJ. Accordingly, RON2- and AMA1-depleted tachyzoites successfully complete the initial step of attachment but fail to invade and eventually detach (Lamarque et al., 2014). How this intimate attachment is then conveyed in an internalization step is currently unclear, but the Cterminal cytosolic tail of AMA1 is likely involved in this process. The AMA1 cytoplasmic tail is necessary for invasion by both T. gondii and P. falciparum (Treeck et al., 2009; Sheiner et al., 2010). In Toxoplasma, mutation of two aromatic residues with the tail is sufficient to abrogate invasion (Sheiner et al., 2010). More specifically, parasites secrete rhoptries, remain attached through a MJ, but fail to proceed further in internalization (Lamarque et al., 2014), a phenotype reminiscent of cytochalasin-treated tachyzoites defective for a fully functional glideosome. Interestingly, Krishnamurthy et al. (2016) identified a role for engagement of AMA1 with RON2 in regulating intramembrane cleavage by ROM4 and ROM5. Engagement of AMA1 with a peptide from RON2 that binds tightly in the AMA1 ligand binding site substantially reduced intramembrane shedding of AMA1 from the parasite surface. Ligand engagement also resulted in less phosphorylation of a particular serine residue in the AMA1 CD.

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A phosphomimetic mutant of this residue showed reduce invasion, suggesting that ligand-dependent dephophorylation of the AMA1 cytosolic tail is important for entry. This work supports a model in which AMA1 engagement of RON2 during invasion reduces intramembrane cleavage and activates outsidein signaling to preserve the TM connection of AMA1 in the MJ during invasion. Altogether the current model proposes that the intimate interaction between the ectodomain of AMA1 and RON2 build up the MJ and maintain the interaction between the parasite and host cell membrane, while the cytosolic tail of AMA1 contributes to internalization process (Fig. 14.2). Although the AMA1 and RON2 primary sequences differ among Apicomplexa, the AMA1-RON2 interaction is evolutionarily conserved in Plasmodium sp., with the same invasion-inhibitory effects of the orthologous region of PfRON2 domain (Lamarque et al., 2011; Vulliez-Le Normand et al., 2012) or of a peptide (R1 peptide) or an antibody which inhibit the PfAMA1
PfRON complex formation (Pizarro et al., 2005; Coley et al., 2006, 2007; Collins et al., 2009; Richard et al., 2010; Yang et al., 2017). No interspecies or intergeneric cross binding was seen, highlighting the separate coevolution of the AMA1-RON2 pair in Apicomplexa and the importance of maintaining this interaction for efficient invasion (Lamarque et al., 2011). By solving the costructure in both Toxoplasma and P. falciparum (Tonkin et al., 2011; Vulliez-Le Normand et al., 2012), researchers have defined the molecular basis of species specificity of the AMA1-RON2 pair. While the overall U-shaped architecture of RON2 in complex with AMA1 appears to be remarkably well maintained between the two parasites, clear specific features playing an influential role in species selectivity are visible in the cystine loop, which is the most divergent region within the RON2s. For example, the

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T. gondii cystine loop is two residues shorter which mirrors the narrower groove of AMA1. An Arg2041 residue, specific to the P. falciparum species, located at the tip of the β-hairpin with its guanidyl group, fits snugly into a preformed cavity of PfAMA1, which is also occupied by an arginine of the invasion-inhibitory immunoglobulin IgNAR141-1 (Henderson et al., 2007), a lysine of the inhibitory monoclonal antibody 1F9 (Coley et al., 2007), or of the R1 peptide (Vulliez-Le Normand et al., 2012). These inhibitors form an extensive interface with this region and impair the binding of the cystine loop of RON2. Key to the specificity of the AMA1
RON2 interaction is also an extended surface loop on AMA1 called the DII loop. This loop is very flexible and required to expose the mature RON2 binding groove (Tonkin et al., 2011; Vulliez-Le Normand et al., 2012) and serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove (Parker and Boulanger, 2015). The role of MJ RON proteins has been evaluated using forward genetics. RON2 and RON5 appear essential for the invasion process of tachyzoites (Beck et al., 2014; Lamarque et al., 2014); parasites lacking RON2 or RON5 are nonviable. In contrast, it has been possible to generate KO mutants for RON4, RON8 and RON4L1, indicating a less critical role during invasion for those proteins (Straub et al., 2011; Guerin et al., 2017b; Wang et al., 2017b); RON8 and RON4-depleted parasites exhibit 70% and 60% reduction of invasion, respectively (Straub et al., 2011; Guerin et al., 2017a; Wang et al., 2017b). For the RON4 mutant, the defect cannot be attributed solely to RON4 because the levels of RON5 and RON2 proteins were also significantly reduced in the mutant (Guerin et al., 2017a). Similarly, in absence of RON2 or RON5, the remaining MJ components RON2/ RON4/RON5 are not correctly targeted to the rhoptries and to the MJ, but successful invaders form a MJ decorated with RON4L1 and

RON8 proteins, supporting the existence of alternate coccidian-specific MJ complexes in tachyzoites of T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, and the remaining RON components form trails behind invading KO parasites, suggesting a role for RON8 in closing the vacuole. No obvious defect in invasion was observed for RON4L1 mutant, but RON4L1-depleted parasites are significantly impaired in virulence in mice (Guerin et al., 2017b). Whether this defect observed in vivo is linked to invasion of peculiar cell type remains to be investigated. Apart from being located at the MJ and playing role for invasion, the precise role of the RON4/RON4L1/RON5/RON8 proteins during invasion remained elusive until recently. Being exposed to the host cytoplasm, RON proteins are ideally positioned to interact with host proteins. A direct interaction of the Toxoplasma parasite rhoptry neck proteins RON2, RON4 and RON5 with host proteins has been described recently by Guerin et al. (2017a), illustrating how RONs subvert host function for invasion. They showed that RON2, RON4, and RON5 cooperate to actively accumulate the host proteins CIN85, CD2AP, and the endosomal sorting complexes required for transport (ESCRT)-I components ALIX and TSG101 to the MJ during invasion, using multiple binding sites within RON proteins. For instance, RON4 contains two binding sites that are responsible for direct binding with ALIX and three sites allowing interaction with CD2AP and CIN85, these two later host proteins being also recruited at the MJ through binding sites present within RON2 and RON5 sequences. Parasite mutants unable to recruit these host proteins show less efficient host cell invasion in culture and attenuated virulence in mice. Furthermore, additive interactions are necessary for efficient invasion, highlighting a cooperative role for these host proteins during invasion of fibroblasts. ALIX and TSG101

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participate in ESCRT-dependent membrane fission events, such as the formation of intraluminal vesicles, virus budding, cytokinesis, and membrane repair, but the rest of the ESCRT machinery is not present at the MJ (Guerin et al., 2017a), consistent with an ESCRT-independent function of ALIX and TSG101 at the MJ. The exact role of ALIX, TSG101, CIN85, and CD2AP at the MJ remains unclear, but these proteins are known to interact with each other and to act as adaptor proteins connecting the cortical cytoskeleton to the CDs of TM proteins involved in homotypic or heterotypic junctions (Dustin et al., 1998; Veiga and Cossart, 2005; Johnson et al., 2008; Tang and Brieher, 2013). In the context of MJ, they might physically bridge the RON proteins with cortical cytoskeleton to provide the tensile strength required during the internalization process. This function is supported by a role of host actin and tubulin during Toxoplasma invasion (Gonzalez et al., 2009; Sweeney et al., 2010). Among other possible function is the selective segregation of host proteins that accessed to the PV (Mordue et al., 1999a). As stated in Section 14.3.1, this process occurs at the MJ and likely contributes to avoid the fusion of the vacuole with endolysosomal host cell compartments. Finally, whether the junctional complex facilitates PV closure by PVM fusion remains to be investigated (Pavlou et al., 2018). 14.6.4.3.2 The rhoptry kinase family (ROPKs): effectors to disarm the host immune response

The ROPK superfamily, also termed ROP2 family (in reference to a representative member of the family), is the best characterized of the ROP effector proteins. These proteins are associated with the bulb part of the rhoptries. The family comprises more than B50 kinases and pseudokinases, also called “rhoptry kinases” (Peixoto et al., 2010; Talevich and Kannan, 2013). The ROP2 family was first described as three rhoptry proteins recognized by a single monoclonal antibody (Sadak et al., 1988). Additional members of the family have been

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successively identified using proteomics, in silico searches, integrative genomic analysis, and gene cloning to obtain a comprehensive view of the family that comprises to date at least 55 members in ME49 strain (Talevich and Kannan, 2013). ROPK genes are often present in expanded loci (gene duplication) (including ROP5 genes, ROP38/ROP29/ROP19 genes) (Peixoto et al., 2010; Reese et al., 2011). These proteins share several common features such as the presence in the C-terminus of a protein kinase-like fold, a similar size (50 kDa range), and a basic amino acid-rich N-terminal area (El Hajj et al., 2006, 2007a, 2007b; Saeij et al., 2006, 2007; Taylor et al., 2006; Peixoto et al., 2010). While annotated as ROPKs, some are not associated with rhoptries, such as ROP21, ROP22, ROP27, ROP33, ROP34, and ROP35 (Peixoto et al., 2010; Jones et al., 2017; Beraki et al., 2019). Complete assignation of each ROPK to a compartment in intracellular parasites awaits further experimentation. The N-terminal portion of the kinase domain encompassing the activation loop and substrate-binding site is the most conserved part. Many members are predicted to be active based on the putative presence of a complete catalytic triad (ROP18, 35, 31, 17, 21, 27, 30, 16, 25, 28, 20, 39, 38, 29, 19, 41, and 42) (Peixoto et al., 2010). However, about half the members of the ROPK family lack the glycine loops responsible for stabilization of the αβ-phosphate of ATP and the conserved aspartic acid in the catalytic loop critical for phosphotransferase activity. These degenerate members, including ROP2, ROP4, ROP5, ROP7, ROP8, and ROP54, are not expected to be enzymatically active and are referred to as pseudokinases. Crystallographic analysis reveals that ROP2, ROP5, and ROP18 maintain a conserved kinase fold with a unique regulatory domain, suggesting that these pseudokinases may function in scaffolding and/or sequestering substrates (Labesse et al., 2009; Qiu et al., 2009; Reese and Boothroyd, 2011).

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Although the ROPK family forms a single clade distinct from preiously characterized kinase families, phylogenetic clustering defined two main clades among Toxoplasma ROPKs (Talevich and Kannan, 2013). A specific subgroup was defined with a homologous Nterminal extension to the kinase domain (ROP2/8, ROP18, ROP17, and ROP5). The second divergent clade included the bradyzoiteexpressed pseudokinase BPK1, ROP33, ROP34, ROP35, and ROP46; two of these were recently experimentally assigned as DGs proteins [ROP34 (WNG2) and ROP35 (WNG1) (Beraki et al., 2019)]. Each clade comprises both predicted active kinases and pseudokinases, suggesting that pseudokinases have repeatedly emerged from ancestral active kinases rather than expansion of pseudokinases. The array of roles played by these kinases and pseudokinases has expanded considerably in the recent years, making them major players in the modulation of the host cell biology to promote a suitable environment for growth and proliferation under immune pressure (see details in Chapter 17: Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages). Upon secretion the ROPKs associate with the PVM or are targeted to the host cell where they play different functions. Parasitophorous vacuole membrane
associated ROPKs Rhoptry-derived vesicles [evacuoles

(Hakansson et al., 2001)] are secreted into the host cell cytoplasm. These vesicles, decorated with ROP1, fuse with the nascent PVM (Hakansson et al., 2001) and many ROPKs become exposed on the host cell side (Beckers et al., 1994; Carey et al., 2004a; Hajj et al., 2005; El Hajj et al., 2007a, 2007b; Kim et al., 2016). While ROP2, ROP4, and ROP5 were first described as a PVM-integral TM proteins (Beckers et al., 1994; El Hajj et al., 2007b), it has been shown that ROPKs are associated with membrane through arginine-rich-amphipathic

helices (RAH domain) conserved in the Nterminal part of the ROP2 family (Labesse et al., 2009; Reese and Boothroyd, 2009). This domain displays a preferential tropism for the PVM rather than host cell membranes and appears to be a good predictor of a Toxoplasma protein’s localization to the PVM. When ectopically expressed in the host cell, this domain does not associate to any specific membrane but is targeted to the PVM in cells infected by T. gondii (Labesse et al., 2009). As the PVM is largely composed of membrane derived from the host cell, the specificity of the RAH domain is probably due to an interaction with an unidentified parasite dependent factor, such as another ROP or a specific lipid associated with the PVM. So far, the most notable functions for ROP kinases and pseudokinases at the PVM (ROP5, ROP17, ROP18, and ROP54) are the disarming of GTP-binding proteins loaded onto the PVM upon interferon (INF)γ stimulation, a class of proteins used as the first line of defense against intracellular pathogens (Saeij et al., 2006; Taylor et al., 2006; El Hajj et al., 2007a; Fentress et al., 2010; Steinfeldt et al., 2010; Behnke et al., 2011; Reese et al., 2011) (for details see Chapter 17: Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages). The PVM-ROPKs are therefore important virulence factors in vivo. They are also used by the parasite to establish chronic infection of the host (Saeij et al., 2006; Taylor et al., 2006; Reese et al., 2011; Fox et al., 2016; Kim et al., 2016; Wang et al., 2017a). The polymorphisms and expression level in PVM-associated ROPKs also explain the pathogenicity differences among laboratory isolates but also account for variation in natural isolates of T. gondii (Saeij et al., 2006; Taylor et al., 2006; Reese et al., 2011; Behnke et al., 2012, 2015; Niedelman et al., 2012; Sanchez et al., 2014). ROPK targeted to the host nucleus By virtue of its direct access to the host cell nucleus,

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ROP16 is uniquely positioned to modulate changes in host transcription. ROP16 was identified as a virulence determinant, distinguishing how type I/III and II strains activate STAT3/6 during parasite infection and in turn the expression of genes involved in the immune response (Saeij et al., 2007). The effector role of ROP16 is described in Chapter 17, Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages. 14.6.4.3.3 Toxofilin: control of host cell actin polymerization

Toxofilin is a 27-kDa protein that has been shown to bind mammalian G-actin (Poupel et al., 2000). It is an actin-sequestering protein that caps actin filaments. Toxofilin was first suggested to be present in the cytosol of the apical end of the parasite and involved in control of parasite actin polymerization during invasion and motility (Poupel et al., 2000). Toxofilin was subsequently shown to localize within the rhoptries (Bradley et al., 2005), both in neck and the bulb (Delorme-Walker et al., 2012). It was also shown to be secreted into the host cell (Lodoen et al., 2010), where it upregulates the host cortical actin cytoskeleton dynamics facilitating Toxoplasma invasion (Delorme-Walker et al., 2012). In vitro, the control of actin dynamics by Toxofilin has been shown to depend on a casein kinase II and a phosphatase 2C (PP2C) (Delorme et al., 2003). 14.6.4.3.4 Other RONs/ROPs with less characterized functions

RON1, RON3, and RON6 are Apicomplexaspecific RON proteins identified in the proteome of rhoptries (Bradley et al., 2005). RON1 is a Sushi-containing protein (also called CCP for complement control protein), found in the neck of the rhoptry (Bradley et al., 2005). RON1 is conserved in all Apicomplexa parasites. Its ortholog in Plasmodium is called PfASP1 (O’Keeffe et al., 2005; Srivastava et al., 2010).

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The Sushi domain spans approximately 60 residues and contains four invariant cysteine residues. It is present in various complement regulator proteins found in mammals. The role played by RON1 is currently unknown. A highly stable hetero-complex formed by the association of RON9 and RON10 was identified in the rhoptry neck of tachyzoites. This complex is distinct from the MJ-complex (Lamarque et al., 2012). Both RON9 and RON10 are conserved in Coccidia and Cryptosporidia. RON10 does not display known domains, while RON9 contains a Sushi domain, repetitions of ankyrin motifs and a set of repetitions enriched in proline (P), glutamic acid (D), aspartic acid (E), and serine (S) or threonine (T) typical of PEST sequences, which are targets for rapid degradation, at least for cytosolic proteins. The ankyrin repeat is a common motif in nature, predominantly found in eukaryotic proteins and involved in protein
protein interactions. Genetic disruption of RON9 or RON10 lead to loss of the entire complex but does not result in defects in development in HFF in vitro or virulence in mice (Lamarque et al., 2012). The conservation of RON9/RON10 in the genome of Cryptosporidia, which does not form a MJ, argues against a participation of this complex in MJ formation. As the primary site of infection for Coccidia, as well as Cryptosporidium spp., is the epithelial cells of the gastrointestinal tract, further studies are needed to determine if the function of the RON9
10 complex might be linked to the interaction with a brush border membrane. RON12 was found in an in silico screen for rhoptry proteins (Camejo et al., 2014). It is a rhoptry neck protein specific to Toxoplasma and Neospora, with a molecular weight of 135 kDa (Camejo et al., 2014). It is cleaved to give a Cterminal product of 40 kDa. Accordingly, it contains a predicted cleavage site SPQE between amino acids 968 and 971. ROP1 was the first ROP protein identified in Toxoplasma (Schwartzman, 1986), encoded by

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the first rhoptry gene cloned, and sequenced (Ossorio et al., 1992). It is a soluble protein localizing to the rhoptry bulb whose function is as yet unknown. ROP1 has no recognizable domain. It is subjected to proteolytic maturation by ASP3 (Dogga et al., 2017). Although it has no TM anchor, it is associated with the PVM after invasion (Saffer et al., 1992) and is completely degraded within 24 hours suggesting that it has fulfilled its role by then. ROP1 KO parasites are not impaired in growth, invasion, or virulence but do show altered rhoptry ultrastructure (Kim et al., 1993; Soldati et al., 1995). Rhoptries are thinner and homogeneously electron-dense compared with the thicker and normally mottled or honeycombed appearance of wild-type rhoptries. Three different classes of protease have been found in the rhoptry lumen, a subtilisin-like (SUB2), a chymotrypsin-like (DegP), and a metalloprotease (Toxolysin-1). A cathepsin Blike protein named toxopain-1 or cathepsin protease B (CPB) was initially identified in T. gondii rhoptries and proposed to play a role in rhoptry protein processing and rhoptry biogenesis (Que et al., 2002). However, more recent findings (Dou et al., 2012) suggest that CPB is principally located in the lysosome-like vacuolar compartment (Parussini et al., 2010), and its targeted deletion did not affect rhoptry ultrastructure or the processing of ROP2 (Dou et al., 2012). A subtilisin-like serine protease or subtilase (SUB2) has been identified in T. gondii rhoptries by homology to P. falciparum SUB2 (Miller et al., 2003). SUB2 is predicted to be a type I TM protein with a conserved catalytic domain. It is autocatalytically processed at the Nterminus. Initially proposed to be the maturase of rhoptries proteins at SφXE/D consensus, this hypothesis has been recently genetically invalidated (Dogga et al., 2017). DegP is a rhoptry protein homologous to high temperature requirement A or Deg-like family of serine proteases that are serine proteases close to the chymotrypsin family. It was

initially identified in a proteomic study (Bradley et al., 2005) and was subsequently validated as a rhoptry bulb protein subjected to proteolytic maturation (Lentini et al., 2017). DegP is dispensable for the lytic cycle of both RH type I and Pru type II strains (Lentini et al., 2017). It appears important for the establishment of a lethal infection, but only in type II parasites. Immunodeficient mice are susceptible to infection with DegP-deficient parasites. Although its secretion into the host cell has not been visualized, these results suggest a role for this serine protease in immune evasion. An insulinase-like protein has been identified in the rhoptry fraction (Bradley et al., 2005) and named Toxolysin-1 (Hajagos et al., 2012). It belongs to the M16 family of metalloproteases that generally depend on divalent cations for their activity (Rawlings et al., 1991). The function of this insulinase is unknown, but it is not essential for the parasite (Hajagos et al., 2012). Toxolysin-1 is subjected to two different processing events. One is ASP3 dependent (as stated previously), the other separates the functional protease domain in two portions that remain tightly associated, in this way reconstituting a mature enzyme. PP2C-type protein phosphatases are monomeric enzymes present in both prokaryotes and eukaryotes. Members of this family of phosphatases are involved in the regulation of several signaling pathways including regulation of the cell cycle, adaptation and cell recovery after DNA double-strand breaks, or environmental-stress response (Schweighofer et al., 2004). The presence of a PP2C in rhoptries and targeted to the host cell nucleus was therefore very attractive (Bradley et al., 2005; Gilbert et al., 2007). However, PP2C is not an essential gene. Parasites knocked out for this phosphatase in the type I RH strain show only a mild growth defect in vitro and no virulence defect in mice (Gilbert et al., 2007). A straindependent function of PP2C had never been investigated.

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Bulb proteins ROP9, ROP10, ROP12, ROP13, ROP14, and ROP15 were identified in the proteome of rhoptries (Bradley et al., 2005). ROP10, 12, 13, and 15 are restricted to Toxoplasma and Neospora, while ROP9 and ROP14 are conserved apicomplexan proteins (Bradley et al., 2005). ROP13 is synthetized as a proprotein (Turetzky et al., 2010) matured by ASP3 (Dogga et al., 2017), and whose processing is not essential for traffic to rhoptries (Turetzky et al., 2010). ROP13 is a soluble effector protein ejected into the host cell, but in type I RH strain, it appears not essential for growth in fibroblasts in vitro or for virulence in vivo. ROP13 does not have any particular domains, but when exogenously expressed in human cells, it appears toxic, suggesting it might interfere with the host function (Turetzky et al., 2010). ROP9 contains a signal sequence but no recognizable domains, while ROP14 is a multipass TM protein, without signal sequence and containing a predicted lipase maturation factor. The genome of T. gondii contains several paralogs of ROP14. Rhoptry bulb proteins ROP47 and ROP48 were found in an in silico screen for rhoptry proteins (Camejo et al., 2014). ROP47 is a small protein (15 kDa) with no known conserved domains, giving no indication about its potential function. However, ROP47 is secreted and traffics to the host cell nucleus upon invasion (Camejo et al., 2014), suggesting that it might modulate the host cell response. Disruption of ROP47 gene in a type II strain does not impair the virulence of the parasite during acute or chronic phases, but since ROP47 is one of the most polymorphic Toxoplasma genes, it might play a role in control of immune response in other type strains. ROP48 has no particular domains. In the Prugnaud type II strain, it is not implicated in evading the INFγ response in MEF cells, or in acute or chronic infections. The characterization of EF-hand containing proteins revealed TGGT1_280480 as a rhoptry bulb protein (Chang et al., 2018). When

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disrupted, the mutant parasite grew at a slower rate than control cells. A search for potential substrates for ASP3 defined new rhoptry proteins named TAILS (Dogga et al., 2017). TAILS5 (TGGT1_273860) and TAILS8 (TGGT1_321650) are rhoptry neck proteins. TAILS5 contains a transmembrane domain (TMD) and a serine threonine kinase domain. The uncleaved form exhibits an altered distribution within the organelle. TAILS8 contains a signal sequence and then likely present in the lumen. TAILS6 (TGGT1_273860) is a rhoptry protein associated with the neck and bulb. It does not contain any TMDs or recognizable domains. TAILS7 (TGGT1_279420) is a rhoptry bulb protein that does not contain any TMDs but displays a TSP-1 domain.

14.6.5 Stage-specific expression of ROPs/RONs Virtually, all the information about the contents or functions of the rhoptries has come from the tachyzoite stage of T. gondii. Only a few stage-specific ROPs and RONs have been described (Schwarz et al., 2005; Fritz et al., 2012a, 2012b). The bradyzoite-specific rhoptry protein 1 (BRP1) was identified by a bioinformatic analysis of previously identified genes that are highly expressed during bradyzoite development and prediction of genes encoding secretory proteins (Cleary et al., 2002; Schwarz et al., 2005). BRP1 is also expressed in the merozoite stages in the gut of infected cats. The only homolog known is in the closely related parasite N. caninum. In vitro and in vivo analysis of BRP1 KO parasites show that BRP1 does not play an essential role in development of the bradyzoite stage, development of brain cysts, or oral infection of new hosts. The quantitative comparison of the transcriptomes and proteomic of three major developmental stages of Toxoplasma, tachyzoites,

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bradyzoites, and oocysts, has revealed a specific sporozoite-rhoptry neck protein 2 (sporoRON2, named also RON2L2) (Fritz et al., 2012a, 2012b), which is one of the two paralogs of RON2. The expression level of this paralog in both tachyzoites and bradyzoites is close to background (Fritz et al., 2012a). SporoRON2 localizes at the apical end of sporozoites and interacts exclusively with the microneme protein sporoAMA1, forming a functional AMA/ RON2 pair crucial for invasion of sporozoites (Poukchanski et al., 2013). Sporozoites also express a third complex formed by RON2L1 and AMA4 (Lamarque et al., 2014) that likely plays a role in invasion by sporozoites (Parker et al., 2016). Interestingly, AMA4 and RON2L1 are highly expressed in both merozoites and bradyzoites (ToxoDB), two stages that also invade enterocytes, suggesting a more specialized function for these proteins in entering the intestinal cells. While the MJ RONs are expressed in both tachyzoites and bradyzoites, they lack expression in the merozoite except RON4L1 gene, which has slight expression in the merozoite but remains downregulated as compared to the tachyzoite/bradyzoite stages (Behnke et al., 2014; Hehl et al., 2015). Moreover, in merozoites, most of the ROPs/ RONs are not expressed. Those that exhibit upregulated expression include ROP21, ROP32, ROP36, ROP42, ROP43, ROP46, and ROP50 (Behnke et al., 2014; Hehl et al., 2015). These observations strengthen the hypothesis of specialization for merozoite invasion and growth in intestine cells.

on a rhoptry-enriched fraction, suggested that these organelles are particularly enriched in cholesterol, with a cholesterol to phospholipid ratio .1 (Foussard et al., 1991) and phosphatidylcholine being the major rhoptry phospholipid. Significant amounts of PA and lysophospholipids were also found, but not phosphatidylserine, phosphatidylinositol, or sphingomyelin (Foussard et al., 1991). It was suggested that secretion of PA and lysophospholipids may facilitate initial vacuole membrane formation (Foussard et al., 1991). Reevaluation of the rhoptry-lipid content using HPLC and capillary GLC shows that that cholesterol is present in lower proportions (Besteiro et al., 2008). Consistent with this, cholesterol in the rhoptries does not appear to be essential for invasion, as parasites depleted of rhoptry cholesterol are still able to invade cells (Coppens and Joiner, 2003; Besteiro et al., 2008). An enrichment of saturated fatty acids has been also observed using these improved technological tools (Besteiro et al., 2008), suggesting an elevated rigidity for membranes derived from rhoptries. This was supported by a lower fluidity of rhoptry-derived membranes than membranes from whole T. gondii cells by fluorescence anisotropy (Besteiro et al., 2008). Overall, total membranes from rhoptries (possibly including internal membranous structure) appear to have high membrane rigidity.

14.7 Dense granules 14.7.1 The dense granule organelles

14.6.6 Rhoptry lipids T. gondii rhoptries not only contain proteins but also lipids, which may form the membranous structures sometimes observed in the organelles. Two studies have attempted to determine the lipid composition of rhoptries. The first one, using TLC and enzymatic assays

DGs are homogenous spherical electron dense vesicles B200 nm in diameter and enclosed by a unit membrane. These organelles are not present in all Apicomplexa. They have been only described in parasites that form tissue cysts, that is, the genera Toxoplasma, Neospora, Sarcocystis, Hammondia, and Besnoitia (Mercier and Cesbron-Delauw, 2015b). In Toxoplasma, the

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number of DGs varies between the different infectious stages. The largest numbers (B15) have been observed in the tachyzoites and sporozoites with intermediate numbers (8
10) in the bradyzoites and few (3
6) in the merozoites. This may correlate with the number of DG proteins (GRAs) expressed and the type of PV formed (discussed next). The existence of subpopulations of DGs storing specific GRAs was examined by double or triple labeling with specific antibodies. All DGs exhibited multiple labeling, showing localization of different GRAs within the same granules (Sibley et al., 1995; Ferguson et al., 1999a; Labruyere et al., 1999). However, facing the growing number of newly described GRA proteins whose subcellular localization is questioned, the existence of a second set of DGs cannot be excluded and has to be explored (see next) (Mercier and Cesbron-Delauw, 2015b; Nadipuram et al., 2016).

14.7.2 The dense granule proteins: GRAs and others Characterization of DG molecules started with the production of monoclonal antibodies against in vitro excreted-secreted antigens and subcellular fractionation of tachyzoites (CesbronDelauw et al., 1989; Charif et al., 1990; Leriche and Dubremetz, 1991). These DG proteins constitute a group of relatively small proteins, most of which contain an N-terminal hydrophobic sequence fitting the characteristics of a signal peptide (Table 14.3). Except for a few where a specific enzymatic or regulatory function has been defined (see next), the majority of DG proteins do not present any significant similarity to each other nor to proteins with known function. These were named GRA proteins solely on the basis of their subcellular localization within the DGs of the tachyzoite stage (Sibley et al., 1991). The list of DG proteins that have been characterized has grown steadily since Cesbron-

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Delauw et al. (1989), first described P23, which was later renamed GRA1 according to the nomenclature proposed by Sibley et al. (1991) (see Table 14.3). A large number of them were more recently identified by proteomic approaches using GRA13, 17, and 25 fused to the biotin ligase BirA* as baits, to purify biotinylated partners by streptavidin chromatography. This allowed the identification (by tandem mass spectrometry) of 13 novel GRA proteins (GRA28 to GRA40). All colabeled with GRA14 within the DGs and are secreted into the PV (Nadipuram et al., 2016). Thereafter, using GRA1 as bait for proximity biotin labeling, three novel GRA candidates were identified and colocalized with both GRA1 and GRA35 (Pan et al., 2019). Altogether, among a total of 43 known GRA proteins (Table 14.3), 32 are dubbed “canonical GRAs” in being colocalized in the same DGs as those containing the first described (i.e., GRA1, 2, or 3); the localization of some others still need to be confirmed (Mercier and Cesbron-Delauw, 2015a). GRA1 is an abundant protein (B2% of the total ESTs derived from a RH strain cDNA library; Ajioka et al., 1998) that is soluble and characterized by two predicted EF-hand domains (AA 149
180 and 197
223, respectively) whose calcium binding property was confirmed experimentally (Cesbron-Delauw et al., 1989). With the exception of GRA1 and GRA38
40, which are found soluble within the vacuole, the canonical GRA proteins are predicted to contain one hydrophobic α-helix or amphipathic α-helix(ces) that correlates with their postsecretory membrane association in the PV, that is, the TVN or the PVM (Table 14.3). Another common feature of GRA proteins is the low apparent molecular weight (20
75 kDa) of the monomeric form with the exception of GRA38
40, which are around 100 kDa (Table 14.3). Differences have been observed between their theoretical molecular weight, calculated from the amino acid

Toxoplasma Gondii

TABLE 14.3

Properties of Toxoplasma secretory proteins: dense granule (GRA) proteins.

GRA protein

ID in ToxoDB Release 43 (25.04.2019)

# AAa Signal Pb

GRA1

TGME49_270250

190

GRA2

TGME49_227620

185

Yes

Yes

H α
helixc

Localization PV/host cellsd

Transcriptomics during the parasite life cyclee

Not predicted

PV lumen PVE

High, maximum in TZ, then SZ and BZ

Hammondia

Not predicted

TVN PVE

High, maximum in TZ, then the BZ

Hammondia

Orthologs in Apicomplexaf

Neospora

Neospora

GRA3

TGME49_227280

220

Yes

160
178

PVM PVE

High in TZ and BZ

Hammondia, Neospora

GRA4

TGME49_310780

345

Yes

276
293

PV

High, in TZ and BZ

Hammondia, Neospora

GRA5

TGME49_286450

120

Yes

75
93

TVN

High, B constant

Not identified

GRA6

TGME49_275440

224

Yes

153
171

TVN PVE

High, maximum in TZ, then SZ

Hammondia, Neospora

GRA7

TGME49_203310

236

Yes

181
202

PVM PVE HOSTs

High, maximum in SZ and Hammondia TZ, then late BZ Neospora

GRA8

TGME49_254720

269

Yes

225
243

PVM

High, maximum in TZ and Neospora SZ

GRA9

TGME49_251540

318

Yes

Not predicted

TVN

Medium, maximum in BZ

Hammondia Neospora, Sarcocystis Eimeria

GRA10

TGME49_268900

894

Yes

Not predicted

PVM

Medium, maximum in SZ

Neospora, Eimeria, Cryptosporidium, Babesia, Theileria, Plasmodium

GRA11

TGME49_212410

752

Yes

114
135, 194
212, 299
315

Not do ne

Low, B constant

Not identified

GRA11 bis

TGME49_237800

471

Yes

112
133, 188
208, 282
300

Not done

Low, B constant

Not identified

GRA12

TGME49_288650

436

Yes

Not predicted

TVN

High, maximum in SZ and Hammondia TZ

GRA12bis TGME49_275850

419

Non-classical

203
226, 237
254

Not done

Low, constant

Neospora Eimeria

GRA13

TGME49_237880

164

Yes

Not predicted

Not done

High maximum in BZ, low Hammondia at SZ

GRA14

TGME49_239740

408

Yes

290
308

PVM PVE

High, maximum in SZ and Hammondia, Neospora then TZ

GRA15

TGME49_275470

550

Not predicted

54
72

PVM HCN

Low, maximum in TZ

Hammondia

GRA16

TGME49_208830

505

Yes

Not predicted

PV

Not reported

Hammondia

Not predicted

PVM

Medium, maximum in TZ and BZ

Neospora

GRA17

TGME49_222170

300

Yes

HCN

TVN (partial)

Hammondia

648

Yes

Not predicted

Not done

Medium, maximum at late Hammondia SZ, then TZ and BZ Neospora

TGME49_200010

413

Not predicted

Not predicted

PV

Medium, maximum in TZ and BZ

Hammondia, Neospora

GRA21

TGME49_241610

501

Yes

Not predicted

PV

Low, maximum in late BZ and the TZ

Neospora,

GRA22

TGME49_215220

629

Not predicted

39
62

PV

High to medium, maximum in SZ, then TZ and late BZ

Neospora, Eimeria

GRA23

TGME49_297880

219

Yes

Not predicted

PV

Medium, maximum in TZ and BZ

Plasmodium

GRA18

TGME49_288840

GRA19

Not identified

GRA20

Neospora Hammondia Sarcocystis

GRA24

TGME49_230180

542

Yes

Not predicted

PV, HCN

Medium, maximum in SZ

not identified

GRA25

TGME49_290700

315

Yes

123
140

PV

Medium, maximum in SZ and TZ

Neospora, Eimeria, Plasmodium (Continued)

TABLE 14.3

(Continued)

GRA protein

ID in ToxoDB Release 43 (25.04.2019)

GRA26

Not identified

GRA27

Not identified

GRA28

TGME49_231960

1904 Yes

GRA29

TGME49_269690

865

GRA30

TGME49_232000

# AAa Signal Pb

637

Yes

Yes

Localization PV/host cellsd

Transcriptomics during the parasite life cyclee

Orthologs in Apicomplexaf

Not predicted

HCN

Medium, maximum in SZ

Not identified

Not predicted

PV

Medium maximum in BZ, then TZ, low in SZ

Hammondia, Neospora

129
148

PV

Medium, maximum in SZ

Hammondia, Neospora

Hammondia, Neospora

H α
helixc

Sarcocystis

214
233 GRA31

TGME49_220240

526

Yes

79
101

PV

Medium to low maximum in TZ

GRA32

TGME49_212300

683

Yes

Not predicted

PV

High in TZ, medium in BZ Hammondia, Neospora and SZ Sarcocystis

GRA33

TGME49_247440

376

Yes

173
190

PV

High in TZ and late BZ, medium in SZ

Hammondia

211
233 303
225 GRA34

TGME49_203290

330

Yes

Not predicted

PV

High, TZ with maximum in BZ low in SZ

Hammondia, Neospora

GRA35

TGME49_226380

378

Yes

119
141

PVM

Medium, maximum in SZ

Hammondia, Neospora

GRA36

TGME49_213067

452

Yes

190
209

PV

Medium, maximum in BZ, Hammondia, Neospora TZ and SZ

GRA37

TGME49_236890

218

Not predicted

41
63

PV

High in TZ, BZ and SZ

Hammondia, Neospora

Note: 2 possible in frame start codons

149
168

PV lumen

Medium, maximum in TZ

Hammondia Neospora

GRA38

TGME49_312420

1086 Yes

Not predicted

Sarcocystis

Eimeria GRA39

GRA40

TGME49_289380

TGME49_219810

921

966

Yes

Not predicted Note: 2 possible in frame start codons

Not predicted

PV lumen

Not predicted

PV lumen

Low, B constant

Hammondia, Neospora Sarcocystis

Medium, B constant

Hammondia, Neospora

TVN

GRA41

No longer annotated in this release (LaFavers et al., 2017)

GRA42

TGME49_236870

214

Nonconventional

155
177

PV lumen

Not reported

Hammondia

GRA43

TGME49_237015

264

Nonconventional

129
148

PV lumen

Not reported

Hammondia

a

Predicted number of amino acids based on the ME49 ortholog. http://www.cbs.dtu.dk/services/SignalP/ and http://www.cbs.dtu.dk/services/SecretomeP/ available on the ExPASy server (http://www.expasy.org/proteomics). c http://embnet.vital-it.ch/software/TMPRED_form.html available on the ExPASy server (http://www.expasy.org/proteomics). d Postsecretory localization performed by immunofluorescence assay. e Expression Profiling of Oocyst, Tachyzoite, and Bradyzoite Development in the M4 strain from H. Fritz, K.R. Buchholz, P. Conrad, J.C. Boothroyd (Fritz et al., 2012a,b). ToxoDB v. 11.0. f ToxoDB. BZ, Bradyzoite; HCN, host cell nucleus; PV, parasitophorous vacuole; PVE, PV extensions; PVM, PV membrane; SZ, sporozoite; TZ, tachyzoite; TVN, tubulo-vesicular network, HOSTS, host organelle-sequestering tubulo-structures; B, almost. Canonical GRA are highlighted in boldface; that is, those that fit conventional criteria (Mercier and CesbronDelauw, 2015a,b). b

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

sequence, and the molecular weight of the native protein, as detected on SDS
PAGE of tachyzoite lysates. This suggests potential posttranslational modifications [see review (Mercier et al., 2005)]. Although numerous Nand O-glycosylation sites are predicted within the GRA amino acid sequences, only a few GRA proteins were shown to be O-glycosylated: GRA2 (Zinecker et al., 1998), GRA4 (Achbarou et al., 1991b), and GRA6 (Travier, Mercier et al., unpublished). Phosphorylation of DG proteins has been shown up to now, to be a postsecretory event occurring within the PV compartment for both GRA2 (Mercier et al., unpublished data) and GRA6 (Labruyere et al., 1999). This phosphorylation event is regulated by the WNG1 kinase, a DG protein secreted into the lumen of the PV (Beraki et al., 2019). Moreover, GRA4 and GRA8, which are typically proline-rich proteins (Carey et al., 2000), may be modified by the peptidyl prolyl cis-isomerase activity of cyclophilin-18 (Cy-18) that was found in both the DGs and the PV (see next) (High et al., 1994). Although other posttranslational modifications cannot be excluded, the relative richness of GRA proteins in charged amino acids and in proline residues also accounts for aberrant migrations (Rauscher and CesbronDelauw, unpublished), like it is the case for other highly charged proteins such as histones. The second group comprises several soluble DG proteins with known (or predictive) functions and therefore that have been named by their function. These include various enzymes, such as two NTPase isoforms, NTPase-I, and NTPase-II (E.C. 3.6.1.3), slightly different at the genetic level (97% of identity) and described in Toxoplasma tachyzoites and one in N. caninum (Asai et al., 1998). In vitro activity of these enzymes was extensively characterized, but their vacuolar function remains unclear (see next): Cathepsins CPC1 and CPC2 exhibit the typical catalytic site of cathepsins in their sequences (Que et al., 2007). Cyclophilins

(Cyp18 and Cyp20) exhibit in vitro peptidylprolyl cis/trans-isomerase (PPIase or rotamase) activity (High et al., 1994) that might help in the folding of proline-rich GRAs for better trafficking (Carey et al., 2000). In vivo, Cyp18 recruits cells and enhances the growth of host cells at the site of infection for maintenance of the interaction (Ibrahim et al., 2010). LCAT, a lipolytic lecithin-cholesterol acyltransferase of 83 kDa, is secreted by the parasite into the PV and contributes to parasite egress (Pszenny et al., 2016; Schultz and Carruthers, 2018). Unlike other LCAT enzymes, it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity (Pszenny et al., 2016). The DGs also contain serine protease inhibitors from the Kazal family (PI-1 and PI-2), the exact function in vivo of which remains unknown. In vitro, PI-1 is a broad-spectrum inhibitor capable of neutralizing trypsin, chymotrypsin, and elastase, and PI-2 appears to be specific for trypsin (Morris et al., 2002; Morris and Carruthers, 2003), Other DG proteins include (1) an osteopontin-like protein (OPN) (Cortez et al., 2008); (2) an inhibitor of STAT1dependent transcription (IST) (Gay et al., 2016; Olias et al., 2016); (3) a MAF1 expressed at both tachyzoite and bradyzoite stages (Pernas et al., 2014; Adomako-Ankomah et al., 2016); (4) a small family of kinases misannotated as ROP33, ROP34, and ROP35 and further dubbed WNG (With-No-Gly-Loop) given the lack of the glycine-rich or P-loop required for ATP binding in the active site of canonical kinases (Beraki et al., 2019); (5) the Toxoplasma E2F4-associated EZH2-inducing gene regulator (TEEGR) (Braun et al., 2019); independently reported as the Host Cyclin E inducer (HCE1) (Panas et al., 2019), and (6) the Myc-regulation-1 (MYR1) identified through a genetic screen for mutants defective in c-Myc regulation. MYR1 together with MYR2 and MYR3 form a complex essential for effector translocation across the PVM (Franco et al., 2016; Marino et al., 2018).

Toxoplasma Gondii

14.7 Dense granules

Finally, particular mention should be made for several “GRA” proteins that once secreted into the PV traverse the PVM to reach the host cell cytosol where they act as effectors modulating host pathways (see Chapter 17: Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages). These “GRA” proteins lack evidence for colocalization with historical DG proteins. Also, while canonical GRAs are highly constitutively expressed, these “GRA” are moderately expressed, in particular during cyst development (Mercier and Cesbron-Delauw, 2015a). It was thus hypothesized, but not yet proven, that they might be stored in a subset of DG population or another type of secretory vesicles (Mercier and Cesbron-Delauw, 2015a). Among these proteins are the effectors GRA16 (Bougdour et al., 2013), GRA24 (Braun et al., 2013), GRA18 (He et al., 2018), and TEEGR/HCE1 (Braun et al., 2019, Panas et al., 2019), which are targeted to the host cell. Unlike canonical GRA proteins, their secretion is dependent on both MYR1 and the aspartyl protease ASP5 (Hammoudi et al., 2015; Curt-Varesano et al., 2016; Coffey et al., 2018) that is a resident of the parasite Golgi (see next, Section 14.7.5). Therefore “GRA” proteins together with the MYR family and LCAT may belong to an emerging distinct subset of “DG” proteins.

14.7.3 Biogenesis of dense granules: features of both constitutive and regulated secretory pathways There are still several unresolved questions regarding both DG formation and the sorting of GRA proteins into this secretory organelle. In particular, whether DGs are functionally analogous to constitutive versus regulated secretory vesicles is not fully established. In other eukaryotic cells, sorting of proteins to constitutive versus regulated secretory pathways occurs in the

675

TGN. Proteins destined for regulated secretion aggregate and are packaged into immature secretory granules (ISG). Clathrin-coated vesicles bud from these ISGs and recycle proteins back to the TGN or endosomes, resulting in further concentration of the secretory proteins. The specific-coated proteins, identified at the TGN, include both the adaptor proteins AP1 and AP3, and adaptor-related proteins GGAs [for a review (Arvan and Castle, 1998)]. In Toxoplasma, immature DGs have never been observed. Moreover, transient and stable expression of several soluble reporter proteins in Toxoplasma showed that any soluble protein, provided it possesses a signal peptide, is delivered to DGs and later secreted into the PV. By contrast, addition of a GPI signal anchor targets the same reporter protein to the plasma membrane through transport vesicles (Karsten et al., 1998). Conversely, the SAG1 protein for which the GPI signal anchor domain has been deleted is routed to the PV via the DGs (Striepen et al., 1998). Thus DGs constitute the default constitutive pathway for soluble proteins in Toxoplasma. Morphologically, however, DGs resemble the dense core granules involved in regulated secretion in mammalian cells, indicating that retention and condensation of secretory products may occur during DG formation. The prevailing model of sorting by retention in higher organisms is the selective aggregation of regulated, but not constitutive secretory proteins, which limits the ability of the former to escape from maturing granules during the process of constitutive vesicle budding (Arvan and Castle, 1998). This condensation may be due to an inherent property of the regulated secretory proteins to aggregate via subtle changes in the forming granule, while trafficking through the different compartments of the secretory pathway. These changes include mild acidification or increasing concentration of bivalent cations such as calcium (Chanat and Huttner, 1991).

Toxoplasma Gondii

676

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Most of the GRA proteins are not intrinsically soluble and are predicted as TM proteins, a state which in turn occurs following their secretion into the PV (Lecordier et al., 1999; Gendrin et al., 2010). While TM surface proteins are targeted to the parasite plasma membrane via an alternative vesicular route (Gendrin et al., 2008), the TM domain-bearing GRA proteins are trafficked to the DG as solubilized aggregates (Sibley et al., 1995; Labruyere et al., 1999; Lecordier et al., 1999; Ruffiot and Cesbron-Delauw, unpublished data). These aggregates include high molecular weight complexes, in which GRA proteins interact together (Braun et al., 2008). The mechanism by which these proteins remain excluded from the endomembranous system is not still fully understood. It was shown that the length of the TM domain is critical in the segregation of membrane proteins to the DG versus the Golgi (Karsten et al., 2004). However, for both GRA5 and GRA6, it was further demonstrated that the N-terminal hydrophilic domain predominates over the TM domain in their DG targeting (Gendrin et al., 2008, 2010). It is plausible that Toxoplasma has developed security systems to ensure targeting of single-pass TM proteins to the DG using characteristics of both the TM domain and the Nt domain as sorting elements. Whether this mechanism relies on properties of these highly charged N-terminal sequences to spontaneously aggregate, that is, without any additional interaction of other cofactor, remains to be investigated. Since DGs have never been reported to constitute an acidic compartment, protein condensation is unlikely to result from a substantial decrease in pH. An AP-1 ortholog was localized at the transmost cisternae of the Golgi apparatus (Ngo et al., 2003) and despite the fact that YXXø motifs were localized in the cytoplasmic tails of two GRA proteins (GRA4 and 7), these were not recognized by the Toxoplasma AP-1 (Ngo et al., 2003). Given that GRA1, the most

abundant and essential DG product, is a soluble calcium binding protein (Cesbron-Delauw et al., 1989), a role for Ca21 in regulating aggregation remains a possibility.

14.7.4 Exocytosis of dense granules The secretion of DGs has been difficult to capture: fusion of the DG membrane with the parasite plasma membrane takes place subapically, at supposed gaps between the plates forming the IMC (Leriche and Dubremetz, 1990; Dubremetz et al., 1993). How the newly formed DG is trafficked from their site of synthesis at the Golgi to release sites at the parasite plasma membrane is not yet fully elucidated. They are transported from the Golgi to the parasite’s periphery by class 27 MyoF motors moving along filamentous actin (Heaslip et al., 2016). If the granule does not encounter a potential exit site between the inner membrane, the MyoF motors linked to the granule would initiate a directed run parallel to the IMC (Heaslip et al., 2016). DG secretion appears to respond to signals associated with both constitutive and regulated pathways of secretion. On the one hand, in favor of constitutive secretion, DG fusion with the target parasite plasma membrane is assisted by small GTPases of the Rab family and by soluble accessory factors [N-ethylmaleimide soluble factor (NSF), soluble NSFassociated protein receptor/soluble NSFassociated protein machinery (SNARE/SNAP)] (Chaturvedi et al., 1999). Also, DG exocytosis is not triggered by an increase in intracellular Ca21 concentration, which usually elicits fusion of mammalian dense core granules with the plasmalemma (Chaturvedi et al., 1999; Liendo and Joiner, 2000). On the other hand, several features are consistent with regulated secretion including (1) the burst of DG secretion into the PV occurring shortly after its formation, peaking within 10
30 minutes

Toxoplasma Gondii

14.7 Dense granules

postinvasion (Carruthers and Sibley, 1997); (2) that brefeldin A has no effect on the release of prestored GRAs (Coppens et al., 1999); and (3) that DG secretion is quantitatively and specifically induced by heat-inactivated serum (Coppens et al., 1999). Interestingly, the rise of cytosolic Ca21 concentration that triggers MIC protein secretion was shown to negatively control GRA secretion (Katris et al., 2019). This work showed that like micronemes, the calcium response of DGs is contingent of the calcium-dependent protein kinases CDPK1 and CDPK3. A reciprocal regulatory mechanism between MIC and GRA secretion provides an elegant way to tightly coordinate these two mutually exclusive secretory events during parasite invasion. Altogether, the two hypothesized mechanisms driving DG secretion (constitutive vs regulated) could coexist, and the type of secretion might be related to the compaction stage of DGs and/or to their secretion phase. The Ca21controlled DG discharge is likely to occur at the time of parasite invasion to form the nascent PV, whereas the constitutive pathway may be operative thereafter to ensure the continuous delivery of GRA proteins to the PV required by its enlargement during intracellular parasite development and used to modulate the host response.

14.7.5 Postsecretory trafficking of GRAs The involvement of GRAs in the maturation of the PV has been examined in detail during tachyzoite development. Shortly after invasion, the burst of DG secretion coincides with the specific structural changes of the PV (see Section 14.3.2). At this stage, only GRA1, 38, 39, and 40, PIs, and NTPases remain primarily in the lumen of the vacuole (Sibley et al., 1994, 1995; Pszenny et al., 2002; Morris and Carruthers, 2003; Nadipuram et al., 2016).

677

Based on immuno-electron microscopy, most of the GRAs are detected associated with the membranous system of the PV that includes the TVN, the HOST, and the PVM; GRA2, 4, 6, 9, and 12 are associated to the TVN (Charif et al., 1990; Dubremetz et al., 1993; Sibley et al., 1994; Lecordier et al., 1995; Bonhomme et al., 1998; Labruyere et al., 1999; Adjogble et al., 2004; Michelin et al., 2008). In the TVN membranes, GRA2, 4, and 6 are components of a multimeric protein complex (Labruyere et al., 1999) and their membrane association correlates to their phosphorylated state in a WNG1 kinase-dependent manner (Beraki et al., 2019). GRA3, 5, 7, 8, 10, 14, and 15 were preferentially detected at both the PVM and its membranous extensions within the host cell cytoplasm (Achbarou et al., 1991b; Dubremetz et al., 1993; Lecordier et al., 1993; Sinai et al., 1997; Bonhomme et al., 1998; Carey et al., 2000; Rome et al., 2008; Rosowski et al., 2011). Up to now, while GRA7 is the sole DG proteins described as specifically associated to the HOSTs (Coppens et al., 2006), GRA15 is the only one that is described to be associated to evacuoles and then targeted to host cell nucleus (Rosowski et al., 2011). The PV-targeted GRAs exhibit various types of membrane association. While both GRA1 and NTPases exhibit a very loose association to the TVN (Sibley et al., 1994, 1995), GRA4 is only displaced by urea treatment (Labruyere et al., 1999), suggesting an association based mainly on hydrogen bonds. In contrast, GRA2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 are only quantitatively displaced from their respective membranes by nonionic detergents, indicating membrane spanning domains that are stabilized by hydrophobic interactions. For both GRA2 (Mercier et al., 1998a) and GRA5 (Lecordier et al., 1999), their respective putative membrane domains (the GRA2 amphipathic α-helices and the GRA5 TM domain) were demonstrated to be responsible for membrane association. Furthermore, topologically, the

Toxoplasma Gondii

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

N-terminal domain of GRA5 is exposed to the host cell cytoplasm, whereas its C-terminal faces the lumen of the PV (Lecordier et al., 1999). This mechanism of posttranslational membrane insertion is unconventional and is not fully elucidated. In comparison to what is observed within the DGs, both the compaction state and the number of interactors of GRA complexes decreases following their secretion into the PV (Braun et al., 2008). This structural change is proposed to favor membrane association (Mercier and Cesbron-Delauw, unpublished; Braun et al., 2008). Moreover, the targeting of either GRA6 to the TVN or GRA5 to the PVM is essentially mediated by their respective N-terminal hydrophilic sequence (Gendrin et al., 2008, 2010). These studies led to the model that the selective targeting to either PVM or TVN relies on specific protein
protein or protein
lipid interactions between the N-terminal domain of GRAs and specific components of these membranes (Cesbron-Delauw et al., 2008). Even if the lipid composition of the different membranous compartments found within the PV remains unknown, their width differences (Magno et al., 2005a) could imply specificities in their lipid composition. One might thus suggest that specific interactions established between GRA proteins and a particular class of lipids could contribute to the specific protein targeting. Preliminary results, such as the specific interaction of GRA7 with phosphoinositides (Coppens et al., 2006) and of GRA6 Nterminal domain with negatively charged lipids (Gendrin et al., 2010), are congruous with this hypothesis. A growing number of DG proteins are targeted to the cytosolic face of the PVM or the host cell nucleus. So far, this is the case of MYR1, MAF1 (cytosolic face of the PVM), GRA18 (cytosol), GRA16, 24, TEEGR/HCE1, and IST (host cell nucleus). How these proteins are transported across the PVM and the mechanisms by which they are targeted to the

host cell nucleus are key questions to understand the host cell/parasite interaction. In Plasmodium, an amino-terminal motif called the Plasmodium export element (PEXEL) directs proteins into the export pathway (Hiller et al., 2004, Marti et al., 2004). It corresponds to the cleavage site of plasmepsin V (PfPMV), an integral membrane protein of the ER, allowing the release of a mature protein, which is competent for export through the PTEX translocon located at the PVM (Spillman et al., 2015). In Toxoplasma, the “PEXEL”-like motif observed in GRA16 was found to mediate its transport into the host cell (Hammoudi et al., 2015; CurtVaresano et al., 2016; Coffey et al., 2018). As mentioned above, this so-called TEXEL motif is cleaved by the aspartyl protease ASP5, a Golgiresident homolog of PfPMV. ASP5 is also implicated in the transport of GRA24 to the host cell nucleus, even if this later lacks TEXEL motif and is not proteolytically cleaved (Hammoudi et al., 2015; Curt-Varesano et al., 2016). It thus seems that, in Toxoplasma, two different routes of protein export may operate: one which is TEXEL-dependent allowing the transport of ASP5-cleaved proteins such as GRA16, 19, and 20, IST, LCAT, and Myr1 across the PV, and the second for the transport of GRA24, HCE1/TGEER, and MAF1 across the PVM that does not require cleavage by ASP5. The PVM-resident protein MYR1, which is crucial for the transport of GRA24 to the host cell nucleus (Franco et al., 2016) and forms a protein complex with MYR2 and MYR3, might be a part of a PVM-translocation machinery (Franco et al., 2016; Marino et al., 2018).

14.7.6 Dense granule protein function At the tachyzoite stage the burst of DG protein secretion into the PV following host cell invasion and their selective targeting within the PV compartment suggest that they might

Toxoplasma Gondii

14.7 Dense granules

contribute significantly to the structural organization of this new compartment and/or have important functions in PV metabolism (Dubremetz et al., 1993; Cesbron-Delauw et al., 1994; Carruthers and Sibley, 1997). 14.7.6.1 GRAs BLAST searches performed on GRA sequences did not reveal significant similarities between the GRAs and other proteins in the databases. However, motif searches indicated some potential biochemical properties. For example, two EF-hands predicted in GRA1 (AA 149
180 and 197
223) suggested calciumbinding activity, which was subsequently demonstrated (Cesbron-Delauw et al., 1989). Additional examples include an ATP/GTP binding site in GRA4 (AA 307
314), an ER binding domain in the C-terminal end of GRA3 (Henriquez et al., 2005), and a RGD adhesion motif in both GRA9 (AA 170
172) and GRA10 (AA 294
296) (Mercier et al., 2005). The construction of genetic KO parasites from the virulent RH background has provided clues on the function of several GRAs. Despite numerous attempts, a GRA1 KO has not been reported, suggesting that GRA1 may be essential for tachyzoite intracellular survival (Rommereim et al., 2016; Mercier and Cesbron-Delauw, unpublished). As mentioned previously, GRA1 is the most abundant EST (http://toxodb.org/News/ Item-3.0-newESTdata-Mf.shtml) and its Ca21binding properties suggest an important role in calcium homeostasis of the PV, or in assisting the packaging of other GRA proteins as proposed earlier. While GRA10 was found to be also refractory to gene deletion (Rommereim et al., 2016), the KO of GRA39 resulted in mutant parasites with a severe growth defect and the appearance of lipid deposits within the PV, suggesting an important role in lipid metabolism for parasite replication (Nadipuram et al., 2016). The disruption of other GRA genes was easily obtained showing that most of the GRA proteins

679

(GRA2
8, 11
14, 20, 21, 28
31, and 33
38) are not essential for the parasite survival in vitro (Rommereim et al., 2016; Bai et al., 2018). The loss of each single GRA gene does not affect the parasite replication rate; however, an effect on replication is seen in some double KOs including Δgra4-gra6, Δ a4-gra6s, and Δgra3-gra7 (Rommereim et al., 2016). Whether these proteins are involved in parasite virulence remains unclear due to contrasting results obtained for parasite virulence in infected mice (Mercier et al., 1998b; Alaganan et al., 2014; Shastri et al., 2014; Rommereim et al., 2016). This highlights the necessity for better standardization of infection procedures (i.e., mouse strain and route of inoculum) to draw conclusions. Disruption of genes encoding several TVNassociated GRAs has led to important findings on their role in the biogenesis of the TVN and consequently, on the function of the TVN itself. Hence, the observation of the Δgra2 and/or Δgra6 KOs by electron microscopy revealed that these proteins contribute to the formation of the TVN (Mercier et al., 1998b; Travier et al., 2008; Muniz-Hernandez et al., 2011; Lopez et al., 2015; Rommereim et al., 2016). GRA2, via its amphipathic alpha helices, induces the tubulation of the vesicular material observed at the posterior end of the parasite shortly after invasion. GRA6 further stabilizes these preformed membranous tubules (Mercier et al., 2002), probably via interaction of its Nterminal hydrophilic domain, which displays affinity for negatively charged phospholipids and is crucial for GRA6’s association with the TVN (Gendrin et al., 2010). Lastly, incubation of recombinant GRA2 and GRA6 with small unilamellar vesicles (SUVs) resulted in SUV deformation into membranous tubules resembling those observed within the PV (Lopez et al., 2015). It was further shown that the TVN formed by Δgra7 parasites was hyperdeveloped (Rommereim et al., 2016). Knowing that GRA7 deforms liposomes into tubular

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membranes (Coppens et al., 2006) and interacts with both GRA2 and GRA6 (Braun et al., 2008), these observations could indirectly indicate that the association of GRA7, 2 and 6 limits the TVN expansion within the PV. Together, these data provide a model wherein GRA2 initiates formation of the TVN, which is thereafter stabilized by GRA6 and modulated by GRA7. The deletion of gra2 and the subsequent destabilization of the TVN lead to several other phenotypes, including vacuoles containing parasites that are gradually discarded and egress erratically (MunizHernandez et al., 2011), and a decrease in the rate at which parasites ingest host cytosolic proteins, suggesting an implication of the TVN in heterophagy (Dou et al., 2014). The study of Δgra2 parasites also provided an interesting and unexpected role of the TVN in the modulation of adaptive immunity to T. gondii (Lopez et al., 2015). During infection of mice strain, CD81 T cells are activated by the recognition of a short antigenic peptide (HF10; 8
10 amino acids) located in the C-terminal sequence of GRA6 from type II parasites and presented at the surface of infected cells by MHC Class I molecules (Blanchard et al., 2008). In the PV formed by Δgra2 parasites, that is, in the absence of a normal TVN, the relocalization of GRA6 to the PVM was associated to an increase in the MHCClass I presentation of the HF10 peptide (Lopez et al., 2015). It is possible that an increased exposure of the GRA6 C-terminal domain to the host cell may facilitate its cleavage and release into the host cell cytosol before being processed by the proteasome (Blanchard et al., 2008; Lopez et al., 2015) or that the proximity of GRA6 with a translocation machinery embedded in the PVM (as stated previously) might facilitate the export of the HF10 peptide to the host cell cytosol and ER (Lopez et al., 2015). Finally, the TVN and calcium homeostasis was found to be associated the phenotype of Δgra41 parasites, which exhibit resistance to calcium ionophore A23187 and defects in both

calcium regulation and the timing of egress (LaFavers et al., 2017). The mechanism underlying the pleotropic function of GRA41 and its connection with the TVN is unclear. GRA41 is a TVN-associated protein but, unlike GRA1, does not present any conserved calcium binding domains and its recombinant form failed to bind calcium. Also, although the TVN produced by Δgra41 parasites is morphologically altered, other mutant parasites with a similarly defective TVN, for example, Δgra2 and Δgra6, appear to have normal calcium homeostasis and egress (LaFavers et al., 2017). Finally, whereas disruption of GRA22 results in early egress, it does not impact the TVN structure. Whether GRA22 and GRA41 interact with one another needs to be examined. Although the PVM is a barrier that protects parasites from fusion with endocytic compartments and host’s innate defense, it also provides an important physical interface allowing hostparasite exchanges. The KOs of several PVMassociated GRAs in the virulent RH background and have provided interesting molecular insight into the PVM function, particularly in the recruitment of host organelles and nutrient scavenging. Host cell lipids are scavenged and delivered into the PV to allow parasite growth and the maturation of both PVM and TVN (Coppens et al., 2006, 2000; Caffaro and Boothroyd, 2011). The host organelle-sequestering tubulo-structures (HOSTs) have been proposed as conduits for the internalization into the PV of hostderived endosomal vesicles enriched in cholesterol (Coppens et al., 2006). The disruption of the GRA7 gene has generated mutant parasites exhibiting the disappearance of HOSTs from the PV and an altered parasite morphology associated with decreased parasite replication when the growth in host cells maintained in culture medium depleted in lipids. Together with the ability of recombinant GRA7 to induce liposome tubulation, it was concluded that GRA7 is a main effector of HOST formation (Coppens et al., 2006).

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14.7 Dense granules

A similar reduced in vitro growth rate under starving conditions was also reported for both Δgra3 and Δgra14 (Craver and Knoll, 2007; Rome et al., 2008). GRA3, whose Cterminal sequence contains an ER retrieval motif and was found in a yeast two-hybrid assay to bind to the host ER calciummodulating ligand (Kim et al., 2008), was further identified among a set of proteins interacting with the host Golgi apparatus (Deffieu et al., 2019). Supporting this later finding, while Δgra3 parasites were not impaired in the recruitment of host ER or mitochondria (Craver and Knoll, 2007), conditional depletion of GRA3 impacted negatively the entry of host Golgi vesicles at the PVM (Deffieu et al., 2019). Host cell mitochondria tightly surround the PVM of type I and III parasites. This so-called host mitochondrial association (HMA) phenomenon has been proposed to be involved in the uptake of host nutrients. Various attempts to determine if PVM-associated protein(s) are involved in this process (Sinai and Joiner, 2001; Pernas et al., 2014) served as the foundation for the eventual discovery of MAF1, a DG protein targeted to the PVM (Pernas et al., 2014; Adomako-Ankomah et al., 2016). Specifically, HMA is mediated by the MAF1b1 paralog which is more abundantly expressed by type I and type III Toxoplasma strains compared to the type II strains, correlating with the observation that HMA is a type I and III
specific event (Pernas et al., 2014; Blank et al., 2018). Another feature of the PVM is its selective permeability, which allows via a putative pore, the passive diffusion of small molecules (Schwab et al., 1994; Desai and Rosenberg, 1997). GRA17, which displays 26% similarity with PfEXP2, a molecular-translocation channel described in the PVM of Plasmodium (de Koning-Ward et al., 2009), has been shown to function as a transporter of small molecules (,1300 Da) (Gold et al., 2015). Hence, the PV of Δgra17 mutants has an aberrant morphology and reduced permeability to small molecules

681

(Gold et al., 2015). GRA17 works in synergy with its paralog GRA23, as either hetero- or homo-multimeric complexes within the PVM (Gold et al., 2015). These are not likely to be involved in the translocation of effectors such as GRA16 and GRA24 across the PVM(Gold et al., 2015). As described previously, in Toxoplasma, a proper translocation system operates in the PVM for the export of effectors. It was discovered thanks to a genetic screen based on the search of mutants defective in host c-myc upregulation and in the export of GRA16 and GRA24 (Franco et al., 2016). This led to the characterization of the PVM-associated proteins, MYR1, which together with MYR2 and MYR3 form a complex that is essential for the export of effectors from the PV to the host cytosol (Marino et al., 2018). Although the overall export blockage obtained by fusing GRA16 to DHFR is in favor of a translocation machinery by analogy to other bacterial systems (Marino et al., 2018), the precise molecular mechanism involved in this process remains to be elucidated. 14.7.6.2 Other dense granule proteins Despite their homology with wellcharacterized proteins, the function of these DG proteins is not yet clearly established. The abundant NTPases are essential and display apyrase activity (Asai et al., 1995; Nakaar et al., 1999). Hence, in a primary model, as Toxoplasma is auxotroph for purines, it was postulated that the presence of vacuolar NTPases, as well as of a 50 -nucleotidase would allow stepwise degradation of ATP into ADP, AMP, and adenosine, with the latter being eventually transported across the parasite plasma membrane via a low affinity adenosine transporter (Stedman and Joiner, 1999). However, it was also demonstrated that in vitro, maximal activation of NTPases requires dithiols (Asai et al., 1983; Bermudes et al., 1994), and this enzyme is minimally

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

active in the PV (Silverman et al., 1998). Moreover, no 50 -nucleotidase activity was detected on the parasite surface suggesting that the parasite is incapable of converting AMP to adenosine for transport across the parasite plasma membrane (Ngo et al., 2000). Thus it appears that the initial hypothesis that NTPases are involved in purine salvage was incorrect. An alternative role for NTPases in parasite egress has been proposed. With the PVM becoming more permeable as parasites develop, intravacuolar activation of NTPases would occur just before parasite egress (Stommel et al., 1997; Silverman et al., 1998), thus resulting in depletion of host ATP, impairment of host ATP-dependent ion transporters, and activation of parasite motility due to ionic flux (Moudy et al., 2001). According to this model, and given their abundance, NTPase activity would be tightly regulated to avoid rapid depletion of host ATP, which would trigger premature egress of the parasite from the vacuole (Silverman et al., 1998). Among the serine protein inhibitors detected within the PV, PI-1 is a broadspectrum inhibitor that is capable of neutralizing trypsin, chymotrypsin, and elastase in vitro, whereas PI-2 appears to be specific for trypsin (Morris et al., 2002; Morris and Carruthers, 2003). The intravacuolar function of these inhibitors is unclear. Because both are active against digestive enzymes, PI-1 and PI-2, possibly released into the extracellular environment during the host cell lysis, may protect the parasite from proteases as it traverses the gastrointestinal tract (Morris et al., 2002). Although it was reported that PI-1 deficient parasites differentiate more readily (Pszenny et al., 2012), precisely how PI-1 regulates differentiation remains to be determined. Cyclophilins (CyPs) are highly conserved proteins associated with an in vitro peptidylprolyl cis/trans-isomerase (PPIase or rotamase) activity and are implicated more broadly in

mediating protein
protein interactions within large protein complexes. As such, CyP-18 could be involved in regulating the assembly of protein complexes within the DGs and/or the PV. In particular, GRA4 and/or GRA8 which are rich in proline residues could be potential substrates (Carey et al., 2000).

14.7.7 Stage-specific expression of dense granule proteins Based on the few studies available (immunofluorescence microscopy and microarray), bradyzoites and sporozoites express nearly the full tachyzoite repertoire of the “canonical” GRA proteins (Ferguson, 2004; Mercier et al., 2005). This is consistent with the “tachyzoitefate” of both bradyzoites and sporozoites into the host. The DG repertoire would be required to adapt the PV for optimal tachyzoite development and facilitate parasite proliferation in many cell types. In contrast, merozoites, which undergo limited proliferation and rapidly differentiate into the sexual stages in the enterocytes of the cat small intestine, express a very limited repertoire of DG proteins (Ferguson, 2004). 14.7.7.1 Bradyzoite tissue cyst and GRA proteins Bradyzoites, quiescent parasites formed in intracellular tissue cysts, are found within muscle cells and within cells of the central nervous system, predominantly neurons. The bradyzoite PV is limited by a unit membrane with numerous shallow invaginations (see Chapter 18: Bradyzoite and sexual stage development). An underlying layer of moderately electron dense fine granular material contributes to the wall of the tissue cyst (Ferguson et al., 1989). At the bradyzoite stage, DGs contain most of canonical GRAs identified in the tachyzoite (Ferguson, 2004), an observation that correlates with their gene expression being

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14.7 Dense granules

maintained during the bradyzoite stage (Table 14.3; Mercier and Cesbron-Delauw, 2015a), although there is evidence for reduced expression of NTPase (Nakaar et al., 1998; Ferguson et al., 1999a, 1999b). Furthermore, GRA4, GRA8, and NTPases were not detected within the cyst wall, which may be a consequence of their degradation or modification during encystment. All the other GRAs examined (GRA1, 2, 3, 5, 6, and 7) were shown by TEM associated to immunogold labeling, to be present in the cyst wall with a location reminiscent of that observed within the tachyzoite PV (Torpier et al., 1993; Ferguson, 2004). Evidence for DG protein trafficking beyond the cyst wall has only been reported for GRA7 (Fischer et al., 1998; Ferguson et al., 1999a). By contrast, effectors such as GRA16 and GRA24, which are secreted within the host cell at the tachyzoite stage, are not transported beyond the cyst wall membrane (Krishnamurthy and Saeij, 2018), indicating that modulation of host signaling pathway by such GRAs is restricted at the bradyzoite stage. The importance of the GRA proteins for the establishment of cyst burden has been demonstrated in the case of both GRA4 and GRA6. The isolation of a KU80 KO mutant (deficient in nonhomologous end joining) in an avirulent, type II cyst-forming strain allowed the subsequent deletion of GRA4 and/or GRA6. Noticeably, while no significant difference was observed in the in vitro growth rate between the mutants and the parental strain, dramatic reductions in cyst burdens (reduction by 91% in the case of single deletions and by 99% in the case of the double deletion) were observed 3 weeks postinfection in mice (Fox et al., 2011). However, the extent to which lower cyst burden might be due to effects during the acute infection was not tested. Mice infected with a Δgra39 mutant also harbored fewer brain cysts, but in this case the lower brain burden was likely due, at least in part, to defects during the acute infection (Nadipuram et al., 2016).

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Whether the lack in cyst burden of the mutants relies on a default in cyst formation or on an interference with the host response that would dramatically prevent parasite dissemination remains to be investigated. 14.7.7.2 Merozoite GRA proteins During the coccidian development of T. gondii in the enterocytes of the cat, the ultrastructural appearance of the PV is very different from that observed for the tachyzoite PV (Ferguson, 2004). The parasites are located in a tightly fitting PV limited by a thickened membrane with a laminated appearance consisting of three closely applied unit membranes. The PV lacks the TVN and there is no association of the host cell ER or mitochondria (Ferguson, 2004). At this stage, only two DG proteins, GRA7 and NTPase, are detected within the DGs. Both are released into the PV shortly after invasion, but their level of staining drops as the parasites mature (Ferguson et al., 1999a, 1999b). It is possible that merozoites might have stage-specific GRAs, but progress in understanding modifications of the merozoite PV would require the development of an in vitro culture system for the cat stages. 14.7.7.3 Sporozoite GRA proteins The DGs of the sporozoite appear to contain at least GRA1-8 with the exception of GRA3 and NTPase (Tilley et al., 1997). In vitro, sporozoites entering into a host cell form an unusual large vacuole (PV1) devoid of DG proteins (Tilley et al., 1997) but leave this compartment to enter a new vacuole (PV2) that more closely resembles a tachyzoite PV. Formation of PV2 is correlated with the secretion of the tachyzoite repertoire of DG proteins (Tilley et al., 1997). GRA3, GRA5, and NTPases are the first to be detected, followed by GRA1, 2, 4, and 6 (Tilley et al., 1997). While these studies have also been limited by the lack of an in vitro culture system for the cat stages, transcriptomic studies have profiled the expression of the GRA gene

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14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

repertoire at different time points of oocysts maturation (Fritz et al., 2012b) (Table 14.3). The results correlated with proteomic evidence that the repertoire of GRA genes is expressed at the sporozoite stage. They also showed that a few of GRA including GRA8 and GRA14 are upregulated compared either to the tachyzoite or the bradyzoite stages (Table 14.3).

14.8 Conclusion Modern genetic analyses in T. gondii including a complete genetic map, -omics analysis, efficient genetic transformation, and KO or knock-down of essential genes have greatly facilitated improvement of our knowledge of the contents and the functions played by secretory organelles. We know the key molecular actors of Toxoplasma invasion of its host, and these studies allowed comparison to Plasmodium, an apicomplexan parasite that is less amenable to genetic manipulation. While we know a lot about the kinetics of secretion and the function of secretion organelles, very little is known about the exocytic mechanisms. Important molecular details have been obtained for the calcium signaling
dependent process of micronemes secretion and the first clues about how DGs proteins are exported across the PVM to reach the host cell. In contrast, where and how secretory organelles fuse with the parasite plasma membrane and how rhoptry proteins cross the host cell membrane remain mysteries. Recent years have been marked by an explosion in the number of new ROPs and GRAs and report of how they hijack host organelles and modulate the host cell response. These proteins are usually specific to Toxoplasma and Neospora, which constitute the tissue cystforming branch of the enteric coccidians, and contribute to establish and maintain a latent infection to favor transmission between hosts. While most early studies focused on the

tachyzoite stage, recent studies address the function of effectors during chronic infection. Meanwhile, new tissue models and organoids hold promise for helping to defining the repertoire and function of secretory proteins used by sporozoites and sexual stages.

Acknowledgments We are grateful to Jean-Franc¸ois Dubremetz to critical review of the manuscript and to David Elliott for providing the electron micrograph shown in Fig. 14.1. We would like to thank Julien Marcetteau for the graphic design of the figures.

References Achbarou, A., Mercereau-Puijalon, O., Autheman, J.M., Fortier, B., Camus, D., Dubremetz, J.F., 1991a. Characterization of microneme proteins of Toxoplasma gondii. Mol. Biochem. Parasitol. 47, 223
233. Achbarou, A., Mercereau-Puijalon, O., Sadak, A., Fortier, B., Leriche, M.A., Camus, D., et al., 1991b. Differential targeting of dense granule proteins in the parasitophorous vacuole of Toxoplasma gondii. Parasitology 3, 321
329. Adjogble, K.D., Mercier, C., Dubremetz, J.F., Hucke, C., Mackenzie, C.R., Cesbron-Delauw, M.F., et al., 2004. GRA9, a new Toxoplasma gondii dense granule protein associated with the intravacuolar network of tubular membranes. Int. J. Parasitol. 34, 1255
1264. Adomako-Ankomah, Y., English, E.D., Danielson, J.J., Pernas, L.F., Parker, M.L., Boulanger, M.J., et al., 2016. Host mitochondrial association evolved in the human parasite Toxoplasma gondii via neofunctionalization of a gene duplicate. Genetics 203, 283
298. Agop-Nersesian, C., Naissant, B., Ben Rached, F., Rauch, M., Kretzschmar, A., Thiberge, S., et al., 2009. Rab11Acontrolled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis. PLoS Pathog. 5, e1000270. Ahn, H.J., Kim, S., Nam, H.W., 2006. Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii. Korean J. Parasitol. 44, 251
254. Aikawa, M., Miller, L.H., Johnson, J., Rabbege, J., 1978. Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite. J. Cell Biol. 77, 72
82. Aikawa, M., Miller, L.H., Rabbege, J.R., Epstein, N., 1981. Freeze-fracture study on the erythrocyte membrane during malarial parasite invasion. J. Cell Biol. 91, 55
62.

Toxoplasma Gondii

References

Ajioka, J.W., Boothroyd, J.C., Brunk, B.P., Hehl, A., Hillier, L., Manger, I.D., et al., 1998. Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa. Genome Res. 8, 18
28. Akhouri, R.R., Bhattacharyya, A., Pattnaik, P., Malhotra, P., Sharma, A., 2004. Structural and functional dissection of the adhesive domains of Plasmodium falciparum thrombospondin-related anonymous protein (TRAP). Biochem. J. 379, 815
822. Alaganan, A., Fentress, S.J., Tang, K., Wang, Q., Sibley, L. D., 2014. Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse. Proc. Natl. Acad. Sci. U.S.A. 111, 1126
1131. Alexander, D.L., Mital, J., Ward, G.E., Bradley, P., Boothroyd, J.C., 2005. Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog. 1, e17. Alexander, D.L., Kapur, S.A., Dubremetz, J.F., Boothroyd, J. C., 2006. Plasmodium falciparum AMA1 (PfAMA1) binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma. Eukaryot. Cell 5 (7), 1169
1173. Andenmatten, N., Egarter, S., Jackson, A.J., Jullien, N., Herman, J.P., Meissner, M., 2013. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods 10, 125
127. Antoine, J.C., Prina, E., Lang, T., Courret, N., 1998. The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages. Trends Microbiol. 6, 392
401. Appella, E., Weber, I.T., Blasi, F., 1988. Structure and function of epidermal growth factor-like regions in proteins. FEBS Lett. 231, 1
4. Arvan, P., Castle, D., 1998. Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem. J. 332, 593
610. Asai, T., O’Sullivan, W.J., Tatibana, M., 1983. A potent nucleoside triphosphate hydrolase from the parasitic protozoan Toxoplasma gondii. Purification, some properties, and activation by thiol compounds. J. Biol. Chem. 258, 6816
6822. Asai, T., Miura, S., Sibley, L.D., Okabayashi, H., Takeuchi, T., 1995. Biochemical and molecular characterization of nucleoside triphosphate hydrolase isozymes from the parasitic protozoan Toxoplasma gondii. J. Biol. Chem. 270, 11391
11397. Asai, T., Howe, D.K., Nakajima, K., Nozaki, T., Takeuchi, T., Sibley, L.D., 1998. Neospora caninum: tachyzoites express a potent type-I nucleoside triphosphate hydrolase. Exp. Parasitol. 90, 277
285.

685

Bai, T., Becker, M., Gupta, A., Strike, P., Murphy, V.J., Anders, R.F., et al., 2005. Structure of AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket. Proc. Natl. Acad. Sci. U.S.A. 102, 12736
12741. Bai, M.J., Wang, J.L., Elsheikha, H.M., Liang, Q.L., Chen, K., Nie, L.B., et al., 2018. Functional characterization of dense granule proteins in Toxoplasma gondii RH strain using CRISPR-Cas9 system. Front. Cell. Infect. Microbiol. 8, 300. Bandini, G., Leon, D.R., Hoppe, C.M., Zhang, Y., AgopNersesian, C., Shears, M.J., et al., 2019. O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites. J. Biol. Chem. 294, 1967
1983. Bannister, L.H., Mitchell, G.H., 1989. The fine structure of secretion by Plasmodium knowlesi merozoites during red cell invasion. J. Protozool. 36, 362
367. Bannister, L.H., Mitchell, G.H., Butcher, G.A., Dennis, E.D., 1986. Lamellar membranes associated with rhoptries in erythrocytic merozoites of Plasmodium knowlesi: a clue to the mechanism of invasion. Parasitology 92 (Pt 2), 291
303. Bargieri, D.Y., Andenmatten, N., Lagal, V., Thiberge, S., Whitelaw, J.A., Tardieux, I., et al., 2013. Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion. Nat. Commun. 4, 2552. Barondes, S.H., Castronovo, V., Cooper, D.N., Cummings, R.D., Drickamer, K., Feizi, T., et al., 1994. Galectins: a family of animal beta-galactoside-binding lectins. Cell 76, 597
598. Barragan, A., Brossier, F., Sibley, L.D., 2005. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell. Microbiol. 7, 561
568. Beck, J.R., Fung, C., Straub, K.W., Coppens, I., Vashisht, A. A., Wohlschlegel, J.A., et al., 2013. A Toxoplasma palmitoyl acyl transferase and the palmitoylated Armadillo Repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress. PLoS Pathog. 9, e1003162. Beck, J.R., Chen, A.L., Kim, E.W., Bradley, P.J., 2014. RON5 is critical for organization and function of the Toxoplasma moving junction complex. PLoS Pathog. 10, e1004025. Beckers, C.J., Dubremetz, J.F., Mercereau-Puijalon, O., Joiner, K.A., 1994. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J. Cell Biol. 127, 947
961.

Toxoplasma Gondii

686

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Behnke, M.S., Wootton, J.C., Lehmann, M.M., Radke, J.B., Lucas, O., Nawas, J., et al., 2010. Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5, e12354. Behnke, M.S., Khan, A., Wootton, J.C., Dubey, J.P., Tang, K., Sibley, L.D., 2011. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc. Natl. Acad. Sci. U.S. A. 108, 9631
9636. Behnke, M.S., Fentress, S.J., Mashayekhi, M., Li, L.X., Taylor, G.A., Sibley, L.D., 2012. The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18. PLoS Pathog. 8, e1002992. Behnke, M.S., Zhang, T.P., Dubey, J.P., Sibley, L.D., 2014. Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development. BMC Genomics 15, 350. Behnke, M.S., Khan, A., Lauron, E.J., Jimah, J.R., Wang, Q., Tolia, N.H., et al., 2015. Rhoptry proteins ROP5 and ROP18 are major murine virulence factors in genetically divergent South American strains of Toxoplasma gondii. PLoS Genet. 11, e1005434. Beraki, T., Hu, X., Broncel, M., Young, J.C., O’Shaughnessy, W.J., Borek, D., et al., 2019. Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole. Proc. Natl. Acad. Sci. U.S.A. 116, 6361
6370. Bermudes, D., Peck, K.R., Afifi, M.A., Beckers, C.J., Joiner, K.A., 1994. Tandemly repeated genes encode nucleoside triphosphate hydrolase isoforms secreted into the parasitophorous vacuole of Toxoplasma gondii. J. Biol. Chem. 269, 29252
29260. Besteiro, S., Bertrand-Michel, J., Lebrun, M., Vial, H., Dubremetz, J.F., 2008. Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries: further insights into the role of cholesterol. Biochem. J. 415, 87
96. Besteiro, S., Michelin, A., Poncet, J., Dubremetz, J.F., Lebrun, M., 2009. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 5, e1000309. Besteiro, S., Dubremetz, J.F., Lebrun, M., 2011. The moving junction of apicomplexan parasites: a key structure for invasion. Cell. Microbiol. 13, 797
805. Bichet, M., Joly, C., Henni, A., Guilbert, T., Xemard, M., Tafani, V., et al., 2014. The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force. BMC Biol. 12, 773. Bichet, M., Touquet, B., Gonzalez, V., Florent, I., Meissner, M., Tardieux, I., 2016. Genetic impairment of parasite

myosin motors uncovers the contribution of host cell membrane dynamics to Toxoplasma invasion forces. BMC Biol. 14, 97. Binder, E.M., Kim, K., 2004. Location, location, location: trafficking and function of secreted proteases of Toxoplasma and Plasmodium. Traffic 5, 914
924. Bisio, H., Lunghi, M., Brochet, M., Soldati-Favre, D., 2019. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. Nat. Microbiol. 4, 420
428. Blanchard, N., Gonzalez, F., Schaeffer, M., Joncker, N.T., Cheng, T., Shastri, A.J., et al., 2008. Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum. Nat. Immunol. 9, 937
944. Blank, M.L., Parker, M.L., Ramaswamy, R., Powell, C.J., English, E.D., Adomako-Ankomah, Y., et al., 2018. A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions. Mol. Microbiol. 108, 519
535. Blumenschein, T.M., Friedrich, N., Childs, R.A., Saouros, S., Carpenter, E.P., Campanero-Rhodes, M.A., et al., 2007. Atomic resolution insight into host cell recognition by Toxoplasma gondii. EMBO J. 26, 2808
2820. Bonhomme, A., Maine, G.T., Beorchia, A., Burlet, H., Aubert, D., Villena, I., et al., 1998. Quantitative immunolocalization of a P29 protein (GRA7), a new antigen of Toxoplasma gondii. J. Histochem. Cytochem. 46, 1411
1422. Bougdour, A., Durandau, E., Brenier-Pinchart, M.P., Ortet, P., Barakat, M., Kieffer, S., et al., 2013. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression. Cell Host Microbe 13, 489
500. Bradley, P.J., Boothroyd, J.C., 1999. Identification of the promature processing site of Toxoplasma ROP1 by mass spectrometry. Mol. Biochem. Parasitol. 100, 103
109. Bradley, P.J., Boothroyd, J.C., 2001. The pro region of Toxoplasma ROP1 is a rhoptry-targeting signal. Int. J. Parasitol. 31, 1177
1186. Bradley, P.J., Hsieh, C.L., Boothroyd, J.C., 2002. Unprocessed Toxoplasma ROP1 is effectively targeted and secreted into the nascent parasitophorous vacuole. Mol. Biochem. Parasitol. 125, 189
193. Bradley, P.J., Li, N., Boothroyd, J.C., 2004. A GFP-based motif-trap reveals a novel mechanism of targeting for the Toxoplasma ROP4 protein. Mol. Biochem. Parasitol. 137, 111
120. Bradley, P.J., Ward, C., Cheng, S.J., Alexander, D.L., Coller, S., Coombs, G.H., et al., 2005. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii. J. Biol. Chem. 280, 34245
34258.

Toxoplasma Gondii

References

Braun, L., Travier, L., Kieffer, S., Musset, K., Garin, J., Mercier, C., et al., 2008. Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway. Mol. Biochem. Parasitol. 157, 13
21. Braun, L., Brenier-Pinchart, M.P., Yogavel, M., CurtVaresano, A., Curt-Bertini, R.L., Hussain, T., et al., 2013. A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation. J. Exp. Med. 210, 2071
2086. Braun, L., Brenier-Pinchart, M.P., Hammoudi, P.M., Cannella, D., Kieffer-Jaquinod, S., Vollaire, J., et al., 2019. The Toxoplasma effector TEEGR promotes parasite persistence by modulating NF-kappaB signalling via EZH2. Nat. Microbiol . Available from: https://doi. org/10.1038/s41564-019-0431-8. Brecht, S., Carruthers, V.B., Ferguson, D.J., Giddings, O.K., Wang, G., Jakle, U., et al., 2001. The Toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. J. Biol. Chem. 276, 4119
4127. Breinich, M.S., Ferguson, D.J., Foth, B.J., van Dooren, G.G., Lebrun, M., Quon, D.V., et al., 2009. A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii. Curr. Biol. 19, 277
286. Brossier, F., Jewett, T.J., Sibley, L.D., Urban, S., 2005. A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc. Natl. Acad. Sci. U.S.A. 102, 4146
4151. Brossier, F., Starnes, G.L., Beatty, W.L., Sibley, L.D., 2008. Microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma gondii. Eukaryot. Cell 7, 664
674. Brown, P.J., Billington, K.J., Bumstead, J.M., Clark, J.D., Tomley, F.M., 2000. A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and plasma pre-kallikrein. Mol. Biochem. Parasitol. 107, 91
102. Brown, P.J., Gill, A.C., Nugent, P.G., McVey, J.H., Tomley, F.M., 2001. Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma prekallikrein and are members of the PAN module superfamily. FEBS Lett. 497, 31
38. Brydges, S.D., Sherman, G.D., Nockemann, S., Loyens, A., Daubener, W., Dubremetz, J.F., et al., 2000. Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii. Mol. Biochem. Parasitol. 111, 51
66. Brydges, S.D., Zhou, X.W., Huynh, M.H., Harper, J.M., Mital, J., Adjogble, K.D., et al., 2006. Targeted deletion of MIC5 enhances trimming proteolysis of Toxoplasma invasion proteins. Eukaryot. Cell 5, 2174
2183.

687

Brydges, S.D., Harper, J.M., Parussini, F., Coppens, I., Carruthers, V.B., 2007. A transient forward-targeting element for microneme-regulated secretion in Toxoplasma gondii. Biol. Cell 100, 253
264. Buchholz, K.R., Fritz, H.M., Chen, X., Durbin-Johnson, B., Rocke, D.M., Ferguson, D.J., et al., 2011. Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii bradyzoites. Eukaryot. Cell 10, 1637
1647. Buguliskis, J.S., Brossier, F., Shuman, J., Sibley, L.D., 2010. Rhomboid 4 (ROM4) affects the processing of surface adhesins and facilitates host cell invasion by Toxoplasma gondii. PLoS Pathog. 6, e1000858. Bullen, H.E., Jia, Y., Yamaryo-Botte, Y., Bisio, H., Zhang, O., Jemelin, N.K., et al., 2016. Phosphatidic acidmediated signaling regulates microneme secretion in Toxoplasma. Cell Host Microbe 19, 349
360. Cabrera, A., Herrmann, S., Warszta, D., Santos, J.M., John Peter, A.T., Kono, M., et al., 2012. Dissection of minimal sequence requirements for rhoptry membrane targeting in the malaria parasite. Traffic 13, 1335
1350. Caffaro, C.E., Boothroyd, J.C., 2011. Evidence for host cells as the major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells. Eukaryot. Cell 10, 1095
1099. Caldas, L.A., Attias, M., de Souza, W., 2009. Dynamin inhibitor impairs Toxoplasma gondii invasion. FEMS Microbiol. Lett. 301, 103
108. Camejo, A., Gold, D.A., Lu, D., McFetridge, K., Julien, L., Yang, N., et al., 2014. Identification of three novel Toxoplasma gondii rhoptry proteins. Int. J. Parasitol. 44, 147
160. Carey, K.L., Donahue, C.G., Ward, G.E., 2000. Identification and molecular characterization of GRA8, a novel, proline-rich, dense granule protein of Toxoplasma gondii. Mol. Biochem. Parasitol. 105, 25
37. Carey, K.L., Jongco, A.M., Kim, K., Ward, G.E., 2004a. The Toxoplasma gondii rhoptry protein ROP4 is secreted into the parasitophorous vacuole and becomes phosphorylated in infected cells. Eukaryot. Cell 3, 1320
1330. Carey, K.L., Westwood, N.J., Mitchison, T.J., Ward, G.E., 2004b. A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 101, 7433
7438. Carruthers, V.B., 2019. Interrupting Toxoplasma’s regularly scheduled program of egress. Trends Parasitol. Available from: https://doi.org/10.1016/j.pt.2019.03.007. Carruthers, V.B., Sibley, L.D., 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur. J. Cell Biol. 73, 114
123.

Toxoplasma Gondii

688

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Carruthers, V.B., Sibley, L.D., 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol. Microbiol. 31, 421
428. Carruthers, V.B., Giddings, O.K., Sibley, L.D., 1999. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell. Microbiol. 1, 225
235. Carruthers, V.B., Sherman, G.D., Sibley, L.D., 2000. The Toxoplasma adhesive protein MIC2 is proteolytically processed at multiple sites by two parasite-derived proteases. J. Biol. Chem. 275, 14346
14353. Cerede, O., Dubremetz, J.F., Bout, D., Lebrun, M., 2002. The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin. EMBO J. 21, 2526
2536. Cerede, O., Dubremetz, J.F., Soete, M., Deslee, D., Vial, H., Bout, D., et al., 2005. Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J. Exp. Med. 201, 453
463. Cesbron-Delauw, M.F., Guy, B., Torpier, G., Pierce, R.J., Lenzen, G., Cesbron, J.Y., et al., 1989. Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 86, 7537
7541. Cesbron-Delauw, M.F., Gendrin, C., Travier, L., Ruffiot, P., Mercier, C., 2008. Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole. Traffic 9, 657
664. Cesbron-Delauw, M.F., Tomavo, S., Beauchamps, P., Fourmaux, M.P., Camus, D., Capron, A., et al., 1994. Similarities between the primary structures of two distinct major surface proteins of Toxoplasma gondii. J. Biol. Chem. 269, 16217
16222. Chanat, E., Huttner, W.B., 1991. Milieu-induced, selective aggregation of regulated secretory proteins in the transGolgi network. J. Cell Biol. 115, 1505
1519. Chang, L., Dykes, E.J., Li, J., Moreno, S.N.J., Hortua Triana, M.A., 2018. Characterization of two EF-hand domaincontaining proteins from Toxoplasma gondii. J. Eukaryot. Microbiol. Available from: https://doi.org/10.1111/ jeu.12675. Charif, H., Darcy, F., Torpier, G., Cesbron-Delauw, M.F., Capron, A., 1990. Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites. Exp. Parasitol. 71, 114
124. Charron, A.J., Sibley, L.D., 2004. Molecular partitioning during host cell penetration by Toxoplasma gondii. Traffic 5, 855
867. Chasen, N.M., Asady, B., Lemgruber, L., Vommaro, R.C., Kissinger, J.C., Coppens, I., et al., 2017. A glycosylphosphatidylinositol-anchored carbonic anhydrase-related protein of Toxoplasma gondii is important for rhoptry biogenesis and virulence. mSphere 2. Available from: https://doi.org/10.1128/ mSphere.00027-17.

Chattopadhyay, R., Rathore, D., Fujioka, H., Kumar, S., de la Vega, P., Haynes, D., et al., 2003. PfSPATR, a Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies. J. Biol. Chem. 278, 25977
25981. Chaturvedi, S., Qi, H., Coleman, D., Rodriguez, A., Hanson, P.I., Striepen, B., et al., 1999. Constitutive calcium-independent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab machinery. J. Biol. Chem. 274, 2424
2431. Chen, W., Feng, Y., Chen, D., Wandinger-Ness, A., 1998. Rab11 is required for trans-Golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. Mol. Biol. Cell 9, 3241
3257. Chen, H., Herndon, M.E., Lawler, J., 2000. The cell biology of thrombospondin-1. Matrix Biol. 19, 597
614. Chen, Z., Harb, O.S., Roos, D.S., 2008. In silico identification of specialized secretory-organelle proteins in apicomplexan parasites and in vivo validation in Toxoplasma gondii. PLoS One 3, e3611. Clarke, L.E., Tomley, F.M., Wisher, M.H., Foulds, I.J., Boursnell, M.E., 1990. Regions of an Eimeria tenella antigen contain sequences which are conserved in circumsporozoite proteins from Plasmodium spp. and which are related to the thrombospondin gene family. Mol. Biochem. Parasitol. 41, 269
279. Cleary, M.D., Singh, U., Blader, I.J., Brewer, J.L., Boothroyd, J.C., 2002. Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryot. Cell 1, 329
340. Coffey, M.J., Dagley, L.F., Seizova, S., Kapp, E.A., Infusini, G., Roos, D.S., et al., 2018. Aspartyl protease 5 matures dense granule proteins that reside at the host-parasite interface in Toxoplasma gondii. MBio 9. Available from: https://doi.org/10.1128/mBio.01796-18. Coleman, B.I., Saha, S., Sato, S., Engelberg, K., Ferguson, D. J.P., Coppens, I., et al., 2018. A member of the ferlin calcium sensor family is essential for Toxoplasma gondii rhoptry secretion. MBio 9. Available from: https://doi. org/10.1128/mBio.01510-18. Coley, A.M., Parisi, K., Masciantonio, R., Hoeck, J., Casey, J.L., Murphy, V.J., et al., 2006. The most polymorphic residue on Plasmodium falciparum apical membrane antigen 1 determines binding of an invasion-inhibitory antibody. Infect. Immun. 74, 2628
2636. Coley, A.M., Gupta, A., Murphy, V.J., Bai, T., Kim, H., Foley, M., et al., 2007. Structure of the malaria antigen AMA1 in complex with a growth-inhibitory antibody. PLoS Pathog. 3, 1308
1319. Collins, C.R., Withers-Martinez, C., Hackett, F., Blackman, M.J., 2009. An inhibitory antibody blocks interactions between components of the malarial invasion machinery. PLoS Pathog. 5, e1000273.

Toxoplasma Gondii

References

Conseil, V., Soete, M., Dubremetz, J.F., 1999. Serine protease inhibitors block invasion of host cells by Toxoplasma gondii. Antimicrob. Agents Chemother. 43, 1358
1361. Coppens, I., Joiner, K.A., 2003. Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge. Mol. Biol. Cell 14, 3804
3820. Coppens, I., Sinai, A.P., Joiner, K.A., 2000. Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J. Cell Biol. 149, 167
180. Coppens, I., Andries, M., Liu, J.L., Cesbron-Delauw, M.F., 1999. Intracellular trafficking of dense granule proteins in Toxoplasma gondii and experimental evidences for a regulated exocytosis. Eur. J. Cell Biol. 78, 463
472. Coppens, I., Dunn, J.D., Romano, J.D., Pypaert, M., Zhang, H., Boothroyd, J.C., et al., 2006. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space. Cell 125, 261
274. Cortez, E., Stumbo, A.C., Saldanha-Gama, R., Villela, C.G., Barja-Fidalgo, C., Rodrigues, C.A., et al., 2008. Immunolocalization of an osteopontin-like protein in dense granules of Toxoplasma gondii tachyzoites and its association with the parasitophorous vacuole. Micron 39, 25
31. Craver, M.P., Knoll, L.J., 2007. Increased efficiency of homologous recombination in Toxoplasma gondii dense granule protein 3 demonstrates that GRA3 is not necessary in cell culture but does contribute to virulence. Mol. Biochem. Parasitol. 153, 149
157. Crawford, J., Tonkin, M.L., Grujic, O., Boulanger, M.J., 2010. Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii. J. Biol. Chem. 285, 15644
15652. Curt-Varesano, A., Braun, L., Ranquet, C., Hakimi, M.A., Bougdour, A., 2016. The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells. Cell. Microbiol. 18, 151
167. Davis, C.G., 1990. The many faces of epidermal growth factor repeats. New Biol. 2, 410
419. de Koning-Ward, T.F., Gilson, P.R., Boddey, J.A., Rug, M., Smith, B.J., Papenfuss, A.T., et al., 2009. A newly discovered protein export machine in malaria parasites. Nature 459, 945
949. de Souza, W., Attias, M., 2015. New views of the Toxoplasma gondii parasitophorous vacuole as revealed by helium ion microscopy (HIM). J. Struct. Biol. 191, 76
85. Deffieu, M.S., Alayi, T.D., Slomianny, C., Tomavo, S., 2019. The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport. Biol. Open 8. Available from: https://doi. org/10.1242/bio.039818. Delorme-Walker, V., Abrivard, M., Lagal, V., Anderson, K., Perazzi, A., Gonzalez, V., et al., 2012. Toxofilin

689

upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion. J. Cell Sci. 125, 4333
4342. Delorme, V., Cayla, X., Faure, G., Garcia, A., Tardieux, I., 2003. Actin dynamics is controlled by a casein kinase II and phosphatase 2C interplay on Toxoplasma gondii Toxofilin. Mol. Biol. Cell 14, 1900
1912. Desai, S.A., Rosenberg, R.L., 1997. Pore size of the malaria parasite’s nutrient channel. Proc. Natl. Acad. Sci. U.S.A. 94, 2045
2049. Dobrowolski, J.M., Carruthers, V.B., Sibley, L.D., 1997. Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii. Mol. Microbiol. 26, 163
173. Dogga, S.K., Mukherjee, B., Jacot, D., Kockmann, T., Molino, L., Hammoudi, P.M., et al., 2017. A druggable secretory protein maturase of Toxoplasma essential for invasion and egress. eLife 6. Available from: https:// doi.org/10.7554/eLife.27480. Donahue, C.G., Carruthers, V.B., Gilk, S.D., Ward, G.E., 2000. The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels. Mol. Biochem. Parasitol. 111, 15
30. Dou, Z., Coppens, I., Carruthers, V.B., 2012. Non-canonical maturation of two papain-family proteases in Toxoplasma gondii. J. Biol. Chem . Available from: https://doi.org/ 10.1074/jbc.M112.443697. Dou, Z., McGovern, O.L., Di Cristina, M., Carruthers, V.B., 2014. Toxoplasma gondii ingests and digests host cytosolic proteins. MBio 5, e01188-01114. Dowse, T.J., Soldati, D., 2005. Rhomboid-like proteins in Apicomplexa: phylogeny and nomenclature. Trends Parasitol. 21, 254
258. Dowse, T.J., Pascall, J.C., Brown, K.D., Soldati, D., 2005. Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion. Int. J. Parasitol. 35, 747
756. Dubois, D.J., Soldati-Favre, D., 2019. Biogenesis and secretion of micronemes in Toxoplasma gondii. Cell. Microbiol. e13018. Dubremetz, J.F., 2007. Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction. Cell. Microbiol. 9, 841
848. Dubremetz, J.F., Ferreira, E., 1978. Capping of cationized ferritin by coccidian zoites. J. Protozool. 25, 9B. Dubremetz, J.F., Achbarou, A., Bermudes, D., Joiner, K.A., 1993. Kinetics and pattern of organelle exocytosis during Toxoplasma gondii/host-cell interaction. Parasitol. Res. 79, 402
408. Dundas, K., Shears, M.J., Sun, Y., Hopp, C.S., Crosnier, C., Metcalf, T., et al., 2018. Alpha-v-containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP. Proc. Natl. Acad. Sci. U.S.A. 115, 4477
4482.

Toxoplasma Gondii

690

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Dunn, J.D., Ravindran, S., Kim, S.K., Boothroyd, J.C., 2008. The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4. Infect. Immun. 76, 5853
5861. Dustin, M.L., Olszowy, M.W., Holdorf, A.D., Li, J., Bromley, S., Desai, N., et al., 1998. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94, 667
677. Dvorak, J.A., Miller, L.H., Whitehouse, W.C., Shiroishi, T., 1975. Invasion of erythrocytes by malaria merozoites. Science 187, 748
750. Egarter, S., Andenmatten, N., Jackson, A.J., Whitelaw, J.A., Pall, G., Black, J.A., et al., 2014. The Toxoplasma ActoMyoA motor complex is important but not essential for gliding motility and host cell invasion. PLoS One 9, e91819. El Hajj, H., Demey, E., Poncet, J., Lebrun, M., Wu, B., Galeotti, N., et al., 2006. The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling. Proteomics 6, 5773
5784. El Hajj, H., Lebrun, M., Arold, S.T., Vial, H., Labesse, G., Dubremetz, J.F., 2007a. ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii. PLoS Pathog. 3, e14. El Hajj, H., Lebrun, M., Fourmaux, M.N., Vial, H., Dubremetz, J.F., 2007b. Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane. Cell. Microbiol. 9, 54
64. El Hajj, H., Papoin, J., Cerede, O., Garcia-Reguet, N., Soete, M., Dubremetz, J.F., et al., 2008. Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes. Eukaryot. Cell 7, 1019
1028. Etheridge, R.D., Alaganan, A., Tang, K., Lou, H.J., Turk, B. E., Sibley, L.D., 2014. The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice. Cell Host Microbe 15, 537
550. Farrell, A., Thirugnanam, S., Lorestani, A., Dvorin, J.D., Eidell, K.P., Ferguson, D.J., et al., 2012. A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis. Science 335, 218
221. Fentress, S.J., Behnke, M.S., Dunay, I.R., Mashayekhi, M., Rommereim, L.M., Fox, B.A., et al., 2010. Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence. Cell Host Microbe 8, 484
495.

Ferguson, D.J., 2004. Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. Int. J. Parasitol. 34, 347
360. Ferguson, D.J., Hutchison, W.M., Pettersen, E., 1989. Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study. Parasitol. Res. 75, 599
603. Ferguson, D.J., Cesbron-Delauw, M.F., Dubremetz, J.F., Sibley, L.D., Joiner, K.A., Wright, S., 1999a. The expression and distribution of dense granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Exp. Parasitol. 91, 203
211. Ferguson, D.J., Jacobs, D., Saman, E., Dubremetz, J.F., Wright, S.E., 1999b. In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii. Parasitology 119, 259
265. Finlay, B.B., Cossart, P., 1997. Exploitation of mammalian host cell functions by bacterial pathogens. Science 276, 718
725. Fischer, H.G., Stachelhaus, S., Sahm, M., Meyer, H.E., Reichmann, G., 1998. GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by infected host cells. Mol. Biochem. Parasitol. 91, 251
262. Fourmaux, M.N., Achbarou, A., Mercereau-Puijalon, O., Biderre, C., Briche, I., Loyens, A., et al., 1996a. The MIC1 microneme protein of Toxoplasma gondii contains a duplicated receptor-like domain and binds to host cell surface. Mol. Biochem. Parasitol. 83, 201
210. Fourmaux, M.N., Garcia-Reguet, N., Mercereau-Puijalon, O., Dubremetz, J.F., 1996b. Toxoplasma gondii microneme proteins: gene cloning and possible function. Curr. Top. Microbiol. Immunol. 219, 55
58. Foussard, F., Leriche, M.A., Dubremetz, J.F., 1991. Characterization of the lipid content of Toxoplasma gondii rhoptries. Parasitology 102 (Pt 3), 367
370. Fox, B.A., Falla, A., Rommereim, L.M., Tomita, T., Gigley, J. P., Mercier, C., et al., 2011. Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryot. Cell 10, 1193
1206. Fox, B.A., Rommereim, L.M., Guevara, R.B., Falla, A., Hortua Triana, M.A., Sun, Y., et al., 2016. The Toxoplasma gondii rhoptry kinome is essential for chronic infection. MBio 7. Available from: https://doi. org/10.1128/mBio.00193-16.

Toxoplasma Gondii

References

Franco, M., Panas, M.W., Marino, N.D., Lee, M.C., Buchholz, K.R., Kelly, F.D., et al., 2016. A novel secreted protein, MYR1, is central to Toxoplasma’s manipulation of host cells. MBio 7, e02231-02215. Fraser, T.S., Kappe, S.H., Narum, D.L., VanBuskirk, K.M., Adams, J.H., 2001. Erythrocyte-binding activity of Plasmodium yoelii apical membrane antigen-1 expressed on the surface of transfected COS-7 cells. Mol. Biochem. Parasitol. 117, 49
59. Frenal, K., Dubremetz, J.F., Lebrun, M., Soldati-Favre, D., 2017a. Gliding motility powers invasion and egress in Apicomplexa. Nat Rev Microbiol 15, 645
660. Frenal, K., Jacot, D., Hammoudi, P.M., Graindorge, A., Maco, B., Soldati-Favre, D., 2017b. Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat. Commun. 8, 15710. Frenal, K., Tay, C.L., Mueller, C., Bushell, E.S., Jia, Y., Graindorge, A., et al., 2013. Global analysis of apicomplexan protein S-acyl transferases reveals an enzyme essential for invasion. Traffic 14, 895
911. Frevert, U., Engelmann, S., Zougbede, S., Stange, J., Ng, B., Matuschewski, K., et al., 2005. Intravital observation of Plasmodium berghei sporozoite infection of the liver. PLoS Biol. 3, e192. Friedrich, N., Santos, J.M., Liu, Y., Palma, A.S., Leon, E., Saouros, S., et al., 2010a. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites. J. Biol. Chem. 285, 2064
2076. Friedrich, R., Yeheskel, A., Ashery, U., 2010b. DOC2B, C2 domains, and calcium: a tale of intricate interactions. Mol. Neurobiol. 41, 42
51. Fritz, H.M., Bowyer, P.W., Bogyo, M., Conrad, P.A., Boothroyd, J.C., 2012a. Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance. PLoS One 7, e29955. Fritz, H.M., Buchholz, K.R., Chen, X., Durbin-Johnson, B., Rocke, D.M., Conrad, P.A., et al., 2012b. Transcriptomic analysis of Toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts. PLoS One 7, e29998. Gaffar, F.R., Yatsuda, A.P., Franssen, F.F., de Vries, E., 2004. Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum apical membrane antigen 1. Infect. Immun. 72, 2947
2955. Gaji, R.Y., Behnke, M.S., Lehmann, M.M., White, M.W., Carruthers, V.B., 2011a. Cell cycle-dependent,

691

intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression. Mol. Microbiol. 79, 192
204. Gaji, R.Y., Flammer, H.P., Carruthers, V.B., 2011b. Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids. Traffic 12, 840
853. Garcia-Reguet, N., Lebrun, M., Fourmaux, M.N., Mercereau-Puijalon, O., Mann, T., Beckers, C.J., et al., 2000. The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite. Cell. Microbiol. 2, 353
364. Garnett, J.A., Liu, Y., Leon, E., Allman, S.A., Friedrich, N., Saouros, S., et al., 2009. Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii. Protein Sci. 18, 1935
1947. Gaskins, E., Gilk, S., DeVore, N., Mann, T., Ward, G., Beckers, C., 2004. Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J. Cell Biol. 165, 383
393. Gay, G., Braun, L., Brenier-Pinchart, M.P., Vollaire, J., Josserand, V., Bertini, R.L., et al., 2016. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-gammamediated host defenses. J. Exp. Med. 213, 1779
1798. Gendrin, C., Bittame, A., Mercier, C., Cesbron-Delauw, M. F., 2010. Post-translational membrane sorting of the Toxoplasma gondii GRA6 protein into the parasitecontaining vacuole is driven by its N-terminal domain. Int. J. Parasitol. 40, 1325
1334. Gendrin, C., Mercier, C., Braun, L., Musset, K., Dubremetz, J.F., Cesbron-Delauw, M.F., 2008. Toxoplasma gondii uses unusual sorting mechanisms to deliver transmembrane proteins into the host-cell vacuole. Traffic 9, 1665
1680. Ghosh, A.K., Devenport, M., Jethwaney, D., Kalume, D.E., Pandey, A., Anderson, V.E., et al., 2009. Malaria parasite invasion of the mosquito salivary gland requires interaction between the Plasmodium TRAP and the Anopheles saglin proteins. PLoS Pathog. 5, e1000265. Gilbert, L.A., Ravindran, S., Turetzky, J.M., Boothroyd, J.C., Bradley, P.J., 2007. Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells. Eukaryot. Cell 6, 73
83. Giovannini, D., Spath, S., Lacroix, C., Perazzi, A., Bargieri, D., Lagal, V., et al., 2011. Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by Apicomplexa. Cell Host Microbe 10, 591
602.

Toxoplasma Gondii

692

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Gold, D.A., Kaplan, A.D., Lis, A., Bett, G.C., Rosowski, E. E., Cirelli, K.M., et al., 2015. The Toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole. Cell Host Microbe 17, 642
652. Goldszmid, R.S., Coppens, I., Lev, A., Caspar, P., Mellman, I., Sher, A., 2009. Host ER-parasitophorous vacuole interaction provides a route of entry for antigen crosspresentation in Toxoplasma gondii-infected dendritic cells. J. Exp. Med. 206, 399
410. Gong, H., Kobayashi, K., Sugi, T., Takemae, H., Kurokawa, H., Horimoto, T., et al., 2012. A novel PAN/apple domain-containing protein from Toxoplasma gondii: characterization and receptor identification. PLoS One 7, e30169. Gonzalez, V., Combe, A., David, V., Malmquist, N.A., Delorme, V., Leroy, C., et al., 2009. Host cell entry by Apicomplexa parasites requires actin polymerization in the host cell. Cell Host Microbe 5, 259
272. Gras, S., Jackson, A., Woods, S., Pall, G., Whitelaw, J., Leung, J.M., et al., 2017. Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo. Wellcome Open Res. 2, 32. Gras, S., Jimenez-Ruiz, E., Klinger, C.M., Lemgruber, L., Meissner, M., 2019. Apicomplexan motility depends on the operation of an endocytic-secretory cycle. bioRxiv 450080. Available from: https://doi.org/10.1101/450080. Groffen, A.J., Martens, S., Diez Arazola, R., Cornelisse, L. N., Lozovaya, N., de Jong, A.P., et al., 2010. Doc2b is a high-affinity Ca2 1 sensor for spontaneous neurotransmitter release. Science 327, 1614
1618. Guerin, A., Corrales, R.M., Parker, M.L., Lamarque, M.H., Jacot, D., El Hajj, H., et al., 2017a. Efficient invasion by Toxoplasma depends on the subversion of host protein networks. Nat. Microbiol. 2, 1358
1366. Guerin, A., El Hajj, H., Penarete-Vargas, D., Besteiro, S., Lebrun, M., 2017b. RON4L1 is a new member of the moving junction complex in Toxoplasma gondii. Sci. Rep. 7, 17907. Guerra, A.J., Zhang, O., Bahr, C.M.E., Huynh, M.H., DelProposto, J., Brown, W.C., et al., 2018. Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress. PLoS Pathog. 14, e1007476. Guo, N.H., Krutzsch, H.C., Negre, E., Vogel, T., Blake, D. A., Roberts, D.D., 1992. Heparin- and sulfatide-binding peptides from the type I repeats of human thrombospondin promote melanoma cell adhesion. Proc. Natl. Acad. Sci. U.S.A. 89, 3040
3044. Haefliger, J.A., Tschopp, J., Vial, N., Jenne, D.E., 1989. Complete primary structure and functional

characterization of the sixth component of the human complement system. Identification of the C5b-binding domain in complement C6. J. Biol. Chem. 264, 18041
18051. Hajagos, B.E., Turetzky, J.M., Peng, E.D., Cheng, S.J., Ryan, C.M., Souda, P., et al., 2012. Molecular dissection of novel trafficking and processing of the Toxoplasma gondii rhoptry metalloprotease toxolysin-1. Traffic 13, 292
304. Hajj, H.E., Lebrun, M., Fourmaux, M.N., Vial, H., Dubremetz, J.F., 2005. Characterization, biosynthesis and fate of ROP7, a ROP2 related rhoptry protein of Toxoplasma gondii. Mol. Biochem. Parasitol . Available from: https://doi.org/10.1016/j.molbiopara.2005.10.011. Hakansson, S., Morisaki, H., Heuser, J., Sibley, L.D., 1999. Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion. Mol. Biol. Cell 10, 3539
3547. Hakansson, S., Charron, A.J., Sibley, L.D., 2001. Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole. EMBO J. 20, 3132
3144. Hammoudi, P.M., Jacot, D., Mueller, C., Di Cristina, M., Dogga, S.K., Marq, J.B., et al., 2015. Fundamental roles of the Golgi-associated Toxoplasma aspartyl protease, ASP5, at the host-parasite interface. PLoS Pathog. 11, e1005211. Hammoudi, P.M., Maco, B., Dogga, S.K., Frenal, K., Soldati-Favre, D., 2018. Toxoplasma gondii TFP1 is an essential transporter family protein critical for microneme maturation and exocytosis. Mol. Microbiol . Available from: https://doi.org/10.1111/mmi.13981. Hanssen, E., Dekiwadia, C., Riglar, D.T., Rug, M., Lemgruber, L., Cowman, A.F., et al., 2013. Electron tomography of Plasmodium falciparum merozoites reveals core cellular events that underpin erythrocyte invasion. Cell. Microbiol. 15, 1457
1472. Harper, J.M., Hoff, E.F., Carruthers, V.B., 2004a. Multimerization of the Toxoplasma gondii MIC2 integrin-like A-domain is required for binding to heparin and human cells. Mol. Biochem. Parasitol. 134, 201
212. Harper, J.M., Zhou, X.W., Pszenny, V., Kafsack, B.F., Carruthers, V.B., 2004b. The novel coccidian micronemal protein MIC11 undergoes proteolytic maturation by sequential cleavage to remove an internal propeptide. Int. J. Parasitol. 34, 1047
1058. Harper, J.M., Huynh, M.H., Coppens, I., Parussini, F., Moreno, S., Carruthers, V.B., 2006. A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex. Mol. Biol. Cell 17, 4551
4563.

Toxoplasma Gondii

References

He, H., Brenier-Pinchart, M.P., Braun, L., Kraut, A., Touquet, B., Coute, Y., et al., 2018. Characterization of a Toxoplasma effector uncovers an alternative GSK3/ beta-catenin-regulatory pathway of inflammation. eLife 7. Available from: https://doi.org/10.7554/eLife.39887. Heaslip, A.T., Nelson, S.R., Warshaw, D.M., 2016. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments. Mol. Biol. Cell 27, 2080
2089. Hehl, A.B., Lekutis, C., Grigg, M.E., Bradley, P.J., Dubremetz, J.F., Ortega-Barria, E., et al., 2000. Toxoplasma gondii homologue of Plasmodium apical membrane antigen 1 is involved in invasion of host cells. Infect. Immun. 68, 7078
7086. Hehl, A.B., Basso, W.U., Lippuner, C., Ramakrishnan, C., Okoniewski, M., Walker, R.A., et al., 2015. Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of nonoverlapping gene families to attach, invade, and replicate within feline enterocytes. BMC Genomics 16, 66. Henderson, K.A., Streltsov, V.A., Coley, A.M., Dolezal, O., Hudson, P.J., Batchelor, A.H., et al., 2007. Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure 15, 1452
1466. Henriquez, F.L., Nickdel, M.B., McLeod, R., Lyons, R.E., Lyons, K., Dubremetz, J.F., et al., 2005. Toxoplasma gondii dense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif. Parasitology 131, 1
11. Herion, P., Hernandez-Pando, R., Dubremetz, J.F., Saavedra, R., 1993. Subcellular localization of the 54kDa antigen of Toxoplasma gondii. J. Parasitol. 79, 216
222. Herm-Gotz, A., Agop-Nersesian, C., Munter, S., Grimley, J. S., Wandless, T.J., Frischknecht, F., et al., 2007. Rapid control of protein level in the apicomplexan Toxoplasma gondii. Nat. Methods 4, 1003
1005. Herwald, H., Renne, T., Meijers, J.C., Chung, D.W., Page, J. D., Colman, R.W., et al., 1996. Mapping of the discontinuous kininogen binding site of prekallikrein. A distal binding segment is located in the heavy chain domain A4. J. Biol. Chem. 271, 13061
13067. High, K.P., Joiner, K.A., Handschumacher, R.E., 1994. Isolation, cDNA sequences, and biochemical characterization of the major cyclosporin-binding proteins of Toxoplasma gondii. J. Biol. Chem. 269, 9105
9112. Hiller, N.L., Bhattacharjee, S., van Ooij, C., Liolios, K., Harrison, T., Lopez-Estrano, C., et al., 2004. A hosttargeting signal in virulence proteins reveals a secretome in malarial infection. Science 306, 1934
1937.

693

Hinds, L., Green, J.L., Knuepfer, E., Grainger, M., Holder, A.A., 2009. Novel putative glycosylphosphatidylinositol-anchored micronemal antigen of Plasmodium falciparum that binds to erythrocytes. Eukaryot. Cell 8, 1869
1879. Ho, D.H., Badellino, K., Baglia, F.A., Walsh, P.N., 1998. A binding site for heparin in the apple 3 domain of factor XI. J. Biol. Chem. 273, 16382
16390. Hodder, A.N., Crewther, P.E., Matthew, M.L., Reid, G.E., Moritz, R.L., Simpson, R.J., et al., 1996. The disulfide bond structure of Plasmodium apical membrane antigen1. J. Biol. Chem. 271, 29446
29452. Hoff, E.F., Cook, S.H., Sherman, G.D., Harper, J.M., Ferguson, D.J., Dubremetz, J.F., et al., 2001. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Exp. Parasitol. 97, 77
88. Holt, G.D., Pangburn, M.K., Ginsburg, V., 1990. Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates. J. Biol. Chem. 265, 2852
2855. Hoppe, H.C., Joiner, K.A., 2000. Cytoplasmic tail motifs mediate endoplasmic reticulum localization and export of transmembrane reporters in the protozoan parasite Toxoplasma gondii. Cell. Microbiol. 2, 569
578. Howell, S.A., Withers-Martinez, C., Kocken, C.H., Thomas, A.W., Blackman, M.J., 2001. Proteolytic processing and primary structure of Plasmodium falciparum apical membrane antigen-1. J. Biol. Chem. 276, 31311
31320. Howell, S.A., Hackett, F., Jongco, A.M., Withers-Martinez, C., Kim, K., Carruthers, V.B., et al., 2005. Distinct mechanisms govern proteolytic shedding of a key invasion protein in apicomplexan pathogens. Mol. Microbiol. 57, 1342
1356. Hume, A.N., Seabra, M.C., 2011. Melanosomes on the move: a model to understand organelle dynamics. Biochem. Soc. Trans. 39, 1191
1196. Huynh, M.H., Carruthers, V.B., 2006. Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS Pathog. 2, e84. Huynh, M.H., Carruthers, V.B., 2016. A Toxoplasma gondii ortholog of Plasmodium GAMA contributes to parasite attachment and cell invasion. mSphere 1. Available from: https://doi.org/10.1128/mSphere.00012-16. Huynh, M.H., Rabenau, K.E., Harper, J.M., Beatty, W.L., Sibley, L.D., Carruthers, V.B., 2003. Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex. EMBO J. 22, 2082
2090. Huynh, M.H., Opitz, C., Kwok, L.Y., Tomley, F.M., Carruthers, V.B., Soldati, D., 2004. Trans-genera reconstitution and complementation of an adhesion complex in Toxoplasma gondii. Cell. Microbiol. 6, 771
782.

Toxoplasma Gondii

694

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Huynh, M.H., Boulanger, M.J., Carruthers, V.B., 2014. A conserved apicomplexan microneme protein contributes to Toxoplasma gondii invasion and virulence. Infect. Immun. 82, 4358
4368. Huynh, M.H., Liu, B., Henry, M., Liew, L., Matthews, S.J., Carruthers, V.B., 2015. Structural basis of Toxoplasma gondii MIC2-associated protein interaction with MIC2. J. Biol. Chem. 290, 1432
1441. Ibrahim, H.M., Xuan, X., Nishikawa, Y., 2010. Toxoplasma gondii cyclophilin 18 regulates the proliferation and migration of murine macrophages and spleen cells. Clin. Vaccine Immunol. 17, 1322
1329. Jacot, D., Daher, W., Soldati-Favre, D., 2013. Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance. EMBO J. 32, 1702
1716. Jacot, D., Tosetti, N., Pires, I., Stock, J., Graindorge, A., Hung, Y.F., et al., 2016. An apicomplexan actin-binding protein serves as a connector and lipid sensor to coordinate motility and invasion. Cell Host Microbe 20, 731
743. Jensen, J.B., Edgar, S.A., 1976. Effects of antiphagocytic agents on penetration of Eimeria magna sporozoites into cultured cells. J. Parasitol. 62, 203
206. Jethwaney, D., Lepore, T., Hassan, S., Mello, K., Rangarajan, R., Jahnen-Dechent, W., et al., 2005. Fetuin-A, a hepatocyte-specific protein that binds Plasmodium berghei thrombospondin-related adhesive protein: a potential role in infectivity. Infect. Immun. 73, 5883
5891. Jewett, T.J., Sibley, L.D., 2003. Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites. Mol. Cell 11, 885
894. Jewett, T.J., Sibley, L.D., 2004. The Toxoplasma proteins MIC2 and M2AP form a hexameric complex necessary for intracellular survival. J. Biol. Chem. 279, 9362
9369. Johnson, A.M., Illana, S., 1991. Cloning of Toxoplasma gondii gene fragments encoding diagnostic antigens. Gene 99, 127
132. Johnson, R.I., Seppa, M.J., Cagan, R.L., 2008. The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning. J. Cell Biol. 180, 1191
1204. Jones, T.C., Hirsch, J.G., 1972. The interaction between Toxoplasma gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J. Exp. Med. 136, 1173
1194. Jones, N.G., Wang, Q., Sibley, L.D., 2017. Secreted protein kinases regulate cyst burden during chronic toxoplasmosis. Cell. Microbiol. 19. Available from: https://doi. org/10.1111/cmi.12651. Kafsack, B.F., Pena, J.D., Coppens, I., Ravindran, S., Boothroyd, J.C., Carruthers, V.B., 2009. Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 323, 530
533.

Kappe, S., Bruderer, T., Gantt, S., Fujioka, H., Nussenzweig, V., Menard, R., 1999. Conservation of a gliding motility and cell invasion machinery in apicomplexan parasites. J. Cell Biol. 147, 937
944. Karasov, A.O., Boothroyd, J.C., Arrizabalaga, G., 2005. Identification and disruption of a rhoptry-localized homologue of sodium hydrogen exchangers in Toxoplasma gondii. Int. J. Parasitol. 35, 285
291. Karsten, V., Qi, H., Beckers, C.J., Reddy, A., Dubremetz, J. F., Webster, P., et al., 1998. The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms. J. Cell Biol. 141, 1323
1333. Karsten, V., Hegde, R.S., Sinai, A.P., Yang, M., Joiner, K.A., 2004. Transmembrane domain modulates sorting of membrane proteins in Toxoplasma gondii. J. Biol. Chem. 279, 26052
26057. Kato, K., Mayer, D.C., Singh, S., Reid, M., Miller, L.H., 2005. Domain III of Plasmodium falciparum apical membrane antigen 1 binds to the erythrocyte membrane protein Kx. Proc. Natl. Acad. Sci. U.S.A. 102, 5552
5557. Katris, N.J., Ke, H., McFadden, G.I., van Dooren, G.G., Waller, R.F., 2019. Calcium negatively regulates secretion from dense granules in Toxoplasma gondii. Cell. Microbiol. 21, e13011. Kawase, O., Nishikawa, Y., Bannai, H., Zhang, H., Zhang, G., Jin, S., et al., 2007. Proteomic analysis of calciumdependent secretion in Toxoplasma gondii. Proteomics 7, 3718
3725. Kawase, O., Nishikawa, Y., Bannai, H., Igarashi, M., Matsuo, T., Xuan, X., 2010. Characterization of a novel thrombospondin-related protein in Toxoplasma gondii. Parasitol. Int. 59, 211
216. Keeley, A., Soldati, D., 2004. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa. Trends Cell Biol. 14, 528
532. Kessler, H., Herm-Gotz, A., Hegge, S., Rauch, M., SoldatiFavre, D., Frischknecht, F., et al., 2008. Microneme protein 8—a new essential invasion factor in Toxoplasma gondii. J. Cell Sci. 121, 947
956. Khurana, S., Coffey, M.J., John, A., Uboldi, A.D., Huynh, M.H., Stewart, R.J., et al., 2019. Protein O-fucosyltransferase 2-mediated O-glycosylation of the adhesin MIC2 is dispensable for Toxoplasma gondii tachyzoite infection. J. Biol. Chem. 294, 1541
1553. Kim, K., Soldati, D., Boothroyd, J.C., 1993. Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. Science 262, 911
914. Kim, J.Y., Ahn, H.J., Ryu, K.J., Nam, H.W., 2008. Interaction between parasitophorous vacuolar membraneassociated GRA3 and calcium modulating ligand of host cell endoplasmic reticulum in the parasitism of Toxoplasma gondii. Korean J. Parasitol. 46, 209
216.

Toxoplasma Gondii

References

Kim, E., Lee, Y., Lee, H.J., Kim, J.S., Song, B.S., Huh, J.W., et al., 2010. Implication of mouse Vps26b-Vps29-Vps35 retromer complex in sortilin trafficking. Biochem. Biophys. Res. Commun. 403, 167
171. Kim, E.W., Nadipuram, S.M., Tetlow, A.L., Barshop, W.D., Liu, P.T., Wohlschlegel, J.A., et al., 2016. The rhoptry pseudokinase ROP54 modulates Toxoplasma gondii virulence and Host GBP2 loading. mSphere 1. Available from: https://doi.org/10.1128/mSphere.00045-16. Kimata, I., Tanabe, K., 1987. Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell. J. Cell Sci. 88 (Pt 2), 231
239. Klein, H., Loschner, B., Zyto, N., Portner, M., Montag, T., 1998. Expression, purification, and biochemical characterization of a recombinant lectin of Sarcocystis muris (Apicomplexa) cyst merozoites. Glycoconj. J. 15, 147
153. Koshy, A.A., Dietrich, H.K., Christian, D.A., Melehani, J.H., Shastri, A.J., Hunter, C.A., et al., 2012. Toxoplasma coopts host cells it does not invade. PLoS Pathog. 8, e1002825. Kremer, K., Kamin, D., Rittweger, E., Wilkes, J., Flammer, H., Mahler, S., et al., 2013. An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes. PLoS Pathog. 9, e1003213. Krishnamurthy, S., Saeij, J.P.J., 2018. Toxoplasma does not secrete the GRA16 and GRA24 effectors beyond the parasitophorous vacuole membrane of tissue cysts. Front. Cell. Infect. Microbiol. 8, 366. Krishnamurthy, S., Deng, B., Del Rio, R., Buchholz, K.R., Treeck, M., Urban, S., et al., 2016. Not a simple tether: binding of Toxoplasma gondii AMA1 to RON2 during invasion protects AMA1 from rhomboid-mediated cleavage and leads to dephosphorylation of its cytosolic tail. MBio 7. Available from: https://doi.org/10.1128/ mBio.00754-16. Labesse, G., Gelin, M., Bessin, Y., Lebrun, M., Papoin, J., Cerdan, R., et al., 2009. ROP2 from Toxoplasma gondii: a virulence factor with a protein-kinase fold and no enzymatic activity. Structure 17, 139
146. Labruyere, E., Lingnau, M., Mercier, C., Sibley, L.D., 1999. Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii. Mol. Biochem. Parasitol. 102, 311
324. LaFavers, K.A., Marquez-Nogueras, K.M., Coppens, I., Moreno, S.N.J., Arrizabalaga, G., 2017. A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii. Cell. Microbiol. 19. Available from: https://doi.org/10.1111/cmi.12749. Lagal, V., Binder, E.M., Huynh, M.H., Kafsack, B.F., Harris, P.K., Diez, R., et al., 2010. Toxoplasma gondii protease

695

TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites. Cell. Microbiol. 12, 1792
1808. Lai, L., Bumstead, J., Liu, Y., Garnett, J., CampaneroRhodes, M.A., Blake, D.P., et al., 2011. The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella. PLoS Pathog. 7, e1002296. Laliberte, J., Carruthers, V.B., 2011. Toxoplasma gondii toxolysin 4 is an extensively processed putative metalloproteinase secreted from micronemes. Mol. Biochem. Parasitol. 177, 49
56. Lamarque, M., Besteiro, S., Papoin, J., Roques, M., VulliezLe Normand, B., Morlon-Guyot, J., et al., 2011. The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites. PLoS Pathog. 7, e1001276. Lamarque, M.H., Papoin, J., Finizio, A.L., Lentini, G., Pfaff, A. W., Candolfi, E., et al., 2012. Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii. PLoS One 7, e32457. Lamarque, M.H., Roques, M., Kong-Hap, M., Tonkin, M.L., Rugarabamu, G., Marq, J.B., et al., 2014. Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites. Nat. Commun. 5, 4098. Larson, E.T., Parussini, F., Huynh, M.H., Giebel, J.D., Kelley, A.M., Zhang, L., et al., 2009. Toxoplasma gondii cathepsin L. is the primary target of the invasioninhibitory compound morpholinurea-leucylhomophenyl-vinyl sulfone phenyl. J. Biol. Chem. 284, 26839
26850. Lawler, J., 1986. The structural and functional properties of thrombospondin. Blood 67, 1197
1209. Lebrun, M., Michelin, A., El Hajj, H., Poncet, J., Bradley, P. J., Vial, H., et al., 2005. The rhoptry neck protein RON4 relocalizes at the moving junction during Toxoplasma gondii invasion. Cell. Microbiol. 7, 1823
1833. Lecordier, L., Mercier, C., Torpier, G., Tourvieille, B., Darcy, F., Liu, J.L., et al., 1993. Molecular structure of a Toxoplasma gondii dense granule antigen (GRA 5) associated with the parasitophorous vacuole membrane. Mol. Biochem. Parasitol. 59, 143
153. Lecordier, L., Moleon-Borodowsky, I., Dubremetz, J.F., Tourvieille, B., Mercier, C., Deslee, D., et al., 1995. Characterization of a dense granule antigen of Toxoplasma gondii (GRA6) associated to the network of the parasitophorous vacuole. Mol. Biochem. Parasitol. 70, 85
94. Lecordier, L., Mercier, C., Sibley, L.D., Cesbron-Delauw, M. F., 1999. Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell. Mol. Biol. Cell 10, 1277
1287. Lee, J.O., Rieu, P., Arnaout, M.A., Liddington, R., 1995. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell 80, 631
638.

Toxoplasma Gondii

696

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Lek, A., Evesson, F.J., Sutton, R.B., North, K.N., Cooper, S. T., 2012. Ferlins: regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair. Traffic 13, 185
194. Lemgruber, L., Lupetti, P., De Souza, W., Vommaro, R.C., 2011. New details on the fine structure of the rhoptry of Toxoplasma gondii. Microsc. Res. Tech. 74, 812
818. Lentini, G., El Hajj, H., Papoin, J., Fall, G., Pfaff, A.W., Tawil, N., et al., 2017. Characterization of Toxoplasma DegP, a rhoptry serine protease crucial for lethal infection in mice. PLoS One 12, e0189556. Leriche, M.A., Dubremetz, J.F., 1990. Exocytosis of Toxoplasma gondii dense granules into the parasitophorous vacuole after host cell invasion. Parasitol. Res. 76, 559
562. Leriche, M.A., Dubremetz, J.F., 1991. Characterization of the protein contents of rhoptries and dense granules of Toxoplasma gondii tachyzoites by subcellular fractionation and monoclonal antibodies. Mol. Biochem. Parasitol. 45, 249
259. Leung, J.M., Rould, M.A., Konradt, C., Hunter, C.A., Ward, G.E., 2014. Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay. PLoS One 9, e85763. Leung, J.M., He, Y., Zhang, F., Hwang, Y.C., Nagayasu, E., Liu, J., et al., 2017. Stability and function of a putative microtubule-organizing center in the human parasite Toxoplasma gondii. Mol. Biol. Cell 28, 1361
1378. Liendo, A., Joiner, K.A., 2000. Toxoplasma gondii: conserved protein machinery in an unusual secretory pathway? Microbes Infect 2, 137
144. Lodoen, M.B., Gerke, C., Boothroyd, J.C., 2010. A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection. Cell. Microbiol. 12, 55
66. Lopez, J., Bittame, A., Massera, C., Vasseur, V., Effantin, G., Valat, A., et al., 2015. Intravacuolar membranes regulate CD8 T cell recognition of membrane-bound Toxoplasma gondii protective antigen. Cell Rep. 13, 2273
2286. Lourenco, E.V., Pereira, S.R., Faca, V.M., Coelho-Castelo, A. A., Mineo, J.R., Roque-Barreira, M.C., et al., 2001. Toxoplasma gondii micronemal protein MIC1 is a lactosebinding lectin. Glycobiology 11, 541
547. Lourido, S., Moreno, S.N., 2015. The calcium signaling toolkit of the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell Calcium 57, 186
193. Magno, R.C., Lemgruber, L., Vommaro, R.C., De Souza, W., Attias, M., 2005a. Intravacuolar network may act as a mechanical support for Toxoplasma gondii inside the parasitophorous vacuole. Microsc. Res. Tech. 67, 45
52.

Magno, R.C., Straker, L.C., de Souza, W., Attias, M., 2005b. Interrelations between the parasitophorous vacuole of Toxoplasma gondii and host cell organelles. Microsc. Microanal. 11, 166
174. Mahajan, B., Jani, D., Chattopadhyay, R., Nagarkatti, R., Zheng, H., Majam, V., et al., 2005. Identification, cloning, expression, and characterization of the gene for Plasmodium knowlesi surface protein containing an altered thrombospondin repeat domain. Infect. Immun. 73, 5402
5409. Marchant, J., Cowper, B., Liu, Y., Lai, L., Pinzan, C., Marq, J.B., et al., 2012. Galactose recognition by the apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 287, 16720
16733. Marino, N.D., Panas, M.W., Franco, M., Theisen, T.C., Naor, A., Rastogi, S., et al., 2018. Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii. PLoS Pathog. 14, e1006828. Marti, M., Good, R.T., Rug, M., Knuepfer, E., Cowman, A. F., 2004. Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306, 1930
1933. Matuschewski, K., Nunes, A.C., Nussenzweig, V., Menard, R., 2002. Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system. EMBO J. 21, 1597
1606. McCormick, C.J., Tuckwell, D.S., Crisanti, A., Humphries, M.J., Hollingdale, M.R., 1999. Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein. Mol. Biochem. Parasitol. 100, 111
124. Meissner, M., Reiss, M., Viebig, N., Carruthers, V.B., Toursel, C., Tomavo, S., et al., 2002. A family of transmembrane microneme proteins of Toxoplasma gondii contain EGF-like domains and function as escorters. J. Cell Sci. 115, 563
574. Mercier, C., Cesbron-Delauw, M.F., 2015a. Toxoplasma secretory granules: one population or more? Trends Parasitol. 31, 604. Mercier, C., Cesbron-Delauw, M.F., 2015b. Toxoplasma secretory granules: one population or more? Trends Parasitol. 31, 60
71. Mercier, C., Cesbron-Delauw, M.F., Sibley, L.D., 1998a. The amphipathic alpha helices of the toxoplasma protein GRA2 mediate post-secretory membrane association. J. Cell Sci. 111, 2171
2180. Mercier, C., Howe, D.K., Mordue, D., Lingnau, M., Sibley, L.D., 1998b. Targeted disruption of the GRA2 locus in Toxoplasma gondii decreases acute virulence in mice. Infect. Immun. 66, 4176
4182.

Toxoplasma Gondii

References

Mercier, C., Dubremetz, J.F., Rauscher, B., Lecordier, L., Sibley, L.D., Cesbron-Delauw, M.F., 2002. Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. Mol. Biol. Cell 13, 2397
2409. Mercier, C., Adjogble, K.D., Daubener, W., Delauw, M.F., 2005. Dense granules: Are they key organelles to help understand the parasitophorous vacuole of all Apicomplexa parasites? Int. J. Parasitol. 35, 829
849. Michelin, A., Bittame, A., Bordat, Y., Travier, L., Mercier, C., Dubremetz, J.F., et al., 2008. GRA12, a Toxoplasma dense granule protein associated with the intravacuolar 1 membranous 2 nanotubular network. Int. J. Parasitol. in press. Miller, L.H., Aikawa, M., Johnson, J.G., Shiroishi, T., 1979. Interaction between cytochalasin B-treated malarial parasites and erythrocytes. Attachment and junction formation. J. Exp. Med. 149, 172
184. Miller, S.A., Binder, E.M., Blackman, M.J., Carruthers, V.B., Kim, K., 2001. A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii. J. Biol. Chem. 276, 45341
45348. Miller, S.A., Thathy, V., Ajioka, J.W., Blackman, M.J., Kim, K., 2003. TgSUB2 is a Toxoplasma gondii rhoptry organelle processing proteinase. Mol. Microbiol. 49, 883
894. Mital, J., Meissner, M., Soldati, D., Ward, G.E., 2005. Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol. Biol. Cell 16, 4341
4349. Mitchell, G.H., Thomas, A.W., Margos, G., Dluzewski, A. R., Bannister, L.H., 2004. Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells. Infect. Immun. 72, 154
158. Mondragon, R., Frixione, E., 1996. Ca(2 1 )-dependence of conoid extrusion in Toxoplasma gondii tachyzoites. J. Eukaryot. Microbiol. 43, 120
127. Monteiro, V.G., Soares, C.P., de Souza, W., 1998. Host cell surface sialic acid residues are involved on the process of penetration of Toxoplasma gondii into mammalian cells. FEMS Microbiol. Lett. 164, 323
327. Mordue, D.G., Sibley, L.D., 1997. Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry. J. Immunol. 159, 4452
4459. Mordue, D.G., Desai, N., Dustin, M., Sibley, L.D., 1999a. Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring. J. Exp. Med. 190, 1783
1792. Mordue, D.G., Hakansson, S., Niesman, I., Sibley, L.D., 1999b. Toxoplasma gondii resides in a vacuole that avoids

697

fusion with host cell endocytic and exocytic vesicular trafficking pathways. Exp. Parasitol. 92, 87
99. Morisaki, J.H., Heuser, J.E., Sibley, L.D., 1995. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J. Cell Sci. 108 (Pt 6), 2457
2464. Morlon-Guyot, J., Pastore, S., Berry, L., Lebrun, M., Daher, W., 2015. Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes, is essential for the biogenesis of secretory organelles. Cell. Microbiol. 17, 1157
1178. Morlon-Guyot, J., Berry, L., Sauquet, I., Singh Pall, G., El Hajj, H., Meissner, M., et al., 2018a. Conditional knockdown of a novel coccidian protein leads to the formation of aberrant apical organelles and abrogates mature rhoptry positioning in Toxoplasma gondii. Mol. Biochem. Parasitol. 223, 19
30. Morlon-Guyot, J., El Hajj, H., Martin, K., Fois, A., Carrillo, A., Berry, L., et al., 2018b. A proteomic analysis unravels novel CORVET and HOPS proteins involved in Toxoplasma gondii secretory organelles biogenesis. Cell. Microbiol. 20, e12870. Morris, M.T., Carruthers, V.B., 2003. Identification and partial characterization of a second Kazal inhibitor in Toxoplasma gondii. Mol. Biochem. Parasitol. 128, 119
122. Morris, M.T., Coppin, A., Tomavo, S., Carruthers, V.B., 2002. Functional analysis of Toxoplasma gondii protease inhibitor 1. J. Biol. Chem. 277, 45259
45266. Moudy, R., Manning, T.J., Beckers, C.J., 2001. The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii. J. Biol. Chem. 276, 41492
41501. Mueller, C., Klages, N., Jacot, D., Santos, J.M., Cabrera, A., Gilberger, T.W., et al., 2013. The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion. Cell Host Microbe 13, 289
301. Mueller, C., Samoo, A., Hammoudi, P.M., Klages, N., Kallio, J.P., Kursula, I., et al., 2016. Structural and functional dissection of Toxoplasma gondii armadillo repeats only protein. J. Cell Sci. 129, 1031
1045. Muller, H.M., Reckmann, I., Hollingdale, M.R., Bujard, H., Robson, K.J., Crisanti, A., 1993. Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum binds specifically to sulfated glycoconjugates and to HepG2 hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes. EMBO J. 12, 2881
2889. Muniz-Feliciano, L., Van Grol, J., Portillo, J.A., Liew, L., Liu, B., Carlin, C.R., et al., 2013. Toxoplasma gondiiinduced activation of EGFR prevents autophagy protein-mediated killing of the parasite. PLoS Pathog. 9, e1003809.

Toxoplasma Gondii

698

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Muniz-Hernandez, S., Carmen, M.G., Mondragon, M., Mercier, C., Cesbron, M.F., Mondragon-Gonzalez, S.L., et al., 2011. Contribution of the residual body in the spatial organization of Toxoplasma gondii tachyzoites within the parasitophorous vacuole. J. Biomed. Biotechnol. 2011, 473983. Nadipuram, S.M., Kim, E.W., Vashisht, A.A., Lin, A.H., Bell, H.N., Coppens, I., et al., 2016. In vivo biotinylation of the Toxoplasma parasitophorous vacuole reveals novel dense granule proteins important for parasite growth and pathogenesis. MBio 7. Available from: https://doi.org/10.1128/mBio.00808-16. Nakaar, V., Bermudes, D., Peck, K.R., Joiner, K.A., 1998. Upstream elements required for expression of nucleoside triphosphate hydrolase genes of Toxoplasma gondii. Mol. Biochem. Parasitol. 92, 229
239. Nakaar, V., Samuel, B.U., Ngo, E.O., Joiner, K.A., 1999. Targeted reduction of nucleoside triphosphate hydrolase by antisense RNA inhibits Toxoplasma gondii proliferation. J. Biol. Chem. 274, 5083
5087. Ngo, H.M., Ngo, E.O., Bzik, D.J., Joiner, K.A., 2000. Toxoplasma gondii: are host cell adenosine nucleotides a direct source for purine salvage? Exp. Parasitol. 95, 148
153. Ngo, H.M., Yang, M., Paprotka, K., Pypaert, M., Hoppe, H., Joiner, K.A., 2003. AP-1 in Toxoplasma gondii mediates biogenesis of the rhoptry secretory organelle from a post-Golgi compartment. J. Biol. Chem. 278, 5343
5352. Ni, T., Williams, S.I., Rezelj, S., Anderluh, G., Harlos, K., Stansfeld, P.J., et al., 2018. Structures of monomeric and oligomeric forms of the Toxoplasma gondii perforin-like protein 1. Sci. Adv. 4, eaaq0762. Nichols, B.A., Chiappino, M.L., O’Connor, G.R., 1983. Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J. Ultrastruct. Res. 83, 85
98. Niedelman, W., Gold, D.A., Rosowski, E.E., Sprokholt, J.K., Lim, D., Farid Arenas, A., et al., 2012. The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferongamma response. PLoS Pathog. 8, e1002784. Nishi, M., Hu, K., Murray, J.M., Roos, D.S., 2008. Organellar dynamics during the cell cycle of Toxoplasma gondii. J. Cell Sci. 121, 1559
1568. Nolan, S.J., Romano, J.D., Coppens, I., 2017. Host lipid droplets: an important source of lipids salvaged by the intracellular parasite Toxoplasma gondii. PLoS Pathog. 13, e1006362. O’Keeffe, A.H., Green, J.L., Grainger, M., Holder, A.A., 2005. A novel Sushi domain-containing protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 140, 61
68. Okada, T., Marmansari, D., Li, Z.M., Adilbish, A., Canko, S., Ueno, A., et al., 2013. A novel dense granule protein,

GRA22, is involved in regulating parasite egress in Toxoplasma gondii. Mol. Biochem. Parasitol. 189, 5
13. Olias, P., Etheridge, R.D., Zhang, Y., Holtzman, M.J., Sibley, L.D., 2016. Toxoplasma effector recruits the Mi2/NuRD complex to repress STAT1 transcription and block IFN-gamma-dependent gene expression. Cell Host Microbe 20, 72
82. Opitz, C., Soldati, D., 2002. ‘The glideosome’: a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii. Mol. Microbiol. 45, 597
604. Opitz, C., Di Cristina, M., Reiss, M., Ruppert, T., Crisanti, A., Soldati, D., 2002. Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii. EMBO J. 21, 1577
1585. Ossorio, P.N., Schwartzman, J.D., Boothroyd, J.C., 1992. A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry. Mol. Biochem. Parasitol. 50, 1
15. Pan, M., Li, M., Li, L., Song, Y., Hou, L., Zhao, J., et al., 2019. Identification of novel dense-granule proteins in Toxoplasma gondii by two proximity-based biotinylation approaches. J. Proteome Res. 18, 319
330. Panas, M.W., Naor, A., Cygan, A.M., Boothroyd, J.C., 2019. Toxoplasma controls host cyclin E expression through the use of a novel MYR1-dependent effector protein, HCE1. MBio 10. Available from: https://doi.org/ 10.1128/mBio.00674-19. Pao, S.S., Paulsen, I.T., Saier Jr., M.H., 1998. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62, 1
34. Parker, M.L., Boulanger, M.J., 2015. An extended surface loop on Toxoplasma gondii apical membrane antigen 1 (AMA1) governs ligand binding selectivity. PLoS One 10, e0126206. Parker, M.L., Penarete-Vargas, D.M., Hamilton, P.T., Guerin, A., Dubey, J.P., Perlman, S.J., et al., 2016. Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA-RON2 pair. Proc. Natl. Acad. Sci. U.S.A. 113, 398
403. Parussini, F., Coppens, I., Shah, P.P., Diamond, S.L., Carruthers, V.B., 2010. Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii. Mol. Microbiol. 76, 1340
1357. Parussini, F., Tang, Q., Moin, S.M., Mital, J., Urban, S., Ward, G.E., 2012. Intramembrane proteolysis of Toxoplasma apical membrane antigen 1 facilitates hostcell invasion but is dispensable for replication. Proc. Natl. Acad. Sci. U.S.A. 109, 7463
7468. Pavlou, G., Biesaga, M., Touquet, B., Lagal, V., Balland, M., Dufour, A., et al., 2018. Toxoplasma parasite twisting motion mechanically induces host cell membrane fission to complete invasion within a protective vacuole. Cell Host Microbe 24, 81
96. e85.

Toxoplasma Gondii

References

Peixoto, L., Chen, F., Harb, O.S., Davis, P.H., Beiting, D.P., Brownback, C.S., et al., 2010. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses. Cell Host Microbe 8, 208
218. Periz, J., Gill, A.C., Knott, V., Handford, P.A., Tomley, F. M., 2005. Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4. Mol. Biochem. Parasitol. 143, 192
199. Periz, J., Whitelaw, J., Harding, C., Gras, S., Del Rosario Minina, M.I., Latorre-Barragan, F., et al., 2017. Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation. eLife 6. Available from: https://doi. org/10.7554/eLife.24119. Pernas, L., Boothroyd, J.C., 2010. Association of host mitochondria with the parasitophorous vacuole during Toxoplasma infection is not dependent on rhoptry proteins ROP2/8. Int. J. Parasitol. 40, 1367
1371. Pernas, L., Adomako-Ankomah, Y., Shastri, A.J., Ewald, S. E., Treeck, M., Boyle, J.P., et al., 2014. Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS Biol. 12, e1001845. Pieperhoff, M.S., Pall, G.S., Jimenez-Ruiz, E., Das, S., Melatti, C., Gow, M., et al., 2015. Conditional U1 Gene Silencing in Toxoplasma gondii. PLoS One 10, e0130356. Pihlajamaa, T., Kajander, T., Knuuti, J., Horkka, K., Sharma, A., Permi, P., 2013. Structure of Plasmodium falciparum TRAP (thrombospondin-related anonymous protein) A domain highlights distinct features in apicomplexan von Willebrand factor A homologues. Biochem. J. 450, 469
476. Pingret, L., Millot, J.M., Sharonov, S., Bonhomme, A., Manfait, M., Pinon, J.M., 1996. Relationship between intracellular free calcium concentrations and the intracellular development of Toxoplasma gondii. J. Histochem. Cytochem. 44, 1123
1129. Pizarro, J.C., Vulliez-Le Normand, B., Chesne-Seck, M.L., Collins, C.R., Withers-Martinez, C., Hackett, F., et al., 2005. Crystal structure of the malaria vaccine candidate apical membrane antigen 1. Science 308, 408
411. Porchet-Hennere, E., Nicolas, G., 1983. Are rhoptries of coccidia really extrusomes? J. Ultrastruct. Res. 84, 194
203. Poukchanski, A., Fritz, H.M., Tonkin, M.L., Treeck, M., Boulanger, M.J., Boothroyd, J.C., 2013. Toxoplasma gondii sporozoites invade host cells using two novel paralogues of RON2 and AMA1. PLoS One 8, e70637. Poupel, O., Boleti, H., Axisa, S., Couture-Tosi, E., Tardieux, I., 2000. Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments. Mol. Biol. Cell 11, 355
368. Pszenny, V., Ledesma, B.E., Matrajt, M., Duschak, V.G., Bontempi, E.J., Dubremetz, J.F., et al., 2002. Subcellular localization and post-secretory targeting of TgPI, a

699

serine proteinase inhibitor from Toxoplasma gondii. Mol. Biochem. Parasitol. 121, 283
286. Pszenny, V., Davis, P.H., Zhou, X.W., Hunter, C.A., Carruthers, V.B., Roos, D.S., 2012. Targeted disruption of Toxoplasma gondii serine protease inhibitor 1 increases bradyzoite cyst formation in vitro and parasite tissue burden in mice. Infect. Immun. 80, 1156
1165. Pszenny, V., Ehrenman, K., Romano, J.D., Kennard, A., Schultz, A., Roos, D.S., et al., 2016. A lipolytic lecithin: cholesterol acyltransferase secreted by Toxoplasma facilitates parasite replication and egress. J. Biol. Chem. 291, 3725
3746. Pytela, R., 1988. Amino acid sequence of the murine Mac-1 alpha chain reveals homology with the integrin family and an additional domain related to von Willebrand factor. EMBO J. 7, 1371
1378. Qiu, W., Wernimont, A., Tang, K., Taylor, S., Lunin, V., Schapira, M., et al., 2009. Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii. EMBO J. 28, 969
979. Qiu, J., Wang, L., Zhang, R., Ge, K., Guo, H., Liu, X., et al., 2016. Identification of a TNF-alpha inducer MIC3 originating from the microneme of non-cystogenic, virulent Toxoplasma gondii. Sci. Rep. 6, 39407. Quadt, K.A., Streichfuss, M., Moreau, C.A., Spatz, J.P., Frischknecht, F., 2016. Coupling of retrograde flow to force production during malaria parasite migration. ACS Nano 10, 2091
2102. Que, X., Ngo, H., Lawton, J., Gray, M., Liu, Q., Engel, J., et al., 2002. The cathepsin B of Toxoplasma gondii, toxopain-1, is critical for parasite invasion and rhoptry protein processing. J. Biol. Chem. 277, 25791
25797. Que, X., Engel, J.C., Ferguson, D., Wunderlich, A., Tomavo, S., Reed, S.L., 2007. Cathepsin Cs are key for the intracellular survival of the protozoan parasite, Toxoplasma gondii. J. Biol. Chem. 282, 4994
5003. Rabenau, K.E., Sohrabi, A., Tripathy, A., Reitter, C., Ajioka, J.W., Tomley, F.M., et al., 2001. TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2. Mol. Microbiol. 41, 537
547. Ravindran, S., Lodoen, M.B., Verhelst, S.H., Bogyo, M., Boothroyd, J.C., 2009. 4-Bromophenacyl bromide specifically inhibits rhoptry secretion during Toxoplasma invasion. PLoS One 4, e8143. Rawlings, N.D., Polgar, L., Barrett, A.J., 1991. A new family of serine-type peptidases related to prolyl oligopeptidase. Biochem. J. 279 (Pt 3), 907
908. Reese, M.L., Boothroyd, J.C., 2009. A helical membranebinding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole. Traffic 10, 1458
1470. Reese, M.L., Boothroyd, J.C., 2011. A conserved noncanonical motif in the pseudoactive site of the ROP5 pseudokinase domain mediates its effect on Toxoplasma virulence. J. Biol. Chem. 286, 29366
29375.

Toxoplasma Gondii

700

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Reese, M.L., Zeiner, G.M., Saeij, J.P., Boothroyd, J.C., Boyle, J.P., 2011. Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence. Proc. Natl. Acad. Sci. U.S.A. 108, 9625
9630. Reichmann, G., Dlugonska, H., Fischer, H.G., 2002. Characterization of TgROP9 (p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified by T cell clone. Mol. Biochem. Parasitol. 119, 43
54. Reiss, M., Viebig, N., Brecht, S., Fourmaux, M.N., Soete, M., Di Cristina, M., et al., 2001. Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii. J. Cell Biol. 152, 563
578. Richard, D., Kats, L.M., Langer, C., Black, C.G., Mitri, K., Boddey, J.A., et al., 2009. Identification of rhoptry trafficking determinants and evidence for a novel sorting mechanism in the malaria parasite Plasmodium falciparum. PLoS Pathog. 5, e1000328. Richard, D., MacRaild, C.A., Riglar, D.T., Chan, J.A., Foley, M., Baum, J., et al., 2010. Interaction between Plasmodium falciparum apical membrane antigen 1 and the rhoptry neck protein complex defines a key step in the erythrocyte invasion process of malaria parasites. J. Biol. Chem. 285, 14815
14822. Riglar, D.T., Richard, D., Wilson, D.W., Boyle, M.J., Dekiwadia, C., Turnbull, L., et al., 2011. Superresolution dissection of coordinated events during malaria parasite invasion of the human erythrocyte. Cell Host Microbe 9, 9
20. Robson, K.J., Hall, J.R., Jennings, M.W., Harris, T.J., Marsh, K., Newbold, C.I., et al., 1988. A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature 335, 79
82. Roger, N., Dubremetz, J.F., Delplace, P., Fortier, B., Tronchin, G., Vernes, A., 1988. Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 27, 135
141. Roiko, M.S., Carruthers, V.B., 2013. Functional dissection of Toxoplasma gondii perforin-like protein 1 reveals a dual domain mode of membrane binding for cytolysis and parasite egress. J. Biol. Chem. 288, 8712
8725. Roiko, M.S., Svezhova, N., Carruthers, V.B., 2014. Acidification activates Toxoplasma gondii motility and egress by enhancing protein secretion and cytolytic activity. PLoS Pathog. 10, e1004488. Romano, J.D., Sonda, S., Bergbower, E., Smith, M.E., Coppens, I., 2013. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole. Mol. Biol. Cell 24, 1974
1995. Romano, J.D., Nolan, S.J., Porter, C., Ehrenman, K., Hartman, E.J., Hsia, R.C., et al., 2017. The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network. J. Cell Biol. 216, 4235
4254.

Rome, M.E., Beck, J.R., Turetzky, J.M., Webster, P., Bradley, P.J., 2008. Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii. Infect. Immun. 76, 4865
4875. Rommereim, L.M., Bellini, V., Fox, B.A., Petre, G., Rak, C., Touquet, B., et al., 2016. Phenotypes associated with knockouts of eight dense granule gene loci (GRA2-9) in virulent Toxoplasma gondii. PLoS One 11, e0159306. Rosowski, E.E., Lu, D., Julien, L., Rodda, L., Gaiser, R.A., Jensen, K.D., et al., 2011. Strain-specific activation of the NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208, 195
212. Rugarabamu, G., Marq, J.B., Guerin, A., Lebrun, M., Soldati-Favre, D., 2015. Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion. Mol. Microbiol. 97, 244
262. Russell, D.G., Sinden, R.E., 1981. The role of the cytoskeleton in the motility of coccidian sporozoites. J. Cell Sci. 50, 345
359. Ryning, F.W., Remington, J.S., 1978. Effect of cytochalasin D on Toxoplasma gondii cell entry. Infect. Immun. 20, 739
743. Sadak, A., Taghy, Z., Fortier, B., Dubremetz, J.F., 1988. Characterization of a family of rhoptry proteins of Toxoplasma gondii. Mol. Biochem. Parasitol. 29, 203
211. Saeij, J.P., Boyle, J.P., Coller, S., Taylor, S., Sibley, L.D., Brooke-Powell, E.T., et al., 2006. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314, 1780
1783. Saeij, J.P., Coller, S., Boyle, J.P., Jerome, M.E., White, M.W., Boothroyd, J.C., 2007. Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445, 324
327. Saffer, L.D., Mercereau-Puijalon, O., Dubremetz, J.F., Schwartzman, J.D., 1992. Localization of a Toxoplasma gondii rhoptry protein by immunoelectron microscopy during and after host cell penetration. J. Protozool. 39, 526
530. Sakura, T., Sindikubwabo, F., Oesterlin, L.K., Bousquet, H., Slomianny, C., Hakimi, M.A., et al., 2016. A critical role for Toxoplasma gondii vacuolar protein sorting VPS9 in secretory organelle biogenesis and host infection. Sci. Rep. 6, 38842. Sanchez, V., de-la-Torre, A., Gomez-Marin, J.E., 2014. Characterization of ROP18 alleles in human toxoplasmosis. Parasitol. Int. 63, 463
469. Sangare, L.O., Alayi, T.D., Westermann, B., Hovasse, A., Sindikubwabo, F., Callebaut, I., et al., 2016. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection. Nat. Commun. 7, 11191. Santi-Rocca, J., Blanchard, N., 2017. Membrane trafficking and remodeling at the host-parasite interface. Curr. Opin. Microbiol. 40, 145
151.

Toxoplasma Gondii

References

Santos, J.M., Ferguson, D.J., Blackman, M.J., Soldati-Favre, D., 2011. Intramembrane cleavage of AMA1 triggers Toxoplasma to switch from an invasive to a replicative mode. Science 331, 473
477. Saouros, S., Edwards-Jones, B., Reiss, M., Sawmynaden, K., Cota, E., Simpson, P., et al., 2005. A novel galectin-like domain from Toxoplasma gondii micronemal protein 1 assists the folding, assembly, and transport of a cell adhesion complex. J. Biol. Chem. 280, 38583
38591. Saouros, S., Dou, Z., Henry, M., Marchant, J., Carruthers, V.B., Matthews, S., 2012. Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain. J. Biol. Chem. 287, 36029
36040. Sasono, P.M., Smith, J.E., 1998. Toxoplasma gondii: an ultrastructural study of host-cell invasion by the bradyzoite stage. Parasitol. Res. 84, 640
645. Sawmynaden, K., Saouros, S., Friedrich, N., Marchant, J., Simpson, P., Bleijlevens, B., et al., 2008. Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition. EMBO Rep. 9, 1149
1155. Schultz, A.J., Carruthers, V.B., 2018. Toxoplasma gondii LCAT primarily contributes to tachyzoite egress. mSphere 3. Available from: https://doi.org/10.1128/ mSphereDirect.00073-18. Schwab, J.C., Beckers, C.J., Joiner, K.A., 1994. The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve. Proc. Natl. Acad. Sci. U.S.A. 91, 509
513. Schwartzman, J.D., 1986. Inhibition of a penetrationenhancing factor of Toxoplasma gondii by monoclonal antibodies specific for rhoptries. Infect. Immun. 51, 760
764. Schwartzman, J.D., Saffer, L.D., 1992. How Toxoplasma gondii gets in and out of hostcells. Subcell. Biochem. 18, 333
364. Schwarz, J.A., Fouts, A.E., Cummings, C.A., Ferguson, D.J., Boothroyd, J.C., 2005. A novel rhoptry protein in Toxoplasma gondii bradyzoites and merozoites. Mol. Biochem. Parasitol. 144 (2), 159
166. Schweighofer, A., Hirt, H., Meskiene, I., 2004. Plant PP2C phosphatases: emerging functions in stress signaling. Trends Plant Sci. 9, 236
243. Seetharaman, J., Kanigsberg, A., Slaaby, R., Leffler, H., Barondes, S.H., Rini, J.M., 1998. X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution. J. Biol. Chem. 273, 13047
13052. Shastri, A.J., Marino, N.D., Franco, M., Lodoen, M.B., Boothroyd, J.C., 2014. GRA25 is a novel virulence factor of Toxoplasma gondii and influences the host immune response. Infect. Immun. 82, 2595
2605. Shaw, M.K., Roos, D.S., Tilney, L.G., 1998. Acidic compartments and rhoptry formation in Toxoplasma gondii. Parasitology 117 (Pt 5), 435
443.

701

Sheiner, L., Dowse, T.J., Soldati-Favre, D., 2008. Identification of trafficking determinants for polytopic rhomboid proteases in Toxoplasma gondii. Traffic 9, 665
677. Sheiner, L., Santos, J.M., Klages, N., Parussini, F., Jemmely, N., Friedrich, N., et al., 2010. Toxoplasma gondii transmembrane microneme proteins and their modular design. Mol. Microbiol . Shen, B., Sibley, L.D., 2014. Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion. Proc. Natl. Acad. Sci. U.S.A. 111 (9), 3567
3572. Shen, B., Buguliskis, J.S., Lee, T.D., Sibley, L.D., 2014. Functional analysis of rhomboid proteases during Toxoplasma invasion. MBio 5, e01795-01714. Sibley, L.D., 2003. Toxoplasma gondii: perfecting an intracellular life style. Traffic 4, 581
586. Sibley, L.D., Andrews, N.W., 2000. Cell invasion by unpalatable parasites. Traffic 1, 100
106. Sibley, L.D., Weidner, E., Krahenbuhl, J.L., 1985. Phagosome acidification blocked by intracellular Toxoplasma gondii. Nature 315, 416
419. Sibley, L.D., Krahenbuhl, J.L., Adams, G.M., Weidner, E., 1986. Toxoplasma modifies macrophage phagosomes by secretion of a vesicular network rich in surface proteins. J. Cell Biol. 103, 867
874. Sibley, L.D., Pfefferkorn, E.R., Boothroyd, J.C., 1991. Proposal for a uniform genetic nomenclature in Toxoplasma gondii. Parasitol Today 7, 327
328. Sibley, L.D., Niesman, I.R., Asai, T., Takeuchi, T., 1994. Toxoplasma gondii: secretion of a potent nucleoside triphosphate hydrolase into the parasitophorous vacuole. Exp. Parasitol. 79, 301
311. Sibley, L.D., Niesman, I.R., Parmley, S.F., Cesbron-Delauw, M.F., 1995. Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J. Cell Sci. 108, 1669
1677. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166, 1423
1435. e1412. Silverman, J.A., Qi, H., Riehl, A., Beckers, C., Nakaar, V., Joiner, K.A., 1998. Induced activation of the Toxoplasma gondii nucleoside triphosphate hydrolase leads to depletion of host cell ATP levels and rapid exit of intracellular parasites from infected cells. J. Biol. Chem. 273, 12352
12359. Silvie, O., Franetich, J.F., Charrin, S., Mueller, M.S., Siau, A., Bodescot, M., et al., 2004. A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites. J. Biol. Chem. 279, 9490
9496. Sinai, A.P., Joiner, K.A., 2001. The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane. J. Cell Biol. 154, 95
108.

Toxoplasma Gondii

702

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Sinai, A.P., Webster, P., Joiner, K.A., 1997. Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J. Cell Sci. 110 (Pt 17), 2117
2128. Singh, S., Alam, M.M., Pal-Bhowmick, I., Brzostowski, J.A., Chitnis, C.E., 2010. Distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites. PLoS Pathog. 6, e1000746. Sloves, P.J., Delhaye, S., Mouveaux, T., Werkmeister, E., Slomianny, C., Hovasse, A., et al., 2012. Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection. Cell Host Microbe 11, 515
527. Sohn, C.S., Cheng, T.T., Drummond, M.L., Peng, E.D., Vermont, S.J., Xia, D., et al., 2011. Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach. PLoS One 6, e18383. Soldati, D., Meissner, M., 2004. Toxoplasma as a novel system for motility. Curr Opin Cell Biol. 16, 32
40. Soldati, D., Kim, K., Kampmeier, J., Dubremetz, J.F., Boothroyd, J.C., 1995. Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection. Mol. Biochem. Parasitol. 74, 87
97. Soldati, D., Lassen, A., Dubremetz, J.F., Boothroyd, J.C., 1998. Processing of Toxoplasma ROP1 protein in nascent rhoptries. Mol. Biochem. Parasitol. 96, 37
48. Spano, F., Putignani, L., Naitza, S., Puri, C., Wright, S., Crisanti, A., 1998. Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the thrombospondin family. Mol. Biochem. Parasitol. 92, 147
162. Speer, C.A., Dubey, J.P., Blixt, J.A., Prokop, K., 1997. Time lapse video microscopy and ultrastructure of penetrating sporozoites, types 1 and 2 parasitophorous vacuoles, and the transformation of sporozoites to tachyzoites of the VEG strain of Toxoplasma gondii. J. Parasitol. 83, 565
574. Speer, C.A., Tilley, M., Temple, M.E., Blixt, J.A., Dubey, J. P., White, M.W., 1995. Sporozoites of Toxoplasma gondii lack dense-granule protein GRA3 and form a unique parasitophorous vacuole. Mol. Biochem. Parasitol. 75, 75
86. Spillman, N.J., Beck, J.R., Goldberg, D.E., 2015. Protein export into malaria parasite-infected erythrocytes: mechanisms and functional consequences. Annu. Rev. Biochem. 84, 813
841.

Srivastava, A., Singh, S., Dhawan, S., Mahmood Alam, M., Mohmmed, A., Chitnis, C.E., 2010. Localization of apical sushi protein in Plasmodium falciparum merozoites. Mol. Biochem. Parasitol. 174, 66
69. Stadler, R.V., White, L.A., Hu, K., Helmke, B.P., Guilford, W.H., 2017. Direct measurement of cortical force generation and polarization in a living parasite. Mol. Biol. Cell 28, 1912
1923. Starnes, G.L., Jewett, T.J., Carruthers, V.B., Sibley, L.D., 2006. Two separate, conserved acidic amino acid domains within the Toxoplasma gondii MIC2 cytoplasmic tail are required for parasite survival. J. Biol. Chem. 281, 30745
30754. Stedman, T.T., Joiner, K.A., 1999. En route to the vacuole. Tracing the secretory pathway of Toxoplasma gondii. In: Gordon, S. (Ed.), Phagocytosis: Microbial Invasion. JAI Press Inc, Stamford, CT, pp. 233
261. Steinfeldt, T., Konen-Waisman, S., Tong, L., Pawlowski, N., Lamkemeyer, T., Sibley, L.D., et al., 2010. Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol. 8, e1000576. Stewart, M.J., Schulman, S., Vanderberg, J.P., 1986. Rhoptry secretion of membranous whorls by Plasmodium falciparum merozoites. Am. J. Trop. Med. Hyg. 35, 37
44. Stommel, E.W., Ely, K.H., Schwartzman, J.D., Kasper, L.H., 1997. Toxoplasma gondii: dithiol-induced Ca2 1 flux causes egress of parasites from the parasitophorous vacuole. Exp. Parasitol. 87, 88
97. Straub, K.W., Cheng, S.J., Sohn, C.S., Bradley, P.J., 2009. Novel components of the apicomplexan moving junction reveal conserved and coccidia-restricted elements. Cell. Microbiol. 11, 590
603. Straub, K.W., Peng, E.D., Hajagos, B.E., Tyler, J.S., Bradley, P.J., 2011. The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii. PLoS Pathog. 7, e1002007. Striepen, B., He, C.Y., Matrajt, M., Soldati, D., Roos, D.S., 1998. Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii. Mol. Biochem. Parasitol. 92, 325
338. Sultan, A.A., Thathy, V., Frevert, U., Robson, K.J., Crisanti, A., Nussenzweig, V., et al., 1997. TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell 90, 511
522. Suss-Toby, E., Zimmerberg, J., Ward, G.E., 1996. Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore. Proc. Natl. Acad. Sci. U.S.A. 93, 8413
8418.

Toxoplasma Gondii

References

Sweeney, K.R., Morrissette, N.S., LaChapelle, S., Blader, I.J., 2010. Host cell invasion by Toxoplasma gondii is temporally regulated by the host microtubule cytoskeleton. Eukaryot. Cell 9, 1680
1689. Talevich, E., Kannan, N., 2013. Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors. BMC Evol. Biol. 13. Available from: https://doi.org/10.1186/1471-214813-117. Tan, K., Duquette, M., Liu, J.H., Dong, Y., Zhang, R., Joachimiak, A., et al., 2002. Crystal structure of the TSP1 type 1 repeats: a novel layered fold and its biological implication. J. Cell Biol. 159, 373
382. Tang, V.W., Brieher, W.M., 2013. FSGS3/CD2AP is a barbedend capping protein that stabilizes actin and strengthens adherens junctions. J. Cell Biol. 203, 815
833. Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., et al., 2006. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314, 1776
1780. Thomas, A.W., Deans, J.A., Mitchell, G.H., Alderson, T., Cohen, S., 1984. The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes. Mol. Biochem. Parasitol. 13, 187
199. Tilley, M., Fichera, M.E., Jerome, M.E., Roos, D.S., White, M.W., 1997. Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins. Infect. Immun. 65, 4598
4605. Tomley, F.M., Billington, K.J., Bumstead, J.M., Clark, J.D., Monaghan, P., 2001. EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats. Int. J. Parasitol. 31, 1303
1310. Tonkin, M.L., Grujic, O., Pearce, M., Crawford, J., Boulanger, M.J., 2010. Structure of the micronemal protein 2 A/I domain from Toxoplasma gondii. Protein Sci. 19, 1985
1990. Tonkin, M.L., Roques, M., Lamarque, M.H., Pugniere, M., Douguet, D., Crawford, J., et al., 2011. Host cell invasion by apicomplexan parasites: insights from the costructure of AMA1 with a RON2 peptide. Science 333, 463
467. Tonkin, M.L., Crawford, J., Lebrun, M.L., Boulanger, M.J., 2012. Babesia divergens and Neospora caninum apical membrane antigen 1 (AMA1) structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion. Protein Sci . Available from: https://doi.org/ 10.1002/pro.2193.

703

Tordai, H., Banyai, L., Patthy, L., 1999. The PAN module: the N-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins. FEBS Lett. 461, 63
67. Torpier, G., Charif, H., Darcy, F., Liu, J., Darde, M.L., Capron, A., 1993. Toxoplasma gondii: differential location of antigens secreted from encysted bradyzoites. Exp. Parasitol. 77, 13
22. Tosetti, N., Dos Santos Pacheco, N., Soldati-Favre, D., Jacot, D., 2019. Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii. eLife 8. Available from: https://doi. org/10.7554/eLife.42669. Travier, L., Mondragon, R., Dubremetz, J.F., Musset, K., Mondragon, M., Gonzalez, S., et al., 2008. Functional domains of the Toxoplasma GRA2 protein in the formation of the membranous nanotubular network of the parasitophorous vacuole. Int. J. Parasitol. 38, 757
773. Treeck, M., Zacherl, S., Herrmann, S., Cabrera, A., Kono, M., Struck, N.S., et al., 2009. Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process. PLoS Pathog. 5, e1000322. Turetzky, J.M., Chu, D.K., Hajagos, B.E., Bradley, P.J., 2010. Processing and secretion of ROP13: a unique Toxoplasma effector protein. Int. J. Parasitol. 40, 1037
1044. Tyler, J.S., Boothroyd, J.C., 2011. The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex. PLoS Pathog. 7, e1001282. Ullrich, O., Reinsch, S., Urbe, S., Zerial, M., Parton, R.G., 1996. Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135, 913
924. Umemoto, K., Leffler, H., Venot, A., Valafar, H., Prestegard, J.H., 2003. Conformational differences in liganded and unliganded states of galectin-3. Biochemistry 42, 3688
3695. Urban, S., Freeman, M., 2003. Substrate specificity of rhomboid intramembrane proteases is governed by helixbreaking residues in the substrate transmembrane domain. Mol. Cell 11, 1425
1434. Vanderberg, J.P., 1974. Studies on the motility of Plasmodium sporozoites. J. Protozool. 21, 527
537. Veiga, E., Cossart, P., 2005. Listeria hijacks the clathrindependent endocytic machinery to invade mammalian cells. Nat. Cell Biol. 7, 894
900.

Toxoplasma Gondii

704

14. Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Venugopal, K., Werkmeister, E., Barois, N., Saliou, J.M., Poncet, A., Huot, L., et al., 2017. Dual role of the Toxoplasma gondii clathrin adaptor AP1 in the sorting of rhoptry and microneme proteins and in parasite division. PLoS Pathog. 13, e1006331. Vulliez-Le Normand, B., Tonkin, M.L., Lamarque, M.H., Langer, S., Hoos, S., Roques, M., et al., 2012. Structural and functional insights into the malaria parasite moving junction complex. PLoS Pathog. 8, e1002755. Walker, M.E., Hjort, E.E., Smith, S.S., Tripathi, A., Hornick, J.E., Hinchcliffe, E.H., et al., 2008. Toxoplasma gondii actively remodels the microtubule network in host cells. Microbes Infect 10, 1440
1449. Wan, K.L., Carruthers, V.B., Sibley, L.D., Ajioka, J.W., 1997. Molecular characterisation of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2. Mol. Biochem. Parasitol. 84, 203
214. Wang, Y., Weiss, L.M., Orlofsky, A., 2010. Coordinate control of host centrosome position, organelle distribution, and migratory response by Toxoplasma gondii via host mTORC2. J. Biol. Chem. 285, 15611
15618. Wang, Z.T., Harmon, S., O’Malley, K.L., Sibley, L.D., 2015. Reassessment of the role of aromatic amino acid hydroxylases and the effect of infection by Toxoplasma gondii on host dopamine. Infect. Immun. 83, 1039
1047. Wang, K., Peng, E.D., Huang, A.S., Xia, D., Vermont, S.J., Lentini, G., et al., 2016. Identification of novel O-linked glycosylated Toxoplasma proteins by Vicia villosa lectin chromatography. PLoS One 11, e0150561. Wang, J.L., Li, T.T., Elsheikha, H.M., Chen, K., Zhu, W.N., Yue, D.M., et al., 2017a. Functional characterization of rhoptry kinome in the virulent Toxoplasma gondii RH strain. Front. Microbiol. 8, 84. Wang, M., Cao, S., Du, N., Fu, J., Li, Z., Jia, H., et al., 2017b. The moving junction protein RON4, although not critical, facilitates host cell invasion and stabilizes MJ members. Parasitology 144, 1490
1497. Weisman, L.S., 2006. Organelles on the move: insights from yeast vacuole inheritance. Nat. Rev. Mol. Cell Biol. 7, 243
252. Werk, R., Fischer, S., 1982. Attempts to infect plant protoplasts with Toxoplasma gondii. J. Gen. Microbiol. 128, 211
213.

Whitelaw, J.A., Latorre-Barragan, F., Gras, S., Pall, G.S., Leung, J.M., Heaslip, A., et al., 2017. Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion. BMC Biol. 15, 1. Whittaker, C.A., Hynes, R.O., 2002. Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. Mol. Biol. Cell 13, 3369
3387. Wright, C.S., Kellogg, G.E., 1996. Differences in hydropathic properties of ligand binding at four independent sites in wheat germ agglutinin-oligosaccharide crystal complexes. Protein Sci. 5, 1466
1476. Wright, H.T., Sandrasegaram, G., Wright, C.S., 1991. Evolution of a family of N-acetylglucosamine binding proteins containing the disulfide-rich domain of wheat germ agglutinin. J. Mol. Evol. 33, 283
294. Yang, A.S.P., Lopaticki, S., O’Neill, M.T., Erickson, S.M., Douglas, D.N., Kneteman, N.M., et al., 2017. AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver. Cell. Microbiol. 19. Ye, S., Xia, N., Zhao, P., Yang, J., Zhou, Y., Shen, B., et al., 2019. Micronemal protein 13 contributes to the optimal growth of Toxoplasma gondii under stress conditions. Parasitol. Res. 118, 935
944. Zhao, Y., Marple, A.H., Ferguson, D.J., Bzik, D.J., Yap, G.S., 2014. Avirulent strains of Toxoplasma gondii infect macrophages by active invasion from the phagosome. Proc. Natl. Acad. Sci. U.S.A. 111, 6437
6442. Zhou, X.W., Blackman, M.J., Howell, S.A., Carruthers, V.B., 2004. Proteomic analysis of cleavage events reveals a dynamic two-step mechanism for proteolysis of a key parasite adhesive complex. Mol. Cell. Proteomics 3, 565
576. Zhou, X.W., Kafsack, B.F., Cole, R.N., Beckett, P., Shen, R.F., Carruthers, V.B., 2005. The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins. J. Biol. Chem. 280, 34233
34244. Zinecker, C.F., Striepen, B., Tomavo, S., Dubremetz, J.F., Schwarz, R.T., 1998. The dense granule antigen, GRA2 of Toxoplasma gondii is a glycoprotein containing Olinked oligosaccharides. Mol. Biochem. Parasitol. 97, 241
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15 Endomembrane trafficking pathways in Toxoplasma Se´bastien Besteiro1, Christen M. Klinger2, Markus Meissner3 and Vern B. Carruthers4 1

DIMNP, UMR5235 CNRS, University of Montpellier, Montpellier, France 2Division of Infectious Diseases, Department of Medicine, University of Alberta, Edmonton, AB, Canada 3Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-University, Munich, Germany 4 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, United States

15.1 Introduction Toxoplasma gondii, like all apicomplexan parasites, is a highly polarized cell (see Chapter 2: The ultrastructure of Toxoplasma gondii), with a complex secretory system comprising three different types of unique secretory organelles (micronemes, rhoptries, and dense granules) that fulfill important roles during host cell invasion, modulation, and egress (see Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection). In addition, other apicomplexan-specific organelles, such as the apicoplast (a nonphotosynthetic secondary plastid of red algal origin) and the inner membrane complex (IMC, a system of flattened Golgi-derived membrane cisternae underlying the entire plasma membrane), are also

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00015-3

connected to the secretory pathway of the parasite. Intriguingly, this complex cellular organization is not reflected by an obvious expansion of trafficking factors (see Section 15.3), leading to the hypothesis that apicomplexans repurposed parts of their endocytic system (Tomavo et al., 2013). Empirical support for this has come from functional characterization of several trafficking factors involved in endocytosis or endocytic trafficking in other eukaryotes, revealing defects in the biogenesis of micronemes, rhoptries, dense granules, or the IMC (see Section 15.4). However, previous electron-microscopic studies suggested clathrin-dependent and independent uptake of exogenous material at a unique structure called the micropore (Nichols et al., 1994) and receptor-mediated uptake of heparin-glycans was demonstrated to occur in

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extracellular parasites (Botero-Kleiven et al., 2001). In good agreement, recent studies demonstrated the uptake of exogenous material by the parasite during intracellular replication (Dou et al., 2014; McGovern et al., 2018) and in extracellular parasites (Gras et al., 2019), strongly suggesting that apicomplexans are well capable of endocytosis. Internalized material accumulates in a vacuolar compartment (VAC), also called the plant-like vacuole (PLV), which contains a set of proteases, such as cathepsins B and L (Miranda et al., 2010; Parussini et al., 2010), demonstrating that the secretory and endocytic pathways intersect (see Sections 15.4 and 15.6). The VAC/PLV also intersects with the parasite’s autophagy pathway (see Section 15.9), which may play an important role during stress responses (Section 15.9.2), as well as replication and differentiation (Section 15.9.3), while part of the autophagy-related (ATG)

machinery also plays a role in apicoplast homeostasis unrelated to this degradative function (Section 15.9.4). The purpose of this chapter is to recapitulate and to integrate key findings regarding the organization of the Toxoplasma endomembrane system, discuss the evolution of the membrane trafficking system (MTS), and to highlight open questions that need to be addressed in future studies to understand the cell biology of this fascinating parasite.

15.2 Sorting signals of secretory proteins Apicomplexan parasites possess a conventional eukaryotic secretory system (Fig. 15.1) consisting of the endoplasmic reticulum (ER), a single Golgi stack (Pelletier et al., 2002), endosomal-like compartments [ELCs (Harper et al., 2006; Parussini et al., 2010)], from

FIGURE 15.1 Schematic representation of the general endomembrane system in Toxoplasma. This illustration is highly schematic and is used to give an overview of the major membrane-bound organelles and components of the exocytic and endocytic pathways typically present in a Toxoplasma gondii tachyzoite. ER, Endoplasmic reticulum; IMC, inner membrane complex; TGN, trans-Golgi network; VAC/PLV, vacuolar compartment/plant-like vacuole.

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15.2 Sorting signals of secretory proteins

where proteins are further transported to the VAC/PLV (see later), and/or the unique secretory organelles localized at the apical pole of the parasite (microneme and rhoptries) (Jimenez-Ruiz et al., 2016). In contrast to micronemes or rhoptries, where sorting signals have been described for their directed transport, a third set of secretory organelles, the dense granules, is assumed to be part of the constitutive, or “default,” secretory pathway, since proteins trafficked to these compartments do not require specific signal sequences, apart from a signal peptide that directs them into the ER (Karsten et al., 1998). Furthermore, the elaborate alveolar IMC system (Mann and Beckers, 2001) and the apicoplast (Fichera and Roos, 1997) are also directly linked to the secretory system of the parasite. Other organelles described in apicomplexans and probably linked to the secretory system include the acidocalcisomes [calcium storage organelles (Miranda et al., 2008)]. While targeting of proteins to the apicoplast and micronemes is facilitated via recognition of conserved sorting signals (Di Cristina et al., 2000; Tonkin et al., 2008), the presence of specific signals involved in the directed transport to other organelles is still enigmatic. As discussed in more detail later, the route for trafficking to the rhoptries and micronemes is almost identical, and it has been speculated that the timing of protein expression is a major determinant for the directed transport to these organelles (Besteiro et al., 2009).

15.2.1 Trafficking of rhoptry proteins The rhoptries are secreted at the onset of host cell invasion. Although the signaling pathway involved in regulated rhoptry secretion is unknown, the micronemal proteins TgMIC8 (Kessler et al., 2008) and TgCLAMP (Sidik et al., 2016), and a cytosolic ferlin calcium sensor (Coleman et al., 2018) have been

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identified as being essential for rhoptry secretion. These organelles contain factors important for host cell modifications and generating the entry portal in the host cell plasma membrane through which the parasite invades (see Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection). Similar to micronemes, rhoptries are thought to be formed de novo late during daughter cell assembly (Nishi et al., 2008), but in contrast to micronemes, these organelles appear to be developed from immature organelles, defined as prerhoptries, which can be found in the apical region of the parasite. The prerhoptries evolve into mature organelles by elongating the rhoptry neck toward the conoid (Dubremetz, 2007). While it has been suggested that subpellicular microtubules are important for anchoring the rhoptries to the apical tip (Nichols et al., 1983), subsequent work indicates that this process is dependent on actin, MyoF, and an armadillo repeat protein, TgAro, since their disruption results in dispersal of rhoptries (Mueller et al., 2013). A similar role in apical positioning of rhoptries has been attributed to an apicomplexan-specific factor, TgCSCHAP, found to interact with subunits of the CORVET/HOPS complex (Morlon-Guyot et al., 2018), although in this case, a more general effect on secretory organelles has been observed, leading also to the disappearance of micronemes (see later). The existence and identity of specific sorting signals for trafficking of proteins to the rhoptries are still under debate. Earlier studies suggested that a dileucine and tyrosine-based signal is present in the cytosolic tail of members of the rhoptry protein (ROP)2 family (Hoppe et al., 2000), which interacts with the adaptor complex AP-1 (Ngoˆ et al., 2003) resulting in transport to the rhoptries. However, subsequent studies suggested that this interaction was an in vitro artifact, despite confirming a role for AP-1 in rhoptry trafficking (see Section 15.3.8).

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15.2.2 Trafficking of micronemal proteins Apicomplexan parasites express a large repertoire of proteins destined for the micronemes, including critical factors required for host cell egress, gliding motility, and invasion (see Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection). Recent studies demonstrated that micronemes exist in different, potentially functionally distinct, subsets (Kremer et al., 2013). Functional interference with either T. gondii Rab GTPase 5A or 5C leads to sorting defects to the rhoptries and of a subset of micronemal proteins, such as MIC3 and MIC8, while other micronemal proteins are not affected, including AMA1, MIC2, and M2AP (Kremer et al., 2013). Such an organization could explain the stepwise action of micronemal proteins during the lytic cycle of the parasite (MIC2 for gliding, MIC8 for rhoptry secretion and AMA1 for invasion), but it remains to be determined if the independent subsets of micronemes are also independently secreted (Kremer et al., 2013). Many micronemal transmembrane proteins contain well-defined tyrosine-based signals in their cytosolic tails, as shown for MIC2 and MIC6, which are required for their directed transport (Di Cristina et al., 2000; Reiss et al., 2001). Mutation of these motifs leads to rerouting to the dense granules and their constitutive secretion. Furthermore, some soluble microneme proteins appear to be transported in complex with transmembrane proteins that act as escorters, as shown for MIC6-1-4 and MIC2M2AP (Reiss et al., 2001; Harper et al., 2006). However, some transmembrane proteins, including MIC8 and AMA1 (Kessler et al., 2008; Sheiner et al., 2010), do not contain welldefined sorting signals, suggesting that they are transported in a complex with other micronemal proteins or that proteolytic maturation during their transport is required for correct sorting to the micronemes.

15.2.3 The role of proteolytic maturation of secretory proteins for their transport More than half of the known MIC proteins appear to undergo processing steps. Propeptide processing has emerged as an important feature of many eukaryotic proteins in order to be activated and sorted to their final destination. This situation appears to be similar in apicomplexan parasites, where several micronemal proteins require a propeptide for correct sorting and cleavage of the propeptide for efficient secretion. An antibody specific to the propeptide of M2AP (proM2AP) not only demonstrated the importance of this processing step, but it also led to the identification of ELCs in the parasite (Harper et al., 2006). Furthermore, since M2AP is transported in complex with the important micronemal adhesin MIC2, deleting the M2AP propeptide also results in mislocalization of MIC2 and a phenotype that is comparable to a mic2 null mutant (Harper et al., 2006). Similar to M2AP, propeptides on MIC5 and MIC3 are necessary for their correct sorting, and domain swap experiments using propeptides from different micronemal proteins from Toxoplasma and Eimeria tenella demonstrated the presence of conserved amino acids required for targeting to the micronemes (Gaji et al., 2011). A noncleavable propeptide mutant of proM2AP correctly localized to the micronemes, indicating that propeptide processing is not necessary for trafficking to the micronemes (Harper et al., 2006). However, the processing of proM2AP is required to form a highly stable MIC2
M2AP complex and the noncleavable mutant was secreted from the micronemes at a moderately lower rate (Harper et al., 2006). A recent study identified aspartylprotease 3 (TgASP3) to be required for the processing of several micronemal and ROPs. These proteins were still efficiently trafficked to the micronemes of asp3 null parasites (Dogga et al., 2017), consistent with propeptide processing being dispensable for correct

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localization. Those asp3 null parasites are substantially impaired in egress and invasion is likely due to additive effects from defective propeptide processing of multiple microneme and ROPs.

15.2.4 Recycling of maternal organelles during replication Apicomplexan parasites replicate in a process best described as internal budding, when daughter cells are formed within the mother. In the case of T. gondii asexual stages only two daughters are formed in a process called endodyogeny. Interestingly, the polarised organisation of the mother cell is maintained until daughter cells are mature, when they suddenly disappear, and daughters hatch out of the mother. Until recently it was believed that formation of the unique secretory organelles is exclusively de novo, while the organelles of the mother are degraded (Jimenez-Ruiz et al. 2016). Using novel imaging approaches it has been demonstrated that formation of the IMC in the daughters involves a combination of de novo synthesis and recycling of maternal IMC at the final stages of replication (Ouologuem and Roos, 2014). Similarly, using a dual labelling strategy it has been demonstrated that maternal micronemes are almost quantitatively recycled from the mother to the daughter parasites in a mechanism that requires F-actin dependent vesicular transport (Periz et al. 2019), leading to the hypothesis that this recycling mechanisms might be in place for other organelles, such as dense granules or rhoptries.

15.3 Coding complement of the Toxoplasma gondii membrane-trafficking system

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and lipid components between distinct subcellular locations. The machinery involved includes proteins for vesicle formation, translocation, tethering, and fusion. Critically, each step is mediated by different members of the same paralogous gene family [e.g., Rabs and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)], with the combinatorial interaction between different members dictating the specificity of the trafficking step. This suggests a straightforward mechanism for the evolution of a complex network of autogenously generated organelles from a single ancestral compartment, termed the organelle paralogy hypothesis (Dacks and Field, 2007). It also suggests what our previous metaanalysis confirmed, that orthologous genes tend to perform the same or similar function, allowing initial hypotheses for gene function based on well-characterized orthologs in other systems (Klinger et al., 2016). In the case of further paralogous expansion, the additional copies allow one or more to be unconstrained and diverge both in sequence and function, a case that has been described in Apicomplexa (see later). Early studies suggested the presence of trafficking homologs in the genomes of apicomplexans, including components mediating all steps of trafficking (Field et al., 2007; Koumandou et al., 2007; Langsley et al., 2008; Leung et al., 2008; Nevin and Dacks, 2009). Subsequent studies expanded these results and also revealed the general pattern of MTS evolution in apicomplexa compared to their free-living relatives: general retention, but with frequent loss of individual paralogs/subunits of larger complexes, and occasional loss of whole complexes as well (Klinger et al., 2013; Woo et al., 2015). We briefly touch on a few key examples.

15.3.1 Overview of trafficking in the apicomplexa

15.3.2 Ras-related protein from brain (Rab) GTPases

The MTS comprises the cellular machinery required for the regulated movement of protein

Rab GTPases are small G-proteins that undergo a guanosine triphosphate-guanosine

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diphosphate (GTP-GDP) cycle: the Rab binds GTP, hydrolyzes the GTP to form GDP, and subsequently releases the GDP to prepare for binding of another GTP molecule [(Stenmark, 2009) inter alia]. The bound nucleotide state of a Rab determines its conformation, which in turn allows it to bind to different sets of effectors. Although normally associated with membranes in their GDP-bound state, Rabs can be found in the cytosol in complex with GDP dissociation inhibitor proteins; reversible binding allows Rabs to become membrane associated again through their prenylated C-terminus and interact with guanine exchange factor (GEF) proteins to release GDP and bind GTP (Edler and Stein, 2019). Langsley et al. reported a complement of 15 Rabs for T. gondii, compared to 11 in yeast and B70 in mammalian cells (Langsley et al., 2008; Langemeyer et al., 2018). In mammalian cells, Rab5 localizes to early endosomes, where it recruits effectors involved in the tethering of endocytic vesicles, maturation of the compartment and the switch to a “late” Rab7-positive compartment, as well as homo/heterotypic fusion of early and late endosomes (Christoforidis et al., 1999; Shin et al., 2005; Peplowska et al., 2007; Poteryaev et al., 2010; Lachmann et al., 2014). In T. gondii, there are three Rab5 paralogs, referred to as TgRab5A, TgRab5B, and TgRab5C (Kremer et al., 2013). The Plasmodium ortholog of Rab5B was noted to be N-terminally myristoylated, bearing some similarity to Arabidopsis thaliana ARA6 (Ezougou et al., 2014), and as predicted for TgRab5B as well (Klinger, unpublished). In T. gondii, all three Rab5 paralogs localize to a subapical region housing the Golgi and endosomal compartments. TgRab5B appears to be localized to the plasma membrane as well (Kremer et al., 2013) and interacts with the retromer subunit TgVps26 (Sangare´ et al., 2016). In other systems, Rab6 localizes primarily to the Golgi/trans-Golgi network (TGN) and plays

diverse roles, including in trafficking through the Golgi, Golgi organization, autophagy, and cytokinesis (Hill et al., 2000; Matanis et al., 2002; Burguete et al., 2008; Ayala et al., 2018). TgRab6 localizes to the Golgi, TGN, and somewhat to dense granules. Golgi localization was further supported by ER-like staining of TgRab6 following treatment with brefeldin A (BFA) a fungal molecule that inhibits a subset of adenosine diphosphate (ADP)-ribosylation factor (ARF) family protein GEFs and typically causes collapse of the Golgi into the ER. Single nucleotide mutants predicted to be either dominantly active or inactive altered bacterial alkaline phosphatase (BAP) and GRA1 staining (both dense granule markers), causing more to be retained in the Golgi; hence, it was suggested that Rab6 is involved in dense granule formation (Stedman et al., 2003). In opisthokonts, Rab7 primarily localizes to late endosomal compartments and is responsible for fusion of these compartments as well as recruitment of retromer for cargo recycling prior to terminal degradation (Seaman et al., 2009; Plemel et al., 2011; McEwan et al., 2015; ˝ et al., 2016). In T. gondii, Rab7 localizes Hegedus to the compartments that stain with TgSORTLR, proM2AP, TgVP1, and TgCPL, all markers of endolysosomal structures (Parussini et al., 2010; Kremer et al., 2013). Overexpression of TgRab7 shows no obvious effects (Kremer et al., 2013), and constitutively active or inactive forms in Plasmodium show no apparent effects either (Krai et al., 2014); as such, Rab7 function in Apicomplexa is currently unclear. Rab11 is perhaps best known for its role in endosome to plasma membrane recycling (Ullrich et al., 1996) but is involved in diverse processes, including cytokinesis, ciliogenesis, and autophagy (Kno¨dler et al., 2010; Szatma´ri et al., 2014; Nakayama, 2016). T. gondii possesses two paralogs, the pan-eukaryotic TgRab11A and the alveolate-specific TgRab11B (AgopNersesian et al., 2010; Klinger, unpublished). TgRab11B localizes apical to the Golgi, interacts

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with the retromer subunit TgVps26, and is involved in the delivery of early and late stage components to the IMC during endodyogeny (Agop-Nersesian et al., 2010; Sangare´ et al., 2016). TgRab11A also localizes apical to the Golgi and colocalizes with ROP5; disruption of TgRab11A results in aberrant localization of the surface protein TgSAG1 in the Golgi region, suggesting TgRab11A mediates trafficking and/ or recycling of cell surface material (AgopNersesian et al., 2009), and may have other roles as well (Venugopal and Marion, 2018). Other Rab GTPases in Apicomplexa are less well characterized; an overexpression screen localized the majority to distinct locations within the cell, including the ER/Golgi (TgRab1B, TgRab2, TgRab18) and the Golgi/ TGN (TgRab4). Of these, TgRab2 and TgRab4 overexpression blocked growth, although in a CRISPR-based genome-wide disruption screen, TgRab2 was predicted to be essential, while TgRab4 was not (Kremer et al., 2013; Sidik et al., 2016). Thus further studies are required to elucidate Rab function in Apicomplexa.

15.3.3 Other GTPases Other important GTPases in the MTS are the ARF and Arf-related (Arl) GTPases. There are six ARFs in mammalian cells divided into three classes, which localize primarily to the Golgi (ARF1
5) and the plasma membrane (ARF6) and are involved in recruitment of enzymes, tethers, adaptors, and coats. Arl protein localization and function are more diverse, but they are generally involved in trafficking and sometimes mediate interaction with the cytoskeleton ((Donaldson and Jackson, 2011) inter alia). The only characterized ARF family GTPase in T. gondii to date is the ARF homolog, TgARF1. This protein localizes to the Golgi complex, and expression of a dominant negative version appears to affect dense granule formation (Liendo et al., 2001).

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15.3.4 Tethers Eukaryotes possess a diverse array of tethers, including the multisubunit tethering complexes: the various TRAPP and VpsC complexes, Dsl1, GARP, COG, and exocyst (Bro¨cker et al., 2010). Although they function throughout the cell, all are involved in the initial long-range interaction of a transport structure with the target membrane, prior to SNARE-mediated fusion. With the exception of the VpsC core complex subunits of Vps11, Vps16, Vps18, and Vps33, no complex is universally conserved in Apicomplexa, despite almost complete retention of most subunits in their free-living chromerid ancestors (Woo et al., 2015; Klinger et al., 2016). Of note, the exocyst complex is involved in diverse-regulated secretion events in mammalian cells (Polgar and Fogelgren, 2018) but is absent in all myzozoa (i.e., Apicomplexa, dinoflagellates, and related basal taxa, but excluding ciliates) datasets studied to date, which we hypothesize suggests a unique, exocystindependent, mode of regulated apical organelle exocytosis (Farrell et al., 2012). The only characterized tether to date in Apicomplexa is the VpsC complex. This core complex additionally binds either Vps3/8 or Vps39/41 on its terminal edges to produce either the early endosomal CORVET or late endosomal HOPS complexes (Kleine Balderhaar and Ungermann, 2013). It appears that the only complex present in canonical form is the CORVET complex; Morlon-Guyot et al. described the lone TgVps3/39-like subunit as Vps39, but robust phylogenetic analysis clearly demonstrates that this subunit is a Vps3 ortholog (Klinger et al., 2013; Morlon-Guyot et al., 2015; Klinger, unpublished). This study also reported the absence of Vps8 (Morlon-Guyot et al., 2015), despite previous informatic prediction of a canonical Vps8 ortholog (Klinger et al., 2013), which was subsequently confirmed as a bona fide VpsC complex subunit by pulling down TgVps11 (Morlon-Guyot et al., 2018). An

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additional subunit, TgBDCP, may represent an analogous replacement for the canonical Vps39 subunit in these parasites, as it interacts with the VpsC complex and knockdown of the protein results in aberrant morphology of the VAC/PLV (Morlon-Guyot et al., 2018). Determining Rab interactions between these subunits may further assist in confirming their identities, although interpretation of such results should be performed carefully, as yeast HOPS directly binds Rab7, while mammalian HOPS appears to do so only through intermediates (Plemel et al., 2011; McEwan et al., 2015).

15.3.5 Soluble N-ethylmaleimidesensitive factor attachment protein receptors SNAREs are coiled-coil proteins, usually with a C-terminal transmembrane domain, that mediate fusion of closely opposing membranes (Jahn and Scheller, 2006). Although T. gondii encodes B26 SNAREs, including members of each major SNARE subfamily (Qa, Qb, Qc, and R; Klinger, unpublished), to date, only a Syntaxin-6 (TgStx6) homolog has been characterized. It localizes to the Golgi/TGN and is potentially involved in fusion of retrograde vesicular traffic from the endosomal system with the TGN, as overexpression causes fragmentation of Golgi and post-Golgi compartments (Jackson et al., 2013).

15.3.6 Endosomal sorting complexes required for transport complexes The endosomal sorting complexes required for transport (ESCRT) are several heteromultimeric complexes (ESCRT0-III) and associated machinery (ESCRT-IIIa) that mediate diverse functions in the cell, all of which rely on membrane budding that is the topological reverse of the invagination involved in vesicular formation (i.e., “away” from the cytosol). They are not

only primarily involved in the formation of intraluminal vesicles in late endosomal structures (“multivesicular bodies,” MVBs) and in cell division but are also hijacked by some viruses to mediate budding of new viral particles [(Henne et al., 2011) inter alia]. Apicomplexa retain few ESCRT subunits; all apicomplexans apart from piroplasmids completely lack ESCRT-I and ESCRT-II and retain only a subset of ESCRT-III/IIIa. Although no eukaryotes outside of opisthokonts encode the “canonical” ESCRT-0 complex, Tom1 is thought to perform similar function and is encoded in most apicomplexan genomes (Leung et al., 2008; Blanc et al., 2009; Woo et al., 2015). Vps4, the lone subunit studied to date, is lethal when overexpressed and was described to result in the formation of unusual “MVBlike” structures (Yang et al., 2004). However, knockout parasites are viable; in general, it appears as though ESCRT in Apicomplexa may play a nonessential role in cell division (E. Jimenez-Ruiz, personal communication).

15.3.7 Coats Three canonical protein coats are described to mediate trafficking in eukaryotes. The coatomer protein (COP) I and COPII complexes mediate retrograde and anterograde transport between the ER and Golgi and throughout the Golgi, respectively (Miller and Schekman, 2013; Jackson, 2014). The clathrin coat is involved in both anterograde and retrograde post-Golgi trafficking (Robinson, 2015). In T. gondii, only the beta subunit of COPI has been characterized, which localizes primarily to the Golgi complex (Hager et al., 1999; Pfluger et al., 2005). Conversely, the T. gondii ortholog of Sec23, which is also typically associated with COPII, was reported to be mostly cytosolic in tachyzoites (Hager et al., 1999). However, various COPII components (Sar1, Sec12, Sec13, and Sec24) have been localized to the ER in

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Plasmodium, suggesting that COPII may localize and function as in other eukaryotes (Adisa et al., 2007; Lee et al., 2008; Struck et al., 2008). Clathrin has been comparatively better studied. Endogenously tagged clathrin heavy chain (TgCHC) localizes to the TGN, and overexpression of a dominant negative fragment (CHCHub) resulted in pleiotropic defects consistent with a generalized role in Golgi maintenance and post-Golgi trafficking, consistent with its role in other systems (Pieperhoff et al., 2013).

15.3.8 Adaptor proteins and cargo adapters There are five adaptor protein complexes across eukaryotes, AP-1
AP-5, each comprising a heterotetramer of two large (γ, α, δ, ε, ζ, and β1
5), one medium (μ1
5), and one small (σ1
5) subunit. As adaptor proteins, they physically link transmembrane cargo to coat proteins during vesicle formation, although with the exception of AP-1, AP-2, and possibly AP-3, which interact with clathrin, the coat involved in all such interactions remains unclear (Robinson, 2004; Hirst et al., 2011). T. gondii encodes orthologs for all subunits of all five complexes, though, like other eukaryotes outside of certain opisthokonts, the beta subunit for the AP-1 and AP-2 complexes is shared (Dacks et al., 2008; Woo et al., 2015). AP-1 was one of the first complexes to be studied in T. gondii and was localized to the Golgi and post-Golgi compartments (Ngoˆ et al., 2003). Genetic disruption and yeast-two-hybrid interaction data suggested that AP-1 could interact directly with ROP2 and mediate its transport to the rhoptries, which was later called into question by the discovery that ROP2 was not actually a transmembrane protein and hence could not bind AP-1 in a physiological manner (Ngoˆ et al., 2003; Labesse et al., 2009). The discovery of TgSORTLR, a Vps10/sortilin homolog, appeared to answer the question of

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how this interaction could be mediated, as TgSORTLR binds both AP-1 and ROP2, as well as numerous other microneme/ROPs (Sloves et al., 2012). Further studies in both Plasmodium falciparum and T. gondii implicated AP-1 in rhoptry trafficking and, at least in T. gondii, microneme trafficking and cell division (Kibria et al., 2015; Venugopal et al., 2017). No extensive characterization of other adaptor protein complexes has been performed in T. gondii to date. Endogenous tagging of AP-2 alpha reveals localization at the plasma membrane, and intracellular puncta (Gras et al., 2019, unpublished). AP-3 has not been characterized, but a class of parasitistatic compounds inhibiting T. gondii targets AP-3β and results in substantial mislocalization of markers for the VAC/PLV, formation of aberrant “empty” dense granules, and less severe defects in micronemes and rhoptries (Fomovska et al., 2012).

15.4 Organization of the Toxoplasma gondii membrane trafficking system 15.4.1 Overview Despite early skepticism regarding the putative nature of the apicomplexan MTS, based largely on the presence of unique organelles in addition to the “absence” of others [e.g., the absence of a stacked Golgi in Plasmodium (Van Wye et al., 1996; Struck et al., 2005)], numerous studies have elucidated a surprisingly conserved organellar complement for the apicomplexan MTS (Fig. 15.1). We will start by discussing the ER and Golgi, which are canonical eukaryotic organelles, before moving on to secretory organelles that are more specialized to apicomplexans and T. gondii (dense granules, micronemes, and rhoptries), and discussing how the biogenesis of micronemes and rhoptries in particular is linked to the endolysosomal system of the parasite.

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15.4.2 The endoplasmic reticulum T. gondii possesses a single ER continuous with the nuclear envelope with a somewhat “branched” morphology on the medial/basal side. Homologs of conserved machinery for retrieval of missorted proteins provided markers for this organelle in the form of fusions with the HDEL motif [e.g., the P30-GFP-HDEL fusion (Hager et al., 1999)].

15.4.3 The Golgi T. gondii possesses a single Golgi apparatus just apical to the nucleus, which typically has between three and six morphologically recognizable cisternae (Pelletier et al., 2002). Early studies used fusions of exogenous proteins, including BAP, to the human low-density lipoprotein receptor (LDLR, thus creating BAPLDLR), to mark this organelle (Hoppe et al., 2000). Subsequently, the T. gondii homolog of ER-retention deficient protein 2 (TgERD2) was identified and fused to a fluorescent protein (FP), which strongly labels the Golgi and weakly labels the ER (Pfluger et al., 2005). Consistent with generalizable conserved function, the mammalian GRASP55 protein also serves as a good marker of the Golgi (Pelletier et al., 2002). Finally, identification of an O-linked glycosylation factor, UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferase (GalNAc or NAGTI) yielded a marker for the TGN (Wojczyk et al., 2003; Harper et al., 2006) and maybe for further post-Golgi compartments (ELCs) and/or TGN subdomains as well (McGovern et al., 2018).

15.4.4 The dense granules T. gondii contains small (B200 nm), electron dense, single membrane-bound organelles appropriately called dense granules that are dispersed throughout the parasite cytoplasm.

Although T. gondii dense granules ultrastructurally resemble dense core granules of certain specialized exocytic mammalian cells, they are not secreted in response to elevated intracellular calcium like their mammalian pseudocounterparts. To the contrary, a recent report concluded that elevation of intracellular calcium suppresses dense granule secretion in T. gondii (Katris et al., 2019). There are potentially two or more distinct “types” of dense granules, each containing specific subsets of the GRA proteins and mediating a variety of functions from formation of the parasitophorous vacuole (PV) and cyst wall to modification of the host cell (reviewed in Mercier and Cesbron-Delauw, 2015). Although dense granules are known to move along actin tracks (Heaslip et al., 2016), little is known about their biogenesis or exocytosis. Early studies suggested that dense granules represent a form of constitutive secretion in the parasite (Karsten et al., 1998), and this may account for mislocalization of apical organelle components with disrupted trafficking, as discussed later. In addition, although poorly defined, disruption of TgRab6, TgARF1, TgDrpB, TgStx6, retromer, or the NSF/SNAP machinery (required for SNARE mediated fusion) adversely affected dense granules (Chaturvedi et al., 1999; Liendo et al., 2001; Stedman et al., 2003; Breinich et al., 2009; Jackson et al., 2013; Sangare´ et al., 2016); in the latter case, subsequent studies have shown inhibition of the SNAP machinery to have broad effects on the Golgi, secretory organelles, and apicoplast (Stewart et al., 2015). Processing of dense granule proteins has been shown to be mediated, at least in part, by the Golgi/TGNlocalized ASP5 (Hammoudi et al., 2015; Coffey et al., 2018). Combined with the observations regarding the mislocalization of microneme/ROPs, which are known to transit the Golgi/TGN, it is likely that dense granule formation occurs from, or downstream of, this compartment.

Toxoplasma Gondii

15.4 Organization of the Toxoplasma gondii membrane trafficking system

15.4.5 The endosomal system (micronemes, rhoptries, and the vacuolar compartment/plant-like vacuole) The organization of the endosomal system is beginning to emerge in T. gondii. For a long time, it was assumed that tachyzoites had a limited endocytic capacity. However, there was some evidence that endocytosis in T. gondii may occur at the micropore, a cup-shaped depression in the plasma membrane present at the junction between the cortical cisternae composing the IMC that underlies the plasma membrane (Nichols et al., 1994) (Fig. 15.2A). Moreover, due to limited metabolic capabilities, as well as the need for recycling during extracellular gliding motility and intracellular replication, it was subsequently recognized that parasites must be able to perform

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internalization/recycling. This was subsequently shown, first for intracellular (Dou et al., 2014; McGovern et al., 2018) and later extracellular (Gras et al., 2019) stages of T. gondii. Early studies localizing a paralog of the early endosomal Rab5 GTPase (then referred to as TgRab51 but now known as TgRab5A) revealed its presence at the trans face of the Golgi and in tubulovesicular extensions and electron-lucent vesicular structures (Robibaro et al., 2002). This marker was subsequently found to colocalize with a vacuolar-H 1 -pyrophosphatase, TgVP1 (Drozdowicz et al., 2003), the propeptide of the micronemal MIC2associated protein (proM2AP), and other markers in a structure that was presumed to be something similar to an early endosome (Harper et al., 2006). However, several

FIGURE 15.2 Ultrastructural view of the endolysosomal system in Toxoplasma tachyzoites. (A) Electron micrograph showing a micropore invaginating from the parasite plasma membrane. Note the characteristic electron-dense collar (arrow) on both sides of the invagination. (B) Immunogold staining of TgCPL associated with the VAC/PLV and other vesicles (arrow) visualized by immunoelectron microscopy. Note the typical position of the VAC/PLV in the mid-apical region. ap, apicoplast; c, conoid; er, endoplasmic reticulum; g, Golgi; m, microneme; mp, micropore; mt, mitochondrion; VAC/PLV, vacuolar compartment/plant-like vacuole.

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observations, including partial colocalization with GalNAc-YFP and partial dispersal of TgRab5A signal upon BFA treatment (Robibaro et al., 2002; Harper et al., 2006; Venugopal et al., 2017), suggest that at least some of this TgRab5A signal is associated to the Golgi/TGN. These “early endosome” markers also colocalize with other markers, including the atypical TgRab5B and late endosomal TgRab7 (Kremer et al., 2013), TgStx6 (Jackson et al., 2013), TgSORTLR (Sloves et al., 2012), the retromer complex (Sangare´ et al., 2016), the AP-1 complex (Venugopal et al., 2017), and the Na 1 /H 1 exchanger TgNHE3 (Francia et al., 2011). In addition, a VAC, the VAC/PLV (Fig. 15.2B), has been described, which is marked by cathepsin B and L proteases (TgCPB or “toxopain-1” and TgCPL, respectively), an aquaporin (TgAQP1), TgVP1, and TgRab7. This organelle is highly dynamic though, and fragments following host cell invasion such that TgCPL and TgVP1 no longer extensively colocalize during parasite intracellular replication (Miranda et al., 2010; Parussini et al., 2010). This and other aspects of the VAC/PLV will be covered more extensively in Section 15.6. In terms of their evolutionary origins, micronemes and rhoptries are squarely placed as endolysosomal in origin. Early studies, on the basis of numerous lines of evidence, suggested that rhoptries may be secretory lysosomes (Ngoˆ et al., 2004) or “lysosome-related organelles” (LROs). Subsequent studies focused on molecular characterization of factors classically associated with the endosomal system, including the dynamin-related protein TgDrpB (Breinich et al., 2009), endocytic Rabs [Rab5 and Rab7 (Kremer et al., 2013)], the clathrin coat (Pieperhoff et al., 2013), the vacuolar sorting receptor TgSORTLR (Sloves et al., 2012), the AP-1 complex (Venugopal et al., 2017), the retromer complex (Sangare´ et al., 2016), and the VpsC tethering complexes (Morlon-Guyot et al., 2015, 2018). In all of these studies,

disruption of these components led to defects in the localization of microneme and rhoptry components, often with the absence of morphologically recognizable organelles. These results supported the hypothesis of rhoptries as a form of endolysosome and also suggested that micronemes represent yet another class of apicomplexan LRO (Klinger et al., 2013a).

15.5 An integrated model of exocytic trafficking through the membrane trafficking system Observing microneme and rhoptry defects from disrupting endosomal trafficking proteins lead to a hypothesis that Apicomplexa “repurposed” their endosomal systems to facilitate secretion and downplayed a potential role for the same system in internalization/endocytosis (Tomavo, 2014). However, as mentioned previously and discussed in greater detail later, classical endocytosis also seems to exist. Hence, endo/exocytic events appear intricately intertwined, and it appears that the organization of the endosomal system in T. gondii is likely more complex than previously envisioned. Herein, we provide a new working model for post-Golgi trafficking and secretory organelle biogenesis, which attempts to reconcile the available data from diverse studies (Fig. 15.3). In this model the TGN provides material to either a single “endosome-like compartment” or an intricately interconnected network of compartments (either way, we referred to this using the common abbreviation ELC throughout this chapter), and likely to nascent dense granules as well. As such, the TGN acts as a sorting station for exo/endocytic cargo, in which this cargo either traverses the system to terminal secretory organelles or degradative compartments, or is trafficked/recycled to other subcellular locations. It is likely that some compartments mature and either fuse, or

Toxoplasma Gondii

FIGURE 15.3 Model of trafficking through the Toxoplasma endosomal/lysosomal system. Organelles are depicted with associated molecular markers. Potential trafficking routes between organelles are also shown, along with known or hypothesized machinery for each step; for simplicity, not all organelles and markers are shown. Dashed lines represent uncertainty in organelle identity or trafficking step. Solid green arrows represent anterograde, and red arrows retrograde, trafficking steps while black arrows represent internalization and light blue arrows putative recycling steps. The thick black arrow between the Golgi and TGN represents cisternal maturation. Dashed gray lines represent trafficking steps that are not described, but that may exist on the basis of other evidence (see main text). Bold text denotes organelle labels, while all other text denotes pathways or trafficking machinery. Markers are shown as filled ovals, with the color corresponding to the type of marker: red, ATG; magenta, various transmembrane proteins; deep blue, proteases/maturases; teal, Rabs; orange, SNAREs; yellow, ARFs. ApC, Apicoplast; AQP, aquaporin; ARF, ADP-ribosylation factor; ASP, aspartyl protease; ATG, autophagy-related; CPL/B, cathepsin protease L/B; CRT, chloroquine resistance transporter; DG, dense granule; ELC, endosome-like compartment; ER, endoplasmic reticulum; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SUB, subtilisin-like protease; TGN, trans-Golgi network; VAC/PLV, vacuolar compartment/plant-like vacuole; VATP, vacuolar ATPase; VP1, vacuolar-H 1 -pyrophosphatase.

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exchange material, with the terminal acidic compartment (VAC/PLV). This model was envisioned to explain the following observations (also discussed further later): 1. Propeptide processing of MICs and ROPs is blocked by BFA, suggesting it occurs downstream of the medial-cis Golgi interface and likely in a post-Golgi compartment (Soldati et al., 1998; Rabenau et al., 2001; Harper et al., 2006; Brydges et al., 2008; El Hajj et al., 2008). 2. TgSORLTR (Vps10) localizes primarily throughout the ER, Golgi, and endosomes and binds microneme and rhoptry resident proteins, as well as AP-1 and retromer. Disruption prevents biogenesis of micronemes and rhoptries (and their resident proteins are mislocalized) but has no apparent effect on dense granules, the IMC, TgSAG1, or other organelles (Sloves et al., 2012). 3. Overexpression of either TgRab5A or TgRab5C causes mislocalization of rhoptry bulb proteins, but not microneme proteins; overexpression of dominant negative versions of either Rab additionally causes mislocalization of MIC3 and MIC8, but not AMA1, M2AP, or MIC2 (Kremer et al., 2013). 4. Retromer binds TgRab5B and TgRab11B and colocalizes with diverse endosomal markers (TgSORTLR, proM2AP, and TgVP1). Disruption causes mislocalization of microneme, dense granule, and rhoptry components, abrogates the processing of ROP1, ROP2, ROP4, M2AP, and MIC5, shifts the localization of TgSORTLR from the Golgi to Rab5/7-positive compartments, and is defective in recycling of transmembrane plasma membrane proteins. The processing defect likely reflects the arrest of proteins in the Golgi/ TGN, prior to a compartment in which they can be cleaved, likely due to the

redistribution of TgSORTLR (Sloves et al., 2012; Sangare´ et al., 2016). 5. AP-1 localizes primarily to the TGN (colocalizes with GalNAc-YFP, TgSORTLR, proMIC3, and proROP4, but not proM2AP) and is partially dispersed by BFA treatment. AP-1 knockout has no effect on dense granules but reroutes MIC3/MIC8 and proROP4/ROP2,3,4 to the vacuolar space, possibly through dense granules. Overexpression has no effect on microneme or ROP processing or microneme protein localization but results in ROP signal in puncta throughout the cytosol. This might be because forming rhoptries require fusion of multiple AP-1derived vesicles, whereas forming micronemes do not, and hence, overexpression of AP-1 leads to increased vesiculation and prevents fusion of ROPpositive vesicles and hence rhoptry maturation (Ngoˆ et al., 2003; Venugopal et al., 2017). In addition, similar to TgRab11A, AP-1 results in defective membrane delivery/recycling and aberrant endodyogeny (Agop-Nersesian et al., 2009; Venugopal et al., 2017). 6. A class of parasitistatic compounds that appear to specifically interact with and/or inhibit the beta subunit of the AP-3 complex in T. gondii result in vacuolar fragmentation, formation of “empty” dense granules, and some mislocalization of both microneme and ROPs (Fomovska et al., 2012). 7. Ingested protein colocalizes with GalNAcYFP, TgDrpB, proM2AP, proMIC5, and two cathepsin proteases (TgCPB/L), but not with TgNHE3 or proRON4. As uptake appears to occur throughout the intracellular lifecycle, this suggests that material is taken up either into a subdomain of the TGN that lacks TgNHE3 or a separate Rab5-positive compartment that contains proMIC, but not proRON,

Toxoplasma Gondii

15.5 An integrated model of exocytic trafficking through the membrane trafficking system

8.

9.

10.

11.

components, prior to trafficking to the VAC/PLV (Dou et al., 2014; McGovern et al., 2018). In extracellular parasites, the uptake of exogenous material occurs concurrently with gliding motility (possibly allowing for membrane recycling). In these parasites a substantial amount of internalized lipid is redistributed to the ER (colocalizes with TgRab18), Golgi (TgRab4, TgCHC), and VAC/PLV (TgCPL), but less is found colocalized with proM2AP-positive compartments. In addition, some internalized material incapable of being sorted (nano-gold particles) localize to the rhoptries (Gras et al., 2019). Concurrently with these two comparable modes of uptake, TgRab11A localizes to rhoptries and mediates trafficking (and possibly recycling) of TgSAG1, and potentially other surface proteins. TgRab11B, alternatively, localizes in the vicinity of the Golgi and subsequently to the IMC and mediates delivery of early- and late-stage IMC components during endodyogeny (Agop-Nersesian et al., 2009, 2010). TgCPL is found in the VAC/PLV, compartments containing proM2AP, and to a lesser extent putative immature micronemes. TgCPL knockout parasites show reduced processing of proM2AP and proMIC3, but the processing of proMIC5, proMIC6, and proAMA1 is normal (Parussini et al., 2010). In addition, M2AP signal in trafficking-deficient parasites localizes differently than MIC5 signal, suggesting that different maturases function at different stages or that different trafficking pathways from the site of maturation to the apical end of the parasite exist (Gaji et al., 2011). TgVps11, a core VpsC subunit, colocalizes with GalNAc-YFP, TgCPL, TgRab5B, TgRab7, proM2AP, and proROP4. Inducible knockdown (iKD) of Vps11

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causes mislocalization of diverse microneme, rhoptry, and dense granule markers, without affecting endosymbiotic organelles (apicoplast and mitochondrion) or the IMC/plasma membrane (MorlonGuyot et al., 2015). BDCP, a putative VpsC interactor in T. gondii, colocalizes with GalNAc-YFP, TgRab5B, proROP4, and TgCPL, but not with proM2AP or TgRab7; iKD causes a change in TgARO and TgCPL staining and increases overlap between TgROP7 and TgCPL. TgVps8, a CORVETspecific subunit, colocalizes well with TgRab5B and somewhat with TgRab7. iKD of TgVps8 causes similarly broad trafficking defects as iKD of TgVps11 (Morlon-Guyot et al., 2018). 12. TgStx6 localizes mainly to the TGN and proM2AP-positive compartments, and occasionally at the cell surface. Overexpression of a dominant negative version causes the membranes in the Golgi region to fragment/vesiculate as assessed by electron microscopy and causes proM2AP and TgVP1 staining in fluorescence microscopy to increase as well as become fragmented. In addition, it disrupts normal cell morphology similar to TgRab11A and likely has an effect on dense granule biogenesis (Jackson et al., 2013). This suggests that this factor acts to tether endosomally derived vesicles at the TGN and possibly also mediates some homo/heterotypic endosomal fusion. 13. Disruption of some factors, including VpsC (Morlon-Guyot et al., 2015) and AP-1 (Venugopal et al., 2017), has resulted in extreme apical staining of a subset of microneme proteins (e.g., MIC1, MIC2, M2AP, MIC4, and MIC6). This was suggested to represent a hitherto unrecognized direct TGN-apical microneme trafficking pathway. However, this phenotype is also observed in parasites lacking TgSORTLR (Sloves et al., 2012).

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Hence, we consider it more likely that this represents aberrant inclusion of a subset of microneme proteins into a different pathway, potentially for the delivery of components to the apical complex, as they likely accumulate in the TGN in these mutants. 14. A dynamin-related protein, TgDrpB, localizes in the vicinity of the Golgi, and overexpression of a dominant negative form causes broad defects on micronemes, rhoptries, and dense granules (Breinich et al., 2009). Almost all models proposed to date involve microneme and rhoptry trafficking proceeding through a post-Golgi/TGN compartment, with micronemes distinct from the endosomal organelles themselves (see, e.g., Venugopal and Marion, 2018); we share this view. Here, we explicitly raise the hypothesis that rhoptries, or at least nascent prorhoptries, represent recycling endosome-like organelles. This is based on several lines of evidence. Recycling endosomes are known to be acidic (Yamashiro et al., 1984), and in T. gondii both forming and mature rhoptries have been described to be acidic (Shaw et al., 1998). Note also that we do not envision that rhoptries are the only acidic compartments in T. gondii, as widespread localization of TgVP1 and the evidence for a V-H 1 -ATPase suggest that other compartments are likely acidified in the parasite (Miranda et al., 2010). The canonical marker for recycling endosomes is Rab11 (Lock and Stow, 2005). T. gondii possesses two Rab11 paralogs, one of which, TgRab11A, represents the canonical pan-eukaryotic paralog (Agop-Nersesian et al., 2010; Klinger, unpublished), localizes primarily to the rhoptries, and is important for myosin-dependent transport of surface material (e.g., TgSAG1) to the IMC/ plasma membrane, much like what is described for canonical recycling endosomes (Gidon et al., 2012). Finally, recycling endosomes are known to be rich in raft lipids [e.g., sphingomyelin,

cholesterol (Gagescu et al., 2000)], and rhoptries are also known to be enriched in such lipid molecules (Besteiro et al., 2008). The emergence of proROPs from a Rab5Apositive compartment(s) has been recently described in detail (Venugopal et al., 2017). The identity of Rab5- and Rab7-compartments has been difficult to pin down precisely, based largely on partially overlapping TGN/endosomal markers, such as TgRab5A/ B/C, TgRab7, TgVP1, proMIC proteins (e.g., proM2AP and proMIC3), and the partial disruption of TgRab5A/AP-1 upon BFA treatment, which suggests that at least some of these “ELC” markers are present in compartments connected with the Golgi/TGN. Here, we envision either a single organelle that is positive for both markers but possesses distinct subdomains, or a single maturing organelle/network that changes from being Rab5- to Rab7-positive. The former is similar to the situation described in Arabidopsis (Dettmer et al., 2006; Viotti et al., 2010), and more recently, yeast (Day et al., 2018), whereas the latter is more similar to mammalian endosomal systems (Huotari and Helenius, 2011). Either way, we envision the TGN as being the first stop for internalized material (i.e., more similar to yeast/plant systems). This is based on several lines of evidence, such as the colocalization of internalized material in both intracellular and extracellular tachyzoites with Golgi/TGN markers (Dou et al., 2014; Gras et al., 2019; McGovern et al., 2018), the presence of TgRab4, which in mammalian cells mediates “fast” recycling of cargo from early endosomes to the plasma membrane (van der Sluijs et al., 1992), at the TGN (Kremer et al., 2013), and the apparent cytokinetic defect observed in AP-1 deficient parasites, which is consistent with a reliance for membrane recycling on an early TGN-ELC step (Venugopal et al., 2017). This will be discussed further in the next section.

Toxoplasma Gondii

15.6 Dynamics of the endolysosomal system

15.6 Dynamics of the endolysosomal system 15.6.1 Overview The endolysosomal system consists of a dynamic series of compartments that communicate with one another by fusion and fission, or by maturation from one compartment to another. As such, this system is in a constant state of change, with cargo and markers often partially occupying more than one compartment at a time or different subdomains of a single compartment. The dynamic nature of the endolysosomal system presents challenges in delineating specific compartments. Despite these constraints, work by several groups has documented morphological changes and intercompartment communication in the system along with charting the course of endocytic material through endolysosomal compartments, as discussed in this section.

15.6.2 Fragmentation and reformation of the vacuolar compartment/plant-like vacuole Limiting the time of tachyzoite invasion (pulse-invasion) permits initial synchronization of the parasite cell cycle to follow changes in the appearance and distribution of organelles and structures. This, coupled with monitoring markers for the centrosome and the IMC, permits charting specific steps in the parasite cell cycle. Parussini et al. (2010) took advantage of these features to document dynamic changes in the VAC/PLV during parasite division based on staining for TgCPL. In extracellular (G0) and newly invaded (early G1) tachyzoites, the VAC/PLV is seen as one or two small puncta, most often localized to the apical region. As the parasite progresses through G1 phase, the VAC/PLV enlarges before fragmenting into multiple clustered puncta during S phase and mitosis. During cytokinesis, each

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daughter cell contains a single VAC/PLV or a tightly spaced cluster of VAC/PLVs. VAC/ PLV fragmentation has also been documented by other studies (Miranda et al., 2010; Warring et al., 2014). Precisely, how or why the VAC/ PLV undergoes such dynamic changes during cell division remains unclear. Parasite endocytosis of host-derived protein occurs throughout the cell cycle (McGovern et al., 2018), making it unlikely that the fragmentation is related to this specific function. One possibility is that fragmentation occurs as part of organellar division to seed a new VAC/PLV in each of the daughter parasites. Alternatively, the VAC/ PLV might be generated de novo in each of the daughters and the mother cell VAC/PLV is discarded in the residual body. Distinguishing between these possibilities will require careful tracking of VAC/PLV structures during parasite cell division.

15.6.3 Interactions between the vacuolar compartment/plant-like vacuole and endosomal-like compartments As described in Section 15.2, microneme and ROPs traffic through ELCs en route to their respective target secretory organelles. The available evidence suggests that proMIC and proROPs are proteolytically processed in the ELCs, or in a preorganellar compartment, prior to packaging in the micronemes or rhoptries. Consistent with this, the essential maturase TgAsp3 resides in the ELCs where it processes several proMICs and proROPs. TgCPL was identified as contributing to the maturation of certain proMICs, including TgM2AP and TgMIC3 (Parussini et al., 2010). Although TgCPL mainly occupies the VAC/PLV, a less abundant subpopulation of this protease resides in the ELCs. This ELC subpopulation of TgCPL might be newly synthesized and en route to the VAC/PLV or it could arise from fusion and fission of the VAC/PLV with ELCs.

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Additional evidence of interactions between the VAC/PLV and ELCs has been reported recently in a careful analysis of tachyzoites deficient in TgCRT (Thornton et al., 2018). Although it had previously been noted that crt null parasites present an enlarged VAC/PLV, presumably because accumulation of TgCRT substrates increases intraluminal osmotic pressure (Warring et al., 2014), where the membrane for enlargement came from remained unknown. Thornton et al. (2018) reported that some intracellular crt null tachyzoites have a compartment that harbors markers of both the VAC/PLV and ELCs. How this hybrid compartment arises remains to be determined. If the VAC/PLV normally undergoes periodic fusion and fission, one possibility is that osmotic pressure in the Δcrt VAC precludes fission into separate organelles. Live imaging of the VAC/PLV and ELCs should provide a clearer view of dynamic interactions between these structures.

15.7 Endocytosis and endocytic trafficking 15.7.1 Overview Although endocytosis is a canonical pathway of all eukaryotes, until recently there was limited evidence that such a pathway existed in T. gondii. It was assumed that because the PV does not fuse with the endocytic or exocytic system of the infected host cell, T. gondii would have a reduced or nonexistent need for endocytosis. Nichols et al. (1994) suggested that tachyzoites undergo endocytosis based on the uptake of the fluid phase marker horseradish peroxidase at the parasite micropore. However, the uptake of this marker was only seen in a minority population of parasites. The same study reported the presence of small vesicles contained in the micropore, the presumptive site of endocytosis, in bradyzoite cysts obtained from infected

mouse brain. As described later, subsequent studies have now more firmly established that T. gondii has an active endocytic pathway as a conduit for a variety of substrates, including macromolecular material, obtained from the infected host cell.

15.7.2 Endocytosis of sulfated glycans Mats Wahlgren’s group reported the first evidence of receptor-mediated endocytosis in T. gondii by using fluorescein isothiocyanate (FITC) conjugated heparin as an endocytic tracer (Botero-Kleiven et al., 2001). This work documented saturable, protein receptordependent binding and internalization of FITC-heparin by extracellular tachyzoites that was competed away by certain sulfated glycans, but not chondroitin sulfates. Although only B25% of the population was competent for binding and uptake of FITC-heparin, less than 5% of tachyzoites internalized the fluid phase marker Lucifer yellow, providing additional evidence that FITC-heparin endocytosis is receptor mediated. The authors implicated TgGRA7 as potentially being involved in uptake of FITC-heparin based on it being surface iodinatable and its ability to bind heparin, but confirmation of its role in endocytosis awaits additional studies.

15.7.3 Endocytosis of lipids and surface proteins The first indication that the T. gondii endocytic system plays a role in uptake of hostderived lipids came from Keith Joiner’s group (Robibaro et al., 2002). This group showed the overexpression of a constitutively active mutant of TgRab5A in intracellular tachyzoites augmented uptake of radiolabeled cholesterol and increased the abundance of lipid bodies in the parasite. Whether cholesterol is internalized by parasite endocytosis remains to be

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15.7 Endocytosis and endocytic trafficking

determined, but augmentation by constitutively active TgRab5A minimally suggests a role for the parasite endolysosomal system as a rate-limiting conduit for host-derived cholesterol in the parasite. More recently, putative endocytic invaginations were observed in intracellular tachyzoites growing in infected monolayers exposed to an excess (0.2 mM) of oleic acid, a C18 monounsaturated fatty acid (Nolan et al., 2017). The invaginations contained material from the PV, were sometimes decorated with a coat reminiscent of clathrin, and they typically occupied the apical end of the parasite, but they were ultrastructurally distinct from micropores. Treated parasites also showed intracellular vesicles containing PV material and TgSAG1 suggesting that they originated from the parasite surface. The extent to which the observed putative endocytic vesicles contribute to endocytosis of oleic acid or is an indirect consequence of exposure to this fatty acid remains to be determined. In good agreement, a recent study demonstrated efficient uptake of fluorescent lipid dyes, such as lysophosphatidyl choline, lysophosphatidic acid, or Bodipy (Gras et al., 2019). Interestingly, an endocytic-secretory cycle appears to operate in extracellular parasites that is linked to retrograde membrane flow and parasite motility, since internalized material can be found to be resecreted upon stimulation of microneme secretion by calcium ionophore. Furthermore, parasite surface proteins, such as TgSAG1, the major surface antigen, are also internalized and appear to transit through the ELCs (Gras et al., 2019), similar to the observations made for host-derived proteins that are endocytosed by intracellular parasites (see later). The process might be similar to the situation in Trypanosoma brucei (Overath et al., 2004; Engstler et al., 2007), where endocytosis is critical for recycling of variant surface glycoproteins and removal of host antibodies bound to these GPI-anchored

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proteins. Together, this led to the hypothesis that a fountain-flow model is operating in T. gondii during motility, where new membrane lipids are delivered to the anterior cell membrane and excess membrane is recycled at the rear, as also observed in other eukaryotes (Tanaka et al., 2017; O’Neill et al., 2018). However, while this process seems to be independent of actin and myosin, the molecules involved in the endocytic process remain to be identified (Gras et al., 2019).

15.7.4 Endocytosis of host-derived protein With the identification of the VAC/PLV in 2010 came the realization that T. gondii has a complete endocytic system, including endosomes and a lysosome/vacuole. Occupation of the VAC/PLV by cathepsin proteases suggested it to be a terminal digestive compartment for the endocytic system. Since these features mirrored those of malaria parasites, which conspicuously internalize and degrade hemoglobin during replication in erythrocytes, Dou et al. (2014) reasoned that T. gondii might also have the ability to access and degrade proteins derived from infected host cells. To test this, they expressed FPs in the cytosol or ER of Chinese Hamster Ovary or HeLa cells, infected them with T. gondii for overnight replication, and examined liberated parasites by fluorescence microscopy. This work revealed that parasite strains (type I and type II) expressing active TgCPL failed to accumulate hostderived FPs, whereas those that lacked TgCPL or its enzymatic activity displayed FPs within the parasite endolysosomal system. Further, cytosolic FPs, but not ER FPs, were internalized by the parasite, suggesting that the parasite does not have access to internalizing proteins from the host ER despite an intimate association of the ER with the PV membrane. Since the pathway would not have evolved to

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selectively target FPs, it is likely that ingestion involves bulk uptake of host cytosolic proteins, similar to the hemoglobin uptake pathway in malaria parasites. In follow up work, McGovern et al. (2018) reported that internalized host-derived FP is associated with certain markers for the ELCs, including proM2AP, but not the sodium/ hydrogen exchanger NHE3. These findings imply that although ingested material transits through the ELCs, it might be restricted to certain subdomains of the endocytic compartments. Unexpectedly, experiments using pulseinfected cells showed that a subpopulation of tachyzoites ingests host-derived FP as early as 7 minutes postinfection. The extent to which early ingestion is mechanistically similar to that of replicating parasites remains to be determined. The study further showed that the ingestion pathway is active throughout the parasite cell cycle and that ingested material is associated with the ELCs at the same time as immature microneme, but not immature rhoptry, proteins. These findings suggest a potential role for receptor-mediated trafficking of ingested material perhaps still enclosed in a PV membrane
derived vesicle. A receptormediated mechanism would assure delivery to the VAC/PLV for degradation and allow for separate trafficking receptor-mediated shuttling of proteins to the micronemes. Finally, the authors reported that TgDrpB, which has been suggested to function in fission of Golgiderived vesicles destined for the ELCs, is not required for trafficking of ingested proteins. Rather, dominant negative interference with TgDrpB function unexpectedly resulted in increased ingestion of host-derived FP. These findings provide further support for the existence of distinct mechanisms of trafficking through the T. gondii endolysosomal system, as discussed in Section 15.5. For what specific purpose T. gondii ingests proteins from the cytosol of infected cells remains to be determined. One possibility is

that they use digestion products (e.g., amino acids and peptides) from the pathway to support parasite replication, mirroring malaria parasites in this regard. The slower replication of TgCPL-deficient parasites potentially supports this prospect (Dou et al., 2014). Alternatively, the pathway could be targeting specific resources from the host such as cofactors for metabolic enzymes, for example, via receptor-mediated ingestion of lipoilated or heme-containing proteins. Defining the role of T. gondii ingestion during intracellular replication awaits identifying and disrupting key proteins in the pathway.

15.8 Comparison of Toxoplasma gondii endosomal trafficking to model systems 15.8.1 Overview of yeast, mammalian, and plant systems Further understanding of the T. gondii MTS can be obtained by comparison with other, better studied, systems, as the underlying function of genes in pathways can be reliably used to infer cellular function across eukaryotes (Klinger et al., 2016). Trafficking in mammalian cells provides perhaps the most canonical “textbook” view, as this system has received the most attention over the longest period of time to date. Although individual cell types display substantial variation, for example, polarized versus nonpolarized cells, in general, secretory traffic follows the canonical pathway of ER import, ER to Golgi transport, sorting and modification by Golgi-resident enzymes, and post-Golgi sorting to the surface, either through constitutive or regulated pathways, or to other organelles, usually through the endosomal system (Barlowe and Miller, 2013). Conversely, internalized material is delivered first to the early endosome before either being recycled to the surface, through rapid direct

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recycling or via a recycling endosome pathway, or transiting the endosomal system for terminal degradation in the vacuole/lysosome (Huotari and Helenius, 2011). It was believed that the MTS of the common yeast system Saccharomyces cerevisiae was similar to the mammalian MTS, including both an early endosomal compartment with recycling capability (either directly or through a recycling endosome), as well as further late endosomes and a terminal vacuole for degradation. However, recent studies challenge this model and propose instead that the yeast Golgi matures, complete with retrograde COPI or AP-1-mediated recycling, and that the terminal cisternae act as both the first stop for endocytic cargo, and also as a sorting hub where material either transits to the plasma membrane or to a single “prevacuolar endosome,” which has properties of both early and late endosomes (Papanikou et al., 2015; Day et al., 2018). This is similar to what has been described for the multicellular plant A. thaliana, namely, lack of a distinct early endosomal compartment, with the TGN serving as the first stop for internalized material (Dettmer et al., 2006; Viotti et al., 2010). Interestingly, the MTS in other organisms, such as T. brucei (Manna et al., 2014), appears at least superficially similar to that in mammalian cells, raising the possibility that reduction in distinct endosomal compartments is a case of convergent evolution across multiple eukaryotic lineages.

15.8.2 Similarities and distinctions of Toxoplasma gondii versus model systems The discovery of the VAC/PLV was the first piece of evidence that the T. gondii MTS may be “plant-like” (Miranda et al., 2010), a sentiment that has been echoed in several subsequent studies (e.g., Jackson et al., 2013). It is unclear if there is a distinct endosomal compartment in T. gondii to which internalized

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material traffics before reaching the TGN, or whether the TGN is the first site of endocytic trafficking, as in A. thaliana. In extracellular tachyzoites, internalized material colocalizes more with markers of the ER/Golgi (TgRab4/ 18, TgCHC) than the endosomes [proM2AP (Gras et al., 2019)], whereas in intracellular parasites, colocalization with both TGN (GalNAc-YFP, TgDrpB) and endosome (proM2AP) markers is observed in approximately equal amounts (McGovern et al., 2018). It is possible that this difference in distribution simply reflects a dynamic reorganization of the organelles between intra/extracellular stages, as it has been described for the VAC/PLV (Miranda et al., 2010; Parussini et al., 2010). Alternatively, it is possible that more than one mode of internalization exists in the parasite, with different trafficking routes. Similarly, it is unclear whether there are distinct “early” and “late” endosomal compartments through which material, including microneme and rhoptry resident proteins, traffics. Partial overlap of a diverse array of markers may suggest that there are distinct compartments, or, at least subdomains, within this system. However, this overlap can be deceptive in static images, as the recruitment of trafficking factors to organelles is often temporally offset, or even nonoverlapping, despite localizing to the same compartment; hence, it will be important to define the nature and extent of such interaction between endosomal markers in T. gondii using live microscopy (Day et al., 2018). Thus our current model is based more on evidence from genetic disruption than colocalization (Fig. 15.3). We envision the T. gondii MTS as being somewhere in-between that of mammalian and yeast/plant cells, with internalization directed first to the TGN, and a single ELC similar to the yeast prevacuolar endosome, but with additional endosomally derived organelles (micronemes and rhoptries) that further complicate the picture.

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Regardless of the exact nature of the endocytic system, it plays a key role in the biogenesis of the secretory organelles. This was previously described as “repurposing” prior to the appreciation of the same system in the uptake of material in both intracellular and extracellular stages, which suggests instead that the system is highly dynamic and has been adapted to achieve both exo- and endocytic traffic simultaneously. Indeed, when the micronemes and rhoptries are viewed as just another class of endosomal compartments, albeit ones specialized for processing and secretion of virulence factors, the overall picture of the apicomplexan MTS is not that different from other canonical systems. The presence of canonical organelles such as ER, Golgi, and a terminal degradative compartment further enforce this view and suggest that the trafficking system in these parasites is derived, rather than truly diverged, from a common ancestral state, as evolutionary mechanisms dictate would be the case (Klinger et al., 2016).

15.9 Autophagy 15.9.1 Coding capacity of the core Toxoplasma gondii autophagy machinery Autophagy is a cellular pathway that targets a variety of cytoplasmic material for degradation in lytic compartments such as lysosomes or plant/yeast vacuoles (Yin et al., 2016). There are several ways for delivering the autophagic cargo to the lytic compartments, which can involve internalization of the cargo by invagination of the lysosomal membrane (microautophagy) or translocation of cytoplasmic proteins at the lysosomal membrane (chaperone-mediated autophagy), but these are only present in some eukaryotes. The most universal (and most studied) form of autophagy is called macroautophagy, and hence it is the one usually referred to when using the generic term

“autophagy.” It involves the formation of a double-membrane compartment termed the autophagosome that will form around the cellular material to be degraded and will subsequently fuse with lytic compartments for degradation and recycling by resident hydrolases. In the 1990s, pioneering genetic screens in the budding yeast model led to the identification of a set of ATG proteins that govern the formation of autophagosomes (Tsukada and Ohsumi, 1993). The core autophagy machinery can be broken down into four functional groups: (1) an induction complex, composed of kinase ATG1 and its regulators; (2) the ATG9 complex, which is involved in the early stages of lipid and protein recruitment to nascent autophagosomes; (3) mediators of autophagosome nucleation, including phosphatidylinositol-3 kinase (PtdIns3k) and its regulators; and (4) mediators of autophagosome elongation, comprising ATG8 and associated machinery regulating its membrane conjugation. This machinery is essential for the formation and expansion of the phagophore (the preautophagosomal structure) and its maturation into a fully functional autophagosome. The high-sequence conservation in most eukaryotic lineages of many core autophagy proteins allowed the identification of ATG orthologs in the genomes of many distant eukaryotes, although there are substantial differences. For instance, there is a high degree of conservation between yeast and mammals, which are relatively close phylogenetically, but there is evidence of losses or duplication in divergent eukaryotes such as parasitic protists (Duszenko et al., 2011). In Toxoplasma specifically, homology searches reveal that this machinery is only partially conserved (Fig. 15.3). Noticeably, the ATG1 kinase complex, an important effector of the formation of autophagosomes in yeast and mammals, is largely incomplete. ATG9 acts early in the formation of

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autophagosomes and may be involved in the recruitment of other ATG components and membrane to the forming organelles. ATG9, as well as its partners ATG18 and ATG2, have potential homologs in Toxoplasma. As the sole integral membrane protein required for forming the phagophore, ATG9 has been considered a likely supplier of initial vesicles for phagophore nucleation. The protein complex formed by protein ATG2 and phosphatidylinositol 3-phosphate (PtdIns3P)-binding protein ATG18 may regulate ATG9 cycling from the preautophagosomal structure in yeast (Suzuki et al., 2013) but is essentially uncharacterized in other eukaryotes. Another important part of the machinery that can be unambiguously identified is related to ATG8, a crucial player in autophagosome formation (Shpilka et al., 2011). ATG8 and most of the proteins regulating its association with autophagosomal membrane are present in Toxoplasma. ATG8 is a small protein with a ubiquitin-like fold, which is expressed in the cytoplasm but associates to membranes by attaching to a lipid substrate through an enzymatic pathway similar to the ubiquitin system. Proteins ATG7, ATG3, and the ATG12/ATG5-ATG16 complex act as E1, E2, and E3 enzymes, respectively, leading to the covalent binding of the C-terminal glycine of ATG8 to the membrane-embedded lipid phosphatidylethanolamine (PE) (Yin et al., 2016). ATG4, a cysteine protease, regulates the conjugation/recycling of ATG8 from membranes. The precise role of ATG8-PE is not completely elucidated; in fact, it might be involved in autophagosome formation at multiple steps, including membrane expansion and closure. Although most ATG proteins were identified in the 1990s and significant progress has been made on the understanding of autophagosome biogenesis through functional studies in yeast and mammals, for several of these proteins their role remains uncharacterized at

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the molecular level. Even at the cellular level, many major conundrums remain, like for instance identifying the source of membrane for autophagosome biogenesis (Tooze, 2013). In Apicomplexa the identification and functional characterization of the pathway started less than 10 years ago and a great deal of work remains to be done to fully uncover the specificities of this machinery.

15.9.2 Autophagy in Toxoplasma gondii 15.9.2.1 Canonical degradative autophagy There is sequence-based, morphological, and functional evidence that indicates that autophagy is broadly present all across the tree of eukaryotes, suggesting this cellular process was a feature of the last eukaryotic common ancestor. The original function of autophagy may have been in promoting survival under nutrient starvation conditions, as such conditions act as a general inducer of autophagy, both in cultured cells and in intact organisms (unicellular and multicellular alike). In addition to starvation, autophagy can also be activated by other physiological stress stimuli such as hypoxia, energy depletion, ER stress, high temperature, or exposure to drugs (Kroemer et al., 2010). Autophagy is generally seen as a prosurvival pathway which is important to maintain cellular homeostasis (Moreau et al., 2010). For instance, under physiological conditions, autophagy has a number of vital roles such as maintenance of the amino acid pool during starvation, clearance of damaged organelles, and remodeling during cellular differentiation. In addition, in complex eukaryotes such as mammals, it has also evolved specialized prosurvival functions for the clearance of intracellular microbes and the regulation of innate and adaptive immunity (Mitchell and Isberg, 2017).

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15.9.2.2 Evidence for stress-activated canonical degradative autophagy Nutrient starvation was logically one of the very first stimuli used in the initial studies on Toxoplasma autophagy. Electron microscopy observation of intracellular parasites grown in diluted medium (Ghosh et al., 2012) or

extracellular parasites incubated for several hours in amino acid
depleted (but glucosecontaining) medium (Besteiro et al., 2011) has shown double and/or multiple membranebound structures containing cytoplasmic material, thus resembling bona fide autophagosomes (Fig. 15.4A). Functional degradative autophagy

FIGURE 15.4 Identification and quantification of autophagic vesicles upon starvation in Toxoplasma. (A) Tachyzoites incubated extracellularly in amino acid
depleted medium (Hank’s balanced salt solution, HBSS) for 6 hours were observed by electron microscopy. Arrowheads show multimembrane compartments containing cytoplasmic material corresponding to autophagosomes. Asterisk shows potentially hybrid compartment with partially digested material. Scale bar is 500 nm. (B) Quantification of the proportion of extracellular parasites displaying puncta-like GFP-TgATG8 signal (arrowhead on the example displayed on the inset) after incubation in complete (Delbecco’s modified Eagle’s medium) or starvation (HBSS) medium. Scale bar is 5 μm. (C) Separation by urea SDS-PAGE and immunoblot analysis of the increase in TgATG8-PE in extracellular tachyzoites after starvation. (D) Immunofluorescence analysis of extracellular tachyzoites incubated for 6 hours in starvation medium. Costaining of GFP-TgATG8 with VAC compartment markers TgVP1 and TgCPL. Scale bar is 5 μm. DIC, differential interference contrast. Toxoplasma Gondii

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implies that after their formation, autophagosomes fuse with a lytic compartment. In mammals, fusion between the autophagosome and lysosomes (which may also involve an intermediate fusion between the autophagosome and an endosome to generate a transient structure called the amphisome) leads to the formation of a hybrid compartment called the autolysosome. In yeast, however, after fusion with the larger lytic vacuole, the inner autophagosome vesicle is directly released into its lumen as an autophagic body. Upon amino acid starvation a number of vacuoles, some of them seemingly containing membrane or organellar material, suggestive of hybrid lytic organelles, are also observed in tachyzoites (Fig. 15.4A). However, these autophagic vesicles which are more advanced in the degradation process are less unequivocally recognizable because part of their content, including the inner membrane of the original autophagosome, might have been degraded. Thus for more accuracy and a more quantitative method, molecular markers can also be used to confirm the morphological hallmarks of autophagy observed in stressed tachyzoites. The most widely used marker to trace autophagy in eukaryotes is the highly conserved ATG8 protein, which associates with the autophagosome membrane and remains associated with the mature autophagosome until it fuses with the vacuole/lysosome. Upon starvation, in extracellular parasites overexpressing a GFP-tagged version of TgATG8, the protein is recruited to puncta likely representing autophagic vesicles (Besteiro et al., 2011); the proportion of parasites displaying TgATG8 puncta, or counting the number of puncta per parasite, can reflect the level of autophagy in the population (Fig. 15.4B). Lipid-conjugated and unconjugated forms of TgATG8 can also be separated by electrophoresis in the presence of urea, as the lipidated form migrates faster in these conditions; thus immunoblot quantification of the TgATG8PE form can also be used as an indication of the

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level of autophagy. As mentioned later in this chapter, TgATG8 also binds to the outermost apicoplast membrane through its association with PE, so there is a relatively high level of endogenous TgATG8-PE already present in basal conditions in tachyzoites, yet a clear upregulation of TgATG8-PE is visible upon induction of autophagy (Fig. 15.4C). These methods are commonly used to measure the steady-state levels of organelles or of TgATG8-PE within a continuous autophagic flux. However, although an increase in autophagosome number or in TgATG8-PE levels is usually interpreted as an increase in autophagic activity, one should keep in mind that it could also represent an accumulation of autophagosomes due to a block in the flux (in the late degradation steps, for example). Morphological observations by electron microscopy are suggestive of the presence of hybrid degradative compartments, and costaining between TgATG8-labeled vesicles and the VAC/PLV compartment shows partial colocalization (Fig. 15.4D), hinting the autophagy and lytic pathways intersect. TgATG9, a transient component of autophagosomes, was also shown to colocalize with the VAC/PLV (Nguyen et al., 2017b). Yet, extensive biochemical evidence of digestion and recycling of autophagy substrates are lacking in Toxoplasma. Yeast and mammalian cells benefit from a long-standing knowledge and identification of autophagy substrates and adapters, so there are a number of markers and assays to monitor autophagic flux in these models (Delorme-Axford et al., 2015); most of these assays are not available for Toxoplasma. However, an indirect method based on assessing the turnover of labeled long-lived proteins, allowing quantitative determination of the bulk autophagic flux, has been successfully implemented in tachyzoites and also suggested that there is an autophagydependent degradation of proteins (Nguyen et al., 2017b).

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15.9.2.3 Autophagy as part of an integrated stress response

15.9.2.4 A role for canonical autophagy in parasite virulence

Besides nutrient deprivation, other stresses have been shown to induce autophagy in tachyzoites, like perturbing ER homeostasis (Nguyen et al., 2017a). When the normal function of the ER in protein synthesis is disrupted, a so-called unfolded protein response is initiated to counter the effects of accumulating unfolded proteins. This includes upregulation of autophagy (Kroemer et al., 2010). Interestingly, signaling pathways involving the α-subunit of eukaryotic initiation factor 2 (TgIF2α) that regulates protein translation in response to both starvation and ER stress (Zhang et al., 2013), are able to modulate autophagosome biogenesis (Nguyen et al., 2017a). This shows that canonical autophagy is finely regulated in response to several stresses, although the TgIF2α-dependent factors modulating autophagy in the parasite remain to be identified. The use of pharmaceutical agents can also trigger autophagy in tachyzoites, like with the ionophore monensin, a very potent antiparasitic drug that is a potential inducer of oxidative stress (Lavine and Arrizabalaga, 2012). Both starvation and the use of monensin can lead to a marked fragmentation of the mitochondrial network that can be prevented by using 3-methyladenine, an inhibitor of autophagy-promoting PtdIns3k, suggesting a form of autophagic cell death is involved (Ghosh et al., 2012; Lavine and Arrizabalaga, 2012). Excessive or uncontrolled levels of autophagy may lead to cell death, but this is still a controversial notion that remains to be confirmed in Toxoplasma by experiments showing increased viability in these conditions upon genetic inactivation of the autophagic pathway in the parasites. Thus it is still unclear whether excessive autophagy might be responsible for cell death or, on the contrary, may be increased as a mechanism trying to prevent the cellular demise. In any case, harsh starvation conditions lead to a significant loss of tachyzoite viability coincidentally with the appearance of autophagosomes (Nguyen et al., 2017b).

The experimental conditions used to trigger autophagy in Toxoplasma in vitro are probably not reflective of a physiological situation. Although Toxoplasma may experience extracellular stress during its developmental cycle, it is mostly an obligate intracellular parasite. It resides in a PV that provides a relatively safe shelter with continuous access to host nutrient sources. A knockout mutant for TgATG9, which is considered to be an upstream factor in the hierarchy of ATG proteins, was generated to get more insights into the functional role of canonical autophagy during the course of infection (Nguyen et al., 2017b). Despite being impaired in the canonical degradative function of autophagy as assessed by quantifying the turnover of long-lived proteins, this mutant is viable in normal growth in vitro conditions (like most eukaryotic autophagy mutants grown in nutrient-rich conditions). However, TgATG9-depleted parasites are more sensitive to stress as extracellular parasites and, importantly, are much less virulent in the mouse model. Interestingly, a Toxoplasma mutant lacking TgCPL, which is a major protease in the VAC/PLV, also has a mild in vitro yet a more prominent in vivo phenotype (Dou et al., 2014). These findings suggest that components of both the autophagic and proteolytic machineries may act synergistically to help surviving specific stresses encountered during the course of infection.

15.9.3 Autophagy and differentiation In metazoans, autophagy not only serves as a crucial intracellular quality control and repair mechanism to eliminate damaged organelles, but it is also involved in cell remodeling during development and cell differentiation (Mizushima and Levine, 2010). Examples include the turnover of organelles during hematopoietic differentiation, or paternal

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mitochondria elimination from fertilized eggs. Like other apicomplexan parasites, Toxoplasma goes through a complex life cycle that includes several developmental forms. In the intermediate host alone, the parasite can switch from the rapidly dividing tachyzoite to the encysted quiescent bradyzoite form upon stress. This cellular differentiation is accompanied by some morphological changes and some extensive metabolic adaptation. Autophagy and proteolysis in the VAC/PLV compartment have been shown to play a role after the transition from tachyzoites to bradyzoites (Di Cristina et al., 2017). It is, however, not known if autophagy following encystation would be required for the recycling of organelles for cellular remodeling purposes, removal of damaged organelles associated with cellular stress, or for adaptation in response to a potentially more limited access to nutrients from the host (as the parasite is enclosed in a potentially less permeable compartment). In any case, disrupting autophagosome turnover in the VAC results in marked accumulation of undigested material prior to loss of bradyzoite viability in vitro and in vivo. These findings imply that the turnover of autophagic material in bradyzoites is crucial for parasite survival following differentiation.

15.9.4 Noncanonical function of autophagy-related proteins at the apicoplast Autophagy arose as a bulk degradation mechanism for meeting the cellular demands in energy, in particular, in the case of nutrient starvation, and this appears to be a universal feature of nearly all eukaryotic cells. However, during the course of evolution, this ancient cellular feature has been modified and adapted by a number of different eukaryotic lineages. For example, budding yeasts use autophagosomal proteins in the cytoplasm-to-vacuole targeting pathway, for delivering cargo proteins to the vacuole and not for degradation purposes (Lynch-Day and Klionsky, 2010).

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In mammalian cells, autophagy has evolved into a mechanism of host defense: the autophagy machinery targets intracellular pathogens for lysosomal degradation directly through autophagosome formation, or though noncanonical modification of microbecontaining vacuoles (Mitchell and Isberg, 2017). Apicomplexan parasites have also evolved unexpected features for the autophagy machinery. In contrast to its recruitment to autophagosomes in stress conditions, in normal intracellular growth conditions TgATG8 was found to localize to the apicoplast (Fig. 15.5), the plastid of secondary endosymbiotic origin present in many apicomplexa. There, ATG8 fulfills an essential function for homeostasis of the organelle, both in Toxoplasma and Plasmodium (Le´veˆque et al., 2015; Walczak et al., 2018). TgATG8 associates to the outermost apicoplast membrane (Le´veˆque et al., 2015), and functional studies on proteins TgATG3 and TgATG4 demonstrated that it involves the same conjugation machinery that regulates TgATG8 binding to the autophagosomal membrane (Kong-Hap et al., 2013). Specific recruitment of this protein during apicoplast replication is important for the proper segregation of the organelle into daughter cells (Le´veˆque et al., 2015). Although the precise molecular mechanism of action is currently unknown, this apicoplast-related function of ATG8 is completely independent from degradative autophagy. Therefore while potentially retaining the capacity to perform canonical degradative autophagy, Toxoplasma seems to have repurposed part of the autophagy machinery centered on ATG8 for performing Apicomplexa-specific functions. Perhaps, a good illustration of this diversification and function specialization is the case of ATG18-related proteins of the PROPPIN family TgPROP1 and TgPROP2 (Nguyen et al., 2018). They both may be involved in early steps of autophagosome biogenesis in the process of canonical autophagy, while TgPROP2 only is essential for parasite survival and may be

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FIGURE 15.5 TgATG8 localizes to the apicoplast in normal intracellular growth conditions. Costaining of GFPTgATG8 with apicoplast marker TgATRX1 and IMC marker TgIMC1 (used to visualize daughter buds) in Toxoplasma gondii tachyzoites undergoing cytokinesis. DNA was stained with 40 ,6-diamidino-2-phenylindole (DAPI). Scale bar is 5 μm. DIC, Differential interference contrast; IMC, inner membrane complex.

involved in an apicoplast-related function, similar to its Plasmodium counterpart (Bansal et al., 2017). The apicoplast is of ultimately prokaryotic origin and the successive double endosymbiotic events that lead to its incorporation into an ancestor of the Apicomplexa imply that its outermost membrane could be of phagosomal origin (Cavalier-Smith, 2000), although it might also have incorporated elements of the host ER (Gould et al., 2015). It is interesting to note that PtdIns3P and ATG8, which are known to be associated with the phagosomal membrane (Lai and Devenish, 2012; Jeschke and Haas, 2016), are both also important for the homeostasis of the plastid (Tawk et al., 2011; Le´veˆque et al., 2015). As an evolutionary twist of fate of some sort, original players potentially involved in prokaryotic pathogen control have been repurposed for controlling replication of an organelle of prokaryotic origin.

15.10 Final remarks The components of the MTS have an ancient origin and are widely conserved across eukaryotes. However, the Apicomplexa constitute a phylum that is very evolutionarily distant from most canonical eukaryotic models and as such, perhaps unsurprisingly, they present a number of divergent features. First, parasites with obligate intracellular stages such as T. gondii are highly adapted to their host. Thus they might have an apparently less complex machinery due to their parasitic lifestyle and the possibility to exploit their host for a number of functions. However, their adaptation to parasitism also leads to some extent to the evolution of lineage-specific proteins. Yet, another strategy for combining reduction of complexity with the capacity to generate novel features is the repurposing a

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number of preexisting proteins or pathways. As an illustration of this, in T. gondii, it seems that the components of the endosomal system have been diverted to specialized secretory organelles involved in virulence and components of the autophagic pathway have been used for maintaining an organelle of endosymbiotic origin and of particular metabolic importance. Because they are quite remote from the canonical textbook models, deciphering some of these particularly original features represents a considerable challenge. Moreover, characterizing the interactions between the components of the MTS is difficult, because they form an intricately interconnected network of highly dynamic compartments. Besides, some organelles, such as dense granules and micronemes, present discrete subpopulations with distinct protein contents (although it is currently unknown if such a diversity in composition is related to different specialized functions). Yet, whereas much remain to be discovered regarding the organization and diverse functions of the MTS in Apicomplexa, a huge amount of progress has been achieved in the last 20 years. As described in this chapter, a number of protein markers have now been identified. Also, recent progress in imaging techniques such as high-resolution microscopy and high-speed live confocal microscopy now allow better tracking and identification of the organelles and should enable further exploration of the MTS in the parasites. In conclusion, a picture is emerging in which the Toxoplasma MTS, which has undergone significant expansions and contractions in both coding complement and organelles, has been modified to achieve robust motility toward, entry into, and exploitation of, host cells. Clearly, the importance of this system for maintaining organelles that are crucial to multiple life cycle stages of the parasite, combined with the differences between apicomplexan and animal systems, certainly suggest that continued

research into the apicomplexan MTS will be invaluable in understanding and combating these parasites.

Acknowledgments The authors thank Drs. Isabelle Coppens and Jean-Franc¸ois Dubremetz for providing electron microscopy pictures displayed in Figs. 15.2 and 15.4A, respectively. CMK wishes to thank Kannan Venugopal and Elena Jimenez-Ruiz for critical reading of part of the manuscript. CMK has been funded by graduate studentships from the Women and Children’s Health Research Institute, Alberta Innovates Health Solutions, and the Canadian Institute for Health Research through the Vanier program. Studies in the authors’ labs related to this chapter are supported by grants from the Agence Nationale de la Recherche (to SB), a Wellcome Trust Senior Fellowship (087582/Z/08/Z to MM), and an European Research Council starting Grant (ERC-2012-StG 309255-EndoTox to MM), and the US National Institutes of Health (R01AI120607 to VBC).

Glossary Acidocalcisomes small, dense, spherical, and acidic organelles containing high concentrations of calcium and phosphate Apicoplast a remnant nonphotosynthetic plastid that compartmentalizes key metabolic pathways Autophagosome a double-membrane structure formed by closure of the phagophore to encapsulate cargo Autophagy a process of encapsulating and delivering cellular proteins, organelles, and structures for lysosomal degradation Dense granules spherical secretory organelles that are distributed throughout the parasite cytoplasm. Dense granule proteins play many roles in nutrient acquisition, immune evasion, and manipulation of host processes Egress the process of parasite exit from infected cells Endocytosis a process of internalizing cell surface components and exogenous material from the environment exterior to the cell

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Inner membrane complex a complex consisting of an intermediate filament-like cytoskeletal structure and flattened cisternae that are sutured together to create a double membrane platform underlying the parasite plasma membrane Micronemes cigar-shaped apical secretory organelles that contain adhesive and membrane disruptive proteins that are discharged in response to elevated cytosolic calcium within the parasite Micropore a putative site of endocytosis consisting of a discrete invagination of the midapical parasite plasma membrane Parasitophorous vacuole a compartment formed during invasion in which the parasite replicates intracellularly Phagophore a double-membrane cup formed as a means of encapsulating autophagic cargo Prerhoptries transient organelles that are observed during parasite cell division and serve as the precursors for rhoptries Rhoptries club-shaped apical secretory organelles that inject their contents into the host cell immediately prior to invasion. Rhoptry proteins function in various ways to mediate invasion and evade host cell intrinsic immune mechanisms Vacuolar compartment/plant-like vacuole a lysosome-like organelle that most often resides in the mid-apical region of the parasites. This organelle is a site for degradation of proteins and likely other macromolecules along with a site for storage of ions and water

References Adisa, A., et al., 2007. Re-assessing the locations of components of the classical vesicle-mediated trafficking machinery in transfected Plasmodium falciparum. Int. J. Parasitol. 37 (10), 1127
1141. Available from: https:// doi.org/10.1016/j.ijpara.2007.02.009. Agop-Nersesian, C., et al., 2009. Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis. PLoS Pathog. 5 (1),

e1000270. Available from: https://doi.org/10.1371/ journal.ppat.1000270. Agop-Nersesian, C., et al., 2010. Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B. PLoS Pathog. 6 (7), e1001029. Available from: https://doi.org/ 10.1371/journal.ppat.1001029. Ayala, C.I., Kim, J., Neufeld, T.P., 2018. Rab6 promotes insulin receptor and cathepsin trafficking to regulate autophagy induction and activity in Drosophila. J. Cell Sci. 131 (17), jcs216127. Available from: https://doi. org/10.1242/jcs.216127. Bansal, P., et al., 2017. Autophagy-related protein ATG18 regulates apicoplast biogenesis in apicomplexan parasites. mBio 8 (5), e01468-17. Available from: https://doi. org/10.1128/mBio.01468-17. Barlowe, C.K., Miller, E.A., 2013. Secretory protein biogenesis and traffic in the early secretory pathway. Genetics 193 (2), 383
410. Available from: https://doi.org/ 10.1534/genetics.112.142810. Besteiro, S., et al., 2008. Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries: further insights into the role of cholesterol. Biochem. J. 415 (1), 87
96. Available from: https://doi.org/10.1042/BJ20080795. Besteiro, S., et al., 2009. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 5 (2), e1000309. Available from: https://doi. org/10.1371/journal.ppat.1000309. Besteiro, S., et al., 2011. Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. PLoS Pathog. 7 (12), e1002416. Available from: https://doi.org/10.1371/journal. ppat.1002416. Blanc, C., et al., 2009. Dictyostelium Tom1 participates to an ancestral ESCRT-0 complex. Traffic 10 (2), 161
171. Available from: https://doi.org/10.1111/j.16000854.2008.00855.x. Botero-Kleiven, S., et al., 2001. Receptor-mediated endocytosis in an apicomplexan parasite (Toxoplasma gondii). Exp. Parasitol. 98 (3), 134
144. Breinich, M.S., et al., 2009. A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii. Curr. Biol. 19 (4), 277
286. Available from: https://doi. org/10.1016/j.cub.2009.01.039. Bro¨cker, C., Engelbrecht-Vandre´, S., Ungermann, C., 2010. Multisubunit tethering complexes and their role in membrane fusion. Curr. Biol. 20 (21), R943
R952. Available from: https://doi.org/10.1016/j. cub.2010.09.015. Brydges, S.D., et al., 2008. A transient forward-targeting element for microneme-regulated secretion in Toxoplasma gondii. Biol. Cell 100 (4), 253
264. Available from: https://doi.org/10.1042/BC20070076.

Toxoplasma Gondii

References

Burguete, A.S., et al., 2008. Rab and Arl GTPase family members cooperate in the localization of the Golgin GCC185. Cell 132 (2), 286
298. Available from: https:// doi.org/10.1016/j.cell.2007.11.048. Cavalier-Smith, T., 2000. Membrane heredity and early chloroplast evolution. Trends Plant Sci. 5 (4), 174
182. Available from: https://doi.org/10.1016/S1360-1385(00) 01598-3. Chaturvedi, S., et al., 1999. Constitutive calciumindependent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab machinery. J. Biol. Chem. 274 (4), 2424
2431. Available from: https://doi.org/10.1074/jbc.274.4.2424. Christoforidis, S., et al., 1999. The rab5 effector EEA1 is a core component of endosome docking. Nature 397 (6720), 621
625. Available from: https://doi.org/ 10.1038/17618. Coffey, M.J., et al., 2018. Aspartyl protease 5 matures dense granule proteins that reside at the host-parasite interface in Toxoplasma gondii. mBio 9 (5), e01796-18. Available from: https://doi.org/10.1128/mBio.01796-18. Coleman, B.I., et al., 2018. A member of the ferlin calcium sensor family is essential for Toxoplasma gondii rhoptry secretion. mBio 9 (5). Available from: https://doi.org/ 10.1128/mBio.01510-18. Dacks, J.B., Field, M.C., 2007. Evolution of the eukaryotic membrane-trafficking system: origin, tempo and mode. J. Cell Sci. 120 (Pt 17), 2977
2985. Available from: https://doi.org/10.1242/jcs.013250. Dacks, J.B., Poon, P.P., Field, M.C., 2008. Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution. Proc. Natl. Acad. Sci. U.S.A. 105 (2), 588
593. Available from: https://doi.org/10.1073/pnas.0707318105. Day, K.J., Casler, J.C., Glick, B.S., 2018. Budding yeast has a minimal endomembrane system. Dev. Cell. 44 (1), 56
72.e4. Available from: https://doi.org/10.1016/j. devcel.2017.12.014. Delorme-Axford, E., et al., 2015. The yeast Saccharomyces cerevisiae: an overview of methods to study autophagy progression. Methods 75, 3
12. Available from: https://doi.org/10.1016/j.ymeth.2014.12.008. Dettmer, J., et al., 2006. Vacuolar H 1 -ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18 (3), 715
730. Available from: https://doi.org/10.1105/tpc.105.037978. Di Cristina, M., et al., 2000. Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii. Mol. Cell. Biol. 20 (19), 7332
7341. Di Cristina, M., et al., 2017. Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection. Nat. Microbiol. 2, 17096. Dogga, S.K., et al., 2017. A druggable secretory protein maturase of Toxoplasma essential for invasion and

735

egress. eLife 6. Available from: https://doi.org/ 10.7554/eLife.27480. Donaldson, J.G., Jackson, C.L., 2011. ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat. Rev. Mol. Cell Biol. 12 (6), 362
375. Available from: https://doi.org/10.1038/ nrm3117. Dou, Z., et al., 2014. Toxoplasma gondii ingests and digests host cytosolic proteins. mBio 5 (4), e01188-14. Available from: https://doi.org/10.1128/mBio.01188-14. Drozdowicz, Y.M., et al., 2003. Isolation and characterization of TgVP1, a type I vacuolar H 1 -translocating pyrophosphatase from Toxoplasma gondii: the dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor. J. Biol. Chem. 278 (2), 1075
1085. Available from: https://doi.org/10.1074/ jbc.M209436200. Dubremetz, J.F., 2007. Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction. Cell. Microbiol. 9 (4), 841
848. Available from: https:// doi.org/10.1111/j.1462-5822.2007.00909.x. Duszenko, M., et al., 2011. Autophagy in protists. Autophagy 7 (2), 127
158. Edler, E., Stein, M., 2019. Recognition and stabilization of geranylgeranylated human Rab5 by the GDP dissociation inhibitor (GDI). Small GTPases 10 (3), 227
242. Available from: https://doi.org/10.1080/ 21541248.2017.1371268. El Hajj, H., et al., 2008. Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes. Eukaryot. Cell 7 (6), 1019
1028. Available from: https://doi.org/10.1128/EC.00413-07. Engstler, M., Pfohl, T., Herminghaus, S., Boshart, M., Wiegertjes, G., Heddergott, N., et al., 2007. Hydrodynamic flow-mediated protein sorting on the cell surface of trypanosomes. Cell 131 (3), 505
515. Nov 2. Ezougou, C.N., et al., 2014. Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite’s plasma and food vacuole membranes. PLoS One 9 (2), e87695. Available from: https://doi.org/10.1371/journal.pone.0087695. Farrell, A., et al., 2012. A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis. Science 335 (6065), 218
221. Available from: https://doi.org/10.1126/science.1210829. Fichera, M.E., Roos, D.S., 1997. A plastid organelle as a drug target in apicomplexan parasites. Nature 390 (6658), 407
409. Available from: https://doi.org/ 10.1038/37132. Field, M.C., Gabernet-Castello, C., Dacks, J.B., 2007. Reconstructing the evolution of the endocytic system: insights from genomics and molecular cell biology. Adv. Exp. Med. Biol. 607, 84
96. Available from: https://doi.org/10.1007/978-0-387-74021-8_7.

Toxoplasma Gondii

736

15. Endomembrane trafficking pathways in Toxoplasma

Fomovska, A., et al., 2012. Novel N-benzoyl-2hydroxybenzamide disrupts unique parasite secretory pathway. Antimicrob. Agents Chemother. 56 (5), 2666
2682. Available from: https://doi.org/10.1128/ AAC.06450-11. Francia, M.E., et al., 2011. A Toxoplasma gondii protein with homology to intracellular type Na 1 /H 1 exchangers is important for osmoregulation and invasion. Exp. Cell Res. 317 (10), 1382
1396. Available from: https://doi. org/10.1016/j.yexcr.2011.03.020. Gagescu, R., et al., 2000. The recycling endosome of MadinDarby canine kidney cells is a mildly acidic compartment rich in raft components. Mol. Biol. Cell. 11 (8), 2775
2791. Available from: https://doi.org/10.1021/ cr00017a024. Gaji, R.Y., Flammer, H.P., Carruthers, V.B., 2011. Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids. Traffic 12 (7), 840
853. Available from: https://doi.org/ 10.1111/j.1600-0854.2011.01192.x. Ghosh, D., et al., 2012. Autophagy is a cell death mechanism in Toxoplasma gondii. Cell. Microbiol. 14 (4), 589
607. Available from: https://doi.org/10.1111/ j.1462-5822.2011.01745.x. Gidon, A., et al., 2012. A Rab11A/Myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane. Traffic 13 (6), 815
833. Available from: https://doi.org/10.1111/ j.1600-0854.2012.01354.x. Gould, S.B., Maier, U.-G., Martin, W.F., 2015. Protein import and the origin of red complex plastids. Curr. Biol. 25 (12), R515
R521. Available from: https://doi. org/10.1016/j.cub.2015.04.033. Gras, S., Jimenez-Ruiz, E., Klinger, C.M., Schneider, K., Klingl, A., et al., 2019. An endocytic-secretory cycle participates in Toxoplasma gondii in motility. PLOS Biology 17 (6). Available from: https://doi.org/ 10.1371/journal.pbio.3000060. Hager, K.M., et al., 1999. The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii. J. Cell Sci. 112 (Pt 16), 2631
2638. Hammoudi, P.-M., et al., 2015. Fundamental roles of the Golgi-associated Toxoplasma aspartyl protease, ASP5, at the host-parasite interface. PLoS Pathog. 11 (10), e1005211. Available from: https://doi.org/10.1371/ journal.ppat.1005211. Harper, J.M., et al., 2006. A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex. Mol. Biol. Cell. 17 (10), 4551
4563. Available from: https:// doi.org/10.1091/mbc.E06-01-0064.

Heaslip, A.T., Nelson, S.R., Warshaw, D.M., 2016. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments. Mol. Biol. Cell. 27 (13), 2080
2089. Available from: https://doi. org/10.1091/mbc.E15-12-0824. ˝ K., et al., 2016. The Ccz1-Mon1-Rab7 module and Hegedus, Rab5 control distinct steps of autophagy. Mol. Biol. Cell. 27 (20), 3132
3142. Available from: https://doi. org/10.1091/mbc.E16-03-0205. Henne, W.M., Buchkovich, N.J., Emr, S.D., 2011. The ESCRT pathway. Dev. Cell. 21 (1), 77
91. Available from: https://doi.org/10.1016/j.devcel.2011.05.015. Hill, E., Clarke, M., Barr, F.A., 2000. The Rab6-binding kinesin, Rab6-KIFL, is required for cytokinesis. EMBO J. 19 (21), 5711
5719. Available from: https://doi.org/ 10.1093/emboj/19.21.5711. Hirst, J., et al., 2011. The fifth adaptor protein complex. PLoS Biol. 9 (10), e1001170. Available from: https://doi. org/10.1371/journal.pbio.1001170. Hoppe, H.C., et al., 2000. Targeting to rhoptry organelles of Toxoplasma gondii involves evolutionarily conserved mechanisms. Nat. Cell Biol. 2 (7), 449
456. Available from: https://doi.org/10.1038/35017090. Huotari, J., Helenius, A., 2011. Endosome maturation. EMBO J. 30 (17), 3481
3500. Available from: https:// doi.org/10.1038/emboj.2011.286. Jackson, L.P., 2014. Structure and mechanism of COPI vesicle biogenesis. Curr. Opin. Cell Biol. 29, 67
73. Available from: https://doi.org/10.1016/j.ceb.2014.04.009. Jackson, A.J., et al., 2013. Toxoplasma gondii Syntaxin 6 is required for vesicular transport between endosomallike compartments and the Golgi complex. Traffic 14 (11), 1166
1181. Available from: https://doi.org/ 10.1111/tra.12102. Jahn, R., Scheller, R.H., 2006. SNAREs
engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7 (9), 631
643. Available from: https://doi.org/10.1038/nrm2002. Jeschke, A., Haas, A., 2016. Deciphering the roles of phosphoinositide lipids in phagolysosome biogenesis. Commun. Integr. Biol. 9 (3), e1174798. Available from: https://doi.org/10.1080/19420889.2016.1174798. Jimenez-Ruiz, E., et al., 2016. Vacuolar protein sorting mechanisms in apicomplexan parasites. Mol. Biochem. Parasitol. 209, 18
25. Karsten, V., et al., 1998. The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms. J. Cell Biol. 141 (6), 1323
1333. Katris, N.J., et al., 2019. Calcium negatively regulates secretion from dense granules in Toxoplasma gondii. Cell. Microbiol. e13011. Available from: https://doi.org/ 10.1111/cmi.13011.

Toxoplasma Gondii

References

Kessler, H., et al., 2008. Microneme protein 8—a new essential invasion factor in Toxoplasma gondii. J. Cell Sci. 121 (Pt 7), 947
956. Kibria, K.M.K., et al., 2015. A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum. Biochim Biophys Acta 1853 (3), 699
710. Available from: https://doi.org/10.1016/j. bbamcr.2014.12.030. Kleine Balderhaar, H.J., Ungermann, C., 2013. CORVET and HOPS tethering complexes
coordinators of endosome and lysosome fusion. J. Cell Sci. 126 (Pt 6), 1307
1316. Available from: https://doi.org/10.1242/ jcs.107805. Klinger, C.M., Klute, M.J., Dacks, J.B., 2013. Comparative genomic analysis of multi-subunit tethering complexes demonstrates an ancient pan-eukaryotic complement and sculpting in Apicomplexa. PLoS One 8 (9), e76278. Available from: https://doi.org/10.1371/journal. pone.0076278. Klinger, C.M., Nisbet, E.R., Ouologuem, D.T., Roos, D.S., Dacks, J.B., 2013a. Cryptic organelle homology in apicomplexan parasites: insights from evolutionary cell biology. Curr. Opin. Microbiol 16 (4), 424
431. ISSN 1369-5274, https://doi.org/10.1016/j.mib.2013.07.015. Klinger, C.M., et al., 2016. Resolving the homologyfunction relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology. Mol. Biochem. Parasitol. 209 (1
2), 88
103. Available from: https://doi.org/10.1016/j. molbiopara.2016.07.003. Kno¨dler, A., et al., 2010. Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc. Natl. Acad. Sci. U.S.A. 107 (14), 6346
6351. Available from: https://doi.org/ 10.1073/pnas.1002401107. Kong-Hap, M.A., et al., 2013. Regulation of ATG8 membrane association by ATG4 in the parasitic protist Toxoplasma gondii. Autophagy 9 (9), 1334
1348. Available from: https://doi.org/10.4161/auto.25189. Koumandou, V.L., et al., 2007. Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins. BMC Evol. Biol. 23 (7), 29. Available from: https://doi.org/10.1186/ 1471-2148-7-29. Krai, P., Dalal, S., Klemba, M., 2014. Evidence for a Golgito-endosome protein sorting pathway in Plasmodium falciparum. PLoS One 9 (2), e89771. Available from: https://doi.org/10.1371/journal.pone.0089771. Kremer, K., et al., 2013. An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes. PLoS Pathog. 9 (3), e1003213. Available from: https://doi.org/10.1371/ journal.ppat.1003213.

737

Kroemer, G., Marin˜o, G., Levine, B., 2010. Autophagy and the integrated stress response. Mol. Cell 40 (2), 280
293. Available from: https://doi.org/10.1016/j. molcel.2010.09.023. Labesse, G., et al., 2009. ROP2 from Toxoplasma gondii: a virulence factor with a protein-kinase fold and no enzymatic activity. Structure 17 (1), 139
146. Available from: https://doi.org/10.1016/j.str.2008.11.005. Lachmann, J., et al., 2014. The Vps39-like TRAP1 is an effector of Rab5 and likely the missing Vps3 subunit of human CORVET. Cell. Logist. 4 (4), e970840. Available from: https://doi.org/10.4161/21592780.2014.970840. Lai, S.-C., Devenish, R.J., 2012. LC3-associated phagocytosis (LAP): connections with host autophagy. Cells 1 (3), 396
408. Available from: https://doi.org/10.3390/ cells1030396. Langemeyer, L., Fro¨hlich, F., Ungermann, C., 2018. Rab GTPase function in endosome and lysosome biogenesis. Trends Cell Biol. 28 (11), 957
970. Available from: https://doi.org/10.1016/j.tcb.2018.06.007. Langsley, G., et al., 2008. Comparative genomics of the Rab protein family in apicomplexan parasites. Microbes Infect. 10 (5), 462
470. Available from: https://doi.org/ 10.1016/j.micinf.2008.01.017. Lavine, M.D., Arrizabalaga, G., 2012. Analysis of monensin sensitivity in Toxoplasma gondii reveals autophagy as a mechanism for drug induced death. PLoS One 7 (7), e42107. Available from: https://doi.org/10.1371/journal.pone.0042107. Lee, M.C.S., et al., 2008. Plasmodium falciparum Sec24 marks transitional ER that exports a model cargo via a diacidic motif. Mol. Microbiol. 68 (6), 1535
1546. Available from: https://doi.org/10.1111/j.1365-2958.2008.06250.x. Leung, K.F., Dacks, J.B., Field, M.C., 2008. Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage. Traffic 9 (10), 1698
1716. Available from: https://doi.org/10.1111/j.16000854.2008.00797.x. Le´veˆque, M.F., et al., 2015. Autophagy-related protein ATG8 has a noncanonical function for apicoplast inheritance in Toxoplasma gondii. mBio 6 (6), e01446-15. Available from: https://doi.org/10.1128/ mBio.01446-15. Liendo, A., et al., 2001. Toxoplasma gondii ADP-ribosylation factor 1 mediates enhanced release of constitutively secreted dense granule proteins. J. Biol. Chem. 276 (21), 18272
18281. Available from: https://doi.org/10.1074/ jbc.M008352200. Lock, J.G., Stow, J.L., 2005. Rab11 in recycling endosomes regulates the sorting and basolateral transport of Ecadherin. Mol. Biol. Cell. 16 (4), 1744
1755. Available from: https://doi.org/10.1091/mbc.E04.

Toxoplasma Gondii

738

15. Endomembrane trafficking pathways in Toxoplasma

Lynch-Day, M.A., Klionsky, D.J., 2010. The Cvt pathway as a model for selective autophagy. FEBS Lett. 584 (7), 1359
1366. Available from: https://doi.org/10.1016/j. febslet.2010.02.013. Mann, T., Beckers, C., 2001. Characterization of the subpellicular network, a filamentous membrane skeletal component in the parasite Toxoplasma gondii. Mol. Biochem. Parasitol. 115 (2), 257
268. Manna, P.T., et al., 2014. Life and times: synthesis, trafficking, and evolution of VSG. Trends Parasitol. 30 (5), 251
258. Available from: https://doi.org/10.1016/j. pt.2014.03.004. Matanis, T., et al., 2002. Bicaudal-D regulates COPIindependent Golgi-ER transport by recruiting the dynein-dynactin motor complex. Nat. Cell Biol. 4 (12), 986
992. Available from: https://doi.org/10.1055/s2005-864830. McEwan, D.G., et al., 2015. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol. Cell 57 (1), 39
54. Available from: https://doi.org/10.1016/j. molcel.2014.11.006. McGovern, O.L., et al., 2018. Intersection of endocytic and exocytic systems in Toxoplasma gondii. Traffic 19 (5), 336
353. Available from: https://doi.org/10.1111/ tra.12556. Mercier, C., Cesbron-Delauw, M.F., 2015. Toxoplasma secretory granules: one population or more? Trends Parasitol. 31 (2), 60
71. Available from: https://doi. org/10.1016/j.pt.2014.12.002. Miller, E.A., Schekman, R., 2013. COPII
a flexible vesicle formation system. Curr. Opin. Cell Biol. 25 (4), 420
427. https://doi.org/10.1016/j. Available from: ceb.2013.04.005. Miranda, K., et al., 2008. Acidocalcisomes in apicomplexan parasites. Exp. Parasitol. 118 (1), 2
9. Available from: https://doi.org/10.1016/j.exppara.2007.07.009. Miranda, K., et al., 2010. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Mol. Microbiol. 76 (6), 1358
1375. Available from: https://doi.org/10.1111/ j.1365-2958.2010.07165.x. Mitchell, G., Isberg, R.R., 2017. Innate immunity to intracellular pathogens: balancing microbial elimination and inflammation. Cell Host Microbe 22 (2), 166
175. Available from: https://doi.org/10.1016/j. chom.2017.07.005. Mizushima, N., Levine, B., 2010. Autophagy in mammalian development and differentiation. Nat. Cell Biol. 12 (9), 823
830. Available from: https://doi.org/10.1038/ ncb0910-823. Moreau, K., Luo, S., Rubinsztein, D.C., 2010. Cytoprotective roles for autophagy. Curr. Opin. Cell Biol. 22 (2),

206
211. Available from: https://doi.org/10.1016/j. ceb.2009.12.002. Morlon-Guyot, J., et al., 2015. Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes, is essential for the biogenesis of secretory organelles. Cell. Microbiol. . Available from: https://doi.org/10.1111/ cmi.12426. Morlon-Guyot, J., et al., 2018. A proteomic analysis unravels novel CORVET and HOPS proteins involved in Toxoplasma gondii secretory organelles biogenesis. Cell. Microbiol. 20 (11), e12870. Available from: https://doi. org/10.1111/cmi.12870. Mueller, C., et al., 2013. The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion. Cell Host Microbe 13 (3), 289
301. Available from: https://doi.org/10.1016/j. chom.2013.02.001. Nakayama, K., 2016. Regulation of cytokinesis by membrane trafficking involving small GTPases and the ESCRT machinery. Crit. Rev. Biochem. Mol. Biol. 51 (1), 1
6. Available from: https://doi.org/10.3109/ 10409238.2015.1085827. Nevin, W.D., Dacks, J.B., 2009. Repeated secondary loss of adaptin complex genes in the Apicomplexa. Parasitol. Int. 58 (1), 86
94. Available from: https://doi.org/ 10.1016/j.parint.2008.12.002. Ngoˆ, H.M., et al., 2003. AP-1 in Toxoplasma gondii mediates biogenesis of the rhoptry secretory organelle from a post-Golgi compartment. J. Biol. Chem. 278 (7), 5343
5352. Available from: https://doi.org/10.1074/ jbc.M208291200. Ngoˆ, H.M., Yang, M., Joiner, K.A., 2004. Are rhoptries in apicomplexan parasites secretory granules or secretory lysosomal granules? Mol. Microbiol. 52 (6), 1531
1541. Available from: https://doi.org/10.1111/j.13652958.2004.04056.x. Nguyen, H.M., Berry, L., et al., 2017a. Autophagy participates in the unfolded protein response in Toxoplasma gondii. FEMS Microbiol. Lett. 364 (15). Available from: https://doi.org/10.1093/femsle/fnx153. Nguyen, H.M., El Hajj, H., et al., 2017b. Toxoplasma gondii autophagy-related protein ATG9 is crucial for the survival of parasites in their host. Cell. Microbiol. 19 (6), e12712. Available from: https://doi.org/10.1111/ cmi.12712. Nguyen, H.M., et al., 2018. Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions. PLoS One 13 (4), e0195921. Available from: https://doi. org/10.1371/journal.pone.0195921. Nichols, B.A., Chiappino, M.L., O’Connor, G.R., 1983. Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J. Ultrastruct. Res. 83 (1), 85
98.

Toxoplasma Gondii

References

Nichols, B.A., Chiappino, M.L., Pavesio, C.E., 1994. Endocytosis at the micropore of Toxoplasma gondii. Parasitol. Res. 80 (2), 91
98. Nishi, M., et al., 2008. Organellar dynamics during the cell cycle of Toxoplasma gondii. J. Cell Sci. 121 (Pt 9), 1559
1568. Available from: https://doi.org/10.1242/ jcs.021089. Nolan, S.J., Romano, J.D., Coppens, I., 2017. Host lipid droplets: an important source of lipids salvaged by the intracellular parasite Toxoplasma gondii. PLoS Pathog. 13 (6). Available from: https://doi.org/10.1371/journal. ppat.1006362. O’Neill, P.R., Castillo-Badillo, J.A., Meshik, X., Kalyanaraman, V., Melgarejo, K., Gautam, N., 2018. Membrane flow drives an adhesion-independent amoeboid cell migration mode. Dev. Cell. 46 (1), 9
22.e4. Available from: https://doi.org/10.1016/j. devcel.2018.05.029. Ouologuem, D.T., Roos, D.S., 2014. Dynamics of the Toxoplasma gondii inner membrane complex. J. Cell Sci. 127 (Pt 15), 3320
3330. Available from: https://doi. org/10.1242/jcs.147736. Overath, P., Engstler, M., 2004. Endocytosis, membrane recycling and sorting of GPI-anchored proteins: Trypanosoma brucei as a model system. Mol. Microbiol. 53 (3), 735
744. Papanikou, E., et al., 2015. COPI selectively drives maturation of the early Golgi. eLife 4, e13232. Available from: https://doi.org/10.7554/eLife.13232. Parussini, F., et al., 2010. Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii. Mol. Microbiol. 76 (6), 1340
1357. Available from: https:// doi.org/10.1111/j.1365-2958.2010.07181.x. Pelletier, L., et al., 2002. Golgi biogenesis in Toxoplasma gondii. Nature 418 (6897), 548
552. Available from: https://doi.org/10.1038/nature00946. Peplowska, K., et al., 2007. The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis. Dev. Cell. 12 (5), 739
750. Available from: https://doi.org/10.1016/j. devcel.2007.03.006. Periz, J., Del Rosario, M., McStea, A., Gras, S., Loney, C., Wang, L., et al., 2019. A highly dynamic F-actin network regulates transport and recycling of micronemes in Toxoplasma gondii vacuoles. Nat. Commun. 10 (1), 4183. Pfluger, S.L., et al., 2005. Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii. Eukaryot. Cell 4 (2), 432
442. Available from: https:// doi.org/10.1128/EC.4.2.432. Pieperhoff, M.S., et al., 2013. The role of clathrin in postGolgi trafficking in Toxoplasma gondii. PLoS One 8 (10),

739

e77620. Available from: https://doi.org/10.1371/journal.pone.0077620. Plemel, R.L., et al., 2011. Subunit organization and Rab interactions of Vps-C protein complexes that control endolysosomal membrane traffic. Mol. Biol. Cell. 22 (8), 1353
1363. Available from: https://doi.org/10.1091/ mbc.E10-03-0260. Polgar, N., Fogelgren, B., 2018. Regulation of cell polarity by exocyst-mediated trafficking. Cold Spring Harb. Perspect. Biol. 10 (3). Available from: https://doi.org/ 10.1101/cshperspect.a031401a031401. Poteryaev, D., et al., 2010. Identification of the switch in early-to-late endosome transition. Cell 141 (3), 497
508. Available from: https://doi.org/10.1016/j. cell.2010.03.011. Rabenau, K.E., et al., 2001. TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2. Mol. Microbiol. 41 (3), 537
547. Reiss, M., et al., 2001. Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii. J. Cell Biol. 152 (3), 563
578. Robibaro, B., et al., 2002. Toxoplasma gondii Rab5 enhances cholesterol acquisition from host cells. Cell. Microbiol. 4 (3), 139
152. Available from: https://doi.org/10.1046/ j.1462-5822.2002.00178.x. Robinson, M.S., 2004. Adaptable adaptors for coated vesicles. Trends Cell Biol. 14 (4), 167
174. Available from: https://doi.org/10.1016/j.tcb.2004.02.002. Robinson, M.S., 2015. Forty years of clathrin-coated vesicles. Traffic 16 (12), 1210
1238. Available from: https://doi.org/10.1111/tra.12335. Sangare´, L.O., et al., 2016. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection. Nat. Commun. 7, 11191. Available from: https://doi.org/ 10.1038/ncomms11191. Seaman, M.N.J., et al., 2009. Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5. J. Cell Sci. 122 (Pt 14), 2371
2382. Available from: https://doi.org/10.1242/jcs.048686. Shaw, M.K., Roos, D.S., Tilney, L.G., 1998. Acidic compartments and rhoptry formation in Toxoplasma gondii. Parasitology 117 (Pt 5), 435
443. Sheiner, L., et al., 2010. Toxoplasma gondii transmembrane microneme proteins and their modular design. Mol. Microbiol. 77, 912
929. Shin, H.-W., et al., 2005. An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway. J. Cell Biol. 170 (4), 607
618. Available from: https://doi.org/10.1083/ jcb.200505128.

Toxoplasma Gondii

740

15. Endomembrane trafficking pathways in Toxoplasma

Shpilka, T., et al., 2011. Atg8: an autophagy-related ubiquitin-like protein family. Genome. Biol. 12 (7), 226. Available from: https://doi.org/10.1186/gb-2011-12-7226. Sidik, S.M., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166 (6), 1423
1435.e12. Available from: https://doi. org/10.1016/j.cell.2016.08.019. Sloves, P.-J., et al., 2012. Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection. Cell Host Microbe 11 (5), 515
527. Available from: https:// doi.org/10.1016/j.chom.2012.03.006. van der Sluijs, P., et al., 1992. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell 70 (5), 729
740. Available from: https://doi.org/10.1016/0092-8674(92)90307-X. Soldati, D., et al., 1998. Processing of Toxoplasma ROP1 protein in nascent rhoptries. Mol. Biochem. Parasitol. 96 (1
2), 37
48. Available from: https://doi.org/10.1016/ S0166-6851(98)00090-5. Stedman, T.T., Sussmann, A.R., Joiner, K.A., 2003. Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex. J. Biol. Chem. 278 (7), 5433
5443. https://doi.org/10.1074/jbc. Available from: M209390200. Stenmark, H., 2009. Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 10 (8), 513
525. Available from: https://doi.org/10.1038/nrm2728. Stewart, R.J., et al., 2015. Phosphorylation of α SNAP is required for secretory organelle biogenesis in Toxoplasma gondii. Traffic 17 (2), 102
116. Available from: https://doi.org/10.1111/tra.12348. Struck, N.S., et al., 2005. Re-defining the Golgi complex in Plasmodium falciparum using the novel Golgi marker PfGRASP. J. Cell Sci. 118 (Pt 23), 5603
5613. Available from: https://doi.org/10.1242/jcs.02673. Struck, N.S., et al., 2008. Spatial dissection of the cis- and trans-Golgi compartments in the malaria parasite Plasmodium falciparum. Mol. Microbiol. 67 (6), 1320
1330. Available from: https://doi.org/10.1111/ j.1365-2958.2008.06125.x. Suzuki, K., et al., 2013. Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae. J. Cell Sci. 126 (11), 2534
2544. Available from: https://doi.org/10.1242/jcs.122960. Szatma´ri, Z., et al., 2014. Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of hook localization. Mol. Biol. Cell. 25 (4), 522
531. Available from: https://doi.org/10.1091/mbc. E13-10-0574. Tanaka, M., Kikuchi, T., Uno, H., Okita, K., KitanishiYumura, T., Yumura, S., 2017. Turnover and flow of the cell membrane for cell migration. Sci. Rep. 11 7 (1),

12970. Available from: https://doi.org/10.1038/s41598017-13438-5. Tawk, L., et al., 2011. Phosphatidylinositol 3monophosphate is involved in toxoplasma apicoplast biogenesis. PLoS Pathog. 7 (2), e1001286. Available from: https://doi.org/10.1371/journal.ppat.1001286. Thornton, L.B., et al., 2018. An ortholog of chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in to facilitate host invasion. bioRxiv. Available from: https://doi.org/10.1101/409904. Tomavo, S., 2014. Evolutionary repurposing of endosomal systems for apical organelle biogenesis in Toxoplasma gondii. Int. J. Parasitol. 44 (2), 133
138. Available from: https://doi.org/10.1016/j.ijpara.2013.10.003. Tomavo, S., et al., 2013. Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion. PLoS Pathog. 9 (10), e1003629. Tonkin, C.J., Kalanon, M., McFadden, G.I., 2008. Protein targeting to the malaria parasite plastid. Traffic 9 (2), 166
175. Available from: https://doi.org/10.1111/ j.1600-0854.2007.00660.x. Tooze, S.A., 2013. Current views on the source of the autophagosome membrane. Essays Biochem. 55, 29
38. Available from: https://doi.org/10.1042/bse0550029. Tsukada, M., Ohsumi, Y., 1993. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333 (1
2), 169
174. Ullrich, O., et al., 1996. Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135 (4), 913
924. Van Wye, J., et al., 1996. Identification and localization of rab6, separation of rab6 from ERD2 and implications for an “unstacked” Golgi, in Plasmodium falciparum. Mol. Biochem. Parasitol. 83 (1), 107
120. Available from: https://doi.org/10.1016/S0166-6851(96)02759-4. Venugopal, K., Marion, S., 2018. Secretory organelle trafficking in Toxoplasma gondii: a long story for a short travel. Int. J. Med. Microbiol. 308 (7), 751
760. Available from: https://doi.org/10.1016/j. ijmm.2018.07.007. Venugopal, K., et al., 2017. Dual role of the Toxoplasma gondii clathrin adaptor AP1 in the sorting of rhoptry and microneme proteins and in parasite division. PLoS Pathog. 13 (4), e1006331. Available from: https://doi. org/10.1371/journal.ppat.1006331. Viotti, C., et al., 2010. Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22 (4), 1344
1357. Available from: https://doi.org/10.1105/tpc.109.072637. Walczak, M., et al., 2018. ATG8 Is essential specifically for an autophagy-independent function in apicoplast biogenesis in blood-stage malaria parasites. mBio 9 (1). Available from: https://doi.org/10.1128/mBio.02021-17.

Toxoplasma Gondii

References

Warring, S.D., et al., 2014. Characterization of the chloroquine resistance transporter homologue in Toxoplasma gondii. Eukaryot. Cell 13 (11), 1360
1370. Available from: https://doi.org/10.1128/EC.00027-14. Wojczyk, B.S., et al., 2003. cDNA cloning and expression of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase T1 from Toxoplasma gondii. Mol. Biochem. Parasitol. 131 (2), 93
107. Available from: https://doi.org/10.1016/S0166-6851 (03)00196-8. Woo, Y.H., et al., 2015. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. eLife 4, e06974. Available from: https://doi.org/10.7554/eLife.06974.

741

Yamashiro, D.J., et al., 1984. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell 37 (3), 789
800. Yang, M., et al., 2004. The Plasmodium falciparum Vps4 homolog mediates multivesicular body formation. J. Cell Sci. 117 (Pt 17), 3831
3838. Available from: https://doi.org/10.1242/jcs.01237. Yin, Z., Pascual, C., Klionsky, D.J., 2016. Autophagy: machinery and regulation. Microb. Cell 3 (12), 588
596. Available from: https://doi.org/10.15698/mic2016.12.546. Zhang, M., et al., 2013. Translational control in Plasmodium and toxoplasma parasites. Eukaryot. Cell 12 (2), 161
167. Available from: https://doi.org/10.1128/ EC.00296-12.

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C H A P T E R

16 The Toxoplasma cytoskeleton: structures, proteins, and processes Naomi Morrissette1 and Marc-Jan Gubbels2 1

Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, United States 2Department of Biology, Boston College, Chestnut Hill, MA, United States

16.1 Morphology 16.1.1 Life cycle and parasite appearance Toxoplasma gondii undergoes a complex life cycle that involves differentiation into distinct forms, which occupy discrete niches (Chapter 1: The history and life cycle of Toxoplasma gondii, Chapter 2: The ultrastructure of Toxoplasma gondii, and Chapter 18: Bradyzoite and sexual stage development). Three asexual invasive “zoite” forms of Toxoplasma (sporozoites, tachyzoites, and bradyzoites) have similar morphological properties and are likely to share many cytoskeletal features (Chobotar and Scholtyseck, 1982). Genetic recombination in Toxoplasma is achieved by a sexual cycle that occurs in the intestinal epithelium of felids and consequently has not been exhaustively studied (Pelster and Piekarski, 1971; Scholtyseck et al., 1971; Ferguson et al., 1974; Ferguson et al., 1975). During this process, Toxoplasma macrogametes are fertilized by microgametes, which are the

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00016-5

only developmental stage that constructs flagella to power motility (Ferguson et al., 1974; Scholtyseck et al., 1972). Although asexual zoite forms lack flagella, they are motile and use an unusual actin and myosin-based gliding motility to invade host cells (Fre´nal et al., 2017a,b; Hakansson et al., 1999; Sibley et al., 1998; Schwartzman, 1998; Dobrowolski et al., 1997). Because the tachyzoite form is most amenable to experimental manipulation, nearly all studies of the cytoskeleton have characterized this stage. Although it is likely that many elements of the tachyzoite cytoskeleton will be conserved with bradyzoites and sporozoites, some cytoskeletal components are expressed or essential in a developmentally specific fashion. Examples include the bradyzoite-enriched expression of the myosins TgMyoB and TgMyoD (Polonais et al., 2011a; Herm-Gotz et al., 2006; Delbac et al., 2001) as well as many proteins that are required to build the microgamete-specific flagellar axoneme (Hodges et al., 2010; Francia et al., 2015).

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16.1.2 Inner membrane complex and pellicle-associated structures Apicomplexans, along with ciliates and dinoflagellates, are classified as alveolate organisms (Leander and Keeling, 2003; Leander et al., 2003; Cavalier-Smith and Chao, 2004; Goodenough et al., 2018). Alveolates have a system of flattened vesicles (alveoli)

that closely underlie the plasma membrane, creating a pellicle structure that is composed of three unit membranes. In apicomplexans the patchwork of plasma membrane associated alveoli is called the inner membrane complex (IMC). This structure is integral to Toxoplasma replication, motility, and invasion of host cells. The apical polar ring (APR) marks the site

FIGURE 16.1

Microtubule and alveolin network organization in Toxoplasma tachyzoites. (A) The Toxoplasma pellicle consists of the plasma membrane and an underlying patchwork of associated vesicular alveoli termed the IMC. The APR marks the site where the IMC begins, leaving the extreme apical region enclosed by plasma membrane. Twenty-two

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L

subpellicular microtubules extend from the APR along the cytoplasmic face of the pellicle to a region just beyond the parasite nucleus. The conoid is a small motile organelle that can be extended from or retracted into the APR. Two fibrous PCRs associate with the conoid while two closely apposed microtubules transit the conoid lumen. Spindle microtubules originate at poles located in the cytoplasm and penetrate to the interior of the nucleus through pores in a specialized region of the nuclear membrane termed the centrocone. A centriole pair, organized in parallel configuration, is located at each spindle pole. During replication, daughter parasite buds emerge above the spindle poles but are omitted from this image for reasons of clarity. (B) IMPs in the IMC exhibit complex organization that reveals association with the underlying subpellicular microtubules and a network of alveolin fibers to create a lattice-like pattern. The apical cap region has 22 rows of IMPs that radiate out from the circular lip of the apical cap, above the 22 subpellicular microtubules. Each row consists of two parallel lines of IMPs separated by a small distance. Below the apical cap, the IMC contains IMPs which are immediately adjacent to each other in double particle columns. Additional single particle rows are interspersed between the double particle rows. (C) A detergent-extracted, negative stained tachyzoite cytoskeleton showing the conoid, PCRs, APR, and 22 subpellicular microtubules. (D) A freeze-fracture tachyzoite IMC membrane showing the IMP rows that extend the length of the parasite. The arrows indicate continuity of the rows across distinct vesicles. Inset shows a magnification of with the dpr marked by asterisks. (E) A proposed filament system linking the IMP lattice (top) is similar to organization of the alveolin network (bottom) revealed by freeze drying a glycerol and detergent-extracted replica for electron microscopy. APR, Apical polar ring; dpr, double particle rows; IMC, inner membrane complex; IMPs, intramembranous particles; PCRs, preconoidal rings; spr, single particle rows; sut, sutures. Source: Panels (C), (D) and the top part of (E) first appeared in Morrissette, N.S., Murray, J.M., Roos, D.S., 1997. Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J. Cell Sci. 110 (Pt 1), 35 42; the lower part of (E) is reproduced from Morrissette, N.S., Sibley, L.D., 2002a. Cytoskeleton of apicomplexan parasites. Microbiol. Mol. Biol. Rev. 66, 21 38 (Morrissette and Sibley, 2002a).

where the IMC begins (Fig. 16.1A and C), an arrangement that leaves the extreme apical region of the parasite enclosed by only plasma membrane, perhaps to facilitate secretion. At the tachyzoite posterior the individual IMC plates join in a turbine-shaped structure (Porchet and Torpier, 1977; Morrissette et al., 1997). The elongated shape of Toxoplasma zoites is maintained by the association of 22 evenly spaced subpellicular microtubules that interact with the cytosolic face of the pellicle (Nichols and Chiappino, 1987). These microtubules extend in a gentle spiral from the APR to a region posterior to the position of the nucleus to impose both an elongated serpentine shape and characteristic apical polarity (Stokkermans et al., 1996; Morrissette and Sibley, 2002b). The minus ends of subpellicular microtubules are inserted into the APR with attachment supported by projections of the APR which resemble a cogwheel in transverse views (Leung et al., 2017; Russell and Burns, 1984). Freeze fracture studies of Toxoplasma and other apicomplexans have shown that IMC membranes are characterized by uniformly

sized intramembranous particles (IMPs) organized into a highly regular lattice (Fig. 16.1B and D). Rows of IMPs are maintained across IMC plates, which represent topologically distinct vesicles, indicating an intimate association with both subpellicular microtubules and a second cytoskeletal network, likely to be imparted by the alveolins (see below). The apical cap (AC) region has a distinct pattern of IMP organization from that of the remaining lateral and posterior regions of the IMC. Twenty-two rows of IMPs radiate out from the circular lip of the AC, in apparent coordination with the APR and 22 subpellicular microtubules. Each row consists of two parallel lines of IMPs separated by a small distance. Below the AC, the IMC contains IMP rows that are immediately adjacent to each other in double particle columns. Additional single particle rows are interspersed between the double particle rows. In order to accommodate increasing diameter along the long axis of the parasite, the distance between double particle rows increases, as does the number of intervening single particle rows, to maintain a constant spacing between

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rows. Fourier analysis of isolated subpellicular microtubules reveals that they are coated with a microtubule-associated protein (MAP) that binds with a 32 nm periodicity (Morrissette et al., 1997). The double IMP rows also exhibit a 32 nm periodicity, suggesting close interaction of these microtubules with the IMC. The 32 nm longitudinal repeat extends across the single rows of IMPs, creating a twodimensional lattice that extends to the parasite posterior. Integrity of the lattice is independent of microtubules and actin filaments, suggesting that other cytoskeletal filaments organize this structure. A filamentous network with similar organization to the IMP lattice can be observed after glycerol or deoxycholate-extraction of Toxoplasma or other apicomplexans (D’Haese et al., 1977). The filaments have a diameter of 8 10 nm and the extracted network maintains the general shape of the parasite (Fig. 16.1E). This network is formed from alveolins to provide tensile strength, akin to intermediate filament proteins found in metazoans. Apicomplexan pellicles are partitioned into apical, central, and basal subdomains which have individual properties conferred by distinct cytoskeletal components (Fig. 16.2). For example, spacing between the IMC and plasma membrane is wider in the AC than in the body of the parasite, possibly to accommodate elongation and contraction of the apex during parasite motility and host cell invasion (Dubremetz and Torpier, 1978). This difference is dictated by the length of a coiled-coil domain in related TgGAP70 and TgGAP45 proteins which are apical and centrally located, respectively (Fre´nal et al., 2014). A set of six apical annuli (Fig. 16.2A) marks the junction between the AC and lower region of the IMC (Suvorova et al., 2015; Hu et al., 2006; Engelberg et al., 2020 See https://www.ncbi.nlm.nih.gov/pubmed/ 31470470). Discrete pellicle compartments are distinguished by proteins that mark individual apical, lateral, and basal regions of the IMC (Fig. 16.2B). Complexes of MAPs exhibit distinct

patterns of binding to the underlying subpellicular microtubules (Fig. 16.2C). The posterior end of mature tachyzoites is defined by a basal cap, which is a specific structure of coccidian apicomplexans that replicate by endodyogeny.

16.1.3 Apical structures Toxoplasma and other members of the phylum Apicomplexa are named for their distinctive polarized apex, which contains organelles that coordinate interaction with host cells. Apical secretory organelles (micronemes and rhoptries) release components that mediate gliding motility, host cell invasion, and establishment of the parasitophorous vacuole. Gregarine and coccidian subsets of apicomplexans (the Conoidasida) have an additional fibrous apical structure termed the conoid (Leander and Keeling, 2003). The Toxoplasma conoid is composed of approximately 10 14 B430 nm long filaments that follow a lefthanded helical path to create a 380 nm diameter funnel-shaped structure that is required for invasion of host cells (Fig. 16.1A and C) (Hu et al., 2002; Morrissette et al., 1997; Nichols and Chiappino, 1987). Although conoid filaments contain tubulin, cross sections through the fibers appear as a nonsymmetric “comma” shape with nine subunits rather than closed tubules (Hu et al., 2002). The conoid is surmounted by two preconoidal rings and two B400 nm long microtubules are located within the conoid (Nichols and Chiappino, 1987). The intraconoid microtubules are closely apposed and appear connected to the preconoidal rings by filaments. A complex composed of the conoid, intraconoid microtubules, and preconoidal rings can extend beyond the APR or retract within it to be surrounded by the corset of subpellicular microtubules. Conoid extension and retraction are visible during invasion and can be triggered in extracellular parasites by

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FIGURE 16.2 Membrane skeleton subdomains in Toxoplasma tachyzoites. (A) The individual IMC plates are joined together by ISCs; transverse junctions have additional TSCs. A set of concentrically organized proteins are located in a set of six apical annuli that mark the lower boundary of the apical cap compartment and the posterior end of mature tachyzoites are closed off by concentric layers of basal cap proteins. The apical annuli are marked by TgCen2, TgPAP1 (also known as TgAAP1) and additional apical annuli proteins (AAPs). Basal cap proteins occupy different widths of the basal cap with the largest coverage marked by MORN1, IMC9, and IMC13, intermediate size areas subsequently occupied by MSC1a and combined IMC5 and IMC8, followed by the smallest area covered by TgCen2. (B) The IMC apical cap (green) is formed from a single ring-shaped compartment that is defined by TgISP1, TgGAP70, and nine apical cap proteins. Below this unitary cap, strips of flattened IMC vesicles spiral following the same path as that of the underlying microtubules to create a lateral compartment (red) and ultimately converge to close off the posterior of mature parasites (blue). The lateral IMC compartment is distinguished by TgISP2 and TgISP4. TgISP3 is found in this compartment but additionally localizes to the basal compartment of the IMC. (C) Protein components of the PCRs, ICMTs, conoid, APR and SPMTs, conoid complex localize to specific substructures (red). These include (1) SAS6L; (2) ICMAP1; (3) CaM1, CaM2, CaM3, CAP1, CPH1, DIP13, DLC1, DCX, MyoH, MyoE, MyoL; (4) APR1, kinesin A, RNG1, RNG2; (5) SPM1; (6) TrxL1, TrxL2; (7) SPM2; (8) TLAP3; (9) Kinesin B; (10) TLAP2; and (11) EB1 (extending daughter buds). APR, Apical polar ring; ICMTs, intraconoid microtubules; IMC, inner membrane complex; ISCs, IMC suture components; PCRs, preconoidal rings; SPMTs, subpellicular microtubules; TSCs, transverse suture components.

pharmacologically raising the intracellular Ca21 concentration (Mondragon and Frixione, 1996; Del Carmen et al., 2009). Coccidian parasites including Toxoplasma construct conoids, while most other apicomplexan

lineages have lost the ability to build this structure. Although the precise origins of the “closed” apicomplexan conoid remain uncertain, it was likely modified from “incomplete” or “pseudoconoid” structures found in closely

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related alveolates. These consist of an opensided cone built from short nearly vertical apical microtubules. Pseudoconoids are found in organisms such as Colpodella vorax and Rastrimonas subtilis that partially or fully invade other free-living aquatic protozoa in order to aspirate their cytoplasmic contents or to grow intracellularly (Brugerolle, 2002; Leander et al., 2003; Okamoto and Keeling, 2014). Although the latter alveolate organisms lack an APR, they assemble subpellicular microtubules that originate in the apical region. The apex harbors secretory organelles and a pseudoconoid adjacent to basal bodies with associated flagellar axonemes. This morphology is most like apicomplexan microgametes that exhibit a set of short microtubules adjacent to apical flagella and lack both APR and conoid (Pelster and Piekarski, 1971; Mehlhorn, 1972; Sinden et al., 2010; Morrissette, 2015). Rastrimonas absorbs its flagella during intracellular growth; this behavior may have become enhanced during evolution of apicomplexan parasites, ultimately leading to the loss of flagella in asexual stages. Perhaps as a consequence, the centrioles (basal bodies without an associated transition zone and axoneme) reside above the nucleus which is centrally located. In some protozoa and unicellular algae, basal bodies associate with several types of fibers (centrin-containing contractile fibers and noncontractile striated fibers) to orient flagella relative to sensory organelles and the nucleus (Harper et al., 2009; Lechtreck and Melkonian, 1991; Salisbury et al., 1984). Remarkably, some of these fiber-forming proteins have been incorporated into the closed conoid (Francia et al., 2012; Hu et al., 2006; Leveque et al., 2016; de Leon et al., 2013). Moreover, centrioles are only observed in the asexual stages of coccidian apicomplexans. Other apicomplexan lineages lack both conoids and centrioles in zoite stages and specifically construct basal bodies and flagellar axonemes de novo during microgametogenesis (Francia et al., 2015; Wall et al., 2016; Sinden et al., 2010).

16.1.4 Basal structures The posterior end of Toxoplasma contains a novel cytoskeletal assembly, the basal complex, which functions in place of a contractile ring to complete cytokinesis at the conclusion of parasite replication (Hu, 2008; Gubbels et al., 2008; Blader et al., 2015). Daughter basal complexes emerge early in mitosis, proximal to duplicated centrioles and extend at the leading edge of the IMC of lengthening daughter buds. Disruption of basal complex maturation causes organelle segregation defects and formation of conjoined daughters (Engelberg et al., 2016; Lorestani et al., 2010; Heaslip et al., 2010a). Importantly, sibling parasites within a parasitophorous vacuole remain connected through posterior junctions to a residual body (RB) located at the center of the tachyzoite rosette. Incomplete basal constriction permits interconnected parasites to share diffusible cytoplasmic contents and maintain replication synchrony but allows partitioning of duplicated organelles. Actin and TgMyoI are required for interparasite transport through the RB and depletion of actin causes loss of this structure (Tardieux, 2017; Fre´nal et al., 2017b; Tosetti et al., 2019). Daughter bud maturation requires contraction of the basal complex and decoration with additional proteins that exclusively mark a permanent basal cap (Fig. 16.2A).

16.1.5 The nucleus Replication in Toxoplasma can occur by endodyogeny (Blader et al., 2015; Francia and Striepen, 2014; Sheffield and Melton, 1968; Striepen et al., 2007), which creates two daughter parasites per replication cycle, or by endopolygeny, where many daughters synchronously bud from a mother parasite (Ferguson et al., 2008; Gubbels et al., 2008; Piekarski et al., 1971). In both processes the nuclear membrane remains intact during

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mitosis and chromosome segregation occurs without chromosome condensation (Fig. 16.3). The centrocone is a specialized apical region of the nuclear envelope that permanently associates with chromosome centromeres and kinetochores, independent of spindle microtubules. During mitosis, spindle microtubules originate in the cytoplasm and pass through nuclear pores embedded in the centrocone, linking replicated centrosomes to duplicated chromosomes (Striepen et al., 2007; Brooks et al., 2011; Morrissette and Sibley, 2002b). Spindle microtubules are essential for association of

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centrosomes with the centrocone and for chromosome partitioning into daughter nuclei (Chen and Gubbels, 2013, 2015; Farrell and Gubbels, 2014; Chen et al., 2015b; Berry et al., 2018).

16.1.6 Centrioles, centrosomes, and basal bodies Centrioles and basal bodies are highly ordered structures that are conserved in organisms ranging from protozoa to vertebrates. Typical centrioles are barrel-shaped,

FIGURE 16.3

Centrioles and cell division by endodyogeny. (A) In organisms ranging from Chlamydomonas to humans centrioles are made of triplet microtubules and are organized in an orthogonal configuration; however, in Toxoplasma tachyzoites two short centrioles consisting of nine singlet microtubules are found in parallel at the spindle poles. Tachyzoite (left) and human (right) centriole structures as diagrams and by transmission electron microscopy. (B) Tachyzoite replication by endodyogeny. Numbered circles 1 and 2 indicated areas of twofold magnification whereas circle 3 is magnified threefold. One chromosome is drawn to communicate the principle of centromere clustering at the centrocone throughout the division cycle. The chromosomes do not condense and are depicted for schematic purpose only, whereas the nuclear envelope does remain intact throughout replication. In magnified Section 16.3, note the annotated bipartite centrosome with the inner-core that organizes the nuclear cycle, and the outer-core that orchestrates daughter bud assembly. Source: (A) The image of the Toxoplasma centriole was first published in Morrissette, N.S., Sibley, L.D., 2002b. Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii. J. Cell Sci. 115, 1017 1025; (B) this panel was modified with permission from Blader, I.J., Coleman, B.I., Chen, C.T., Gubbels, M.J., 2015. Lytic cycle of Toxoplasma gondii: 15 years later. Annu. Rev. Microbiol. 69, 463 485.

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100 250 nm in diameter and 100 400 nm in length (Francia et al., 2015; Dutcher, 2004; Preble et al., 2000). Most centrioles have a “9 1 0” structure of triplet microtubules, exist in pairs, and are arranged orthogonally after duplication. Atypical centriole organization occurs in some organisms: centrioles in Caenorhabditis elegans have nine singlet microtubules, while those in Drosophila melanogaster embryos have nine doublets. Toxoplasma tachyzoite centrioles consist of a central tubule surrounded by nine singlet microtubules (Morrissette and Sibley, 2002b). This organization also occurs in other coccidians such as Besnoitia jellisoni and Eimeria bovis; however, noncoccidian apicomplexans such as Plasmodium species lack asexual stage centrioles altogether. Transmission electron microscopy images of thin sections through coccidian centrioles indicate that they are quite short and are arranged in a parallel rather than orthogonal configuration (Fig. 16.3A). In metazoan organisms the term “centrosome” refers to a centriole pair surrounded by pericentriolar matrix which contains γ-tubulin, pericentrin, and ninein (Chen and Gubbels, 2013; MorlonGuyot et al., 2017; Hodges et al., 2010; Rieder et al., 2001). Although Toxoplasma lacks pericentrin and ninein genes, the term “centrosome” has been adopted to convey its signaling platform and microtubule organizing capacities. The Toxoplasma centrosome has a bipartite structure containing separate but associated domains to flexibly link nuclear division and budding (see next and Fig. 16.3B). Basal bodies are structurally identical to centrioles but are continuous with a transition zone and associated flagellar axoneme. The typical flagellar axoneme has a central pair of microtubules surrounded by nine doublet microtubules (9 1 2 organization). The conversion from 9 1 0 triplet microtubules in the basal body segment to 9 1 2 doublets in the axoneme occurs in the transition zone, which contains machinery for intraflagellar transport required

to assemble the flagellum. In most flagellated eukaryotes, formation of an axoneme requires a centriole containing triplet microtubules and prior construction of a transition zone. Therefore the singlet microtubule centrioles found in asexual stage Toxoplasma may be converted into a structure with triplet blades. In Plasmodium spp., asexual stage parasites lack centrioles altogether and microgamete flagella are extended from de novo assembled basal bodies (Sinden et al., 2010; Francia et al., 2015; Wall et al., 2016). To date, it is unclear if Toxoplasma zoite (singlet) centrioles mature to template axoneme formation in microgametes or whether microgamete basal bodies are separately formed de novo as in Plasmodium spp.

16.2 Cytoskeletal elements Like most eukaryotes, Toxoplasma has three classes of cytoskeletal filaments: microtubules, actin, and intermediate filament like proteins. Each of these elements employs associated proteins to carry out specific cytoskeleton-driven processes and to mediate interactions between filament systems.

16.2.1 Tubulin, microtubules, microtubule-associated proteins, motors, and MTOC Microtubules, built by polymerization of α β tubulin heterodimers, are essential components of the Toxoplasma spindle that is required for nuclear division (Morrissette and Sibley, 2002b). Microtubules are also key components of the zoite membrane cytoskeleton, intimately associating with the pellicle to confer apical polarity and a rigid elongated cell shape. During sexual replication, microgametes construct flagella to drive motility required for fertilization. Gametes exclusively develop in the cat intestinal epithelium and

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characterization to date consists of rare electron microscopy images and identification of homologous flagellar components in the genome. Nearly all experimental manipulation of the cytoskeleton has been performed on tachyzoites, which share many cytoskeletal structures with other zoite forms. Tachyzoites contain at least five distinct tubulin-containing structures: the spindle, subpellicular and intraconoid microtubules, the centrioles, and the conoid (Morrissette, 2015). Each of these structures contains a unique set of associated proteins. Specificity of individual microtubulebased structures may involve expression of particular α- and β-tubulin isotypes, select posttranslational modifications, and binding of distinct associated proteins. The Toxoplasma genome contains genes for three α- and three β-tubulin isotypes (Nagel and Boothroyd, 1988; Morrissette et al., 2004; Morrissette, 2015). A genome-wide CRISPR screen indicates that only α1- and β1-tubulins are essential for in vitro tachyzoite growth (Sidik et al., 2016). Toxoplasma microtubule polymerization is inhibited by dinitroanilines which selectively bind to protozoan tubulin but not vertebrate tubulins (Stokkermans et al., 1996; Morrissette and Sibley, 2002b; Morrissette et al., 2004). Diverse single point mutations to Toxoplasma α1-tubulin confer dinitroaniline resistance by reducing tubulin affinity for dinitroanilines and/or increasing subunit subunit affinity within the microtubule lattice (LyonsAbbott et al., 2010). The α1-tubulin isotype is abundantly represented in tachyzoite proteomes and is mutated in all cases of dinitroaniline resistance (Morrissette et al., 2004; Ma et al., 2007; Ma et al., 2008). The β1-tubulin gene is mutated in a subset of parasite lines that have acquired compensatory mutations to modulate fitness defects conferred by dinitroaniline resistance mutations in α1-tubulin, indicating that α1- and β1-form the predominant heterodimer population in tachyzoites (Ma et al., 2008).

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Studies in a variety of eukaryotes have established that tubulin dimers can be altered by posttranslational modifications that differentially and reversibly mark microtubule subpopulations (Wloga and Gaertig, 2010). For example, α-tubulin can be acetylated on K40 and its C-terminal tail can be reversibly modified by deletion of the terminal tyrosine (detyrosination) or irreversibly modified by deletion of the last two residues (Δ2). The C-terminal tails of both α- and β-tubulins can be reversibly modified by glutamylation and glycylation. Although the K40 posttranslational modification directly modifies microtubule stability (Janke and Montagnac, 2017), C-terminal posttranslational modifications alter microtubule interactions with associated proteins to influence sensitivity to microtubule targeting drugs. Toxoplasma tachyzoite tubulin is subject to diverse posttranslational modifications (Xiao et al., 2010). Modifications to α1-tubulin include acetylation of K40 by the activity of an α-tubulin acetyltransferase (ATAT). ATAT is essential to complete nuclear division and acetylated α-tubulin is enriched during daughter bud formation (Varberg et al., 2016). Other modifications to Toxoplasma tubulin include detyrosination (removal of the C-terminal Y453) and truncation of the last five amino acids (ΔYGDEY) of α1-tubulin, an apparently novel modification (Xiao et al., 2010). Both α1and β1-tubulins are polyglutamylated, a modification observed on axoneme, centriole, and some spindle microtubules. Glycylation, a modification that is restricted to ciliated cells and enriched on axonemes and basal bodies, was not detected, consistent with the lack of flagellar structures in tachyzoites. Significantly, a novel modification (methylation) is detected in the C-terminal tails of α1- and β1-tubulin (Xiao et al., 2010). Toxoplasma tubulin acetylation requires activity of the ATAT. ATAT is essential to complete nuclear division and acetylated α-tubulin is enriched during daughter bud formation (Varberg et al., 2016).

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MAPs influence microtubule stability and endow particular microtubule populations with specific properties. While microtubule motors (dyneins and kinesins), centriole components (SAS-6, centrin, CEP250) and regulatory proteins (EB1) are fairly conserved with other eukaryotes, proteins associated with the subpellicular microtubules and conoid are predominantly novel, reflecting the unusual roles of these structures. Toxoplasma zoites exhibit a rigid and elongated shape conferred by a set of 22 cortical microtubules that are intimately associated with the overlying pellicle (Nichols and Chiappino, 1987; Morrissette and Sibley, 2002b). Mature subpellicular microtubules are nondynamic and unusually stable to detergent and cold treatments that typically induce disassembly. This stability is imparted by heavy coating with MAPs. Proteins that have been shown to localize to the subpellicular microtubules include TgSPM1, TgSPM2, TgTrxL1, TgTrxL2, TgTLAP1, TgTLAP2, TgTLAP3, and TgTLAP4. While some of these MAPs label the full length of the subpellicular microtubules, others localize to distinct subregions (Fig. 16.2C). TgSPM1 has six copies of a 32 amino acid repeat (a characteristic of many MAPs) and localizes along the full length of subpellicular microtubules (Tran et al., 2012). Loss of TgSPM1 causes reduced fitness and subpellicular microtubules become detergentsensitive. TgSPM1 is required for microtubule localization of thioredoxin-like proteins TgTrxL1 and TgTrxL2 (Liu et al., 2013). TrxL1 protein pulldowns identify four TrxL1associating proteins (TLAPs 1 4), which localize to the subpellicular microtubules (Liu et al., 2016). During replication, TgTLAP4 is enriched at the spindle, which implies direct binding to microtubules. TgTLAP3 localizes to an apical stretch of the subpellicular microtubules and to the intraconoid microtubules, again implying direct microtubule binding. TgTLAP2 is present in a ring-like structure adjacent to the APR and is absent from a subapical stretch that

partially coincides with the AC domain of the overlying IMC. TgTLAP2 coats the remainder of the length of the subpellicular microtubules distal to the cap domain. Although tachyzoites null for TgTLAP2 do not have an obvious growth defect, the combined loss of TgTLAP2 and TgSPM1 sensitizes subpellicular microtubules to cold-induced microtubule disassembly. An additional MAP, TgSPM2, associates with the middle portion of the subpellicular microtubules and is nonessential for in vitro growth (Tran et al., 2012). Lastly, a small population of TgEB1 briefly associates with the tips of extending daughter subpellicular microtubules during bud development (Chen et al., 2015b). Subpellicular microtubules originate at the APR which has been considered an MTOC because microtubule plus-ends are distal to this structure. However, the broadly conserved MTOC marker γ-tubulin does not localize to the APR (Suvorova et al., 2015). TgRNG1, TgRNG2, TgAPR1, and TgKinesin A are defined constituents of this structure. Characterization of these components indicates that distinct proteins are required for templating 22 subpellicular microtubules, tensile strength, interaction with the mature pellicle and the close connection between the APR, subpellicular microtubules, and conoid. TgRNG1 is a small, low-complexity, detergentinsoluble protein that appears at the mature APR only after completion of nuclear division as daughter buds emerge from the maternal plasma membrane (Tran et al., 2010). TgAPR1 is similarly a marker of mature APR structures and null parasites exhibit a defect in lytic growth (Leung et al., 2017). In contrast the microtubule motor TgKinesin A labels emergent daughter buds and localizes immediately apical to APR1 at the APR of mature parasites (Leung et al., 2017). Tachyzoites lacking kinesin A have a modest reduction in growth and parasites lacking both TgKinesin A and TgAPR1 have greater defects that severely

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compromise host cell invasion. Although double knockout parasites build daughters with 22 subpellicular microtubules, the APR is fragmented, detergent-labile and detaches from the subpellicular microtubules. Lastly, TgRNG2 is a large protein with similarity to CEP250 and homology to a family of charged repeat motif proteins identified in the pellicle of Tetrahymena (Gould et al., 2011; Katris et al., 2014). During replication, TgRNG2 is recruited to spots proximal to newly duplicated centrosomes and subsequently resolves into rings that mark the APR of developing daughters. When the N- and C-termini of TgRNG2 are distinctly tagged, the extremities appear as discrete rings, indicating that the protein forms a cuff of vertically organized subunits. The relative position of these rings depends on whether the conoid is extended or retracted, revealing that TgRNG2 connects the APR to the conoid. In addition to its close but mobile connection with the APR, the Toxoplasma conoid is part of a larger complex that contains two centrally located, B400 nm long microtubules and two preconoidal rings. Although these are distinct structures, they remain associated after detergent extraction, indicating that they are part of a larger, physically connected complex. TgICMAP1, an SMC domain containing protein is a specific marker of the intraconoid microtubules (Heaslip et al., 2009). Homologs are restricted to coccidians, consistent with loss of the conoid in other apicomplexan lineages. TgTrxL1, TgTrxL2, and TgTLAP3 localize to the intraconoid microtubules in addition to the subpellicular microtubules (Liu et al., 2013; Liu et al., 2016). The fibrous preconoid rings contain a “SAS-6-like” (TgSAS6L) protein that shares homology with the centriole spoke protein SAS-6 (de Leon et al., 2013). A SAS6L homolog is localized at the flagellar basal plate in trypanosomes, suggesting that this protein has been repurposed for function in the conoid. As noncoccidian lineages have lost the ability

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to construct a conoid, it is notable that Plasmodium expresses SAS6L protein in the mosquito asexual stages. PbSAS6L forms an apical ring at the tip of ookinetes and sporozoites, indicating that a conoid-associated component and ring persist after loss of a discrete conoid in this lineage (Wall et al., 2016). The tubulin-based conoid filaments require distinct proteins to specifically accomplish key functions including structural integrity, invasion, and segregation of nuclei in daughter buds. Both TgDCX and TgCPH1 are required for structural integrity, as parasites that lack either protein have shortened and partially collapsed conoids and profound defects in invasion, egress and plaque formation (Nagayasu et al., 2017; Long et al., 2017a). The conoidassociated myosin motor, TgMyoH, requires a rigid conoid for penetration of host cells (Graindorge et al., 2016). Upon productive contact with a host cell the tachyzoite inserts a circular moving junction into the host plasma membrane that links it to the tip of the extended conoid: TgMyoH immobilized at the extended conoid transports this moving junction to a subapical region where it is handed off to the TgMyoA motor that transports it along the remainder of the parasite pellicle (Jacot et al., 2016). Two other myosins, TgMyoE and TgMyoL, localize to the conoid, although these have not yet been extensively characterized (Fre´nal et al., 2017b). In addition to conventional light chains, MyoH interacts with three conoid-localized calmodulin homology proteins (TgCaM1, TgCaM2, and TgCaM3) that are also key to invasion (Long et al., 2017b). These CaMs are mislocalized or degraded in the absence of MyoH and the defects associated with their loss involve impairment of invasion, gliding motility and egress, phenocopying to loss of MyoH. Other conoid components include dynein light chain type 1 (TgDLC1, also known as TgDLC8a), conoid-associated protein 1 (TgCAP1), and a set of as yet uncharacterized proteins

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identified by BioID experiments using TgMyoH, TgRNG2, and TgCPH1 fused to a biotin ligase (Long et al., 2017a; Qureshi et al., 2013; Hu et al., 2006; Skariah et al., 2012). The Toxoplasma conoid complex harbors homologs of fiber-forming proteins that have been demonstrated to orient flagella relative to sensory organelles and the nucleus in green algae such as Chlamydomonas (Francia et al., 2012; Harper et al., 2009; Lechtreck, 2003; Lechtreck and Melkonian, 1991; Salisbury et al., 1984). These include centrin (contractile fibers) and striated fibers assemblin (SFA). Other proteins such as SAS6L (which localizes to the trypanosome flagellar plate) and DIP13 are found in both flagella and the conoid complex. Although SAS6L and DIP13 have not been directly observed as polymers, ectopic expression of either induces filament formation, potentially unmasking an underlying property of these proteins (Leveque et al., 2016; de Leon et al., 2013). Chlamydomonas DIP13 is a deflagellation-inducible protein that localizes to basal bodies, the flagellar axoneme, and cortical (rootlet) microtubules. A vertebrate homolog, SSNA, is located at sperm basal bodies and axonemes. The presence of DIP13 genes is correlated with the construction of conventional centrioles, and axonemes and genes are absent from organisms which lack conventional centrioles or that no longer build flagella. Therefore it is notable that Toxoplasma DIP13 is expressed in tachyzoites, a stage that harbors unconventional singlet centrioles and lacks flagella. TgDIP13 localizes to the conoid as a ring with a more apical dot that coincides with an apical TgCen2 spot (Leveque et al., 2016). It appears in early daughter bud formation, both at the conoid and at the posterior edge of the extending basal complex. The role of centrin, SAS6L, and DIP13 at the conoid may be related to tethering apical organelles to each other and to the nucleus during replication by endodyogeny. This is explicitly the role of two SFA proteins during tachyzoite replication (Francia et al., 2012). Toxoplasma has three

SFA homologs, with TgSFA2 and TgSFA3 expressed in tachyzoites. These proteins colocalize to fiber-like structures during mitosis and are undetectable at interphase. SFA fibers emerge from centrosomes to tether them to the emerging apical daughter buds at the APR and conoid. Loss of either SFA blocks initiation of daughter APRs and subsequent creation of daughter buds. Since nuclear division is not inhibited, multiple nuclei accumulate. This suggests that although tachyzoites have dispensed with building flagella, they retain SFAs to link inheritance of apical complex organelles with individual nuclei. Both centrioles and conoids are present in asexual stages coccidian apicomplexans, but lineages that replicate by schizogony (which bud from the plasma membrane rather than in the cytoplasm) apparently lack both structures in zoite forms. Coccidian retention of centrioles rather than a simplified MTOC may simply reflect the need to maintain a critical centrocone-SFA linkage during replication by internal budding. Centrosomes, centrioles, and basal bodies are highly ordered microtubule-organizing organelles with conserved structural properties and protein components. Despite conservation across eukaryotic kingdoms, different lineages show variation in organization and specific protein constituents. Toxoplasma tachyzoite centrioles are simplified to a short barrel of nine singlet microtubules and comparative bioinformatics indicate that conserved components, including CEP192/SPD-2, PCM1, ninein, rootletin, and asterless, are missing from the Toxoplasma genome (Suvorova et al., 2015; Hodges et al., 2010). Of the three Toxoplasma centrins, TgCen1 exclusively localizes to the centrosomes, TgCen3 is predominantly at the centrosome, and TgCen2 is found at both apical and basal ends of the parasite, six apical annuli and the centrosomes (Suvorova et al., 2015; Hu, 2008; Hartmann et al., 2006; Striepen et al., 2000). Knock-in epitope tags and antibodies have identified juxtanuclear labeling with conserved centriole and centrosome

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components, including TgSas-6 and TgSfi1, which identify a single spot that duplicates when parasites enter S phase (Suvorova et al., 2015; de Leon et al., 2013). In contrast, the Toxoplasma homolog of CEP250/c-Nap labels four closely spaced foci (Fig. 16.3A). The two apically oriented spots exhibit strong γ-tubulin labeling and correspond to the structure labeled by TgSAS-6 and TgSfi1. The two CEP250 spots closest to the nucleus exhibit weak γ-tubulin labeling and are exclusively labeled by a CEP250-related protein, TgCEP250-L1 (Suvorova et al., 2015). This “bipartite” pattern identifies distinct outer (TgSas-6, TgSfi1, TgCEP250, and γ-tubulin positive) and inner (TgCEP250-L1 and TgCEP250 positive, γ-tubulin low) cores. The centrosome components and the associated centrocone complex forms a “super-club-sandwich” structure (Morlon-Guyot et al., 2017). Beginning with the chromosomes, it consists of the following layers: CENPA (Brooks et al., 2011), the kinetochore (Farrell and Gubbels, 2014), a SUN-like protein (TGME49_250010; Chen and Gubbels, unpublished), the inner-core marker Cep250L1 (Suvorova et al., 2015), the core connector Cep530 (Courjol and Gissot, 2018), the outer-core marked by Cep250 (Suvorova et al., 2015), a further out outer-core marked by TgCen1, and lastly the SFA fiber (Francia et al., 2012) that anchors the centrosome and nuclear complex in the daughter bud (Chen and Gubbels, 2019). The bipartite centrosome provides distinct nuclear- and bud-facing platforms to coordinate nuclear division with daughter cell formation (see next). In addition to conserved centrosome components, a variety of parasite-specific proteins migrate onto or close to the centrosomes during replication. This localization may serve to bring them into proximity with signaling molecules to license them for future functions or to sequester them until other structures are assembled during replication. Examples of this include TgRNG2, TgIMC15, and Tg14-3-3 that go on to localize to the APR, the daughter IMC and the basal

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complex (Anderson-White et al., 2011; Katris et al., 2014; Lorestani et al., 2012). Nuclear division in tachyzoites occurs without nuclear envelope breakdown. This process is heavily dependent upon the centrocone, a specialized domain of the nuclear envelope that houses regulatory components (TgMORN1, TgECR1, and TgCrk5) similar to a yeast spindle pole body. Spindle microtubules originate in the cytoplasm and pass through nuclear pores embedded in the centrocone to link replicated centrosomes to duplicated chromosomes (Naumov et al., 2017; Farrell and Gubbels, 2014). The Toxoplasma homolog of EB1 localizes to the centrocone upon spindle assembly (Chen et al., 2015b). EB1 binds to the plus-ends of dynamic microtubules to promote microtubule stability and elongation. A C-terminal extension of B70 amino acids in TgEB1 houses a nuclear localization signal, which (unlike other EB1 proteins) makes TgEB1 a nuclear protein. During G1, it is evenly distributed in the nucleoplasm but relocates to the centrocone and associated microtubules upon spindle assembly. A small population of cytoplasmic TgEB1 briefly associates with the tips of daughter bud subpellicular microtubules after completion of nuclear division, while nuclear TgEB1 remains concentrated at the centrocone until completion of cytokinesis. TgEB1 is not essential, but null parasites display some replication defects due to reduced mitotic integrity. To date, there are no other examples of specific spindle microtubule markers in tachyzoites, although the subpellicular microtubule MAP TLAP4 is highly enriched at the spindle (Liu et al., 2016). While it is likely that homologs of conserved eukaryotic spindle components will be identified in the tachyzoite spindle, the role of microtubules in chromosome segregation is reduced relative to other eukaryotes. Toxoplasma centromeres with associated kinetochores associate with the centrocone throughout the cell cycle, independent of spindle microtubules (Brooks et al., 2011; Farrell and Gubbels, 2014). Spindle microtubules are essential for connecting

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centrosomes with the centrocone and for chromosome partitioning into daughter nuclei. Given that the Toxoplasma spindle is required to separate centrocone-linked duplicated chromosomes, it is reasonable to predict that the spindle will house microtubule motors to drive separation of spindle poles, but specific spindle motors have not been characterized at this time. Microtubule motors are multisubunit complexes that move cargo on microtubule tracks. The Toxoplasma genome has homologs of many kinesin and dynein subunits. Kinesins are typically composed of two heavy and two light chains (Verhey et al., 2011; Miki et al., 2005). Kinesin heavy chains (KHCs) harbor a distinct motor domain-containing head connected to a region that dimerizes and interacts with light chains to coordinate association with cargo. The Toxoplasma genome encodes approximately 19 KHCs. To date, two KHCs have been localized in tachyzoites: TgKinesin A functions at the APR and TgKinesin B localizes to the distal two-thirds of the subpellicular microtubules (Leung et al., 2017). Dynein motors are substantially larger than kinesins; they typically contain 12 subunits, including the dynein heavy chains (DHCs) that contain a motor domain and different types function in the cytoplasm and in flagellar beating (Hook and Vallee, 2006; Asai and Wilkes, 2004). Although some subunits are shared by cytoplasmic and axonemal dyneins, other components are unique to each motor type. In the case of cytoplasmic dyneins, two identical cytoplasmic DHCs associate with two intermediate chains, which anchor dynein to its cargo. Axonemal dyneins may contain one, two, or three distinct DHCs that bridge two microtubules to coordinate the sliding that underlies flagellar motility. The Toxoplasma genome contains 10 DHCs and several types of intermediate and light chains. To date, four dynein light chains have been localized. Dynein light chain type 1 (TgDLC1/ TgDLC8a) localizes to the conoid region of tachyzoites (Qureshi et al., 2013; Hu

et al., 2006). Knock-in tagging of three other members of this subfamily (TgDLC8b, TgDLC8c, and TgDLC8d) establishes that these share a cytoplasmic localization (Qureshi et al., 2013). A genome-wide CRISPR screen data indicates that TgDLC8a, but not the other tagged light chains, is essential for in vitro tachyzoite growth (Sidik et al., 2016). Toxoplasma has genes that encode obvious orthologs of the Arp1, p25, p27, and p62 subunits of the dynactin complex that is essential for motor activity and linkages to cargo (Gordon and Sibley, 2005).

16.2.2 Alveolins, glideosome-associated proteins with multiple membrane spans, and other inner membrane complex proteins The IMC is composed of flattened vesicles that underlie the plasma membrane to create a pellicle composed of three unit membranes (Fig. 16.2A and B). This structure is intimately associated with actin, myosin, microtubules, and intermediate filament like alveolins and is integral to Toxoplasma replication, motility, and invasion of host cells. IMC vesicles are stitched together by suture proteins that either bridge transverse segments or associate with both lateral and longitudinal boundaries of individual plates (Chen et al., 2015a, 2017; Lentini et al., 2015; Tilley et al., 2014). As the IMC plates are distinct vesicles, polarity and pellicle subdomains are governed by occupancy of proteins to one or more subcompartments (Fig. 16.2B) that define the AC, lateral and basal zones (Fung et al., 2012; Beck et al., 2010). Furthermore, replication by endodyogeny requires that the maternal, plasma membrane associated IMC is distinguished from unassociated bud IMC that encloses developing daughters. Many IMClocated proteins associate with maternal or daughter IMCs in an exclusive or biased fashion (Fig. 16.4A) to direct development, maturation, and emergence of daughters. The IMC also

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FIGURE 16.4 Organization of glideosome components in replication and motility. (A) Assembly of the mature pellicle, including assembly of functional glideosome complexes, is regulated during daughter bud formation. (Left) The glideosome-associated integral membrane proteins GAP40 and GAP50, GAPM1 and GAPM2 and a number of IMC proteins, including IMC1, IMC4, IMC19, IMC21, and IMC25 are located in mature pellicles as well as emerging daughter buds. The APR marker RNG2 is also located in both mother and daughter structures. (Center) IMC16 and IMC29 are both located in daughter buds and disappear from maturing pellicles. (Right) The mature APR is specifically marked by APR1 and RNG1 and the mature pellicle contains peripheral glideosome components GAP70/GAP45/GAP80 and associated myosin motors. Other components that selectively localize at the maternal pellicle are IMC7, IMC12, IMC14, IMC17, IMC18, IMC20, and two homologous proteins MSC1b (which localizes to the mature pellicle) and MSC1a (which localizes to the mature basal cap). (B) Location of Toxoplasma FRM proteins and myosins reveals key aspects of their function in tachyzoites. FRM1 is located at the tachyzoites apex and controls actin assembly for gliding motility. FRM2 is found in the apical juxtanuclear region and regulates actin assembly for organelle trafficking and segregation. FRM3 is located in the RB and coordinates actin assembly at the posterior end for cell cell communication between sibling parasites. MyoH is located at the conoid, as are two other uncharacterized myosins, MyoE, and MyoL. The IMC-associated motor, MyoA, is critical for gliding motility and invasion, while MyoF mediates apicoplast segregation and organelle trafficking. MyoK is located at the centrocones and MyoC and MyoJ are positioned at the posterior polar ring. While MyoC is part of a novel posterior glideosome, MyoJ constricts the posterior end of daughter buds. MyoI is located in the RB and is required for trafficking of contents between sibling parasites through the posterior polar ring and associated RB. (C) Toxoplasma tachyzoite gliding motility and invasion require F-actin, surface adhesins, and several distinct myosin motor complexes which are located in the narrow space between the IMC and PM. (1) Activation of motility requires FRM1-mediated actin polymerization at the parasite apex. (2) The conoid-anchored myosin, MyoH engages F-actin to move apically secreted adhesins to the parasite posterior. The cytoplasmic domains of membrane-spanning adhesins such as Mic2 are cross-linked to F-actin by the GAC. MyoH binds to MLC5 and its activity is regulated by three conoid-localized CaM proteins. (3) The adhesin GAC F-actin complex is transferred to the IMC anchored MyoA glideosome which contains integral membrane proteins GAP40 and GAP50 and a peripheral protein, GAP70, which spans the IMC PM stretch. MyoA associates with the IMC via an extension to MLC1. Its activity is regulated by the activity of a second light chain, either ELC1 or ELC2. (4) Below the apical cap region, GAP70 is replaced by GAP45, a homologous protein that has a shorter central region, bringing the PM and IMC spacing closer together. (5) A distinct MyoC glideosome is tethered at the posterior polar ring. It consists of GAP80, a GAP70/GAP45 homolog, which interacts with MyoC, GAP40, and GAP50. A novel component, IAP1, restricts this complex to the posterior pole. APR, Apical polar ring; IMC, inner membrane complex; PM, plasma membrane; RB, residual body.

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contains a strongly cross-linked membrane skeleton consisting of membrane-embedded glideosome-associated proteins with multiple membrane spans (GAPM), adjoining alveolin filaments and other proteins that provide tensile strength and association with the subpellicular microtubules (Bullen et al., 2009; Harding et al., 2019). These components serve as a rigid substrate to anchor the glideosome complex so that the myosin A motor can move associated actin filaments and adhesins to the posterior to drive forward movement and host cell invasion. The IMC begins immediately below the extreme apical tip of the parasite which is solely limited by plasma membrane. The AC is formed from a single ring-shaped IMC compartment that is defined by TgISP1 and nine AC proteins (Beck et al., 2010; Chen et al., 2015a, 2017; Fung et al., 2012). Below this unitary cap, strips of flattened IMC vesicles gently spiral around the zoite body following the same path as that of the underlying microtubules. The lateral IMC compartment is distinguished by TgISP2 and TgISP4. TgISP3 is found in this compartment but additionally localizes to a basal compartment of the IMC where individual IMC plates coalesce to close off the posterior of mature parasites. Targeting of ISPs requires palmitoylation and/or myristoylation and TgISP1 occupancy of the AC excludes the other ISPs from this compartment. Loss of TgISP2 is associated with replication defects, indicating that compartment identity is important for bud maturation. The single apical IMC plate is joined to the lateral and posterior plates by IMC suture components (ISCs) at both lateral and longitudinal plate boundaries. TgISC1, TgISC4, and TgISC5 are detergent insoluble and TgISC3 is enriched in daughter buds. An additional set of proteins is specifically associated with transverse sutures. Transverse suture component (TSC) proteins include TgTSC1 (also known as CBAP and SIP), and TgTSC2 6. TgTSC2, TgTSC3, and TgTSC4 are detergent

insoluble (Tilley et al., 2014; Lentini et al., 2015; Chen et al., 2015a, 2017). Five to six apical annuli marked by TgCen2 and TgAAP1 (also known as TgPAP1) are embedded at the junction between the AC and lateral seams of the IMC plates (Fig. 16.2A). Newly defined apical annuli proteins (AAPs) are organized into four concentric rings with diameters of 200 400 nm (Suvorova et al., 2015; Hu et al., 2006; Engelberg et al., 2020). AAPs are specific to coccidians and harbor protein signatures that are characteristic of centrosomal proteins, suggesting that annuli are derived from centrosomes. The annuli form a pore-like structure that may participate in signaling and exchange of material across the IMC. To date, 29 proteins have been designated as “IMC” proteins as a descriptor for their localization to this compartment. IMC proteins encompass diverse subsets: they may be integral or peripheral membrane proteins or be components of the closely associated alveolin cytoskeleton. In addition, other IMC-localizing proteins (TgPhIL1, TgILP1, and TgMSC1b) have distinct names that reflect their underlying properties (Chen et al., 2015a, 2017; Beck et al., 2010; Gilk et al., 2006; Mann and Beckers, 2001; Gubbels et al., 2004; Anderson-White et al., 2011; Lorestani et al., 2012). Once the Toxoplasma community has acquired a clearer understanding of the roles and categories that individual IMC proteins fall into, it may be useful to assign revised names that reflect specific subcategories (alveolins, IMC-embedded cytoskeletal proteins, and detergent-soluble IMC-anchored components). Fourteen of the first 15 proteins designated as “IMC” proteins are intermediate-filament like alveolins. The exception (TgIMC2) encodes a larger protein than originally reported (Mann and Beckers, 2001) which is adjacent to an unnamed paralog (TGGT1_228160, 187 kDa). Both proteins contain an N-terminal transmembrane domain, followed by metallophosphatase superfamily and SMC modules. More

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recently, a large set of nonalveolin components have been enumerated as IMC proteins 16 29 (Chen et al., 2015a, 2017; Butler et al., 2014). Except for TgIMC28 which labels the apical, lateral, and basal subcompartments, the remaining proteins are located to the lateral and basal compartments. Of this set, TgIMC16, TgIMC17, TgIMC21, and TgIMC25 have been identified as detergent-insoluble, suggesting that they are membrane cytoskeleton components. Although some of this set label both mature IMC and developing daughter buds (TgIMC19, TgIMC21, and TgIMC25), others are specific to maternal (TgIMC17, TgIMC18, and TgIMC20) or daughter (TgIMC16 and TgIMC29) IMCs. The AC IMC is separately defined by AC proteins (above). Of the nine AC proteins, TgAC2, TgAC3, TgAC4, TgAC5, and TgAC7 are detergent insoluble, an attribute of many membrane cytoskeleton components (Beck et al., 2010; Chen et al., 2015a, 2017; Fung et al., 2012). Pellicle tensile strength is provided by a detergent-resistant, IMC-associated network of 14 related alveolin family proteins (AndersonWhite et al., 2011; Mann and Beckers, 2001; Gubbels et al., 2004; Hu et al., 2006). These harbor conserved “EKIVEVP” and “EVVR” or “VPV” subrepeats in valine- and proline-rich domains. While orthologs of some alveolin motif proteins are found in a wide variety of alveolates, this family is expanded in Toxoplasma, perhaps because nonconserved IMC proteins coordinate Toxoplasma-specific functions such as replication by endodyogeny. Alveolin motifs are also present in euglenoids and algae, which, like the alveolates, have a cortical membrane skeleton (Goodenough et al., 2018). The name “epiplastins” was proposed for this new class of proteins that can be distinguished from conventional intermediate filaments by a distinctive low-complexity medial domain (at the center of the polypeptide sequence) and a predicted predominance of β-strand secondary structure. Individual

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alveolin proteins display distinct localization to mother and daughter cytoskeletal structures. TgIMC1 and TgIMC4 are equally distributed in mothers and developing daughters. TgIMC3, TgIMC6, and TgIMC10 distribution is skewed toward developing daughter parasites and as daughter parasites emerge and mature, these proteins diminish. TgIMC7, TgIMC12, and TgIMC14 exclusively associate with the mature cortical cytoskeleton in G1-phase and are critical to the structural integrity of extracellular tachyzoites. TgIMC11 is found at the AC and basal regions, and TgIMC5, TgIMC8, TgIMC9, and TgIMC13 relocate from the entire developing bud to the basal extremity of the IMC (i.e., the basal complex) halfway through daughter assembly. TgIMC15 associates with duplicated centrosomes, transitions to daughter buds and is ultimately located at the basal cap, marking the stages of nuclear division, daughter budding and completion of cytokinesis. Both TgIMC14 and TgIMC15 influence key developmental decisions that distinguish replication by endodyogeny from endopolygeny: loss of either induces formation of multiple daughter buds within the mother parasite (Dubey et al., 2017). In order for daughter buds to enclose segregated contents, a basal complex assembles at the advancing edge of daughter IMC to constrict the posterior region of daughter cytoskeletons (Hu, 2008; Heaslip et al., 2010a; Lorestani et al., 2012; Engelberg et al., 2016). The MORN motif is often found in proteins that coordinate membrane membrane or membrane cytoskeleton interactions. TgMORN1 is a key scaffolding protein of the basal complex (Heaslip et al., 2010a; Ferguson et al., 2008; Lorestani et al., 2010). Early in endodyogeny, TgMORN1 rings encircle duplicated centrosomes, marking the primordial daughter buds. The TgMORN1 splits in a narrow apical and wider basal ring defining the polar ends of the two nascent IMCs. Loss of TgMORN1 inhibits assembly of the basal complex leading to

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aberrant replication (Heaslip et al., 2010a; Lorestani et al., 2010). MORN1 at the emerging IMC buds is conserved across apicomplexans that replicate by diverse mechanisms including endodyogeny, endopolygeny, and schizogony (Ferguson et al., 2008). TgCen2, actin (TgACT1), and the MyoJ myosin are required to constrict the basal complex to separate the daughters (Heaslip et al., 2010a; Tosetti et al., 2019; Fre´nal et al., 2017b). However, this contraction does not induce complete separation of daughters from each other. Prior to host cell lysis, sibling parasites within a vacuole remain connected to a common RB through posterior junctions which permit exchange of diffusible components while partitioning complete sets of essential organelles. The pore between the parasite base and the RB is maintained by a mature basal complex characterized by the additional recruitment of FIKK and MSC1a (Skariah et al., 2016; Lorestani et al., 2012) (Fig. 16.4B). Notably, individual components of the basal complex occupy distinct areas of the basal cap: the largest region is covered by TgIMC9, TgIMC13, and TgMORN1. TgIMC5 and TgIMC8 cover a smaller region and the extreme basal tip is delimited by TgCen2 (Fig. 16.2A). In addition to its key role in daughter cell development during endodyogeny, the IMC is critical to motility and invasion. The motilityassociated characteristics of the IMC are shared with many apicomplexans, which use IMCtethered myosin to move adhesin-associated Factin to the apical pole of zoites. In order to productively move adhesins rearward, IMCtethered myosins must resist displacement. The subpellicular microtubules and alveolin network are key to stiffening the IMC for this purpose and a series of integral membrane proteins, the GAPMs, links these filaments with glideosome components. GAPMs are a family of conserved apicomplexan proteins that fall into three orthologous subsets (GAPM1, GAPM2, and GAPM3) (Harding et al., 2019; Bullen et al., 2009). GAPMs are characterized by

five or six membrane-spanning domains and are localized to the IMC in both mature parasites and developing daughter buds. The sequences of the second and fourth loops that link adjacent transmembrane domains are highly conserved across apicomplexan lineages, suggesting that they are constrained by interactions with other proteins. The N- and C-termini are highly conserved within but not between orthologous groups, suggesting that individual subsets have distinct roles, again constrained by interactions with other proteins. Toxoplasma GAPM1a, GAPM2a, and GAPM3 are highly expressed in tachyzoites and GAPM1b and GAPM2b are present at considerably lower levels. Loss of GAPM1a, GAPM2a, or GAPM3 reduces parasite fitness in vitro, while GAPM1b and GAPM2b are likely dispensable in this context. Induced depletion of GAPM1a causes severe replication defects leading to progressive disorganization and disassembly of subpellicular microtubules. GAPM proteins are robust candidates for the IMC-associated IMPs that link the subpellicular microtubules and alveolin network to the IMC to confer shape, tensile strength, and rigidity.

16.2.3 Actin, actin-like and actin-related proteins, and actin-binding proteins A variety of studies with pharmacological agents that perturb actin or myosin function provided some of the first evidence that actin and myosin propel Toxoplasma tachyzoite motility and invasion (Shaw et al., 2000; Haraldsen et al., 2009; Caldas et al., 2013; Dobrowolski et al., 1997; Dobrowolski and Sibley, 1996; Poupel and Tardieux, 1999; Shaw and Tilney, 1999; Wetzel et al., 2003). These processes are blocked by treatment with the myosin inhibitors butanedione monoxime (BDM) and KT5926 or agents that disrupt (cytochalasin D and mycalolide b) or stabilize (phalloidin and jasplakinolide) actin polymers. Since all of these drugs are

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nonselective, they also interfere with actin or myosin in host cells. Subsequent genetic studies conclusively demonstrated that Toxoplasma actin and myosin power tachyzoite gliding motility and host cell invasion. Genetic depletion of the primary motility-associated myosin (TgMyoA) profoundly impairs tachyzoite motility and invasion (Meissner et al., 2002). Tachyzoites expressing a cytochalasin-resistant parasite actin (A136G), but not wild type actin, are able to invade host cells in the presence of cytochalasin, indicating that parasite actin polymerization is essential for this process (Dobrowolski and Sibley, 1996). Nonetheless, under physiological conditions, Toxoplasma actin polymers are peculiarly ephemeral, making it difficult to detect the timing and location of microfilaments. This impediment has recently been overcome by expression of an actin chromobody, a fluorescent protein fused to a single-chain antibody that specifically binds to polymerized actin, in living parasites (Periz et al., 2017). The chromobody detects dynamic cytosolic filaments and a network that connects sibling parasites within the parasitophorous vacuole. Prior to this technical advance, actin filaments were only reliably observed in jasplakinolide-treated parasites, where they often form an aberrant, extended protrusion of the plasma membrane at the apical end (Shaw and Tilney, 1999). Although jasplakinolide treatment stabilizes actin filaments and increases the rate of gliding, it disrupts normal motility and reversibly inhibits host cell invasion (Poupel and Tardieux, 1999; Wetzel et al., 2003; Skillman et al., 2011). Collectively, these observations indicate that precise control of filament availability is key to tachyzoite motility and invasion and imply that Toxoplasma actin and myosin have unusual properties relative to other homologs (Skillman et al., 2011, 2013; Sahoo et al., 2006). Actin is an essential and conserved component of nearly all eukaryotic cells. This 42 kDa protein can exist as a free subunit (globular, Gactin) or polymerize to form microfilaments

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(filamentous, F-actin). Actin binds to ATP which is hydrolyzed during polymerization: Gactin ATP monomers are preferentially added to the fast-growing filament plus (barbed) end and F-actin ADP monomers are lost from the minus (pointed) end. Polymerized actin is essential to several conserved cellular processes, including motility, vesicular trafficking, shape, and cytokinesis. The Toxoplasma genome has genes for several actin-like and actinrelated proteins (ALPs and Arps) and a single actin isoform (TgACT1). Arps are broadly conserved in eukaryotes, have sequence similarity to actin, and are critical components of distinct protein complexes that carry out diverse functions, including regulation of microtubule motor activity, actin polymerization, and chromatin remodeling. Toxoplasma encodes homologs of Arp1 (part of the dynactin complex), as well as Arp4 and Arp6, which are nuclear proteins. Arp4 is part of chromatin remodeling and histone acetyltransferase complexes; a temperature-sensitive mutation in TgArp4a induces tachyzoite growth arrest due to chromosome loss during nuclear division (Suvorova et al., 2012). Apicomplexans lack most or all components of the Arp2/3 complex, a key promoter of actin polymerization in many eukaryotes (Gordon and Sibley, 2005). Since homologs of a canonical Arp2/3 complex are apparent in Tetrahymena thermophila and some apicomplexans harbor remnant subunits, it is likely that the complex was lost in the apicomplexan lineage. Remarkably, most apicomplexan actin superfamily proteins are actin-like proteins (ALPs) that are unique to this group and likely have parasite-specific roles. TgALP1 is most closely related to conventional actin (Gordon et al., 2008, 2010). It readily interconverts between soluble and bound forms and localizes to a discrete region of the nuclear envelope, on transport vesicles, and on the early IMC of emerging daughter cells prior to appearance of IMC1. Overexpression of TgALP1 disrupts daughter cell IMC formation

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and causes defects in nucleus and apicoplast segregation. Monomeric G-actin assembles head to tail into two helical strands that form an actin filament. Polymerization requires that G-actin be at or above its critical concentration, a value at which F-actin remains in equilibrium, without net growth or disassembly. Above the critical concentration, net F-actin assembly occurs and below this value net disassembly prevails. In nearly all organisms (yeast, amoeba, and vertebrate actin, as well as bacterial actin-like proteins), assembly is cooperative and consists of an unfavorable rate limiting nucleation step followed by efficient elongation from these seeds until G-actin reaches the critical concentration. Nucleation is inefficient because dimer and trimer intermediates are unstable. Once this threshold has been crossed, G-actin is incorporated into extending polymers until the critical concentration is reached. In general, actin has remained highly conserved throughout evolution. However, actins from Toxoplasma and other apicomplexans represent some of the most divergent members of this family characterized to date. Moreover, in striking contrast to other eukaryotes, where the bulk of actin is polymerized, apicomplexan actin is largely unassembled (Skillman et al., 2011, 2013; Sahoo et al., 2006). Computational modeling and biochemical characterization of recombinant actin suggest that sequence differences, particularly the unusual residues G200 and K270, are key to the intrinsically unstable nature of apicomplexan microfilaments. Substitution of conserved amino acids at these positions modestly (K270M) or significantly (G200S) increases polymerization of recombinant TgACT1, which only forms short, unstable filaments in the absence of jasplakinolide (Skillman et al., 2011). Expression of K270M or G200S substituted TgACT1 genes in tachyzoites impairs parasite proliferation and causes aberrant motility, indicating that the system is optimized for intrinsically short-lived

microfilaments. A structure of jasplakinolidestabilized Plasmodium falciparum ACT1 polymers also implicates amino acid differences in the plug and D-loop regions as underlying reduced affinity at contact sites as the basis of unstable filaments in apicomplexan actin (Pospich et al., 2017). The polymerization kinetics of recombinant TgACT1 and PfACT1 are distinct from the expected cooperative nucleation-elongation behavior of actin and are most consistent with isodesmic assembly (Skillman et al., 2013). In this kinetic model, all interactions occur with equal affinity and the amounts of both G- and F-actin increase with total actin concentration. This is distinct from conventional actin that partitions assembly into nucleation and elongation kinetic states and where the G-actin concentration remains constant (driving net assembly) above the critical concentration. The unfavorable first phase of cooperative polymerization makes F-actin assembly dependent upon exogenous nucleators, such as the Arp2/ 3 complex or formins. The observation that apicomplexan G-actin nucleation and elongation are indistinguishable may explain why apicomplexans dispensed with the Arp2/3 complex which mimics a G-actin dimer to overcome the rate limiting nucleation step during cooperative assembly. Nonetheless, tachyzoites use three distinct formins as well as other actin-binding proteins to regulate actin assembly. Toxoplasma has homologs of formins, profilin, ADF/cofilin, cyclase-associated protein (CAP), and coronin and many are critical to regulation of actin polymerization, motility, invasion, and replication. Conserved actinbinding proteins from Toxoplasma have modified properties relative to homologs from other eukaryotes, most likely a parallel adaptation to the altered properties of Toxoplasma actin. Formins are large proteins that promote actin filament nucleation and remain attached to the fast-growing end of new filaments to block binding of capping proteins which

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interfere with subunit addition (Goode and Eck, 2007; Kovar, 2006). Formins are defined by an FH2 (formin homology) domain which is required for formin dimerization, actin nucleation, and barbed end binding. Some formins also contain FH1 domains, characterized by polyproline stretches that recruit profilin and mediate interactions with SH3 or WW domain proteins. The Toxoplasma genome contains three formin genes that participate in motility, apicoplast segregation and cell cell communication between sibling parasites (Daher et al., 2010, 2012; Tosetti et al., 2019). TgFRM1 and TgFRM2 are conserved in other apicomplexans, while TgFRM3 is restricted to the coccidian lineage. TgFRM1 has four tetratricopeptide repeats in the N-terminal half of the protein and an FH2 domain at the C-terminus. Although TgFRM1 lacks a clear-cut FH1 domain, a proline-rich region adjacent to the N-terminal side of the FH2 domain may provide this function. TgFRM1 is located at the conoid and is essential for stimulating actin polymerization to drive motility, invasion, and egress. TgFRM2 and TgFRM3 also have proline-rich domains proximal to an FH2 domain located in the C-terminal half of the protein. TgFRM2 localizes adjacent to the apical nucleus and stimulates actin assembly to drive apicoplast segregation. TgFRM3 is restricted to the RB where participates in RB-based communication between replicating sibling parasites. Consistent with other formins, TgFRM1 and TgFRM2 bundle actin filaments (Skillman et al., 2012). Although bundling has not been implicated as critical for in vivo gliding, dynamic F-actin filaments appear thick, suggesting that they are bundled (Periz et al., 2017). In many eukaryotes, profilin and ADF/ cofilin perform complex roles in actin polymerization to either promote assembly or disassembly, depending on other circumstances (Kovar, 2006; Goode and Eck, 2007; Poukkula et al., 2011). Profilins sequester actin monomers to promote depolymerization but promote

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F-actin assembly by binding formins and by enhancing nucleotide exchange to increase the pools of ATP-bound G-actin. ADF proteins also sequester G-actin to promote depolymerization but also exhibit PIP2-inhibited F-actin severing activity which can promote disassembly or can create actin seeds to bypass nucleation and promote new F-actin assembly. Apicomplexan ADF homologs lack an F-loop which has been thought to be required for F-actin binding and do not bind PIP2 (Haase et al., 2015). Recombinant TgADF induces F-actin disassembly in a dose-dependent manner, suggesting it predominantly functions to sequester G-actin (Mehta and Sibley, 2010, 2011). Loss of TgADF has been variously reported to cause slowed, nonproductive serpentine movement and rocking (Mehta and Sibley, 2011) and to impair apicoplast segregation and parasite replication (Haase et al., 2015; Jacot et al., 2013). Like TgADF, the monomersequestering activity of TgPRF is retained while other properties are reduced or absent. Unlike other profilins, TgPRF inhibits actin nucleotide exchange and the ability of TgFRM1 and TgFRM2 FH2 domains to stimulate actin polymerization, consistent with a dominant or exclusive role in G-actin sequestration. Conditional loss of profilin impairs tachyzoite motility and invasion (Skillman et al., 2012; Plattner et al., 2008) and causes progressive loss of the apicoplast (Jacot et al., 2013). Considering the inherently destabilized nature of apicomplexan microfilaments, it is striking that both TgPRF and TgADF have been streamlined to accentuate-monomer sequestering activity with other traits minimized or eliminated, a trait that is apparently shared with TgCAP. CAPs are a family of conserved actinbinding proteins that regulate actin remodeling in response to signals. Most CAP homologs contain CAP, CARP, and HFD domains that bind and sequester G-actin (using CAP and CARP domains) or regulate filament

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disassembly by promoting ADF-mediated severing (using the HFD). Apicomplexan CAP homologs are considerably smaller and lack CAP and HFD domains, suggesting that they bind G-actin, either to sequester it or to promote nucleotide exchange (Hunt et al., 2019; Lorestani et al., 2012). Toxoplasma uses alternative translation start sites to synthesize two CAP isoforms: short CAP (TgCAPS) is cytosolic, while long CAP (TgCAPL) shuttles between the cytoplasm in extracellular parasites and the tachyzoite apex in intracellular parasites. The dynamic behavior of TgCAPL is regulated by palmitoylation and phosphorylation of a unique N-terminal extension that is specific to coccidian CAP proteins. Tachyzoites lacking CAP have defects in invasion, egress, motility, correct daughter cell orientation, and dense granule trafficking. Since directed dense granule transport is dependent on F-actin and parasites lacking TgCAP have significantly increased run length and velocity TgCAP is likely to be a negative regulator of actin polymerization, similar to TgADF and TgPRF. The tethered apical location of TgCAPL in intracellular tachyzoites may serve to inhibit premature initiation of motility which is relieved by its release after stimulation of motility. Surprisingly, the TgCAPL isoform is dispensable: expression of the short cytoplasmic isoform (TgCAPS) complements the bulk of defects observed in null parasites. Coronins are conserved F-actin-binding proteins that contain a characteristic Nterminal WD40 domain that binds to actin and a C-terminal coiled-coil domain mediates protein protein interactions, including homodimer formation (Gandhi and Goode, 2008). Like other actin-binding proteins, coronins typically play complex, context-dependent roles in actin dynamics. Coronin protects new (ATP bound) F-actin from ADF-induced disassembly and recruits the Arp2/3 complex to filaments to expand actin networks (Gandhi and Goode, 2008). Coronin also synergizes with cofilin to

induce disassembly of old (ADP bound) F-actin. In addition to WD40 and coiled-coil domains, Toxoplasma coronin has a large novel C-terminal extension (Salamun et al., 2014). In vitro experiments indicate that purified TgCor increases actin polymerization but has negligible filament bundling activity. TgCor is cytoplasmic in intracellular parasites and relocates to the posterior pole during motility or invasion in a Ca21-dependent fashion. Loss of TgCor modestly reduces tachyzoite invasion and egress rates. In Plasmodium, where coronin localization dynamics are comparable to Toxoplasma, coronin is critical to organize F-actin for productive motility (Bane et al., 2016). This study also implicates protein kinase A (PKA) as critical for F-actin disassembly and motility. Although PKA is critical for Toxoplasma egress, to date, its role has not been directly connected to motility or actin disassembly (Uboldi et al., 2018; Jia et al., 2017).

16.2.4 Myosin motors, the glideosome, and other associated factors The unusual relationship between F-actin and myosin activity suggests that most or all Factin functions in tachyzoites also involve myosin motors. Myosins move on F-actin and are essential for diverse processes (cytokinesis, vesicle transport, and organelle movement) in nearly all eukaryotes (O’Connell et al., 2007). They consist of motor domain-containing heavy chains that power motility and regulatory light chains. Myosin heavy chains (MHCs) typically contain head, neck, and tail domains: the head domain binds actin and couples ATP hydrolysis to generation of movement; the neck domain typically interacts with regulatory light chains, and the tail region binds cargo. To date, MHCs have been grouped into B35 phylogenetic classes. The Toxoplasma genome encodes 11 MHC homologs, most of which are members of the class XIV MHC subset that is

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found in apicomplexans and ciliates (Heintzelman and Schwartzman, 1999, 2001; Foth et al., 2006). Of the 11 myosins, only TgMyoA and TgMyoF are conserved across the phylum. In addition to the existing data on MHCs in Toxoplasma, seven myosin light chains (MLCs) have been annotated, including TgMLC6 that is identical to TgCaM2 (above) (Long et al., 2017b; Graindorge et al., 2016; Polonais et al., 2011a; Herm-Gotz et al., 2002). A broadly conserved cochaperone, UNC45, is critical for myosin folding and assembly both in an endogenous setting and for heterologous expression of functional TgMyoA (Bookwalter et al., 2014; Fre´nal et al., 2017b). UNC45 homologs have an N-terminal tetratricopeptide repeat domain, a central armadillo repeat domain, and a C-terminal UCS domain, which also contains armadillo repeats. UNC45 brings the myosin head and a general chaperone (HSP90 or HSP70) together by binding each with the UCS and TPR domains, respectively. Loss of TgUNC destabilizes all Toxoplasma myosins to completely block parasite proliferation. This phenotype represents the aggregate effects of loss of all myosin-driven processes including motility, invasion, and egress, defective apicoplast inheritance, impaired dense granule secretion, asynchronous division due to RB defects, and failure to constrict the basal polar ring. The first myosin-driven behaviors to be documented in Toxoplasma are the related processes of tachyzoite gliding motility and host cell invasion. Apicomplexan zoites, including Toxoplasma, exhibit an unusual substratedependent gliding motility that involves apical secretion of plasma membrane-spanning adhesins which are translocated along the pellicle to the parasite posterior by actin and myosinbased machinery (Sibley et al., 1998; Fre´nal et al., 2017a,b). The cytoplasmic tails of adhesins are linked to F-actin beneath the plasma membrane by a glideosome-associated connector (TgGAC). TgGAC is a large armadillo-

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repeat and plextrin homology domaincontaining protein that is conserved in apicomplexans (Jacot et al., 2016). Tachyzoite invasion of host cells links gliding motility to a circular moving junction that is inserted into the host cell plasma membrane at the site of contact. Adhesins bind to moving junction components (rhoptry neck proteins; RON proteins) and, as the action of actin and myosin move adhesins rearward, the tachyzoite is driven through the moving junction into the host cell (see Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection). Remarkably, most moving junction components as well as the primary motor (MyoA) that drives penetration are conserved in apicomplexans. Toxoplasma tachyzoite invasion of host cells involves two myosins, the conoid-associated TgMyoH, which initiates translocation of the moving junction, and TgMyoA, which accepts the adhesin-moving junction complex from TgMyoH at the origin of the IMC and translocates it to the posterior of the parasite. TgMyoA is a fast, single-headed class XIVa myosin that is located between the plasma membrane and the IMC (Herm-Gotz et al., 2002; Meissner et al., 2002; Hettmann et al., 2000). It associates with two light chains: TgMLC1 and either TgELC1 or TgELC2 (Gaskins et al., 2004; Heaslip et al., 2010b; Herm-Gotz et al., 2002; Nebl et al., 2011; Williams et al., 2015). The TgMyoA heavy chain lacks a tail region that characteristically serves to link MHCs to cargo. This function is supplied in trans by a novel N-terminal extension of TgMLC1 that associates with gliding-associated proteins (GAPs) to tether TgMyoA at the IMC; this protein complex is known as the glideosome and is conserved in many apicomplexans. TgMyoA interaction with TgMLC1 is stabilized by Ca21-dependent activity of TgELC1 and TgELC2 that influence motor activity (Nebl et al., 2011; Williams et al., 2015). TgMyoA is critical for efficient gliding motility and host cell invasion: parasites lacking TgMyoA are

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nonmotile, exhibit reduced invasion and egress, and cannot form plaques in culture (Meissner et al., 2002). Moreover, tachypleginA, a small molecule inhibitor of Toxoplasma tachyzoite invasion and motility, irreversibly binds to TgMLC1 and decreases motor activity (Heaslip et al., 2010b). Coccidian parasites, including Toxoplasma, additionally require TgMyoH, a conoid-tethered myosin, to initiate invasion. Of the seven MLCs identified in the Toxoplasma genome, knock-in tags demonstrate that TgMLC3, TgMLC5, and TgMLC7 localize to the conoid, suggesting they might regulate TgMyoH. TgMyoH depletion causes TgMLC5 to relocate to the cytoplasm, indicating that it binds to TgMyoH. TgMLC7 remains at the conoid which may indicate that it associates with the other conoid-associated myosins, TgMyoE or TgMyoL, which have not been extensively characterized to date (Fre´nal et al., 2017b). TgMLC5 and TgMLC7 are dispensable for TgMyoH localization at the conoid and parasite fitness. However, three conoid-localized calmodulin homology proteins (TgCaM1, TgCaM2/TgMLC6, and TgCaM3) regulate invasion, gliding motility, and egress and are mislocalized or degraded in the absence of TgMyoH, indicating that they may regulate this motor (Long et al., 2017b). TgMyoH depleted parasites are incapable of plaque formation, although microneme secretion, host cell attachment, and redistribution of TgAKMT are unaltered, indicating that signaling and secretion are intact. Whereas TgMyoH is immobilized by direct interaction with the conoid, TgMyoA localizes to the IMC of mature parasites through a stable complex with the GAPs TgGAP40, TgGAP50, and TgGAP45 or TgGAP70 (Fre´nal et al., 2014; Rees-Channer et al., 2006; Gilk et al., 2009). TgGAP40 and TgGAP50 are integral IMC proteins that stably anchor the motor complex to the IMC. These glideosome components and the GAPM proteins reach the IMC through the secretory pathway early during daughter bud formation and are essential for formation of

normal daughter IMC (Ouologuem and Roos, 2014; Bullen et al., 2009; Gaskins et al., 2004). TgGAP40 is an integral membrane protein that spans the IMC nine times and is uniformly distributed in the IMC, including apical and basal ends not covered by the alveolin IMC proteins (Fre´nal et al., 2010; Ouologuem and Roos, 2014). TgGAP50 has transmembrane domains at both termini. The N-terminal 50 amino acids target it to the ER and are removed for TgGAP50 maturation and localization to the IMC. The Cterminal transmembrane helix serves to anchor TgGAP50 in the IMC. GAP50 shares B25% identity and overall structural similarity with members of the calcineurin-like/purple acid phosphatase superfamily but lacks key catalytic residues and detectable enzymatic activity (Bosch et al., 2012; Gaskins et al., 2004). Mature TgGAP50 contains a 351-residue luminal domain followed by a transmembrane domain and a six-residue cytoplasmic domain at the Cterminus. Since the bulk of TgGAP50 resides in the IMC lumen, interactions with other components of the glideosome machinery are limited to the C-terminal membrane-spanning helix and short cytoplasmic domain. TgGAP45 contains an N-terminal coiled-coil domain and a Cterminal globular domain. A subcomplex consisting of TgMyoA, TgMLC1 and TgGAP45 assembles in the cytoplasm (Gaskins et al., 2004) and subsequently associates with TgGAP50 and TgGAP40 to form a stable, immobilized complex anchored between the plasma membrane and the IMC. TgGAP45 is posttranslationally modified by N- and C-terminal acylations (ReesChanner et al., 2006; Fre´nal et al., 2010; Johnson et al., 2007) as well as serine phosphorylation (Gilk et al., 2009) to regulate targeting and assembly of the mature glideosome. Depletion of TgGAP45 causes TgMLC1 to be redistributed to the cytoplasm, disrupts the intimate association of the plasma membrane and underlying IMC and causes tachyzoites to have dramatically reduced gliding motility, invasion, and egress (Fre´nal et al., 2010).

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In addition to GAP45, the coccidian subset of apicomplexans expresses two related proteins, GAP70 and GAP80, which mediate apical and posterior interactions of modified glideosome complexes (Fre´nal et al., 2010, 2014). TgGAP70 has a conserved N-terminal region with predicted acylation sites, a longer coiled-coil domain, and a conserved C-terminus. It is dually anchored to the plasma membrane through N-terminal acylation and restricted to the apical pellicle through interaction of its Cterminal domain with the AC IMC compartment. Although TgGAP70 is not essential and null tachyzoites do not have discernible defects, overexpression of TgGAP70 (or TgGAP80) can partially complement depletion of TgGAP45. Substitution of TgGAP70 for TgGAP45 increases the spacing between IMC and plasma membrane, consistent with its extended coiled-coil domain. This suggests that TgGAP70 facilitates the looser cohesion of IMC and plasma membrane in the apical region to permit greater pellicle flexibility required for conoid extension and retraction. A third member of the TgGAP45 family, TgGAP80, exhibits C-terminal conservation with TgGAP45 and TgGAP70, a domain that coordinates interactions between TgGAP45 and TgMLC1. In contrast, the central domain of TgGAP80 is not predicted to adopt a coiled-coil conformation due to its high content of proline residues in this region. TgGAP80 recruits a distinct motor, TgMyoC, to the tachyzoite posterior polar ring (Fre´nal et al., 2014). TgMyoC uses the same MLCs as TgMyoA and interacts with TgGAP40 and TgGAP50 as well as TgGAP80. It is restricted to the posterior pole by the IMCassociated protein 1 (IAP1) which interacts with the IMC by N-terminal palmitoylation. TgMyoC is not essential and null tachyzoites do not have defects in replication or egress. TgGAP80 null tachyzoites also have normal replication and egress. TgMyoC and TgIAP1 localization is unaltered in these parasites because TgGAP45 can substitute for TgGAP80 in the TgMyoC glideosome. In TgIAP null parasites, TgGAP80 is

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destabilized and no longer detected at the posterior polar ring and TgMyoC is found in the cytoplasm, at the pellicle and at the APR. Finally, in the absence of TgMyoA, TgMyoC associates with TgMLC1 and TgGAP45 and expands its localization beyond the posterior polar ring to the lateral pellicle periphery. This plasticity of glideosome functions (Fre´nal et al., 2014) explains the puzzling residual invasion of TgMyoA null parasites (Meissner et al., 2002; Bichet et al., 2016; Andenmatten et al., 2013; Egarter et al., 2014). A separate group of myosins is required for events during tachyzoite replication. Among these, MyoF is conserved across diverse apicomplexan parasites, while TgMyoI and TgMyoJ are restricted to the coccidian lineage (Foth et al., 2006). TgMyoF is a class XXII myosin that contains seven WD40 domains, a characteristic of class XXII myosins restricted to apicomplexan motors. TgMyoF localizes to the cytoplasm and is enriched above the nucleus and at the pellicle (Jacot et al., 2013). During endodyogeny, it associates with the pellicle, within daughter buds, and at the extremities of the apicoplast. TgMyoF is required for correct positioning of the centrosomes and emerging buds to capture the apicoplast, micronemes, and rhoptries during daughter parasite formation. It is also required for directed transport of dense granules, secretory vesicles that release components that are required to form a mature parasitophorous vacuole (Heaslip et al., 2016; Jacot et al., 2013). Disruption of TgMyoF function causes a defect in apicoplast inheritance and increased accumulation of rhoptries, micronemes and fragmented mitochondria in the RB. Depleted parasites have enlarged RBs, do not form plaques and exhibit a delayed death phenotype akin to that observed after drug-induced inhibition of apicoplast function. These results complement observations that loss of TgPRF or TgADF, overexpression of the FH2 domain of TgFRM2, or treatment of intracellular parasites with cytochalasin D cause

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apicoplast segregation defects (Jacot et al., 2013; Tosetti et al., 2019; Haase et al., 2015). In addition, the coccidian-specific motors TgMyoJ and TgMyoI are required for endodyogenyspecific processes: constriction of the posterior end of daughter buds and cell cell communication between sibling parasites (Fre´nal et al., 2017b). TgMyoJ is a class VI myosin; motors in this class exhibit unique minus-end directionality (Wells et al., 1999). During endodyogeny, a ring of TgMyoJ delineates the posterior margin of the lengthening bud IMC and contracts at the basal cap coincident with TgCen2 (Fre´nal et al., 2017b; Hu, 2008). Cytochalasin D treatment increases the TgMyoJ ring diameter and localization of TgCen2, suggesting that actin, TgMyoJ and TgCen2 constrict the basal complex at the completion of cytokinesis. Tachyzoites lacking TgCen2, TgMyoJ, or TgMORN1 (Heaslip et al., 2010a; Lorestani et al., 2010; Leung et al., 2019; Fre´nal et al., 2017b) also exhibit an enlarged posterior ring. TgMyoJ deficient parasites fail to develop into organized rosettes. The class XXIV myosin TgMyoI is located in the RB at the center of tachyzoite rosettes and powers intercellular traffic through the RB (Foth et al., 2006; Fre´nal et al., 2017b). In the absence of TgMyoJ, TgMyoI accumulates at the tachyzoites posterior end. Although TgMyoJ and TgMyoI deficient tachyzoites form RBs, loss of either motor disrupts the durable connection between sibling parasites and causes a progressive defect in synchronized division. These defects uncover a role for soluble regulators or metabolites to coordinate cell cycle checkpoints during tachyzoite replication. While the functions of several Toxoplasma myosins remain to be dissected, two additional motors, TgMyoB and TgMyoD, have been studied in some detail (Delbac et al., 2001; HermGotz et al., 2006). TgMyoD is a class XIVa myosin that is similar to TgMyoA but is smaller and restricted to closely related coccidians (Hettmann et al., 2000). Like TgMyoA, the

TgMyoD heavy chain associates with the plasma membrane through an N-terminal extension to its coccidian-specific associated light chain, TgMLC2, which is necessary and sufficient for pellicle targeting (Polonais et al., 2011a; Herm-Gotz et al., 2006). In contrast to TgMyoA, pellicle association of TgMyoD is mediated by palmitoylation of TgMLC2 rather than an interaction with GAP proteins. The TgMLC2 protein disappears when TgMyoD is knocked out, indicating that it is stabilized by association with the TgMyoD heavy chain. Depletion of TgMyoD demonstrates that it is not essential for in vitro tachyzoite growth and gliding motility. Transcripts for TgMyoB, like those for TgMyoD, are more abundant in bradyzoite stage organisms, suggesting that both motors may have more significant roles in this stage. The TgMyoB motor is encoded by the same gene as TgMyoC (above), but with a distinct 21 rather than 118 amino acid C-terminal tail specified by alternative mRNA splicing. While the TgMyoC motor is known to be a component of the posterior polar ring glideosome (above), the role of TgMyoB is unclear. TgMyoB transcripts are significantly outnumbered by TgMyoC transcripts and its localization is complicated by low expression levels and a nonantigenic unique tail. Although its overexpression causes replication defects, this may be a consequence of titration of light chains that are shared with other myosins because TgMyoB/C null tachyzoites do not show defects in replication or egress (Fre´nal et al., 2014). Collectively, technical advances and biological discoveries have increased our understanding of the unconventional roles of actin and myosin in Toxoplasma tachyzoites. As described previously, Toxoplasma actin polymers are extremely short-lived and reliable detection of native F-actin has required expression of an actin chromobody, which detects cytosolic filaments and a network that connects sibling parasites within the parasitophorous vacuole (Periz et al., 2017). These sites correspond to

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the location of two of the three Toxoplasma formins which are critical to F-actin assembly. The formins TgFRM1 and TgFRM2 are broadly conserved in apicomplexans and regulate activity of two conserved and essential myosins, TgMyoA and TgMyoF, as well as the conoid-located coccidian motor TgMyoH (Tosetti et al., 2019; Heaslip et al., 2016; Jacot et al., 2013; Meissner et al., 2002; Graindorge et al., 2016). By regulating availability of Factin, TgFRM1 and TgFRM2 control myosin activity to carry out the broadly conserved and essential behaviors of gliding motility and organelle segregation. TgFRM3 is restricted to the coccidian lineage and regulates assembly of F-actin required for the endodyogeny-specific processes of constriction of the basal complex and formation of an RB-based network to connect sibling parasites (Tosetti et al., 2019; Fre´nal et al., 2017b). These behaviors also require coccidian-specific myosins, TgMyoI and TgMyoJ. Finally, although the role of actin-binding proteins was initially explored in the context of gliding motility, invasion and egress, it is now appreciated that disrupting the function of myosins also influences apicoplast segregation, dense granule trafficking, rosette maintenance and organelle inheritance during endodyogeny, among others (Tosetti et al., 2019; Fre´nal et al., 2017b; Jacot et al., 2013; Heaslip et al., 2016). The close relationship between activity of formins, actin-binding proteins, F-actin, and myosins in tachyzoites suggests that most or all roles for F-actin are intimately associated with the activity of specific myosin motors.

16.3 Putting it all together: processes 16.3.1 Replication 16.3.1.1 Endodyogeny and endopolygeny Toxoplasma tachyzoites replicate by endodyogeny (Goldman et al., 1958; Sheffield and

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Melton, 1968), whereas presexual stage merozoites in the feline intestine replicate by endopolygeny (Ferguson et al., 1974). Endodyogeny involves a single nuclear division coupled to formation of two daughter parasites per replication cycle. In contrast, in endopolygeny, a multinucleated mother cell is created by several rounds of S-phase and mitosis (S/M) unlinked to cytokinesis prior to the synchronous emergence of many daughter parasites. In both processes, mitosis proceeds without nuclear membrane breakdown (closed mitosis) and employs formation of internal buds to package a complete set of organelles for each daughter within IMC cytoskeletal scaffolds. In both cases, the maternal IMC-plasma membrane organization is maintained until completion of bud formation. At this point, the plasma membrane dissociates from the maternal IMC and subsequently associates with daughter IMC buds to create mature daughter pellicles. By comparison, other apicomplexans replicate by schizogony, a process that involves disassembly of the mother pellicle prior to multiple rounds of nuclear division with subsequent budding of daughter parasites from the plasma membrane (Francia and Striepen, 2014). The uncoupling of cell division (cytokinesis) from S/M is coordinated by the unique bipartite centrosome structure (Chen and Gubbels, 2019, 2015; Suvorova et al., 2015). The distinct components of the inner- and outer-cores independently regulate the nuclear cycle (S-phase, mitosis) and daughter budding, respectively (Fig. 16.3B). Since the nuclear cycle can proceed without activation of the outer-core, apicomplexan parasites can accommodate accumulation of polyploid intermediates. A mutant that disconnects the inner- and outer-cores confirms that each platform executes its function independently (Chen and Gubbels, 2019). Based on the nongeometric expansion of Plasmodium merozoites in the red blood cell, inner-core activation is controlled at the individual core-

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level (Arnot et al., 2011). At the conclusion of schizogony, all outer-cores are activated simultaneously, to permit synchronous budding of all nuclei produced during a final mitotic cycle. This indicates that the outer-core cannot be activated unless the inner-core is activated as well. Several kinases that control cell cycle progression (Naumov et al., 2017; Alvarez and Suvorova, 2017; Berry et al., 2016), centrosome replication (Chen and Gubbels, 2013), and centrosome activation (Suvorova et al., 2015; Berry et al., 2018) have been identified in recent years but are not discussed in detail here. It is clear that the centrosome serves as a physical and signaling hub to orchestrate the various modes of apicomplexan cell division. 16.3.1.2 Nuclear division Toxoplasma mitosis has a number of features that distinguish it from mitosis in metazoan cells. The tachyzoite nuclear membrane remains intact and chromosome segregation occurs without chromosome condensation. Preceding spindle assembly, the centrosome rotates from the apical to basal side of the nucleus where it duplicates (Chen et al., 2015b; Hartmann et al., 2006). Assembly of spindle microtubules begins at the basally located, newly duplicated centrosomes, and these microtubules are stabilized by acetylation of α-tubulin K40 upon return of the duplicated centrosomes to the apical side of the nucleus (Chen et al., 2015b). Studies using YFP-tagged α1-tubulin to measure tubulin content are consistent with the association of each chromosome with a single B1 μm long spindle microtubule (Swedlow et al., 2002). Spindle microtubules are decorated by TgEB1, which does not associate with other microtubule populations, save for a transient association with the tips of newly assembled daughter subpellicular microtubules (Chen et al., 2015b). The mitotic spindle traverses a membrane tunnel between the two centrocones to separate the spindle poles and associated centrosomes.

The spindle poles never extend to the opposite ends of the nucleus but remain in close proximity at the apical side of the nucleus (pleuromitosis). Completion of spindle pole separation is marked by reorientation of polar spindle microtubules from an apical horizontal alignment to a nearly vertical orientation such that they project into the nucleoplasm (Chen et al., 2015b). Although karyokinesis is not concluded until the enclosing daughter cytoskeletons are partially assembled, it is generally assumed that for signaling purposes (permitting initiation of daughter bud assembly) mitosis is complete at this time. Although several components of the eukaryotic chromosomal passenger complex are missing from the Toxoplasma genome, a complex containing the aurora kinase ortholog TgArk1 is critical for mitotic progression (Berry et al., 2018). Homologs of several spindle assembly checkpoint components are encoded in the genome but remain largely unstudied. Many questions regarding mitosis and nuclear division remain unanswered, including how chromosomes are captured (or remain clustered at the centrocone) and how the two sets of chromosomes are distinguished and segregated. 16.3.1.3 Assembly of daughter cytoskeleton buds Like nuclear division, Toxoplasma cytokinesis is distinct from the process in metazoan cells. Rather than partitioning cellular components by formation of a contractile ring immediately prior to abscission, Toxoplasma daughter buds form in a mother parasite which retains functional apical organelles, a pellicle and cortical cytoskeleton until daughters are sufficiently mature to emerge from the mother, adopting her plasma membrane as their own. Prior to S phase, the first indication that tachyzoites have committed to cell division is duplication of the Golgi apparatus (Nishi et al., 2008). Development of daughter buds occurs in a series of orderly steps that are intimately

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associated with the cytoskeleton (Fig. 16.3). Each daughter is endowed with a complete set of apical complex organelles as well as a mitochondrion, Golgi apparatus, apicoplast, and nucleus that are acquired with characteristic timing during replication. All major events revolve around the assembling daughter cytoskeleton scaffold, which initiates at the centrosome outer-core (Anderson-White et al., 2011; Chen and Gubbels, 2019; Suvorova et al., 2015). The earliest sign of daughter scaffold assembly is the appearance of TgIMC15 on newly duplicated centrosomes (Anderson-White et al., 2011). TgIMC15 then shifts to the new daughter scaffolds together with the appearance of Rab11b (Agop-Nersesian et al., 2009), suggesting that Rab11b mediates membrane-based delivery of IMC15 (Harding and Meissner, 2014). Immediately following these events, SFA fibers appear and anchor each centrosome to a daughter bud. Subsequently, bud subpellicular microtubules appear: nascent daughter buds are marked by tubulin arranged in five spots surrounding a central spot in flower petal-like arrangement (Nagayasu et al., 2017). Although the timing relative to microtubules is unknown, TgALP1 appears at the bud prior to TgMORN1 or other members of the IMC family, suggestive of a role in early daughter formation (Gordon et al., 2010). TgMORN1 first appears as a cloud but coalesces into a ring that marks the nascence of the basal complex (Ferguson et al., 2008). The ISP proteins are subsequently deposited in the IMC, beginning with TgISP1 which occupies the cap compartment to exclude other ISPs (Beck et al., 2010). Several alveolin family proteins (TgIMC1, 3 6, 8 11, and 13) are then integrated into this structure (Anderson-White et al., 2011). Once the cap alveolus is established, the apical annuli are assembled at sutures between the cap and median alveoli (Hu et al., 2006; Engelberg et al.,2020; Suvorova et al., 2015). An IMC-located palmitoyl transferase (TgDHHC14) modifies many of these proteins to anchor them to the IMC membrane

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(Fre´nal et al., 2010, 2013). As the lengthening daughter buds envelop the two halves of the horse-shoe shaped nucleus, subpellicular microtubules with associated MAPs extend along the growing daughter IMC structures (Tran et al., 2012; Morrissette and Sibley, 2002b). Both acylanchored and integral membrane proteins such as TgGAP40, TgGAP50, and the TgGAPMs are continuously deposited throughout bud growth (Harding et al., 2019; Harding and Meissner, 2014; Gaskins et al., 2004; Johnson et al., 2007). When the bud reaches its widest point, several events indicate a critical transition in the maturation state. TgCaM1, TgCaM2, and TgDLC are deposited onto the conoid (Anderson-White et al., 2011; Hu et al., 2006). At the posterior end of the bud, TgIMC5, 8, 9, and 13 are recruited to the basal complex which has been marked by TgMORN1 since the onset of bud formation (Anderson-White et al., 2011). At this point, TgSSNA1/DIP13 (Leveque et al., 2016), TgMyoJ (Fre´nal et al., 2017b), and TgCen2 (Hu, 2008) together with a phosphatase, TgHAD2a (Engelberg et al., 2016), are recruited to the basal complex. The posterior tapering of daughter buds relies on contractile activity driven by actin (Periz et al., 2017), TgMyoJ (Fre´nal et al., 2017b) and TgCen2 (Hu et al., 2006). 16.3.1.4 Emergence of daughter parasites Once daughter buds contain a complete set of organelles, their emergence requires several coordinated processes to substitute daughter IMC for mother IMC at the plasma membrane, to separate the plasma membrane at the interface between daughters, and to detach the posterior of daughters from remnants of the mother cell. A key event is the destruction of the maternal cytoskeleton, while the daughters’ cytoskeletons are stabilized. In a zippering process the mother’s cytoskeleton disassembles in an organized apical to basal direction (Morrissette and Sibley, 2002b), which simultaneously deposits the plasma membrane on the emerging daughter. How differential stability is achieved is not

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unequivocally determined but ubiquitination of the mother’s cytoskeleton likely destabilizes the mother (Dhara and Sinai, 2016; Silmon de Monerri et al., 2015). Alternatively, components differentially localizing to mother and daughter could confer differential stability, for example, TgIMC16 and 29 are specific to the daughter, whereas TgMSC1b, TgIMC7, 12, 14, 18, and 20 are unique to the mother cytoskeleton (Anderson-White et al., 2011; Lorestani et al., 2012; Chen et al., 2015a, 2017). The distinct composition of mother and daughters may contribute to differential stability during daughter emergence, although a critical function appears to be additional rigidification of the cytoskeleton (Dubey et al., 2017). Furthermore, a regulated proteolysis removes the C-terminus of the major network component, TgIMC1, which coincides with conversion of the network from a detergent-labile to a detergent-resistant state late in daughter cell development (Mann et al., 2002). TgASP1, a coccidian-specific aspartic protease, is found in close proximity to the emerging IMC of daughter cells during replication (Polonais et al., 2011b). Interestingly, some of the maternal components (e.g., IMC and glideosome proteins) are recycled in the final growth spurt (Ouologuem and Roos, 2014). It is not known whether maturation and recycling processes occur concurrently or are organized spatially. Although the maternal plasma membrane is inherited by emerging daughters, additional membrane is added in a Rab11a-dependent fashion to separate the lateral surfaces of daughter buds (Agop-Nersesian et al., 2009). The association of the plasma membrane with the daughter IMC is interconnected with maturation of the glideosome. TgMyoA, TgMLC1, and TgGAP45 assemble as a soluble “protoglideosome” complex which must interact with TgGAP50 in the daughter IMC to form a functional, pellicle-associated glideosome (Gaskins et al., 2004; Fre´nal et al., 2010; Powell et al., 2017). Once TgGAP45 is correctly located between the plasma membrane and IMC,

depalmitoylation of the N-terminus and palmitoylation of the C-terminus tacks TgGAP45 to the IMC, linking the IMC and plasma membrane. Conditional depletion of TgGAP45 disrupts the intimate association of the plasma membrane and IMC supporting its dual role in pellicle formation and assembly of the MyoA motor complex (Fre´nal et al., 2010). During the process of daughter IMC association with maternal plasma membrane, maternal IMC and apical organelles are discarded in the RB. These remnants are rapidly broken down and the RB contracts; sibling parasites remain tethered to the RB through cytoplasmic bridges that permit cytoplasmic exchange. This bridge must be severed prior to egress to activate parasite motility which requires the activity of an atypical guanylate cyclase fused to a flippase residing in the basal complex (Bisio et al., 2019; Yang et al., 2019). 16.3.1.5 The mature basal complex Nearly all proteins recruited to the basal complex during daughter bud formation are retained at the basal cap of mature parasites. Exceptions to this are TgSSNA1/DIP13 (Leveque et al., 2016) and to a lesser degree TgHAD2a (Engelberg et al., 2016). Several additional proteins such as TgMSC1a (Lorestani et al., 2012) and TgFIKK, the only representative of the FIKK kinase family in Toxoplasma (Skariah et al., 2016), are recruited to the basal complex following completion of cell division. These proteins, together with many additional proteins with the same basal complex dynamics (Engelberg and Gubbels, unpublished data), suggest that this structure harbors additional functions. However, since these proteins are not essential for completion of the lytic cycle, their roles are nonessential or required in other developmental stages. One logical role for these proteins would be a structural role in cytoskeletal organization, but since not all apicomplexan zoites maintain a basal complex following

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completion of cell division, this function is unlikely the complete story. The mature basal complex has been recently demonstrated to be essential for maintaining cytoplasmic bridges to the RB that maintain cytoplasmic exchange between parasites in the same vacuole (Fre´nal et al., 2017b). In fact, TgMyoI is dedicated to this function (Fre´nal et al., 2017b) whereas FRM3 and actin are also critical (Tosetti et al., 2019). F-actin forms a network that connects tachyzoites within the same vacuole (Periz et al., 2017). This network permits communication between parasites and keeps the cell division cycles between sibling parasites synchronized (Periz et al., 2017; Fre´nal et al., 2017b). In larger vacuoles and during bradyzoite replication these connections can get lost, likely due to mechanical forces on the infected cell, which additionally underscores that these connections are not essential. The strict need for this cytoplasmic bridge as well as the mature basal complex is unclear, although previous observations suggest a variety of roles for this portal. For example, host cell vesicles captured by the parasite and transported into the vacuole aggregate near the basal complex suggesting a putative uptake or digestive role (Romano et al., 2017). In addition, the basal complex of the parasites appears twisted and invaginated following invasion (Morisaki et al., 1995), possibly as a result of torsion to sever the parasitophorous vacuole membrane from the host cell plasma membrane (Pavlou et al., 2018). Moreover, the intravacuolar network (IVN) is assembled at this site; IVN assembly relies on dense granule secretion (Mercier et al., 1998) which may also require basal complex functions.

16.3.2 Motility, invasion, and egress Apicomplexan gliding motility is unique to this lineage and is distinct from amoeboid or cilia/flagella-based propulsive mechanisms

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(Fre´nal et al., 2017a,b). Acquisition of gliding motility permitted ancestral apicomplexans to cross biological barriers, greatly expanding their host range and tissue access prior to diversification of this lineage (Leander, 2008). In addition to extracellular movement, gliding motility drives host cell invasion (Fre´nal et al., 2017a,b), scission of the vacuolar membrane from the host cell plasma membrane (Pavlou et al., 2018), and efficient egress from host cell debris (Fre´nal et al., 2017a,b). Gliding motility requires the transport of apically secreted, membrane-spanning microneme proteins to the posterior end of the parasite where they are released from the pellicle by the activity of a rhomboid protease (Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection). Tachyzoites are capable of moving external beads in an apical to posterior direction along their surface, revealing the underlying behavior of secreted adhesins (Stadler et al., 2017). Gliding requires adhesin-substrate engagement and the parasite always moves in an apical (forward) direction. Motile forces are influenced by cytoplasmic calcium levels, F-actin assembly, and activity of TgAKMT. In the artificial twodimensional context of protein-coated glass slides, tachyzoites exhibit three distinct modes of motility: circular gliding, helical gliding, and twirling (Hakansson et al., 1999; Sibley et al., 1998). However, when tachyzoites are embedded in a three-dimensional matrix that better simulates a natural tissue environment, these behaviors are resolved as a uniform corkscrew motility pattern (Leung et al., 2014a). Studies in Toxoplasma have demonstrated that gliding motility requires the concerted action of signaling molecules, cytoskeletal components, secreted proteins, and a rhomboid protease. While myosins characteristically move along fixed F-actin tracks, motilityassociated myosins are immobilized at the conoid (TgMyoH) and the IMC (TgMyoA) and

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move short actin filaments along the space between the plasma membrane and the outer membrane of the IMC (Periz et al., 2017) to the tachyzoite posterior. The short cytoplasmic tail of the TgMIC2 adhesin interacts with TgGAC which also binds to F-actin. Initiation of motility requires apical secretion of TgMIC2, FRM1-induced polymerization of F-actin at the tachyzoite apex, and engagement of a TgMIC2GAC-F-actin complex by the conoid-localized TgMyoH (Fig. 16.4C). Once the TgMIC2GAC-F-actin complex has been moved to the base of the extended conoid, it is handed off to the IMC-immobilized TgMyoA motor which transports adhesins along the length of the parasite to its posterior end (Jacot et al., 2016). Productive motility requires posterior proteolysis of microneme-secreted adhesins (see Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection) to permit localized substrate release for continuous forward motion (Brossier et al., 2005; Shen et al., 2014). In addition to the necessary elements for gliding motility, tachyzoites require conoid extrusion and rhoptry secretion for the successful invasion of host cells (Mondragon and Frixione, 1996; Carey et al., 2004; Fre´nal et al., 2017a,b). Conoid extrusion is a reversible Ca21-mediated process that occurs during gliding motility and at the time of host cell invasion. To date, the mechanism underlying conoid extrusion has not been identified, although the critical role of TgMyoH in initiating host cell invasion explains at least in part why extrusion is essential. While microneme secretion is required for gliding motility, secretion of RONs (rhoptry neck proteins) from rhoptries is essential for host cell invasion. RONs are inserted into the host cell plasma membrane at the site of contact to form a moving junction which serves as an interface between parasite and host cell plasma membranes through which the parasite enters the host cell

(Chapter 14 “Toxoplasma secretory proteins and their roles in parasite cell cycle and infection”). Rhoptry secretion requires TgFER2, a member of the Ferlin family which is involved in membrane fusion and vesicle trafficking (Coleman et al., 2018). Once a host cell has been successfully invaded, modulation of TgPKA activity turns off all aspects of motility (Uboldi et al., 2018; Jia et al., 2017). Toxoplasma tachyzoites egress may occur after multiple rounds of replication as a consequence of parasite-mediated changes in the vacuolar compartment (Bisio et al., 2019) or be triggered prematurely by externally triggered environmental insults to the host cell (Tomita et al., 2009). Both pathways involve signaling cascades to activate egress from the vacuole and host cell (Chapter 13 “Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii”). Egress requires microneme secretion to facilitate parasitophorous vacuole and host plasma membrane disintegration as well as host cell cytoskeleton remodeling (Blackman and Carruthers, 2013; Chandramohanadas et al., 2009; Millholland et al., 2013). Gliding motility participates in effective egress to bring the lytic cycle to completion and to drive successive invasion events. 16.3.2.1 Glideosome assembly, activation, and regulation Gliding motility is controlled at multiple levels beginning with assembly of the glideosome and ending with signals required to engage motor activity. As the first level of control, all myosins require TgUNC for correctly folding (Fre´nal et al., 2017b; Bookwalter et al., 2014). As discussed above, glideosome assembly at the IMC is only completed when the plasma membrane is connected to the IMC. After a soluble complex of TgMyoA, TgMLC1, and TgGAP45 assembles, modifications of TgGAP45 by phosphorylation of S163 and S167, N-terminal depalmitoylation,

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and C-terminal palmitoylation are required to position this complex in its mature, pellicle-associated form (Powell et al., 2017;Gilk et al., 2009; Gaskins et al., 2004; Fre´nal et al., 2010). Furthermore, components of the glideosome vary along the tachyzoite longitudinal axis (Fig. 16.4C). From anterior to posterior, the MHC composition changes (TgMyoH to TgMyoA to TgMyoC) along with associated MLCs. Specific peripheral GAP components that connect the plasma membrane to the IMC and tether glideosome complexes also change (TgGAP70 to TgGAP45 to TgGAP80). Lastly, the most basal TgMyoC glideosome includes a novel component (IAP1) that tethers it to the posterior polar ring. Differences in component function likely contribute to the site-specific efficiency of the various glideosomes. For example, the TgGAP45 and TgGAP70 connectors that link the IMC and plasma membrane are of different lengths: TgGAP70 spans B33 nm at the AC region, while TgGAP45 occupies the remainder of the pellicle, which has closer plasma membrane IMC spacing of B25 nm (Fre´nal et al., 2010). A possible explanation for this difference relates to optimal curvature of the pellicle at the apical pole. Consistent with this theory, parasites lacking TgPhIL1 become shorter and wider, which decreases their trajectory length and mean and maximum velocity (Leung et al., 2014a). At the basal end of the pellicle the predominant TgMyoA glideosome is replaced by a somewhat parallel complex: the TgGAP45 homolog is replaced by TgGAP80 and TgMyoA is replaced by TgMyoC (both heavy chains share the same light chains). Although different glideosomes are tailored for specific spatial functions, there is some level of plasticity between the different complexes, such as the ability of TgMyoC to provide motility in the absence of TgMyoA (Fre´nal et al., 2014; Egarter et al., 2014). The unique role of TgMyoC at the basal complex is unclear. Gliding progresses at the same speed for

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parasites relying on either TgMyoA or TgMyoC; however, host cell invasion is impaired and progresses in a stop-and-go fashion for TgMyoA-deficient tachyzoites that use TgMyoC in its place (Egarter et al., 2014). TgMyoA glideosome activity is controlled by the direct binding of Ca21 to TgELC1 and TgELC2 which stabilize the interaction of TgMLC1 with TgMyoA (Nebl et al., 2011; Williams et al., 2015). Although these proteins are functionally redundant, their presence and Ca21-bound state contribute to the stability of the TgMyoA lever arm, thereby regulating the quality, displacement, and speed of gliding (Williams et al., 2015). The critical importance of TgMLC1 for glideosome activity is further evident from characterization of a small molecule inhibitor of motility, TachypleginA (Heaslip et al., 2010b), which covalently conjugates with thiol groups to modify TgMLC1 (Heaslip et al., 2010b; Leung et al., 2014b). Data from two studies that captured Ca21dependent phosphorylation events suggest that TgMyoA glideosome activity is further regulated by phosphorylation (Nebl et al., 2011; Treeck et al., 2011). Notably, Ca21dependent phosphorylation of six residues in TgGAP45, TgMLC1, and TgMyoA was correlated with motor activity and Ca21 signaling changes the structure or assembly of the TgMyoA glideosome (Nebl et al., 2011). Direct evidence for TgMyoA regulation by Ca21dependent phosphorylation is derived from a small molecule enhancer of motility and invasion (Tang et al., 2014). Reciprocally, parasites expressing a nonphosphorylatable mutant myosin exhibit slower host cell egress after treatment with calcium ionophore. In summary, assembly, localization, and specific components for distinct glideosomes combined with direct Ca21 binding and posttranslational modifications, such as phosphorylation, provide ample modulatory flexibility to tailor control of gliding at specific times, places, and functional niches.

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16.3.2.2 Actin polymerization for gliding motility in particular Spatiotemporal control of actin polymerization is critical to activation of gliding motility. Relative to many eukaryotes, apicomplexan genomes contain genes for a limited set of actin-interacting proteins (Baum et al., 2006). Furthermore, differences in the requirement for these proteins throughout the parasite life cycle indicate that they are not equally critical during all cellular programs (Sattler et al., 2011; Matuschewski and Schuler, 2008). In vitro, Toxoplasma actin polymerizes into extremely short (100 nm), unstable filaments (Sahoo et al., 2006; Skillman et al., 2011; Pospich et al., 2017). Current observations are consistent with a model whereby filaments are formed at the conoid and spatiotemporal polymerization dynamics control the directionality and timing of motility as well as other processes requiring F-actin (Periz et al., 2017; Stadler et al., 2017; Tosetti et al., 2019). TgCAP phosphorylation has been associated with a role in the speed of egress indicating it is a critical regulator (Treeck et al., 2014). Apical F-actin assembly is exclusively driven by TgFRM1 which is anchored at the conoid (Jacot et al., 2016; Tosetti et al., 2019). TgFRM1 produces actin filaments that translocate along the pellicle, through the successive activity of MyoH and MyoA, to accumulate at the basal pole. Inhibitor and protein depletion studies indicate that Ca21-dependent signaling drives egress through activity of CDPK1 and TgAKMT to induce F-actin polymerization (Heaslip et al., 2011; Tosetti et al., 2019). During apical to basal transport, F-actin is stabilized by external contact of the pellicle. During invasion, this is visible as a ring of F-actin that moves from the apical tip to accumulate at the posterior pole (Tosetti et al., 2019). Similarly, F-actin is visible at sites of contact between the parasite and the substratum and this adhesion is required for gliding (Periz et al., 2017; Tosetti et al., 2019). Two G-actin sequestering proteins,

TgCAP and TgADF, likely function to regulate motility by limiting TgFRM1-driven F-actin assembly. Loss of TgCAP severely reduces tachyzoite fitness and significantly impairs gliding motility as well as invasion and egress capacity (Hunt et al., 2019). As many actindependent processes (except apicoplast division) are affected, TgCAP may promote actin turnover in diverse contexts. The observation that parasites lacking TgCAP have dense granule motility suggests that TgCAP is likely to be a negative regulator of actin polymerization. Like TgCAP, TgADF likely regulates F-actin in many contexts. It functions to sequester ADPloaded G-actin and influences both motility and apicoplast segregation (Mehta and Sibley, 2010; Haase et al., 2015). Taken together, these results indicate that motility is regulated by controlling motor assembly and activity and by restricting access to F-actin. 16.3.2.3 Mechanism of conoid extrusion During gliding motility the conoid continuously extrudes and retracts, likely in response to fluctuations in cytoplasmic Ca21 levels (Wetzel et al., 2004). A range of pharmacological agents that interfere with Ca21-signaling and homeostasis blocks conoid extrusion (Del Carmen et al., 2009; Mondragon and Frixione, 1996). Since kinase inhibitors inhibit conoid extrusion, CDPKs may also be required for this process. Inhibition of F-actin (with cytochalasin D) or myosin (with BDM) suggests that extrusion requires the actinomyosin machinery. Although the conoid provides the cytoskeletal anchor for TgMyoH, depletion or manipulation of the TgMyoH motor has minimal effects on conoid extrusion (Long et al., 2017b; Graindorge et al., 2016) but TgMyoE and/or TgMyoL residing in the conoid might be relevant for extrusion (Fig. 16.4B). Conoid extrusion is specifically inhibited by a small molecule, named conoidin A (Carey et al., 2004; Haraldsen et al., 2009). Interestingly,

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conoidin A inhibits invasion without affecting microneme secretion or motility and thus implicates conoid extrusion as a critical element in host cell invasion. Recent characterization of two conoid proteins, TgDCX and TgCPH1, indicates that structural integrity of the conoid is required for parasite motility and host cell invasion (Long et al., 2017a; Nagayasu et al., 2017). 16.3.2.4 The role of the host cell in invasion and egress In addition to regulating its endogenous cytoskeleton, Toxoplasma tachyzoites manipulate the host cytoskeleton to enhance its ability to enter and exploit host cells. The most dramatic example of this is the insertion of a moving junction in the host cell plasma membrane at the site of entry (see Chapter 14 “Toxoplasma secretory proteins and their roles in parasite cell cycle and infection”, for details on the moving junction). RON proteins in the moving junction interface with the host cell actin cytoskeleton as well as with CIN85, CD2AP and the ESCRT-I components ALIX and TSG101 (Guerin et al., 2017). Parasite mutants unable to recruit these host proteins show a significant reduction in host cell invasion as does host cell depletion of ALIX and TSG101 (Pavlou et al., 2018; Guerin et al., 2017). In addition, during host cell invasion tachyzoites secrete toxofilin from rhoptries to induce host actin depolymerization in the vicinity of the parasite, to locally reduce the host cell cortical actin network (Bichet et al., 2014; Czimbalek et al., 2015; Delorme-Walker et al., 2012; Lodoen et al., 2010; Poupel et al., 2000). Local disruption of cortical actin facilitates plasma membrane invagination at the point of entry to enable parasitophorous vacuole biogenesis (Delorme-Walker et al., 2012). A screen for host cell genes influencing tachyzoite invasion identified six antagonists of the actin cytoskeleton, further supporting the critical role of host cell actin (Gaji et al., 2013). Studies with TgMyoA-deficient tachyzoites

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reveal that during host cell entry, the host cell forms actin-containing membrane protrusions around the parasite that are parasite-initiated and not due to host cell micropinocytosis (Bichet et al., 2016). Host microtubules also accumulate around the moving junction (Sweeney et al., 2010) and may be tethered to RON4 (Takemae et al., 2013). Although pharmacological disruption of host microtubules reduces parasite invasion efficiency, this effect is most pronounced for parasites that invade rapidly (Sweeney et al., 2010). Host microtubules may assist in forming or tethering the moving junction, may act as a scaffold to resist compression induced by parasite contact with the host cell, or operate as a fulcrum so that gliding parasites can reorient for apical contact prior to host cell entry. Toxoplasma tachyzoites interact with host cells in diverse and incompletely understood ways to mediate dissemination within the host organism, obtain nutrients for growth and modify immune response to infection. In particular, Toxoplasma uses motility to interact with its host cell to separate the nascent parasitophorous vacuole from invaginated host plasma membrane and to efficiently egress from host cell debris. Completion of host cell invasion requires membrane scission to seal and separate the nascent vacuole from host plasma membrane. Although host cell ESCRT or dynamin machinery has been implicated in moving junction formation (Guerin et al., 2017), membrane scission is exclusively mediated by the parasite (Pavlou et al., 2018). Membrane fission at the site of the RON protein complex is mechanically driven by the newly invaded parasite completing a 360degree rotation inside the vacuole. The specific mechanism of how the parasite generates this torsion remains unknown, but it is likely is actin-myosin mediated. Finally, parasite egress requires the activity of host cell calpains, which remodel the host plasma membrane and cytoskeleton to create an escape tunnel for the

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parasites (Subramanian et al., 2018). Since host cell modifying behaviors are only beginning to be identified, it is likely that future research will identify additional parasite behaviors that manipulate the host cell during invasion, intracellular growth, and egress.

16.3.3 Other critical roles for Toxoplasma actin In addition to the roles of Toxoplasma actin in gliding motility, RB formation and cell cell communication, parasite F-actin is also associated with centrosome positioning, apicoplast division, cell division, localization and trafficking of organelles and bradyzoite differentiation. These functions have been discerned through the use of genetic strategies to discern the roles of actin (Whitelaw et al., 2017; Periz et al., 2017; Drewry and Sibley, 2015), myosins (Fre´nal et al., 2014, 2017b; Graindorge et al., 2016), formins (Tosetti et al., 2019), and TgCAP (Hunt et al., 2019). Intracellular growth and replication necessitate complex interactions between Toxoplasma cytoskeletal elements, signaling proteins and organelles. Indeed, recent data suggest that correct regulation of actinbased processes is critical for establishing chronic infection in mice (Hunt et al., 2019). An example of this complexity of function is provided by the numerous factors that influence apicoplast replication and segregation during endodyogeny. Centrosomes associate with the apicoplast in a cell cycle dependent fashion (Vaishnava et al., 2005; Chen et al., 2015a) that is required for apicoplast division (Striepen et al., 2000). A dynamin-related protein, TgDrpA, is required for apicoplast membrane scission (van Dooren et al., 2009), but correct partitioning to daughter buds relies on additional factors including TgMORN1 (Lorestani et al., 2010), TgMyoF (Jacot et al., 2013), and TgFRM2 (Tosetti et al., 2019). Perturbations of either TgMyoF or TgFRM2 also induce

mis-localization of the centrosomes with respect to the nucleus (Jacot et al., 2013; Tosetti et al., 2019). This causes mis-oriented daughter buds and downstream defects in organelle segregation that cause excess deposition of organelles in the RB. Moreover, it is likely that errors in apicoplast inheritance are a consequence of defects stemming from centrosome mis-orientation. The centrosome disengages from the apicoplast prior to transiting from the apical to basal side of the nucleus where it duplicates (Chen et al., 2015b; Hartmann et al., 2006). Once duplicated centrosomes return to an apical position, they must reestablish an apicoplast connection. The centrosome position defects in TgMyoF and TgFRM2 mutants have been interpreted as direct effects on the centrosome, but since the centrosome remains anchored to the centrocone during its division cycle (Chen et al., 2015b), it is likely that the whole nucleus rotates by a TgMyoF- and TgFRM2-dependent process. In this case, both the bud orientation and apicoplast division phenotypes would be consequences of a defective nucleus and centrosome migration process. Regardless of the exact mechanism, centrosome orientation and apicoplast segregation require TgMyoF and TgFRM2. Lastly, TgMyoF also has a role in transport and positioning of a variety of parasite organelles (Heaslip et al., 2016). It drives directed transport of dense granules though the cytoplasm (Heaslip et al., 2016). Dense granule contents are not secreted at the apical end; proposed sites for their release include through IMC apertures at the apical annuli or the basal end (Dubremetz et al., 1993), which might correspond with demand and require the observed dynamic nature of dense granules. Dense granule motility corresponds with the highly dynamic cytoplasmic F-actin filaments visualized with the actin chromobody tool (Periz et al., 2017). In fact, chromobody overexpression causes hyperstabilized F-actin and arrests dense granule motility. In association with

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TgARO (Mueller et al., 2013), TgMyoF also plays a role in anchoring rhoptries at the apical end of the tachyzoite; there is no evidence of a role for actin or TgFRM2 in microneme localization or secretion.

16.4 Summary: a story of adaptation, loss, and novel components Research on the Toxoplasma cytoskeleton has revealed that this parasite employs a mixture of conserved and novel proteins in order to replicate, move and invade cells. Analysis of the genome databases for Toxoplasma and other apicomplexans has identified a reduced subset of the repertoire of defined cytoskeletal proteins used by other eukaryotes. Moreover, in many cases, apicomplexan homologs of conserved cytoskeletal elements have evolved altered biochemical properties to best serve the particular requirements of these parasites. Given the unusual structures and behaviors of the Toxoplasma cytoskeleton, it is likely that we have only scraped the surface of this divergent biology. Future research is certain to uncover additional examples of cytoskeletal elements with modified biochemistry or novel functions, novel proteins, and surprising losses during the evolution of Toxoplasma and other apicomplexans. Differences between the Toxoplasma cytoskeleton and the cytoskeleton of metazoans represent key targets that can be exploited by future therapeutic agents.

Acknowledgments We would like to thank our colleagues for useful discussions and/or feedback on this chapter. We thank Izra Abbaali for critically reviewing the manuscript. This work was supported by National Institutes of Health grants AI122923 and AI128136.

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References Agop-Nersesian, C., Naissant, B., Ben rached, F., Rauch, M., Kretzschmar, A., Thiberge, S., et al., 2009. Rab11Acontrolled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis. PLoS Pathog. 5, e1000270. Alvarez, C.A., Suvorova, E.S., 2017. Checkpoints of apicomplexan cell division identified in Toxoplasma gondii. PLoS Pathog. 13, e1006483. Andenmatten, N., Egarter, S., Jackson, A.J., Jullien, N., Herman, J.P., Meissner, M., 2013. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods 10, 125 127. Anderson-White, B.R., Ivey, F.D., Cheng, K., Szatanek, T., Lorestani, A., Beckers, C.J., et al., 2011. A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii. Cell. Microbiol. 13, 18 31. Arnot, D.E., Ronander, E., Bengtsson, D.C., 2011. The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony. Int. J. Parasitol. 41, 71 80. Asai, D.J., Wilkes, D.E., 2004. The dynein heavy chain family. J. Eukaryot. Microbiol. 51, 23 29. Bane, K.S., Lepper, S., Kehrer, J., Sattler, J.M., Singer, M., Reinig, M., et al., 2016. The actin filament-binding protein coronin regulates motility in Plasmodium sporozoites. PLoS Pathog. 12, e1005710. Baum, J., Richard, D., Healer, J., Rug, M., Krnajski, Z., Gilberger, T.W., et al., 2006. A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. J. Biol. Chem. 281, 5197 5208. Beck, J.R., Rodriguez-Fernandez, I.A., de Leon, J.C., Huynh, M.H., Carruthers, V.B., Morrissette, N.S., et al., 2010. A novel family of Toxoplasma IMC proteins displays a hierarchical organization and functions in coordinating parasite division. PLoS Pathog. 6, e1001094. Berry, L., Chen, C.T., Reininger, L., Carvalho, T.G., El hajj, H., Morlon-Guyot, J., et al., 2016. The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence. Cell. Microbiol. 18, 1106 1120. Berry, L., Chen, C.T., Francia, M.E., Guerin, A., Graindorge, A., Saliou, J.M., et al., 2018. Toxoplasma gondii chromosomal passenger complex is essential for the organization of a functional mitotic spindle: a prerequisite for productive endodyogeny. Cell Mol. Life Sci. 75, 4417 4443. Bichet, M., Joly, C., Henni, A.H., Guilbert, T., Xemard, M., Tafani, V., et al., 2014. The Toxoplasma-host cell junction

Toxoplasma Gondii

780

16. The Toxoplasma cytoskeleton: structures, proteins, and processes

is anchored to the cell cortex to sustain parasite invasive force. BMC Biol. 12, 773. Bichet, M., Touquet, B., Gonzalez, V., Florent, I., Meissner, M., Tardieux, I., 2016. Genetic impairment of parasite myosin motors uncovers the contribution of host cell membrane dynamics to Toxoplasma invasion forces. BMC Biol. 14, 97. Bisio, H., Lunghi, M., Brochet, M., Soldati-Favre, D., 2019. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. Nat. Microbiol. 4, 420 428. Blackman, M.J., Carruthers, V.B., 2013. Recent insights into apicomplexan parasite egress provide new views to a kill. Curr. Opin. Microbiol. 16, 459 464. Blader, I.J., Coleman, B.I., Chen, C.T., Gubbels, M.J., 2015. Lytic cycle of Toxoplasma gondii: 15 years later. Annu. Rev. Microbiol. 69, 463 485. Bookwalter, C.S., Kelsen, A., Leung, J.M., Ward, G.E., Trybus, K.M., 2014. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility. J. Biol. Chem. 289, 30832 30841. Bosch, J., Paige, M.H., Vaidya, A.B., Bergman, L.W., Hol, W.G., 2012. Crystal structure of GAP50, the anchor of the invasion machinery in the inner membrane complex of Plasmodium falciparum. J. Struct. Biol. 178, 61 73. Brooks, C.F., Francia, M.E., Gissot, M., Croken, M.M., Kim, K., Striepen, B., 2011. Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 108, 3767 3772. Brossier, F., Jewett, T.J., Sibley, L.D., Urban, S., 2005. A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc. Natl. Acad. Sci. U.S.A. 102, 4146 4151. Brugerolle, G., 2002. Colpodella vorax: ultrastructure, predation, life-cycle, mitosis, and phylogenetic relationships. Eur. J. Protistol. 38, 113 125. Bullen, H.E., Tonkin, C.J., O’donnell, R.A., Tham, W.-H., Papenfuss, A.T., Gould, S., et al., 2009. A novel family of apicomplexan glideosome-associated proteins with an inner membrane-anchoring role. J. Biol. Chem. 284, 25353 25363. Butler, C.L., Lucas, O., Wuchty, S., Xue, B., Uversky, V.N., White, M., 2014. Identifying novel cell cycle proteins in Apicomplexa parasites through co-expression decision analysis. PLoS One 9, e97625. Caldas, L.A., Seabra, S.H., Attias, M., De souza, W., 2013. The effect of kinase, actin, myosin and dynamin inhibitors on host cell egress by Toxoplasma gondii. Parasitol. Int. 62, 475 482. Carey, K.L., Westwood, N.J., Mitchison, T.J., Ward, G.E., 2004. A small-molecule approach to studying invasive

mechanisms of Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 101, 7433 7438. Cavalier-Smith, T., Chao, E.E., 2004. Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.). Eur. J. Protistol. 40, 185 212. Chandramohanadas, R., Davis, P.H., Beiting, D.P., Harbut, M.B., Darling, C., Velmourougane, G., et al., 2009. Apicomplexan parasites co-opt host calpains to facilitate their escape from infected cells. Science 324, 794 797. Chen, C.T., Gubbels, M.J., 2013. The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant. J. Cell Sci. 126, 3344 3355. Chen, C.T., Gubbels, M.J., 2015. Apicomplexan cell cycle flexibility: centrosome controls the clutch. Trends Parasitol. 31, 229 230. Chen, C.-T., Gubbels, M.-J., 2019. TgCep250 is dynamically processed through the division cycle and is essential for structural integrity of the Toxoplasma centrosome. Mol. Biol. Cell 30, 1160 1169. Available from: https://doi. org/10.1091/mbc.E18-10-0608. Chen, A.L., Kim, E.W., Toh, J.Y., Vashisht, A.A., Rashoff, A.Q., Van, C., et al., 2015a. Novel components of the Toxoplasma inner membrane complex revealed by BioID. MBio 6, e02357 14. Chen, C.T., Kelly, M., Leon, J., Nwagbara, B., Ebbert, P., Ferguson, D.J., et al., 2015b. Compartmentalized Toxoplasma EB1 bundles spindle microtubules to secure accurate chromosome segregation. Mol. Biol. Cell 26, 4562 4576. Chen, A.L., Moon, A.S., Bell, H.N., Huang, A.S., Vashisht, A.A., Toh, J.Y., et al., 2017. Novel insights into the composition and function of the Toxoplasma IMC sutures. Cell. Microbiol. 19, e12678. Chobotar, W., Scholtyseck, E., 1982. Ultrastructure. In: Long, P.L. (Ed.), The Biology of the Coccidia. University Park Press, Baltimore. Coleman, B.I., Saha, S., Sato, S., Engelberg, K., Ferguson, D. J.P., Coppens, I., et al., 2018. A member of the ferlin calcium sensor family is essential for Toxoplasma gondii rhoptry secretion. mBio 9, e01510 e01518. Courjol, F., Gissot, M., 2018. A coiled-coil protein is required for coordination of karyokinesis and cytokinesis in Toxoplasma gondii. Cell. Microbiol. 20, e12832. Czimbalek, L., Kollar, V., Kardos, R., Lorinczy, D., Nyitrai, M., Hild, G., 2015. The effect of toxofilin on the structure and dynamics of monomeric actin. FEBS Lett. 589, 3085 3089. Daher, W., Plattner, F., Carlier, M.F., Soldati-Favre, D., 2010. Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii. PLoS Pathog. 6, e1001132.

Toxoplasma Gondii

References

Daher, W., Klages, N., Carlier, M.F., Soldati-Favre, D., 2012. Molecular characterization of Toxoplasma gondii formin 3, an actin nucleator dispensable for tachyzoite growth and motility. Eukaryot. Cell. 11, 343 352. de Leon, J.C., Scheumann, N., Beatty, W., Beck, J.R., Tran, J. Q., Yau, C., et al., 2013. A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved from flagellar components. Eukaryot. Cell 12, 1009 1019. Del carmen, M.G., Mondragon, M., Gonzalez, S., Mondragon, R., 2009. Induction and regulation of conoid extrusion in Toxoplasma gondii. Cell. Microbiol. 11, 967 982. Delbac, F., Sanger, A., Neuhaus, E.M., Stratmann, R., Ajioka, J.W., Toursel, C., et al., 2001. Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division. J. Cell Biol. 155, 613 623. Delorme-Walker, V., Abrivard, M., Lagal, V., Anderson, K., Perazzi, A., Gonzalez, V., et al., 2012. Toxofilin upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion. J. Cell Sci. 125, 4333 4342. D’haese, J., Mehlhorn, H., Peters, W., 1977. Comparative electron microscope study of pellicular structures in coccidia (Sarcocystis, Besnoitia and Eimeria). Int. J. Parasitol. 7, 505 518. Dhara, A., Sinai, A.P., 2016. A cell cycle-regulated Toxoplasma deubiquitinase, TgOTUD3A, targets polyubiquitins with specific lysine linkages. mSphere 1, e00085 16. Dobrowolski, J.M., Sibley, L.D., 1996. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933 939. Dobrowolski, J.M., Carruthers, V.B., Sibley, L.D., 1997. Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii. Mol. Microbiol. 26, 163 173. Drewry, L.L., Sibley, L.D., 2015. Toxoplasma actin is required for efficient host cell invasion. mBio 6, e00557 15. Dubey, R., Harrison, B., Dangoudoubiyam, S., Bandini, G., Cheng, K., Kosber, A., et al., 2017. Differential roles for inner membrane complex proteins across Toxoplasma gondii and Sarcocystis neurona development. mSphere 2, e00409 17. Dubremetz, J.F., Torpier, G., 1978. Freeze fracture study of the pellicle of an eimerian sporozoite (Protozoa, Coccidia). J. Ultrastruct. Res. 62, 94 109. Dubremetz, J.F., Achbarou, A., Bermudes, D., Joiner, K.A., 1993. Kinetics and pattern of organelle exocytosis during Toxoplasma gondii/host-cell interaction. Parasitol. Res. 79, 402 408. Dutcher, S.K., 2004. Dissection of basal body and centriole function in the unicellular green alga Chlamydomonas

781

reinhardtii. In: Nigg, E.A. (Ed.), Centrosomes in development and disease. Wiley-VCH. Egarter, S., Andenmatten, N., Jackson, A.J., Whitelaw, J.A., Pall, G., Black, J.A., et al., 2014. The Toxoplasma ActoMyoA motor complex is important but not essential for gliding motility and host cell invasion. PLoS One 9, e91819. Engelberg, K., Ivey, F.D., Lin, A., Kono, M., Lorestani, A., Faugno-Fusci, D., et al., 2016. A MORN1-associated HAD phosphatase in the basal complex is essential for Toxoplasma gondii daughter budding. Cell. Microbiol. 18, 1153 1171. Engelberg, K., Chen, C.T., Bechtel, T., Guzman, V., Stoneburger, E., Drozda, A., et al., 2020. The apical annuli of Toxoplasma gondii are embedded in IMC sutures and composed of centrosome-ralated coiled-coil and signalling proteins. Cell Microbiol. 22(1), e13112. https://doi.org/10.1111/cmi.13112. Farrell, M., Gubbels, M.J., 2014. The Toxoplasma gondii kinetochore is required for centrosome association with the centrocone (spindle pole). Cell. Microbiol. 16, 78 94. Ferguson, D.J., Hutchison, W.M., Dunachie, J.F., Siim, J.C., 1974. Ultrastructural study of early stages of asexual multiplication and microgametogony of Toxoplasma gondii in the small intestine of the cat. Acta Pathol. Microbiol. Scand., B: Microbiol. Immunol. 82, 167 181. Ferguson, D.J., Hutchison, W.M., Siim, J.C., 1975. The ultrastructural development of the macrogamete and formation of the oocyst wall of Toxoplasma gondii. Acta Pathol. Microbiol. Scand., B: Microbiol. Immunol. 83, 491 505. Ferguson, D.J., Sahoo, N., Pinches, R.A., Bumstead, J.M., Tomley, F.M., Gubbels, M.J., 2008. MORN1 has a conserved role in asexual and sexual development across the apicomplexa. Eukaryot. Cell 7, 698 711. Foth, B.J., Goedecke, M.C., Soldati, D., 2006. New insights into myosin evolution and classification. Proc. Natl. Acad. Sci. U.S.A. 103, 3681 3686. Francia, M.E., Striepen, B., 2014. Cell division in apicomplexan parasites. Nat. Rev. Microbiol. 12, 125 136. Francia, M.E., Jordan, C.N., Patel, J.D., Sheiner, L., Demerly, J.L., Fellows, J.D., et al., 2012. Cell division in apicomplexan parasites is organized by a homolog of the striated rootlet fiber of algal flagella. PLoS Biol. 10, e1001444. Francia, M.E., Dubremetz, J.F., Morrissette, N.S., 2015. Basal body structure and composition in the apicomplexans Toxoplasma and Plasmodium. Cilia 5, 3. Fre´nal, K., Polonais, V., Marq, J.B., Stratmann, R., Limenitakis, J., Soldati-Favre, D., 2010. Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe 8, 343 357.

Toxoplasma Gondii

782

16. The Toxoplasma cytoskeleton: structures, proteins, and processes

Fre´nal, K., Tay, C.L., Mueller, C., Bushell, E.S., Jia, Y., Graindorge, A., et al., 2013. Global analysis of apicomplexan protein S-acyl transferases reveals an enzyme essential for invasion. Traffic 14, 895 911. Fre´nal, K., Marq, J.B., Jacot, D., Polonais, V., Soldati-Favre, D., 2014. Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion. PLoS Pathog. 10, e1004504. Fre´nal, K., Dubremetz, J.F., Lebrun, M., Soldati-Favre, D., 2017a. Gliding motility powers invasion and egress in Apicomplexa. Nat. Rev. Microbiol. 15, 645 660. Fre´nal, K., Jacot, D., Hammoudi, P.-M., Graindorge, A., Maco, B., Soldati-Favre, D., 2017b. Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat. Commun. 8, 15710. Fung, C., Beck, J.R., Robertson, S.D., Gubbels, M.J., Bradley, P.J., 2012. Toxoplasma ISP4 is a central IMC subcompartment protein whose localization depends on palmitoylation but not myristoylation. Mol. Biochem. Parasitol. 184, 99 108. Gaji, R.Y., Huynh, M.H., Carruthers, V.B., 2013. A novel high throughput invasion screen identifies host actin regulators required for efficient cell entry by Toxoplasma gondii. PLoS One 8, e64693. Gandhi, M., Goode, B.L., 2008. Coronin: the double-edged sword of actin dynamics. Subcell Biochem. 48, 72 87. Gaskins, E., Gilk, S., Devore, N., Mann, T., Ward, G., Beckers, C., 2004. Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J. Cell Biol. 165, 383 393. Gilk, S.D., Raviv, Y., Hu, K., Murray, J.M., Beckers, C.J., Ward, G.E., 2006. Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide. Eukaryot. Cell 5, 1622 1634. Gilk, S.D., Gaskins, E., Ward, G.E., Beckers, C.J., 2009. GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex. Eukaryot. Cell 8, 190 196. Goldman, M., Carver, R.K., Sulzer, A.J., 1958. Reproduction of Toxoplasma gondii by internal budding. J. Parasitol. 44, 161 171. Goode, B.L., Eck, M.J., 2007. Mechanism and function of formins in the control of actin assembly. Annu. Rev. Biochem. 76, 593 627. Goodenough, U., Roth, R., Kariyawasam, T., He, A., Lee, J.H., 2018. Epiplasts: membrane skeletons and epiplastin proteins in euglenids, glaucophytes, cryptophytes, ciliates, dinoflagellates, and apicomplexans. MBio 9, e02020 18. Gordon, J.L., Sibley, L.D., 2005. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites. BMC Genomics 6, 179.

Gordon, J.L., Beatty, W.L., Sibley, L.D., 2008. A novel actinrelated protein is associated with daughter cell formation in Toxoplasma gondii. Eukaryot. Cell 7, 1500 1512. Gordon, J.L., Buguliskis, J.S., Buske, P.J., Sibley, L.D., 2010. Actin-like protein 1 (ALP1) is a component of dynamic, high molecular weight complexes in Toxoplasma gondii. Cytoskeleton (Hoboken) 67, 23 31. Gould, S.B., Kraft, L.G., Van dooren, G.G., Goodman, C.D., Ford, K.L., Cassin, A.M., et al., 2011. Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates. Mol. Biol. Evol. 28, 1319 1331. Graindorge, A., Fre´nal, K., Jacot, D., Salamun, J., Marq, J.B., Soldati-Favre, D., 2016. The conoid associated motor MyoH is indispensable for Toxoplasma gondii entry and exit from host cells. PLoS Pathog. 12, e1005388. Gubbels, M.J., Wieffer, M., Striepen, B., 2004. Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among Apicomplexa. Mol. Biochem. Parasitol. 137, 99 110. Gubbels, M.J., White, M., Szatanek, T., 2008. The cell cycle and Toxoplasma gondii cell division: tightly knit or loosely stitched? Int. J. Parasitol. 38, 1343 1358. Guerin, A., Corrales, R.M., Parker, M.L., Lamarque, M.H., Jacot, D., El hajj, H., et al., 2017. Efficient invasion by Toxoplasma depends on the subversion of host protein networks. Nat. Microbiol. 2, 1358 1366. Haase, S., Zimmermann, D., Olshina, M.A., Wilkinson, M., Fisher, F., Tan, Y.H., et al., 2015. Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites. Mol. Biol. Cell 26, 3001 3012. Hakansson, S., Morisaki, H., Heuser, J., Sibley, L.D., 1999. Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion. Mol. Biol. Cell 10, 3539 3547. Haraldsen, J.D., Liu, G., Botting, C.H., Walton, J.G.A., Storm, J., Phalen, T.J., et al., 2009. Identification of conoidin A as a covalent inhibitor of peroxiredoxin II. Org. Biomol. Chem. Org. Biomol. Chem. 7, 3040 3048. Harding, C.R., Meissner, M., 2014. The inner membrane complex through development of Toxoplasma gondii and Plasmodium. Cell. Microbiol. 16, 632 641. Harding, C.R., Gow, M., Kang, J.H., Shortt, E., Manalis, S.R., Meissner, M., et al., 2019. Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii. Nat. Commun. 10, 401. Harper, J.D., Thuet, J., Lechtreck, K.F., Hardham, A.R., 2009. Proteins related to green algal striated fiber assemblin are present in stramenopiles and alveolates. Protoplasma 236, 97 101. Hartmann, J., Hu, K., He, C.Y., Pelletier, L., Roos, D.S., Warren, G., 2006. Golgi and centrosome cycles in Toxoplasma gondii. Mol. Biochem. Parasitol. 145, 125 127.

Toxoplasma Gondii

References

Heaslip, A.T., Ems-Mcclung, S.C., Hu, K., 2009. TgICMAP1 is a novel microtubule binding protein in Toxoplasma gondii. PLoS One 4, e7406. Heaslip, A.T., Dzierszinski, F., Stein, B., Hu, K., 2010a. TgMORN1 is a key organizer for the basal complex of Toxoplasma gondii. PLoS Pathog. 6, e1000754. Heaslip, A.T., Leung, J.M., Carey, K.L., Catti, F., Warshaw, D.M., Westwood, N.J., et al., 2010b. A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity. PLoS Pathog. 6, e1000720. Heaslip, A.T., Nishi, M., Stein, B., Hu, K., 2011. The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase. PLoS Pathog. 7, e1002201. Heaslip, A.T., Nelson, S.R., Warshaw, D.M., 2016. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments. Mol. Biol. Cell 27, 2080 2089. Heintzelman, M.B., Schwartzman, J.D., 1999. Characterization of myosin-A and myosin-C: two class XIV unconventional myosins from Toxoplasma gondii. Cell Motil. Cytoskeleton 44, 58 67. Heintzelman, M.B., Schwartzman, J.D., 2001. Myosin diversity in Apicomplexa. J. Parasitol. 87, 429 432. Herm-Gotz, A., Weiss, S., Stratmann, R., Fujita-Becker, S., Ruff, C., Meyhofer, E., et al., 2002. Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor. Embo. J. 21, 2149 2158. Herm-Gotz, A., Delbac, F., Weiss, S., Nyitrai, M., Stratmann, R., Tomavo, S., et al., 2006. Functional and biophysical analyses of the class XIV Toxoplasma gondii myosin D. J. Muscle Res. Cell Motil. 27, 139 151. Hettmann, C., Herm, A., Geiter, A., Frank, B., Schwarz, E., Soldati, T., et al., 2000. A dibasic motif in the tail of a class XIV apicomplexan myosin is an essential determinant of plasma membrane localization. Mol. Biol. Cell 11, 1385 1400. Hodges, M.E., Scheumann, N., Wickstead, B., Langdale, J. A., Gull, K., 2010. Reconstructing the evolutionary history of the centriole from protein components. J. Cell Sci. 123, 1407 1413. Hook, P., Vallee, R.B., 2006. The dynein family at a glance. J. Cell Sci. 119, 4369 4371. Hu, K., 2008. Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii. PLoS Pathog. 4, e10. Hu, K., Roos, D.S., Murray, J.M., 2002. A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J. Cell Biol. 156, 1039 1050. Hu, K., Johnson, J., Florens, L., Fraunholz, M., Suravajjala, S., Dilullo, C., et al., 2006. Cytoskeletal components of

783

an invasion machine—the apical complex of Toxoplasma gondii. PLoS Pathog. 2, e13. Hunt, A., Wagener, J., Kent, R., Carmeille, R., Russell, M., Peddie, C., et al., 2019. Differential requirements of cyclase associated protein (CAP) for actin turnover during the lytic cycle of Toxoplasma gondii, eLife. 8, e50598. Jacot, D., Daher, W., Soldati-Favre, D., 2013. Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance. EMBO J. 32, 1702 1716. Jacot, D., Tosetti, N., Pires, I., Stock, J., Graindorge, A., Hung, Y.F., et al., 2016. An apicomplexan actin-binding protein serves as a connector and lipid sensor to coordinate motility and invasion. Cell Host Microbe 20, 731 743. Janke, C., Montagnac, G., 2017. Causes and consequences of microtubule acetylation. Curr. Biol. 27, R1287 R1292. Jia, Y., Marq, J.B., Bisio, H., Jacot, D., Mueller, C., Yu, L., et al., 2017. Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii. EMBO J. 36, 3250 3267. Johnson, T.M., Rajfur, Z., Jacobson, K., Beckers, C.J., 2007. Immobilization of the type XIV myosin complex in Toxoplasma gondii. Mol. Biol. Cell 18, 3039 3046. Katris, N.J., Van dooren, G.G., Mcmillan, P.J., Hanssen, E., Tilley, L., Waller, R.F., 2014. The apical complex provides a regulated gateway for secretion of invasion factors in Toxoplasma, PLoS Pathog., 10. p. e1004074. Kovar, D.R., 2006. Molecular details of formin-mediated actin assembly. Curr. Opin. Cell Biol. 18, 11 17. Leander, B.S., 2008. Marine gregarines: evolutionary prelude to the apicomplexan radiation? Trends Parasitol. 24, 60 67. Leander, B.S., Keeling, P.J., 2003. Morphostasis in alveolate evolution. Trends Ecol. Evol. 18, 395 402. Leander, B.S., Kuvardina, O.N., Aleshin, V.V., Mylnikov, A.P., Keeling, P.J., 2003. Molecular phylogeny and surface morphology of Colpodella edax (Alveolata): insights into the phagotrophic ancestry of apicomplexans. J. Eukaryot. Microbiol. 50, 334 340. Lechtreck, K.F., 2003. Striated fiber assemblin in apicomplexan parasites. Mol. Biochem. Parasitol. 128, 95 99. Lechtreck, K.F., Melkonian, M., 1991. Striated microtubuleassociated fibers: identification of assemblin, a novel 34kD protein that forms paracrystals of 2-nm filaments in vitro. J. Cell Biol. 115, 705 716. Lentini, G., Kong-Hap, M., El hajj, H., Francia, M., Claudet, C., Striepen, B., et al., 2015. Identification and characterization of Toxoplasma SIP, a conserved apicomplexan cytoskeleton protein involved in maintaining the shape, motility and virulence of the parasite. Cell. Microbiol. 17, 62 78.

Toxoplasma Gondii

784

16. The Toxoplasma cytoskeleton: structures, proteins, and processes

Leung, J.M., Rould, M.A., Konradt, C., Hunter, C.A., Ward, G.E., 2014a. Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay. PLoS One 9, e85763. Leung, J.M., Tran, F., Pathak, R.B., Poupart, S., Heaslip, A. T., Ballif, B.A., et al., 2014b. Identification of T. gondii myosin light chain-1 as a direct target of TachypleginA2, a small-molecule inhibitor of parasite motility and invasion. PLoS One 9, e98056. Leung, J.M., He, Y., Zhang, F., Hwang, Y.C., Nagayasu, E., Liu, J., et al., 2017. Stability and function of a putative microtubule-organizing center in the human parasite Toxoplasma gondii. Mol. Biol. Cell 28, 1361 1378. Leung, J.M., Liu, J., Wetzel, L.A., Hu, K., 2019. Centrin2 from the human parasite Toxoplasma gondii is required for its invasion and intracellular replication. J. Cell Sci. 132, jcs228791. Leveque, M.F., Berry, L., Besteiro, S., 2016. An evolutionarily conserved SSNA1/DIP13 homologue is a component of both basal and apical complexes of Toxoplasma gondii. Sci. Rep. 6, 27809. Liu, J., Wetzel, L., Zhang, Y., Nagayasu, E., Ems-Mcclung, S., Florens, L., et al., 2013. Novel thioredoxin-like proteins are components of a protein complex coating the cortical microtubules of Toxoplasma gondii. Eukaryot. Cell 12, 1588 1599. Liu, J., He, Y., Benmerzouga, I., Sullivan Jr., W.J., Morrissette, N.S., Murray, J.M., et al., 2016. An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii. Mol. Biol. Cell 27, 549 571. Lodoen, M.B., Gerke, C., Boothroyd, J.C., 2010. A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection. Cell. Microbiol. 12, 55 66. Long, S., Anthony, B., Drewry, L.L., Sibley, L.D., 2017a. A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii. Nat. Commun. 8, 2236. Long, S., Brown, K.M., Drewry, L.L., Anthony, B., Phan, I. Q.H., Sibley, L.D., 2017b. Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog 13, e1006379. Lorestani, A., Sheiner, L., Yang, K., Robertson, S.D., Sahoo, N., Brooks, C.F., et al., 2010. A Toxoplasma MORN1 null mutant undergoes repeated divisions but is defective in basal assembly, apicoplast division and cytokinesis. PLoS One 5, e12302. Lorestani, A., Ivey, F.D., Thirugnanam, S., Busby, M.A., Marth, G.T., Cheeseman, I.M., et al., 2012. Targeted proteomic dissection of Toxoplasma cytoskeleton sub-

compartments using MORN1. Cytoskeleton (Hoboken), 69, 1069 1085. Lyons-Abbott, S., Sackett, D.L., Wloga, D., Gaertig, J., Morgan, R.E., Werbovetz, K.A., et al., 2010. alpha-Tubulin mutations alter oryzalin affinity and microtubule assembly properties to confer dinitroaniline resistance. Eukaryot. Cell 9, 1825 1834. Ma, C., Li, C., Ganesan, L., Oak, J., Tsai, S., Sept, D., et al., 2007. Mutations in alpha-tubulin confer dinitroaniline resistance at a cost to microtubule function. Mol. Biol. Cell 18, 4711 4720. Ma, C., Tran, J., Li, C., Ganesan, L., Wood, D., Morrissette, N., 2008. Secondary mutations correct fitness defects in Toxoplasma gondii with dinitroaniline resistance mutations. Genetics 180, 845 856. Mann, T., Beckers, C., 2001. Characterization of the subpellicular network, a filamentous membrane skeletal component in the parasite Toxoplasma gondii. Mol. Biochem. Parasitol. 115, 257 268. Mann, T., Gaskins, E., Beckers, C., 2002. Proteolytic processing of TgIMC1 during maturation of the membrane skeleton of Toxoplasma gondii. J. Biol. Chem. 277, 41240 41246. Matuschewski, K., Schuler, H., 2008. Actin/myosin-based gliding motility in apicomplexan parasites. Subcell Biochem. 47, 110 120. Mehlhorn, H., 1972. Ultrastructural study of developmental stages of Eimeria maxima (Sporozoa, Coccidia). II. Fine structure of microgametes. Z. Parasitenkd. 40, 151 163. Mehta, S., Sibley, L.D., 2010. Toxoplasma gondii actin depolymerizing factor acts primarily to sequester G-actin. J. Biol. Chem. 285, 6835 6847. Mehta, S., Sibley, L.D., 2011. Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii. Mol. Biol. Cell 22, 1290 1299. Meissner, M., Schluter, D., Soldati, D., 2002. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837 840. Mercier, C., Howe, D.K., Mordue, D., Lingnau, M., Sibley, L.D., 1998. Targeted disruption of the GRA2 locus in Toxoplasma gondii decreases acute virulence in mice. Infect. Immun. 66, 4176 4182. Miki, H., Okada, Y., Hirokawa, N., 2005. Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol. 15, 467 476. Millholland, M.G., Mishra, S., Dupont, C.D., Love, M.S., Patel, B., Shilling, D., et al., 2013. A host GPCR signaling network required for the cytolysis of infected cells facilitates release of apicomplexan parasites. Cell Host Microbe 13, 15 28.

Toxoplasma Gondii

References

Mondragon, R., Frixione, E., 1996. Ca(2 1 )-dependence of conoid extrusion in Toxoplasma gondii tachyzoites. J. Eukaryot. Microbiol. 43, 120 127. Morisaki, J.H., Heuser, J.E., Sibley, L.D., 1995. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J. Cell Sci. 108 (Pt 6), 2457 2464. Morlon-Guyot, J., Francia, M.E., Dubremetz, J.F., Daher, W., 2017. Towards a molecular architecture of the centrosome in Toxoplasma gondii. Cytoskeleton (Hoboken) 74, 55 71. Morrissette, N., 2015. Targeting Toxoplasma tubules: tubulin, microtubules, and associated proteins in a human pathogen. Eukaryot. Cell 14, 2 12. Morrissette, N.S., Sibley, L.D., 2002a. Cytoskeleton of apicomplexan parasites. Microbiol. Mol. Biol. Rev. 66, 21 38. table of contents. Morrissette, N.S., Sibley, L.D., 2002b. Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii. J. Cell Sci. 115, 1017 1025. Morrissette, N.S., Murray, J.M., Roos, D.S., 1997. Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J. Cell Sci. 110 (Pt 1), 35 42. Morrissette, N.S., Mitra, A., Sept, D., Sibley, L.D., 2004. Dinitroanilines bind alpha-tubulin to disrupt microtubules. Mol. Biol. Cell 15, 1960 1968. Mueller, C., Klages, N., Jacot, D., Santos, J.M., Cabrera, A., Gilberger, T.W., et al., 2013. The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion. Cell Host Microbe 13, 289 301. Nagayasu, E., Hwang, Y.C., Liu, J., Murray, J.M., Hu, K., 2017. Loss of a doublecortin (DCX)-domain protein causes structural defects in a tubulin-based organelle of Toxoplasma gondii and impairs host-cell invasion. Mol. Biol. Cell 28, 411 428. Nagel, S.D., Boothroyd, J.C., 1988. The alpha- and betatubulins of Toxoplasma gondii are encoded by single copy genes containing multiple introns. Mol. Biochem. Parasitol. 29, 261 273. Naumov, A., Kratzer, S., Ting, L.M., Kim, K., Suvorova, E. S., White, M.W., 2017. The Toxoplasma centrocone houses cell cycle regulatory factors. MBio 8, e00579 17. Nebl, T., Prieto, J.H., Kapp, E., Smith, B.J., Williams, M.J., Yates 3rd, J.R., et al., 2011. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex. PLoS Pathog. 7, e1002222. Nichols, B.A., Chiappino, M.L., 1987. Cytoskeleton of Toxoplasma gondii. J. Protozool. 34, 217 226. Nishi, M., Hu, K., Murray, J.M., Roos, D.S., 2008. Organellar dynamics during the cell cycle of Toxoplasma gondii. J. Cell Sci. 121, 1559 1568.

785

O’connell, C.B., Tyska, M.J., Mooseker, M.S., 2007. Myosin at work: motor adaptations for a variety of cellular functions. Biochim. Biophys. Acta 1773, 615 630. Okamoto, N., Keeling, P.J., 2014. The 3D structure of the apical complex and association with the flagellar apparatus revealed by serial TEM tomography in Psammosa pacifica, a distant relative of the Apicomplexa. PLoS ONE 9, e84653. Ouologuem, D.T., Roos, D.S., 2014. Dynamics of the Toxoplasma gondii inner membrane complex. J. Cell Sci. 127, 3320 3330. Pavlou, G., Biesaga, M., Touquet, B., Lagal, V., Balland, M., Dufour, A., et al., 2018. Toxoplasma parasite twisting motion mechanically induces host cell membrane fission to complete invasion within a protective vacuole. Cell Host Microbe 24, 81 96 e5. Pelster, B., Piekarski, G., 1971. Electron microscopical studies on the microgametogeny of Toxoplasma gondii. Z. Parasitenkd. 37, 267 277. Periz, J., Whitelaw, J., Harding, C., Gras, S., Del Rosario minina, M.I., Latorre-Barragan, F., et al., 2017. Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation. Elife 6, e24119. Piekarski, G., Pelster, B., Witte, H.M., 1971. Endopolygeny in Toxoplasma gondii. Z. Parasitenkd. 36, 122 130. Plattner, F., Yarovinsky, F., Romero, S., Didry, D., Carlier, M.F., Sher, A., et al., 2008. Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response. Cell Host Microbe 3, 77 87. Polonais, V., Javier foth, B., Chinthalapudi, K., Marq, J.B., Manstein, D.J., Soldati-Favre, D., et al., 2011a. Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii. Traffic 12, 287 300. Polonais, V., Shea, M., Soldati-Favre, D., 2011b. Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites. Exp. Parasitol. 128, 454 459. Porchet, E., Torpier, G., 1977. Freeze fracture study of Toxoplasma and Sarcocystis infective stages (author’s transl). Z. Parasitenkd. 54, 101 124. Pospich, S., Kumpula, E.P., von Der ecken, J., Vahokoski, J., Kursula, I., Raunser, S., 2017. Near-atomic structure of jasplakinolide-stabilized malaria parasite F-actin reveals the structural basis of filament instability. Proc. Natl. Acad. Sci. U.S.A. 114, 10636 10641. Poukkula, M., Kremneva, E., Serlachius, M., Lappalainen, P., 2011. Actin-depolymerizing factor homology domain: a conserved fold performing diverse roles in cytoskeletal dynamics. Cytoskeleton (Hoboken) 68, 471 490.

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Poupel, O., Tardieux, I., 1999. Toxoplasma gondii motility and host cell invasiveness are drastically impaired by jasplakinolide, a cyclic peptide stabilizing F-actin. Microbes Infect. 1, 653 662. Poupel, O., Boleti, H., Axisa, S., Couture-Tosi, E., Tardieux, I., 2000. Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments. Mol. Biol. Cell 11, 355 368. Powell, C.J., Jenkins, M.L., Parker, M.L., Ramaswamy, R., Kelsen, A., Warshaw, D.M., et al., 2017. Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex. J. Biol. Chem. 292, 19469 19477. Preble, A.M., Giddings Jr., T.M., Dutcher, S.K., 2000. Basal bodies and centrioles: their function and structure. Curr. Top. Dev. Biol. 49, 207 233. Qureshi, B.M., Hofmann, N.E., Arroyo-Olarte, R.D., Nickl, B., Hoehne, W., Jungblut, P.R., et al., 2013. Dynein light chain 8a of Toxoplasma gondii, a unique conoidlocalized beta-strand-swapped homodimer, is required for an efficient parasite growth. FASEB J. 27, 1034 1047. Rees-Channer, R.R., Martin, S.R., Green, J.L., Bowyer, P.W., Grainger, M., Molloy, J.E., et al., 2006. Dual acylation of the 45 kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites. Mol. Biochem. Parasitol. 149, 113 116. Rieder, C.L., Faruki, S., Khodjakov, A., 2001. The centrosome in vertebrates: more than a microtubuleorganizing center. Trends Cell Biol. 11, 413 419. Romano, J.D., Nolan, S.J., Porter, C., Ehrenman, K., Hartman, E.J., Hsia, R.-C., et al., 2017. The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network. J. Cell Biol. 216, 4235 4254. Russell, D.G., Burns, R.G., 1984. The polar ring of coccidian sporozoites: a unique microtubule-organizing centre. J. Cell Sci. 65, 193 207. Sahoo, N., Beatty, W., Heuser, J., Sept, D., Sibley, L.D., 2006. Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii. Mol. Biol. Cell. 17, 895 906. Salamun, J., Kallio, J.P., Daher, W., Soldati-Favre, D., Kursula, I., 2014. Structure of Toxoplasma gondii coronin, an actin-binding protein that relocalizes to the posterior pole of invasive parasites and contributes to invasion and egress. FASEB J. 28, 4729 4747. Salisbury, J.L., Baron, A., Surek, B., Melkonian, M., 1984. Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle. J. Cell Biol. 99, 962 970. Sattler, J.M., Ganter, M., Hliscs, M., Matuschewski, K., Schuler, H., 2011. Actin regulation in the malaria parasite. Eur. J. Cell Biol. 90, 966 971.

Scholtyseck, E., Mehlhorn, H., Hammond, D.M., 1971. Fine structure of macrogametes and oocysts of Coccidia and related organisms. Z. Parasitenkd. 37, 1 43. Scholtyseck, E., Mehlhorn, H., Hammond, D.M., 1972. Electron microscope studies of microgametogenesis in Coccidia and related groups. Z. Parasitenkd. 38, 95 131. Schwartzman, J.D., 1998. Gliding into the cell: myosins hold the key to invasion by Toxoplasma gondii. Trends Microbiol. 6, 98. Shaw, M.K., Tilney, L.G., 1999. Induction of an acrosomal process in Toxoplasma gondii: visualization of actin filaments in a protozoan parasite. Proc. Natl. Acad. Sci. U. S.A. 96, 9095 9099. Shaw, M.K., Compton, H.L., Roos, D.S., Tilney, L.G., 2000. Microtubules, but not actin filaments, drive daughter cell budding and cell division in Toxoplasma gondii. J. Cell Sci. 113 (Pt 7), 1241 1254. Sheffield, H.G., Melton, M.L., 1968. The fine structure and reproduction of Toxoplasma gondii. J. Parasitol. 54, 209 226. Shen, B., Buguliskis, J.S., Lee, T.D., Sibley, L.D., 2014. Functional analysis of rhomboid proteases during Toxoplasma invasion. mBio 5, e01795 14. Sibley, L.D., Hakansson, S., Carruthers, V.B., 1998. Gliding motility: an efficient mechanism for cell penetration. Curr. Biol. 8, R12 R14. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166, 1423 1435.e12. Silmon De monerri, N.C., Yakubu, R.R., Chen, A.L., Bradley, P.J., Nieves, E., Weiss, L.M., et al., 2015. The ubiquitin proteome of Toxoplasma gondii reveals roles for protein ubiquitination in cell-cycle transitions. Cell Host Microbe 18, 621 633. Sinden, R., Talman, A., Marques, S., Wass, M., Sternberg, M., 2010. The flagellum in malarial parasites. Curr. Opin. Microbiol. 13, 491 500. Skariah, S., Bednarczyk, R.B., Mcintyre, M.K., Taylor, G.A., Mordue, D.G., 2012. Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates. J. Immunol. 188, 3404 3415. Skariah, S., Walwyn, O., Engelberg, K., Gubbels, M.J., Gaylets, C., Kim, N., et al., 2016. The FIKK kinase of Toxoplasma gondii is not essential for the parasite’s lytic cycle. Int. J. Parasitol. 46, 323 332. Skillman, K.M., Diraviyam, K., Khan, A., Tang, K., Sept, D., Sibley, L.D., 2011. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites. PLoS Pathog. 7, e1002280.

Toxoplasma Gondii

References

Skillman, K.M., Daher, W., Ma, C.I., Soldati-Favre, D., Sibley, L.D., 2012. Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro. Biochemistry 51, 2486 2495. Skillman, K.M., Ma, C.I., Fremont, D.H., Diraviyam, K., Cooper, J.A., Sept, D., et al., 2013. The unusual dynamics of parasite actin result from isodesmic polymerization. Nat. Commun. 4, 2285. Stadler, R.V., White, L.A., Hu, K., Helmke, B.P., Guilford, W.H., 2017. Direct measurement of cortical force generation and polarization in a living parasite. Mol. Biol. Cell 28, 1912 1923. Stokkermans, T.J., Schwartzman, J.D., Keenan, K., Morrissette, N.S., Tilney, L.G., Roos, D.S., 1996. Inhibition of Toxoplasma gondii replication by dinitroaniline herbicides. Exp. Parasitol. 84, 355 370. Striepen, B., Crawford, M.J., Shaw, M.K., Tilney, L.G., Seeber, F., Roos, D.S., 2000. The plastid of Toxoplasma gondii is divided by association with the centrosomes. J. Cell Biol. 151, 1423 1434. Striepen, B., Jordan, C.N., Reiff, S., Van dooren, G.G., 2007. Building the perfect parasite: cell division in apicomplexa. PLoS Pathog. 3, e78. Subramanian, G., Belekar, M.A., Shukla, A., Tong, J.X., Sinha, A., Chu, T.T.T., et al., 2018. Targeted phenotypic screening in Plasmodium falciparum and Toxoplasma gondii reveals novel modes of action of medicines for malaria venture malaria box molecules. mSphere 3, e00534 17. Suvorova, E.S., Lehmann, M.M., Kratzer, S., White, M.W., 2012. Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii. Mol. Biochem. Parasitol. 181, 7 16. Suvorova, E.S., Francia, M., Striepen, B., White, M.W., 2015. A novel bipartite centrosome coordinates the apicomplexan cell cycle. PLoS Biol. 13, e1002093. Swedlow, J.R., Hu, K., Andrews, P.D., Roos, D.S., Murray, J.M., 2002. Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and widefield fluorescence microscopy. Proc. Natl. Acad. Sci. U. S.A. 99, 2014 2019. Sweeney, K.R., Morrissette, N.S., Lachapelle, S., Blader, I.J., 2010. Host cell invasion by Toxoplasma gondii is temporally regulated by the host microtubule cytoskeleton. Eukaryot. Cell 9, 1680 1689. Takemae, H., Sugi, T., Kobayashi, K., Gong, H., Ishiwa, A., Recuenco, F.C., et al., 2013. Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin. Sci. Rep. 3, 3199. Tang, Q., Andenmatten, N., Hortua triana, M.A., Deng, B., Meissner, M., Moreno, S.N., et al., 2014. Calcium-

787

dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii. Mol. Biol. Cell 25, 2579 2591. Tardieux, I., 2017. Actin nanobodies uncover the mystery of actin filament dynamics in Toxoplasma gondii. Trends Parasitol. 33, 579 581. Tilley, L.D., Krishnamurthy, S., Westwood, N.J., Ward, G. E., 2014. Identification of TgCBAP, a novel cytoskeletal protein that localizes to three distinct subcompartments of the Toxoplasma gondii pellicle. PLoS ONE 9, e98492. Tomita, T., Yamada, T., Weiss, L.M., Orlofsky, A., 2009. Externally triggered egress is the major fate of Toxoplasma gondii during acute infection. J. Immunol. 183, 6667 6680. Tosetti, N., Dos Santos pacheco, N., Soldati-Favre, D., Jacot, D., 2019. Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii. Elife 8, e42669. Tran, J.Q., de Leon, J.C., Li, C., Huynh, M.H., Beatty, W., Morrissette, N.S., 2010. RNG1 is a late marker of the apical polar ring in Toxoplasma gondii. Cytoskeleton (Hoboken) 67, 586 598. Tran, J.Q., Li, C., Chyan, A., Chung, L., Morrissette, N.S., 2012. SPM1 stabilizes subpellicular microtubules in Toxoplasma gondii. Eukaryot. Cell 11, 206 216. Treeck, M., Sanders, J.L., Elias, J.E., Boothroyd, J.C., 2011. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries. Cell Host Microbe 10, 410 419. Treeck, M., Sanders, J.L., Gaji, R.Y., Lafavers, K.A., Child, M.A., Arrizabalaga, G., et al., 2014. The calciumdependent protein kinase 3 of Toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis. PLoS Pathog. 10, e1004197. Uboldi, A.D., Wilde, M.-L., Mcrae, E.A., Stewart, R.J., Dagley, L.F., Yang, L., et al., 2018. Protein kinase A negatively regulates Ca21 signalling in Toxoplasma gondii. PLoS Biol. 16, e2005642. Vaishnava, S., Morrison, D.P., Gaji, R.Y., Murray, J.M., Entzeroth, R., Howe, D.K., et al., 2005. Plastid segregation and cell division in the apicomplexan parasite Sarcocystis neurona. J. Cell Sci. 118, 3397 3407. Van dooren, G.G., Reiff, S.B., Tomova, C., Meissner, M., Humbel, B.M., Striepen, B., 2009. A novel dynaminrelated protein has been recruited for apicoplast fission in Toxoplasma gondii. Curr. Biol. 19, 267 276. Varberg, J.M., Padgett, L.R., Arrizabalaga, G., Sullivan Jr., W.J., 2016. TgATAT-mediated alpha-tubulin acetylation is required for division of the protozoan parasite Toxoplasma gondii. mSphere 1.

Toxoplasma Gondii

788

16. The Toxoplasma cytoskeleton: structures, proteins, and processes

Verhey, K.J., Kaul, N., Soppina, V., 2011. Kinesin assembly and movement in cells. Annu. Rev. Biophys. 40, 267 288. Wall, R.J., Roques, M., Katris, N.J., Koreny, L., Stanway, R. R., Brady, D., et al., 2016. SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector. Sci. Rep. 6, 28604. Wells, A.L., Lin, A.W., Chen, L.Q., Safer, D., Cain, S.M., Hasson, T., et al., 1999. Myosin VI is an actin-based motor that moves backwards. Nature 401, 505 508. Wetzel, D.M., Hakansson, S., Hu, K., Roos, D., Sibley, L.D., 2003. Actin filament polymerization regulates gliding motility by apicomplexan parasites. Mol. Biol. Cell 14, 396 406. Wetzel, D.M., Chen, L.A., Ruiz, F.A., Moreno, S.N., Sibley, L.D., 2004. Calcium-mediated protein secretion potentiates motility in Toxoplasma gondii. J. Cell Sci. 117, 5739 5748. Whitelaw, J.A., Latorre-Barragan, F., Gras, S., Pall, G.S., Leung, J.M., Heaslip, A., et al., 2017. Surface attachment,

promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion. BMC Biol. 15, 1. Williams, M.J., Alonso, H., Enciso, M., Egarter, S., Sheiner, L., Meissner, M., et al., 2015. Two essential light chains regulate the MyoA lever arm to promote Toxoplasma gliding motility. mBio 6, e00845 15. Wloga, D., Gaertig, J., 2010. Post-translational modifications of microtubules. J. Cell Sci. 123, 3447 3455. Xiao, H., El bissati, K., Verdier-Pinard, P., Burd, B., Zhang, H., Kim, K., et al., 2010. Post-translational modifications to Toxoplasma gondii alpha- and beta-tubulins include novel C-terminal methylation. J. Proteome Res. 9, 359 372. Yang, L., Uboldi, A.D., Seizova, S., Wilde, M.L., Coffey, M. J., Katris, N.J., et al., 2019. An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca21 signalling and motility in Toxoplasma gondii. J Biol Chem. 294, 8959 8972.

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C H A P T E R

17 Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages John C. Boothroyd1 and Mohamed-Ali Hakimi2 1

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, CA, United States 2Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions and Immunity to Infection, INSERM U1209, University Grenoble Alpes, Grenoble, France

17.1 Background The major invasive forms of Toxoplasma gondii possess at least three organelles capable of regulated protein secretion: micronemes, rhoptries, and dense granules (Fig. 17.1). The apical micronemes deliver micronemal proteins (MICs) to the parasite surface soon after the parasites egress from the parasitophorous vacuole (PV) (Dubois and Soldati-Favre, 2019). Most secreted MICs so far identified are either integral membrane proteins that ultimately reside on the parasite’s plasma membrane, such as apical membrane antigen 1 (Hehl et al., 2000) or MIC2 (Carruthers and Sibley, 1997), or proteins bound to other proteins, such as MIC2-associating protein or M2AP (Rabenau

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00017-7

et al., 2001). As discussed elsewhere in this book, these membrane proteins play a key role in attachment to the host cell and subsequent invasion (see also Dubois and Soldati-Favre, 2019; Carruthers and Tomley, 2008; Boothroyd and Dubremetz, 2008). They can be released from the surface by the action of proteases such as the rhomboid proteases (Kim, 2004; Brossier et al., 2005; Dowse et al., 2005) but, as yet, none are known to cross the host plasma membrane (HPM) or PV membrane (PVM) to reach the host cytosol. Some MICs are released as soluble proteins [e.g., MIC10 (Hoff et al., 2001) and MIC11 (Harper et al., 2004)], but their functions are not known and they have not yet been found to specifically interact with their host cell. Thus none of these MICs are

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17. Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages

Legend

MIC secretion and parasite attachment

RON secretion

ROP secretion

Invasion Moving junction

Dense granule

Nascent PVM GRA7

Rhoptry neck Microneme Rhoptry bulb

Host membrane

GRA secretion and PVM maturation

AMA1

RON2 Actin

Parasite nucleus

MIC protein RON protein ROP protein GRA protein MYR protein

GRA1 ROP17 MYR1 GRA6

IVN

MAF1

Host nucleus

ROP16

GRA16

GRA18

Mature PVM

FIGURE 17.1 A model showing the different classes of effectors. Only one representative molecule from each of the classes of effectors discussed in this chapter is shown, including ones that originate in micronemes (MICs/AMA1; red), rhoptry necks (RONs; green) rhoptry bulbs (ROPs; blue), and dense granules (GRAs; orange). There are, for example, at least four GRA effectors that eventually reach the host nucleus: GRA16, GRA24, TgIST, TEEGR/HCE1. MYR proteins are GRA proteins that form a translocation that transports GRA proteins beyond the PVM. AMA1, Apical membrane antigen; GRAs, dense granule proteins; HCE1, host cyclin E 1; MIC, micronemal protein; MYR, c-Myc regulation; RONs, rhoptry neck proteins; ROPs, rhoptry bulb proteins; TEEGR, Toxoplasma E2F4-associated EZH2-inducing gene regulator. Source: Credit for image: Suchita Rastogi. Adapted from Rastogi et al., 2019, submitted. Curr. Opin. Microbiol.

considered “effectors” in the sense used by those studying bacterial pathogens, that is, they are not proteins introduced into a host cell which alter host cell functions. The one MIC that might meet this definition is perforin (PF)-like protein (PLP)1, so named because it is a PLP (Kafsack et al., 2009; Roiko and Carruthers, 2013). This protein has a conserved membrane attack complex/PF domain and is released from micronemes just prior to egress, resulting in disruption of the PVM and parasite release (Kafsack et al., 2009). All other proteins that are known to be associated with the PVM and/or cross it to reach the host cytosol derive from either the rhoptries or dense granules and so these are the focus of this chapter. Rhoptries are large, club-shaped organelles that, like micronemes, are apically located and

form a part of the “apical complex” from which the phylum gets its name. Toxoplasma tachyzoites, bradyzoites, and sporozoites generally have about 4 10 rhoptries (Fig. 17.1), with a minimum of 2 and a maximum of 11 observed (Speer et al., 1998, 1999). The rhoptries have at least two subcompartments, the basal “bulb,” and the more apical “neck” regions. Several early studies showed that the newly identified rhoptry proteins [generically designated as rhoptry bulb proteins (ROPs)], ROP1, and the ROP2 family (discussed further later) were associated with the PV “space” between parasites and the PVM surrounding them (Ossorio et al., 1992; Herion et al., 1993; Beckers et al., 1994b). At that time, these images were interpreted to be indicative of ROP proteins being secreted into the PV where

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17.1 Background

they floated freely and/or were bound to the PV-lumen side of the PVM or nanotube-like intravacuolar network (IVN). The first indication that rhoptries might actually inject their contents into the host cell came from electron micrographs showing a possibly contiguous channel between the rhoptries and the host cytosol (Nichols et al., 1983). The first demonstration that this was in fact true, however, came in 1987 when Kimata and Tanabe used immuno-electron microscopy to show that antibodies to a rhoptry protein stain a diffuse “cloud” within the host cytosol extending well beyond the nascent PVM (Kimata and Tanabe, 1987). Combined with the data on ROP1 and ROP2, these results presented something of a conundrum; in that, this one secretory organelle appeared to be releasing some proteins (ROP1/2) by simple secretion into the PV, while it injected others across the HPM and into the host cytosol. A priori, three possible explanations seemed reasonable: (1) two mechanisms are indeed operating with an unknown process for moving discrete subsets of their cargo in the two processes; (2) all proteins are secreted by conventional fusion of the rhoptries to the parasite plasma membrane (PPM) where some, but not others, continue to transit via unknown means across the nascent PVM; or (3) all proteins are actually introduced directly into the host cell during invasion where a subset then orients in a way that makes them appear to be free within the PV spaces. Current data strongly favor explanation number three. First, the extreme tip of an invading parasite is reproducibly seen to be in direct contact with the invaginating HPM (i.e., nascent PVM) and, in certain selected micrographs, a direct continuity between the parasite rhoptries and host cytosol is evident (Nichols et al., 1983). That is, an apparent pore can be seen spanning the nascent PVM and PPM. The identity and nature of this pore have yet to be determined but evidence for it can also be seen in freeze-fracture micrographs of invading

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parasites where a “dimple” is evident at the extreme apical end of the invading parasites, where they make contact with the nascent PVM (Dubremetz, 2007). Second, several rhoptry proteins have now been definitively located inside the host cell. These include ROPs and several rhoptry neck proteins (RONs) that are introduced into the host cell at the very start of invasion and that play a crucial role in formation of the moving junction that migrates down the length of the parasite during invasion (Lebrun et al., 2005; Alexander et al., 2006; Besteiro et al., 2009, 2011; Lamarque et al., 2011; Tyler and Boothroyd, 2011). These are described further in Chapter 14, Toxoplasma secretory proteins and their roles in parasite cell cycle and infection. In addition, several ROP proteins are now known to be associated with the hostcytosolic face of the PVM, especially several members of the ROP2 family of kinases and pseudokinases, including the original ROP2 (Herion et al., 1993; Beckers et al., 1994a) for which the family is named (Sadak et al., 1988; El hajj et al., 2006). These ROP2 family proteins have also been dubbed rhoptry kinase (ROPK) for their kinase function and presumptive rhoptry localization (Peixoto et al., 2010; Talevich and Kannan, 2013); the finding, however, that some ROPK proteins are in fact dense granule proteins (Jones et al., 2017; Coffey et al., 2018; Beraki et al., 2019) makes this designation potentially confusing and so we will stick with the use of ROP2 family here. Many members of this family face into the host cytosol and they are now known to have many important functions related to the host (Boothroyd and Dubremetz, 2008; Wei et al., 2013; Talevich and Kannan, 2013; Kemp et al., 2013; Hakimi et al., 2017). Lastly, some members of the ROP2 family are found within the host nucleus where they directly impact host transcription; these are described later in detail. The model that all rhoptry proteins are injected into the host cell during invasion can explain the apparent presence of some ROPs

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within the PV space “between” parasites if these ROPs are insinuating themselves into the lumens of IVN nanotubes, thereby appearing to fill the space between parasites (Sibley and Krahenbuhl, 1986; Achbarou et al., 1991; Sibley, 1993). Topologically speaking, these invaginations are extensions of the host cytosol and several ROP2 family members strongly associated with this IVN (Herion et al., 1993; Carey et al., 2004). Data strongly arguing for this association being on the host cytosolic side of the IVN and PVM came from experiments with ROP5 (a member of the ROP2 family), where differential permeabilization was used to localize the protein to the cytosolic side of the PVM (El hajj et al., 2007b). Confirmation of this conclusion came when a fluorescent reporter protein was fused to the N-terminal domain of ROP2 family member, ROP17, and then expressed in an uninfected host cell. If such cells were then infected, the reporter protein was found to localize to the PVM and IVN, thereby appearing to fill the “space” between the parasites (Reese and Boothroyd, 2009). Given that there is no known mechanism for translocation of proteins from the host cytosol into the PV space, this result has been interpreted as being due to sequestration of these proteins deep within the IVN but still topologically within the host cytosol. Thus while certainly not proven, the data strongly argue that all soluble rhoptry proteins are injected into the host cell where some associate with the PVM and IVN and others, such as ROP16 (Saeij et al., 2006) and PP2C-hn (Gilbert et al., 2007) traffic to the host nucleus.

17.2 Rhoptry effectors—a potent class of host manipulators Remarkably, the function of the first identified ROP proteins, ROP1, and several members of the ROP2 family, for example, ROP2/3/4/ 7/8, remains unknown. ROP1 is a novel

protein with unusual charge asymmetry (Ossorio et al., 1992) but, as yet, there is no known biological function assigned to this protein. Mutants lacking it show an ultrastructural alteration in that the rhoptries are somewhat thinner and more electron dense (Soldati et al., 1995), but no biological phenotype has yet been found. Even preliminary host transcriptomic studies using microarrays failed to show any convincing difference between infection with wild-type RH and RHΔrop1 parasites (L. Pernas and J. Boothroyd, unpublished results). Perhaps, more sensitive methods of RNA-Seq, as well as other “omics” approaches will yield information on ROP1’s role; alternatively, work with strains that are less virulent in the mouse model than the supervirulent RH (in which the previously described mutants were all made) might also provide new insight. ROP2 is the founding member of an extremely large family of related proteins, all of which share a conserved kinase “fold” (El hajj et al., 2006; Peixoto et al., 2010). Some of these are active kinases but many, including ROP2, are strongly predicted to be noncatalytic or “pseudokinases” based on their lack of key residues necessary for catalysis in most known kinases. While early studies suggested that ROP2 might be spanning the PVM (Beckers et al., 1994b, Sinai and Joiner, 2001) and play a role in host mitochondrial recruitment (Nakaar et al., 2003), subsequent work showed that this protein is fully on the host cytosolic side of the PVM (El hajj et al., 2006) and knock-outs of the cluster of three tandem genes (ROP2A/2B/8) showed no difference in host mitochondrial association (Pernas and Boothroyd, 2010). As with ROP1, host microarray studies showed no significant difference between RH wild type and RHΔrop2/8 (L. Pernas and J. Boothroyd, unpublished) parasites and no difference in host virulence has so far been described for this or selected other members of the ROP2 family (ROP11/20/23/24/26/28/32/39/42/ 43/44/45) in a mouse model of chronic

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17.2 Rhoptry effectors—a potent class of host manipulators

infection using cyst load as a read-out (Fox et al., 2016). Given current thinking that the large host range of Toxoplasma is likely selected for expansion of the ROP2 family, the function of individual members maybe specific to a certain cell type and/or host species. To date, almost all in vivo works with Toxoplasma have been done within a few strains of lab mice (e.g., Balb/c, C57/black, CD1) and most in vitro works have been done in only a very few host cell types (human foreskin fibroblasts, mouse macrophages, and a handful of others). Chances seem high that when the right experimental context is used and the right assay is deployed, effectors such as the ROP2 family members mentioned previously will be found to play important roles. The challenge to the community is to find those conditions! Despite this limitation, and as a result of some exhaustive searching by various means, the function of several other ROP2 family members has now been determined. One of the first to reveal itself was ROP16, a tyrosine kinase that interacts with host STAT proteins (signal transducer and activator of transcription), particularly STAT3, STAT5, and STAT6 (Saeij et al., 2006; Yamamoto et al., 2009; Ong et al., 2010; Butcher et al., 2011; Jensen et al., 2013). This protein’s crucial role was first recognized by genetic mapping of the transcriptional response of host cells to different progeny stemming from a genetic cross between Type II and Type III parasites; such parasites differ dramatically in the impact they have on the host transcriptome and much of this difference proved to be a Mendelian trait (Saeij et al., 2006). Subsequent studies showed that ROP16 directly phosphorylates STATs and is thus acting as a parasite mimic of the Janus kinases (JAKs) that normally fill this role; moreover, the allele present in Type III (and Type I) strains is highly active, whereas the allele in Type II strains is much less so (Yamamoto et al., 2009; Ong et al., 2010; Butcher et al., 2011; Jensen et al., 2013).

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Remarkably, Type I/III ROP16 can phosphorylate and thereby activate the STATs even faster than the normal route involving crosslinking of the IL4 receptor followed by JAK2 dimerization and then STAT phosphorylation (Ong et al., 2010). Just one amino acid residue appears responsible for the type-specific differences in ROP16 activity (Yamamoto et al., 2009) and this appears to explain the differences in ROP16 activity in a large number of different strains isolated from nature (Rosowski and Saeij, 2012; Minot et al., 2012). In terms of biological impact of this type-specific difference in ROP16 activity, it appears to substantially affect the host’s immune response; depending on the allelic variant of ROP16 carried by a strain, the macrophage response can be driven toward an M1 (“classical”) or M2 (“alternative”) activation state (Yamamoto et al., 2009; Jensen et al., 2011; Butcher et al., 2011). The result is a profound difference in the degree of inflammation and, thus, immunopathology. Interestingly, the deletion of ROP16 from a Type II strain can lead to an increase in cyst load in the brain of infected mice, suggesting that there is a complex interplay between this effector and the progress of an infection (Fox et al., 2016). Despite all these molecular details, the context in which one or other allele of ROP16 evolved is not known but there presumably exist natural hosts of Toxoplasma in which a highly active version is optimal and another in which much less activity provides a selective advantage. The possibility that this difference might be between mammals and birds was tested by comparing the host response between human, mouse, and chicken cells; interestingly, all three showed a similar response to the highversus low-activity versions of ROP16 (Ong et al., 2011), suggesting the selective pressures operating on these allelic forms are not as simple as mammals versus birds. Clearly, however, such a limited study of just one cell type in just three species leaves plenty of rooms for other

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cell types and/or species to hold the answer to this question of what drove the evolution of these different forms of ROP16. Another ROP2 family member that has now been well studied is ROP18, an active serine/ threonine protein kinase that is secreted into the infected cell where it associates with the host cytosolic side of the PVM (El hajj et al., 2007a). Studies on the genetic basis for virulence differences between Types I, II, and III strains of Toxoplasma quickly led to the identification of ROP18 as a key locus, with different alleles of the gene being strongly correlated with virulence (Saeij et al., 2006; Taylor et al., 2006; El hajj et al., 2007a). Subsequent work showed the basis for this impact on virulence as being due to the coordinated action of ROP5/17/18 in defending against a mouse’s immunity-related GTPases (IRGs) (Khaminets et al., 2010; Behnke et al., 2011; Reese et al., 2011; Behnke et al., 2012; Fleckenstein et al., 2012; Niedelman et al., 2012). The IRGs are a family of rodent-specific GTPases that are expressed in response to interferon (IFN)-γ and that attack the vacuole within which certain intracellular pathogens grow. Failure to inactivate the IRGs results in the rapid destruction of the PVM and the parasites inside and so defending against this is a key role for these ROPs. Much is now known about how the trio of ROP5/17/18 functions. ROP5 is a pseudokinase that binds to the IRGs, resulting in allosteric changes in the structure of the bound IRG (Reese et al., 2014). This is necessary for the phosphorylation by ROP17 and ROP18 of sites in the IRGs near the nucleotide-binding pocket. The result is a change in the GDP dissociation from the pocket and, consequently, an inactivation of the GTPase function of the IRG. Interestingly, in some strains, such as Types I and II, ROP18 seems to play a major role in this phosphorylation (Saeij et al., 2006; Taylor et al., 2006; El hajj et al., 2007a). In Type III strains, however, the ROP18 gene is essentially not transcribed (Taylor et al., 2006; Saeij et al.,

2006; Boyle et al., 2008), suggesting that in the natural host of Type III strains, this protein must be more of a liability than an asset. Presumably, that host is not one where IRGs are a major host defense and the presence of such a potent protein kinase phosphorylating other host proteins would do more harm than good. In such strains, ROP17 may be sufficient for the occasional encounter with the host proteins that need (in)activation by phosphorylation (Etheridge et al., 2014). Direct expression of ROP17 within host cells (i.e., without infection) has substantial impacts on the host cell transcriptome unrelated to its activity against IRGs (Li et al., 2019), although the exact mechanism underlying this effect has yet to be determined. Most recently, ROP17 has been shown to be needed for effective translocation of dense granule effectors across the PVM (M. Panas, A. Ferrel and J. Boothroyd, unpublished results). This was dependent on catalytic activity of the ROP17 and the presence of the enzyme within the host cell cytosol. Given its location at the PVM, which is where the c-Myc regulation (MYR) complex functions to translocate GRA proteins into the host cytosol (discussed later), this strongly suggests that one or more MYR components is a target of ROP17. An attractive model is that the MYR components can only function when they encounter a functional ROP17 and ATP, a situation only present within the host cytosol, rather than in the PV space or compartments of the secretory pathway within the parasites (e.g., endoplasmic reticulum, Golgi, or dense granules). As for ROP17, ROP18’s role is not limited to its action on IRGs; it also is involved in modulating the activity of a transcriptional activator known as ATF6beta (Yamamoto et al., 2011). Mice deficient in ATF6beta are more susceptible to an infection with Toxoplasma indicating that this is related to an important host immune response, although the exact function involved has not yet been determined.

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ROP38 is a particularly intriguing member of the ROP2 family; it is predicted to be an active kinase and, as with ROP18, the expression of this gene varies greatly between strains with Type III having the highest expression [relative to Types I and II; (Peixoto et al., 2010)]. Unusually, ROP38 is strongly upregulated in bradyzoites relative to tachyzoites suggesting a key role in the chronic stages of an infection. Engineering a Type I strain to overexpress ROP38, that is, to levels seen in Type III strains, revealed an effect on the downregulation of host genes associated with mitogenactivated protein kinase (MAPK) pathways, including those involved in apoptosis and proliferation (Peixoto et al., 2010). X-ray crystallography has been used to study the structure of three members of the ROP2 family, ROP2 (Labesse et al., 2009; Qiu et al., 2009), ROP8 (Qiu et al., 2009), and ROP5 (Reese et al., 2014). Although all three are pseudokinases, these results indicate a conventional “kinase fold” but with some Toxoplasma-specific variations. These data have been used to make predictions about compounds that might block their function in vivo, opening up a promising area for future research (Qiu et al., 2009). ROP33/34/35 are an interesting trio of related protein kinases that are a distinct clade of the ROP2 family. Structurally, they are distinguished by their lack of a Gly-loop that is common to most protein kinases and, that is, present in the other members of the family. These have therefore been dubbed “with-nogly-loop” kinases or WNG; that is, WNG1/2/3 for ROP33/34/35, respectively (Coffey et al., 2018; Beraki et al., 2019). Clues to their different role in the biology of the parasite came from the fact that they are expressed at a different time in the cell cycle of tachyzoites from the time that the majority of the ROP2 family, including ROP16/17/18, is expressed (Behnke et al., 2010). The rhoptries form over a very limited time in the cell cycle, that is, S (DNA Synthesis) and M (mitosis), which is the same

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period as when the ROP16/17/18 mRNAs are most abundant. This suggested that genes expressed at different times in the cell cycle might encode proteins that are destined for a different compartment than the rhoptries. This prompted further examination of the WNG1/ 2/3 set of proteins which now appear to, in fact, be trafficked to dense granules rather than rhoptries, providing further motivation for their being renamed WNG proteins to prevent confusion from the “ROP” moniker (Coffey et al., 2018; Beraki et al., 2019). As mentioned previously, dense granules release their contents into the PV after invasion, rather than injecting their cargo into the host cell during invasion, as the rhoptries do. It now appears that, indeed, WNG1/2/3 function within the PV lumen, phosphorylating other dense granule proteins that combine to create the IVN by invagination of the PVM (see chapter by Lebrun et al.). It may be that since ATP is abundant within the PV but is likely limiting within the lumen of the dense granules, this is a way to regulate the activity of the IVNforming GRAs such that they are only active once they reach the PV space. Of note, these members of the ROP2 family serve to make an important point: despite being paralogs, the ROP2 family has evolved to include proteins in compartments other than the rhoptries and so the moniker “ROP” or “ROPK” should not be taken as implying rhoptry localization and confusion can be avoided by adopting new names that do not make such an implication. Like WNG1/2/3, ROP21/27/30 are also expressed at a different cell-cycle stage from the well-characterized ROP family members, ROP16/17/18; but in this case, they are not expressed at the same time as known dense granule proteins (Jones et al., 2017). As predicted from this result, these proteins appear to be in fact secreted via another route, “constitutive secretory vesicles.” Like GRA proteins, including the WNG kinases, ROP21/27/30 find themselves within the PV space but, as

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yet, their targets are not known. Interestingly, they appear to be the most important for the chronic stage of the asexual cycle, with peak expression in bradyzoites relative to tachyzoites. Consistent with this, deletion of these genes results in little, if any, effect on in vitro growth of tachyzoites or even the acute stages of in vivo infection; instead, their absence results in a decreased cyst load indicating that they play a role in the chronic stages of an infection (Jones et al., 2017). Of course, not all rhoptry proteins are members of the ROP2 family. One notable exception is one of the first rhoptry proteins to have been shown to reach the host nucleus, a protein phosphatase in the 2C class called PP2C-hn [for host nucleus; (Gilbert et al., 2007)]. The precise role of this protein, whose biochemical function appears to be the exact opposite of the protein kinases discussed previously, is not known, although its absence does result in a mild growth defect, at least in vitro. As yet, transcriptomic analyses revealed no significant changes suggesting that this protein is operating on aspects not related to gene expression, per se (Gilbert et al., 2007). Another nonkinase rhoptry protein is a strong binder of host actin that has thus been dubbed toxofilin (analogous to “profilins” described in other systems). This protein was first identified as a result of its actin-binding properties (Delorme et al., 2003) and was later found to localize, at least in large part, to the rhoptries (Bradley et al., 2005). Like other rhoptry proteins, it is injected into the host cell early during invasion (Lodoen et al., 2010) where it binds host actin (Delorme et al., 2003; Jan et al., 2007; Lee et al., 2007; Delorme-Walker et al., 2012), apparently as a way to open up the cytoskeleton for the passage of the parasite into the host cell (Jan et al., 2007; Delorme-Walker et al., 2012; Czimbalek et al., 2015). Additional possible rhoptry proteins have been identified in a proteomic analysis of purified rhoptries (Bradley et al., 2005). Such

analyses are imperfect in sometimes detecting contaminating molecules while also missing bona fide ROPs that are of low abundance, but this list represents an excellent starting point for pursuing additional candidates that may represent as yet unknown effectors with their own, specific impact on the invaded host cell. In a similar vein, bioinformatic methods have been used to identify possible rhoptry proteins based on the timing of their expression during the tachyzoite cell cycle. As mentioned previously, rhoptries are made in the S/M phases of the cell cycle and so one can search in ToxoDB for proteins that are predicted to be secreted (based on their possession of a predicted signal peptide) and whose cognate mRNA shows peak expression at these times in the cell cycle, based on published transcriptomic studies with synchronized parasites (Behnke et al., 2010). Using this approach, three novel proteins have been identified as rhoptry proteins (Camejo et al., 2014), ROP47, ROP48, and RON12. Although the precise function of these proteins has yet to be determined, ROP47 reaches the host cell nucleus and RON12 is found within the rhoptry necks (the other two are being ROPs). Deletion of these three genes individually had no significant effect on growth in vitro or virulence in vivo, but as stated repeatedly in this review, this likely reflects the limited and very specific assay conditions used (cell type, host species, route of infection, dose, read-out, etc.), rather than the true absence of an important role for these proteins.

17.3 Dense granule effectors—a second wave of manipulation Dense granules secrete their contents after tachyzoite invasion has commenced (Fig. 17.1). Their cargo includes an ever-growing list of “GRA” proteins that until relatively recently were thought to function exclusively within

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17.3 Dense granule effectors—a second wave of manipulation

the PV, helping to create the IVN nanotubes. In recent years, however, it has been discovered that GRA proteins can also function as crucial, extravascular effectors. These fall into two classes, those that function at the PVM and those that cross into the host cell as apparently soluble proteins. Examples of the first class are GRA15 that is key to the induction of the NF-κB pathway (Rosowski et al., 2011), mitochondrial association factor 1 (MAF1) that mediates recruitment of host mitochondria (Pernas et al., 2014; Adomako-Ankomah et al., 2016), and GRA6 that, by interacting with calciummodulating ligand, activates calcineurin and stimulates the transcription factor nuclear factor of activated T-cells 4 (Ma et al., 2014). All three of these proteins appear to operate at the PVM and none has been detected free within the host cell. The second class of GRA effectors is comprised of proteins that transit across the PVM as soluble entities; once on the other side, they should be able to travel to almost any cellular compartment so long as they have the appropriate host-specific signals. The first such protein to be identified, GRA16, came from a bioinformatic screen of parasite proteins that have a signal peptide, a conventional nuclearlocalization signal and a high abundance of intrinsically disordered regions, suggesting that they might cross the PVM and end up in the nucleus of the host cell. This indeed proved correct and the surprise was that instead of being a rhoptry protein, the protein in question trafficked via dense granules and was thus the first GRA to be reported as being able to cross the PVM as a soluble protein (Bougdour et al., 2013; Bougdour et al., 2014). GRA16 migrates to the host cell nucleus, while bound to a high-molecular-weight complex, gathering the PP2A-B55 holoenzyme phosphatase and the herpesvirus-associated ubiquitin-specific protease [HAUSP, also known as USP7; (Bougdour et al., 2013)]. In response to oncogenic insults, HAUSP

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deubiquitinates the tumor suppressor p53 in addition to Mdm2 and thus protects p53 from Mdm2-mediated ubiquitylation and degradation. Exogenous expression of GRA16 in host cells was shown to markedly increase the steady-state protein levels of p53 in an HAUSP-dependent fashion, opposite to the Epstein Barr virus protein EBNA1 which sequesters HAUSP from p53 in vivo, thereby destabilizing p53 (Saridakis et al., 2005). In line with its impact on HAUSP activity, GRA16 significantly modulates the expression of host cell genes involved in serine/glycine, monounsaturated fatty acids, and cholesterol metabolism, three pathways that have been linked recently to the MDM2-p53 pathway (Riscal et al., 2016; Moon et al., 2019). As such, p53 acts as a guardian of metabolic balance. For instance, acute loss of glutamine triggers PP2Amediated activation of p53, thereby linking levels of a critical metabolite to an important regulator of cell survival and proliferation (Reid et al., 2013). In this respect, GRA16 was shown to induce the nuclear translocation of the PP2A-B55 holoenzyme as well as to regulate the expression of glutaminase 2, a p53 target gene (Bougdour et al., 2013). Overall, those data underscore a critical role for GRA16 in coopting HAUSP and PP2A to modulate p53 levels thereby promoting host cell survival under stress conditions. GRA16 has no equivalent in mammalian genomes suggesting that over millions of years of coevolution with its hosts, GRA16 has been selected to interfere with the p53 pathway to contribute to Toxoplasma infections. A role in herpes simplex virus evasion from the host innate immune response has been proposed for HAUSP (Daubeuf et al., 2009). Whether GRA16 hijacks HAUSP and PP2A functions to evade an antimicrobial host response is currently unknown. Nevertheless, GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis (Bougdour et al., 2013).

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GRA24 shares with GRA16 the ability to accumulate in host cell nuclei, while the PV continues to enlarge along with parasite multiplication (Braun et al., 2013). Once released in the host cell cytoplasm, GRA24 binds tightly to the host p38α MAPK and promotes its sustained autophosphorylation which, in turn, triggers both the activation and the nuclear translocation of the host kinase. In this respect, GRA24 behaves as a parasite-derived agonist that bypasses the canonical MAPK phosphorylation cascade to activate transcription factors, such as Egr1 and c-Fos (Braun et al., 2013). GRA24 was determined to be the only component in this context needed to activate p38α. The parasite protein operates through two atypical kinase-interacting motifs (KIMs), embedded in an intrinsically disordered protein, to scaffold two molecules of p38α, promoting autoactivation via transphosphorylation. Those structural features were clearly evidenced by small-angle X-ray scattering and atomic force microscopy (Pellegrini et al., 2017). What was also learned is that the binding of the KIMs to p38α confers allosteric changes in the kinase domain, which, in turn, activate the kinase. Moreover, those atypical KIMs evolved in a way that they are preventing deactivation by regulatory phosphatases, thereby promoting a sustained host kinase activation during Toxoplasma infection (Braun et al., 2013). Strict control of inflammatory signaling prevents a response that is too weak leading to the death of the host or too strong that would prevent the spread and persistence of the parasite. In this regard, GRA24 was shown to control, at the transcriptional level, an impressive network of genes encoding for cytokines/chemokines. Thus the protein elicits a strong inflammatory response by turning on the production of proinflammatory cytokines and chemokines, including IL-12 and MCP-1/CCL2, which enhance macrophage phagocytic activity at the site of infection and accordingly limit parasite burden in the

gut (Braun et al., 2013). GRA24 is a prime example of how molecular mimicry is used by Toxoplasma to hijack a canonical host signaling pathway and somehow to contribute to shaping and modulating the immune response. Interestingly, GRA24 and GRA15 intersect to regulate IL-12 expression, while GRA24 and GRA25 (Shastri et al., 2014) are both involved in CCL2 activation. Significant crosstalk also occurs between proteins originating from rhoptries and dense granules, as GRA24 and ROP38 both shortcut the canonical host MAPK cascade by directly phosphorylating p38 and ERK1,2, respectively (Peixoto et al., 2010). GRA18 differs from GRA16 and GRA24 in that after it crosses the PVM, it remains within the cytoplasm of the infected cell. Once there, the protein forms versatile complexes with regulatory elements of the β-catenin destruction complex (He et al., 2018). The conserved and ancient wingless-int1 (Wnt)/β-catenin pathway regulates stem cell pluripotency and cell fate decisions during development. Under normal conditions (off-state), the cytosolic β-catenin, an integral E-cadherin cell cell adhesion adaptor protein and transcriptional coregulator, is kept at low levels through continual phosphorylation by the S/T kinases glycogen synthase kinase 3 (GSK3) and the casein kinase I-α, which promote its ubiquitination and subsequent proteasomal degradation (Niehrs, 2012). Activating Wnt ligands (on-state) triggers displacement of the multifunctional kinase GSK-3β from a regulatory APC/Axin/GSK-3β destruction complex, leaving β-catenin unphosphorylated. Stabilized β-catenin is then translocated to the nucleus, where it binds to T cell factor/lymphoid enhancer factor transcription factors, displacing corepressors and recruiting additional coactivators to Wnt target genes. While usually associated with embryonic development and tumorigenesis, β-catenin is now well-recognized for its role in immunity (Staal et al., 2008). GRA18 targets the Wnt/β-Catenin pathway in an original and noncanonical fashion.

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17.3 Dense granule effectors—a second wave of manipulation

The parasite effector is embedded in a highmolecular-weight complex that includes β-catenin, GSK3 α/β, and the PR56/B’containing PP2A holoenzyme that prevents the continual elimination of β-catenin (He et al., 2018). Following tachyzoite infection of a cell, cytoplasmic β-catenin travels to and accumulates in the host cell nucleus in a GRA18dependent manner, where it activates otherwise repressed target genes. Thereby, GRA18 acts as a direct regulator of β catenin and although the exact modus operandi of GRA18 awaits clarification, the GRA18-GSK3-β-catenin axis induces the expression of antiinflammatory chemokines, including Ccl17, Ccl22, and Ccl24 (He et al., 2018). Interestingly, those chemokines are expressed by alternatively activated M2-polarized macrophages or tolerant macrophages and their release results in the recruitment of Treg cells and amplification of a Th2 response (Biswas and Mantovani, 2010). By counterbalancing the Th1-induced inflammatory effects, those Th2 chemokines may be involved in dampening the inflammatory response to avoid immunopathology and foster host and parasite survival. Overall, GRA18 unveils another original strategy by which Toxoplasma tachyzoites reshuffle the evolutionarily conserved β-catenin destruction complex to selectively reprogram immune gene expression. From the previous, it is clear that Toxoplasma has evolved an unusual set of effectors that target hubs of the host immune signaling pathways and that, by acting synergically or antagonistically, these generate the appropriate response. IFN pathways are, per se, central hubs involved in host and parasite survival. IFN-γ appears to be the pivotal mediator, inducing anti-Toxoplasma cell-autonomous immunity, in both humans and mice (Jones et al., 1986; Suzuki et al., 1988). The JAK— STAT pathway has a central function in the transmission of cytokine signals from the cell membrane to the nucleus and subsequent gene activation. Upon IFN-γ stimulation, STAT1 is

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thus phosphorylated on Y701, undergoes dimerization, translocates to the nucleus, and regulates gene expression by binding to gamma-activated sequence (GAS) elements in the promoters of IFN-γ-regulated genes [e.g., interferon regulatory factor 1 (IRF1)]. The full activation of STAT1 is achieved by a second independent phosphorylation event on S727 that elicits the recruitment of the CBP/p300 histone acetyltransferases that promotes chromatin opening and the formation of an elongationcompetent RNA polymerase II. Early studies reported that Toxoplasma tachyzoites have evolved mechanisms to timely counteract host defenses by making them unresponsive to IFN-γ, specifically impeding the nuclear-cytoplasmic cycling of STAT1 (Kim et al., 2007; Rosowski et al., 2014). The parasite-derived effector responsible for this impressive feat was eventually identified as a novel dense granule protein named inhibitor of STAT1 transcriptional activity (TgIST; Gay et al., 2016; Olias et al., 2016). Like GRA16 and GRA24, TgIST crosses the PVM in an MYR1-dependent manner and accumulates in the host cell nucleus. TgIST was shown to be responsible for inhibiting the STAT1dependent responsiveness of the host cell to IFN-γ, in particular the IRF1 gene (Gay et al., 2016; Olias et al., 2016). Likewise, TgIST by inhibiting indoleamine 2,3-dioxygenase 1 (IDO1) mRNA expression antagonizes the IFN-γ-induced IDO1-mediated antiparasite cell-autonomous immunity in human cells (Bando et al., 2018). Some targets, however, including iNOS, escape TgIST-mediated repression (Cabral et al., 2018), suggesting that another yet-to-be-identified parasite effector is involved. Once delivered to the host nucleus, TgIST forms a high-molecular-weight complex gathering an activated (phosphorylated Y701) STAT1, the transcriptional corepressors Cterminal binding protein 1 (CtBP1) and CtBP2, NuRD, a chromatin complex coupling lysine

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deacetylation (HDAC1 and HDAC2), and ATPase-mediated chromatin remodeling (CHD3 and CHD4). The partnership of TgIST with NuRD and CtBPs was shown to be STAT1independent (Gay et al., 2016). Although TgIST was shown to bind to STAT1 dimers, it more efficiently assembled with STAT1 tetramers that are essential for effective IFN-γ responsiveness (Nast et al., 2018; Vinkemeier, 2004). Upon IFN-γ stimulation, STAT1 nuclear relocation occurs normally as suggested previously (Kim et al., 2007; Rosowski et al., 2014) but it remains transcriptionally silent despite the dual Y701-S727 phosphorylation. Intriguingly, in the absence of IFN-γ stimulation, Toxoplasma infection still triggers the activation of STAT1 in a TgISTmediated manner but independently of JAKs, suggesting the recruitment by TgIST of host kinases able to shortcut the JAK/STAT pathway (Gay et al., 2016). Chromatin immunoprecipitation revealed that the dually phosphorylated STAT1 was markedly enriched in the vicinity of GAS-containing promoters at the same level in both resting and IFN-γ-stimulated cells (Gay et al., 2016). The sustained retention of STAT1 within chromatin by TgIST is clearly challenging the current model where activated STATs have a short nuclear half-life and are rapidly dephosphorylated and returned to the cytoplasm (Vinkemeier, 2004). One of the hypotheses would be that TgIST-bound HDACs (NuRD subunits), by competing with CBP/p300 HAT, are preventing STAT1 acetylation and DNA dissociation, thereby interfering with STAT1 recycling (Kramer and Heinzel, 2010). Alternatively, NuRD subunits in partnership with ATP-dependent chromatin-remodeling enzymes CHD3 and CHD4 may promote chromatin condensation and consequently gene silencing. However, class I and class II HDAC inhibitors did not prevent TgIST from inhibiting the IFN-γ-induced expression of IRF1 (Gay et al., 2016; Olias et al., 2016), making the hypothesis that TgIST operates by influencing

nucleosome positioning to create a nonpermissive chromatin state very unlikely. At the cellular level the absence of TgIST leads to enhanced clearance and reduced growth of Type II tachyzoites in cells that are infected and then activated with IFN-γ but not when naive cells are prestimulated. Therefore TgIST acts prior to the host cell receiving the IFN-γ signal by blocking at the transcriptional level the potent interferon-stimulated genemediated parasite killing (Gay et al., 2016; Olias et al., 2016). This time-restricted activity of TgIST results in reduced mouse virulence which is restored in IFN-γ-receptor-deficient mice (Olias et al., 2016). How Toxoplasma inhibits the IFN-γ responsive genes has been of long-standing interest to investigators studying immune response evasion; TgIST provides a molecular explanation for the unresponsiveness to IFN-γ of the tachyzoite-infected cells, thereby complementing the function of ROP17, ROP18, and ROP5 in interfering with host defenses. The most recent example of a Toxoplasma effector interfering with host transcription factors is a novel GRA protein dubbed TEEGR (Toxoplasma E2F4-associated EZH2-inducing gene regulator; Braun et al., 2019) or inducer of host cyclin E (HCE1; Panas et al., 2019) that directly modulates E2F transcription factor activity. TEEGR/HCE1 shares with GRA16, GRA24, and TgIST the ability to cross the PVM in an MYR1-dependent manner and to accumulate in the host cell nucleus. Biochemistry uncovered a multivalent partnership between TEEGR/HCE1 and the E2F3 and E2F4 transcription factors in association with their DPs, DP-1 and DP-2 (Braun et al., 2019; Panas et al., 2019). Those chimeric complexes are functional, since Toxoplasma activates the expression of E2F3/4-regulated host genes in a TEEGR/HCE1 manner, including the HCE (Panas et al., 2019) and the epigenetic factor EZH2 (Braun et al., 2019). EZH2 is the catalytic subunit of the polycomb repressive complex 2

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17.4 Conclusion

that catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27me3) and thereby mediates epigenetic transcriptional silencing. Although transcriptomics unambiguously identified TEEGR/HCE1 as a transcriptional activator, it also pointed out that TEEGR silenced host genes in cells infected by Type II cystogenic strains (Braun et al., 2019). TEEGRmediated gene silencing is achieved by switching on EZH2 that, in turn, promotes the shaping of a nonpermissive chromatin. Thus TEEGR from the Type II genetic background specifically silences a subset of NF-κB-regulated cytokines (IL-1β, IL-6, IL-23A, IL-15, granulocytemacrophage colony-stimulating factor) and chemokines (IL-8, CXCL2, CXCL3, CCL20) but intriguingly without affecting the expression of others (IL-12, IL-18) (Braun et al., 2019). A possible explanation is that the TEEGRinduced EZH2 may contribute to a silenced chromatin landscape that would restrict NF-κB accessibility in a cell-specific manner, in agreement with the current model (Bhatt and Ghosh, 2014). Teegr deficiency was associated with a drastic reduction of tachyzoite burden in mice and a significant decrease in cyst load, suggesting that TEEGR contributes to the host immune equilibrium and promotes parasite persistence in mice (Braun et al., 2019). By coopting epigenetic regulators, TEEGR/ HCE1 and TgIST can be seen as “epigenator signals,” according to the definition of Berger et al. (2009). If the analogy is pushed further, these parasite-derived effectors may elicit and sustain a “transcriptional memory” in cells infected by tachyzoites (macrophage, dendritic, and T cells), preventing immunological clearance. Toxoplasma effectors reveal much about the regulation and interaction of intrinsic cellular signaling pathways by serving as probes to dissect their functions. It seems certain that novel activities of these proteins will be found, and there will be new insights into both how pathogens remodel host cells for their own benefit, and how hosts recognize effectors and mount

an immune response. Studies of effectors also continue to offer opportunities for the development of tools to probe host cell biology in the absence of disease. GRA16 is a striking example where a parasite protein is able to interfere with a tumor suppressor p53 protein stability and activity through interaction with HAUSP, the main ubiquitin-protease regulating the p53Mdm2 pathway (Bougdour et al., 2013). How these GRA proteins are translocated across the PVM is incompletely understood but involves at least three parasite-derived proteins named MYRs for the genetic screen that identified them, that is, a defect in host MYR (Franco et al., 2016; Marino et al., 2018). These MYR proteins are discussed in further detail in Chapter 14, Toxoplasma secretory proteins and their roles in parasite cell cycle and infection, but, as yet, little information is available for which if any of these proteins is the actual translocon. All that is known is that all three localize to the PVM and deletion of any one of them essentially eliminates translocation of the GRA effectors describe previously.

17.4 Conclusion For those interested in the interaction between a pathogen and the host cell in which it grows, the last decade or so has been an exciting one in Toxoplasma research. We now know that multiple rhoptry and dense granule proteins are introduced by Toxoplasma into the host cell where they perform many key roles important for the host pathogen interaction. Some appear to be clear counter-defenses that neutralize innate immune responses, while others are more subtle in their effects, tweaking host functions to optimize the intracellular niche for parasite growth. Although we have good data on several such effectors, the list will undoubtedly grow over the coming years and much is likely to be learned as a result about both the parasite and the host cells in

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which they replicate. This is especially so as new, more sensitive techniques allow more effectors to be identified, and as studies expand beyond the usual starting point of lab strains being studied as tachyzoites in established in vitro models, to less studied strains, developmental stages, host cell types, and animal models where the more “natural” biology lies!

Acknowledgments We thank our co-workers for helpful comments on the manuscript and our many colleagues in the field who, through many conversations over the years, have provided important insight and data underlying the biology described here. We especially thank Suchita Rastogi for permission to reproduce her illustration as figure 1. JCB is supported by USA National Institutes of Health (RO1 A!021423 and RO1 AI129529); MAH is supported by the Laboratoire d’Excellence (LabEx) ParaFrap [ANR11-LABX-0024], and the European Research Council [ERC Consolidator Grant N 614880 Hosting TOXO].

References Achbarou, A., Mercereau-Puijalon, O., Sadak, A., Fortier, B., Leriche, M.A., Camus, D., et al., 1991. Differential targeting of dense granule proteins in the parasitophorous vacuole of Toxoplasma gondii. Parasitology 103 (Pt 3), 321 329. Adomako-Ankomah, Y., English, E.D., Danielson, J.J., Pernas, L.F., Parker, M.L., Boulanger, M.J., et al., 2016. Host mitochondrial association evolved in the human parasite Toxoplasma gondii via neofunctionalization of a gene duplicate. Genetics 203, 283 298. Alexander, D.L., Arastu-Kapur, S., Dubremetz, J.F., Boothroyd, J.C., 2006. Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma gondii. Eukaryot. Cell 5, 1169 1173.

Bando, H., Sakaguchi, N., Lee, Y., Pradipta, A., Ma, J.S., Tanaka, S., et al., 2018. Toxoplasma effector TgIST targets host IDO1 to antagonize the IFN-gamma-Induced antiparasitic response in human cells. Front. Immunol. 9, 2073. Beckers, C.J., Dubremetz, J.F., Mercereau-Puijalon, O., Joiner, K.A., 1994a. The Toxoplasma gondii rhoptry protein ROP2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J. Cell Biol. 127, 947 961. Beckers, C.J.M., Dubremetz, J.F., Mercereau-Puijalon, O., Joiner, K.A., 1994b. The Toxoplasma gondii rhoptry protein ROP2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J. Cell Biol. 127, 947 961. Behnke, M.S., Wootton, J.C., Lehmann, M.M., Radke, J.B., Lucas, O., Nawas, J., et al., 2010. Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5, e12354. Behnke, M.S., Khan, A., Wootton, J.C., Dubey, J.P., Tang, K., Sibley, L.D., 2011. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc. Natl. Acad. Sci. U.S. A. 108, 9631 9636. Behnke, M.S., Fentress, S.J., Mashayekhi, M., Li, L.X., Taylor, G.A., Sibley, L.D., 2012. The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18. PLoS Pathog. 8, e1002992. Beraki, T., Hu, X., Broncel, M., Young, J.C., O’shaughnessy, W.J., Borek, D., et al., 2019. Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole. Proc. Natl. Acad. Sci. U.S.A. 116, 6361 6370. Berger, S.L., Kouzarides, T., Shiekhattar, R., Shilatifard, A., 2009. An operational definition of epigenetics. Genes Dev. 23, 781 783. Besteiro, S., Michelin, A., Poncet, J., Dubremetz, J.F., Lebrun, M., 2009. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 5, e1000309. Besteiro, S., Dubremetz, J.F., Lebrun, M., 2011. The moving junction of apicomplexan parasites: a key structure for invasion. Cell. Microbiol. 13, 797 805. Bhatt, D., Ghosh, S., 2014. Regulation of the NF-kappaBmediated transcription of inflammatory genes. Front. Immunol. 5, 71. Biswas, S.K., Mantovani, A., 2010. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat. Immunol. 11, 889 896.

Toxoplasma Gondii

References

Boothroyd, J.C., Dubremetz, J.F., 2008. Kiss and spit: the dual roles of Toxoplasma rhoptries. Nat. Rev. Microbiol. 6, 79 88. Bougdour, A., Durandau, E., Brenier-Pinchart, M.P., Ortet, P., Barakat, M., Kieffer, S., et al., 2013. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression. Cell Host Microbe 13, 489 500. Bougdour, A., Tardieux, I., Hakimi, M.A., 2014. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression. Cell. Microbiol. 16, 334 343. Boyle, J.P., Saeij, J.P., Harada, S.Y., Ajioka, J.W., Boothroyd, J.C., 2008. Expression quantitative trait locus mapping of Toxoplasma genes reveals multiple mechanisms for strain-specific differences in gene expression. Eukaryot. Cell 7, 1403 1414. Bradley, P.J., Ward, C., Cheng, S.J., Alexander, D.L., Coller, S., Coombs, G.H., et al., 2005. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii. J. Biol. Chem. 280, 34245 34258. Braun, L., Brenier-Pinchart, M.P., Yogavel, M., CurtVaresano, A., Curt-Bertini, R.L., Hussain, T., et al., 2013. A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation. J. Exp. Med. 210, 2071 2086. Braun, L., Brenier-Pinchart, M.P., Hammoudi, P.M., Cannella, D., Kieffer-Jaquinod, S., Vollaire, J., et al., 2019. The Toxoplasma effector TEEGR promotes parasite persistence by modulating NF-kappaB signalling via EZH2. Nat Microbiol 4, 1208 1220. Brossier, F., Jewett, T.J., Sibley, L.D., Urban, S., 2005. A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc. Natl. Acad. Sci. U.S.A. 102, 4146 4151. Butcher, B.A., Fox, B.A., Rommereim, L.M., Kim, S.G., Maurer, K.J., Yarovinsky, F., et al., 2011. Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1dependent growth control. PLoS Pathog. 7, e1002236. Cabral, G.R.A., Wang, Z.T., Sibley, L.D., Damatta, R.A., 2018. Inhibition of nitric oxide production in activated macrophages caused by Toxoplasma gondii infection occurs by distinct mechanisms in different mouse macrophage cell lines. Front. Microbiol. 9, 1936. Camejo, A., Gold, D.A., Lu, D., Mcfetridge, K., Julien, L., Yang, N., et al., 2014. Identification of three novel Toxoplasma gondii rhoptry proteins. Int. J. Parasitol. 44, 147 160. Carey, K.L., Jongco, A.M., Kim, K., Ward, G.E., 2004. The Toxoplasma gondii rhoptry protein ROP4 is secreted into

803

the parasitophorous vacuole and becomes phosphorylated in infected cells. Eukaryot. Cell 3, 1320 1330. Carruthers, V.B., Sibley, L.D., 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur. J. Cell Biol. 73, 114 123. Carruthers, V.B., Tomley, F.M., 2008. Microneme proteins in apicomplexans. Subcell. Biochem. 47, 33 45. Coffey, M.J., Dagley, L.F., Seizova, S., Kapp, E.A., Infusini, G., Roos, D.S., et al., 2018. Aspartyl protease 5 matures dense granule proteins that reside at the host-parasite interface in Toxoplasma gondii. mBio 9, 1 21. Czimbalek, L., Kollar, V., Kardos, R., Lorinczy, D., Nyitrai, M., Hild, G., 2015. The effect of toxofilin on the structure and dynamics of monomeric actin. FEBS Lett. 589, 3085 3089. Daubeuf, S., Singh, D., Tan, Y., Liu, H., Federoff, H.J., Bowers, W.J., et al., 2009. HSV ICP0 recruits USP7 to modulate TLR-mediated innate response. Blood 113, 3264 3275. Delorme, V., Cayla, X., Faure, G., Garcia, A., Tardieux, I., 2003. Actin dynamics is controlled by a casein kinase II and phosphatase 2C interplay on Toxoplasma gondii toxofilin. Mol. Biol. Cell 14, 1900 1912. Delorme-Walker, V., Abrivard, M., Lagal, V., Anderson, K., Perazzi, A., Gonzalez, V., et al., 2012. Toxofilin upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion. J. Cell. Sci. 125, 4333 4342. Dowse, T.J., Pascall, J.C., Brown, K.D., Soldati, D., 2005. Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion. Int. J. Parasitol. 35, 747 756. Dubois, D.J., Soldati-Favre, D., 2019. Biogenesis and secretion of micronemes in Toxoplasma gondii. Cell. Microbiol. 21, e13018. Dubremetz, J.F., 2007. Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction. Cell. Microbiol. 9, 841 848. El hajj, H., Demey, E., Poncet, J., Lebrun, M., Wu, B., Galeotti, N., et al., 2006. The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling. Proteomics 6, 5773 5784. El hajj, H., Lebrun, M., Arold, S.T., Vial, H., Labesse, G., Dubremetz, J.F., 2007a. ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii. PLoS Pathog. 3, e14. El Hajj, H., Lebrun, M., Fourmaux, M.N., Vial, H., Dubremetz, J.F., 2007b. Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane. Cell. Microbiol. 9, 54 64.

Toxoplasma Gondii

804

17. Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages

Etheridge, R.D., Alaganan, A., Tang, K., Lou, H.J., Turk, B. E., Sibley, L.D., 2014. The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice. Cell Host Microbe 15, 537 550. Fleckenstein, M.C., Reese, M.L., Konen-Waisman, S., Boothroyd, J.C., Howard, J.C., Steinfeldt, T., 2012. A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins. PLoS Biol. 10, e1001358. Fox, B.A., Rommereim, L.M., Guevara, R.B., Falla, A., Hortua triana, M.A., Sun, Y., et al., 2016. The Toxoplasma gondii rhoptry kinome is essential for chronic infection. mBio 7, 1 12. Franco, M., Panas, M.W., Marino, N.D., Lee, M.C., Buchholz, K.R., Kelly, F.D., et al., 2016. A novel secreted protein, MYR1, is central to Toxoplasma’s manipulation of host cells. mBio 7, 1 16. Gay, G., Braun, L., Brenier-Pinchart, M.P., Vollaire, J., Josserand, V., Bertini, R.L., et al., 2016. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-gammamediated host defenses. J. Exp. Med. 213, 1779 1798. Gilbert, L.A., Ravindran, S., Turetzky, J.M., Boothroyd, J.C., Bradley, P.J., 2007. Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells. Eukaryot. Cell 6, 73 83. Hakimi, M.A., Olias, P., Sibley, L.D., 2017. Toxoplasma effectors targeting host signaling and transcription. Clin. Microbiol. Rev. 30, 615 645. Harper, J.M., Zhou, X.W., Pszenny, V., Kafsack, B.F., Carruthers, V.B., 2004. The novel coccidian micronemal protein MIC11 undergoes proteolytic maturation by sequential cleavage to remove an internal propeptide. Int. J. Parasitol. 34, 1047 1058. He, H., Brenier-Pinchart, M.P., Braun, L., Kraut, A., Touquet, B., Coute, Y., et al., 2018. Characterization of a Toxoplasma effector uncovers an alternative GSK3/betacatenin-regulatory pathway of inflammation. eLife 7, 1 28. Hehl, A.B., Lekutis, C., Grigg, M.E., Bradley, P.J., Dubremetz, J.F., Ortega-Barria, E., et al., 2000. Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells. Infect. Immun. 68, 7078 7086. Herion, P., Hernandez-Pando, R., Dubremetz, J.F., Saavedra, R., 1993. Subcellular localization of the 54kDa antigen of Toxoplasma gondii. J. Parasitol. 79, 216 222. Hoff, E.F., Cook, S.H., Sherman, G.D., Harper, J.M., Ferguson, D.J., Dubremetz, J.F., et al., 2001. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Exp. Parasitol. 97, 77 88.

Jan, G., Delorme, V., David, V., Revenu, C., Rebollo, A., Cayla, X., et al., 2007. The toxofilin-actin-PP2C complex of Toxoplasma: identification of interacting domains. Biochem. J. 401, 711 719. Jensen, K.D., Wang, Y., Wojno, E.D., Shastri, A.J., Hu, K., Cornel, L., et al., 2011. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation. Cell Host Microbe 9, 472 483. Jensen, K.D., Hu, K., Whitmarsh, R.J., Hassan, M.A., Julien, L., Lu, D., et al., 2013. Toxoplasma gondii rhoptry 16 kinase promotes host resistance to oral infection and intestinal inflammation only in the context of the dense granule protein GRA15. Infect. Immun. 81, 2156 2167. Jones, T.C., Alkan, S., Erb, P., 1986. Spleen and lymph node cell populations, in vitro cell proliferation and interferon-gamma production during the primary immune response to Toxoplasma gondii. Parasite Immunol. 8, 619 629. Jones, N.G., Wang, Q., Sibley, L.D., 2017. Secreted protein kinases regulate cyst burden during chronic toxoplasmosis. Cell. Microbiol. 19, 1 13. Kafsack, B.F., Pena, J.D., Coppens, I., Ravindran, S., Boothroyd, J.C., Carruthers, V.B., 2009. Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 323, 530 533. Kemp, L.E., Yamamoto, M., Soldati-Favre, D., 2013. Subversion of host cellular functions by the apicomplexan parasites. FEMS Microbiol. Rev. 37, 607 631. Khaminets, A., Hunn, J.P., Konen-Waisman, S., Zhao, Y.O., Preukschat, D., Coers, J., et al., 2010. Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole. Cell. Microbiol. 12, 939 961. Kim, K., 2004. Role of proteases in host cell invasion by Toxoplasma gondii and other Apicomplexa. Acta Trop. 91, 69 81. Kim, S.K., Fouts, A.E., Boothroyd, J.C., 2007. Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: insights from a genome-wide transcriptional profiling. J. Immunol. 178, 5154 5165. Kimata, I., Tanabe, K., 1987. Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell. J. Cell. Sci. 88, 231 239. Kramer, O.H., Heinzel, T., 2010. Phosphorylationacetylation switch in the regulation of STAT1 signaling. Mol. Cell. Endocrinol. 315, 40 48. Labesse, G., Gelin, M., Bessin, Y., Lebrun, M., Papoin, J., Cerdan, R., et al., 2009. ROP2 from Toxoplasma gondii: a virulence factor with a protein-kinase fold and no enzymatic activity. Structure 17, 139 146. Lamarque, M., Besteiro, S., Papoin, J., Roques, M., Vulliez-Le normand, B., Morlon-Guyot, J., et al., 2011.

Toxoplasma Gondii

References

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites. PLoS Pathog. 7, e1001276. Lebrun, M., Michelin, A., El hajj, H., Poncet, J., Bradley, P.J., Vial, H., et al., 2005. The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion. Cell. Microbiol. 7, 1823 1833. Lee, S.H., Hayes, D.B., Rebowski, G., Tardieux, I., Dominguez, R., 2007. Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer. Proc. Natl. Acad. Sci. U.S.A. 104, 16122 16127. Li, J.X., He, J.J., Elsheikha, H.M., Chen, D., Zhai, B.T., Zhu, X.Q., et al., 2019. Toxoplasma gondii ROP17 inhibits the innate immune response of HEK293T cells to promote its survival. Parasitol. Res. 118, 783 792. Lodoen, M.B., Gerke, C., Boothroyd, J.C., 2010. A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection. Cell. Microbiol. 12, 55 66. Ma, J.S., Sasai, M., Ohshima, J., Lee, Y., Bando, H., Takeda, K., et al., 2014. Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6. J Exp Med 211 (10), 2013 2032. Available from: https://doi.org/10.1084/ jem.20131272. Epub 2014 Sep 15. PubMed PMID: 25225460; PubMed Central PMCID: PMC4172224. Marino, N.D., Panas, M.W., Franco, M., Theisen, T.C., Naor, A., Rastogi, S., et al., 2018. Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii. PLoS Pathog. 14, e1006828. Minot, S., Melo, M.B., Li, F., Lu, D., Niedelman, W., Levine, S.S., et al., 2012. Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity. Proc. Natl. Acad. Sci. U.S.A. 109, 13458 13463. Moon, S.H., Huang, C.H., Houlihan, S.L., Regunath, K., Freed-Pastor, W.A., Morris, J.P.T., et al., 2019. p53 Represses the mevalonate pathway to mediate tumor suppression. Cell 176, 564 580.e19. Nakaar, V., Ngo, H.M., Aaronson, E.P., Coppens, I., Stedman, T.T., Joiner, K.A., 2003. Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii. J. Cell. Sci. 116, 2311 2320. Nast, R., Staab, J., Meyer, T., Luder, C.G.K., 2018. Toxoplasma gondii stabilises tetrameric complexes of tyrosine-phosphorylated signal transducer and activator of transcription-1 and leads to its sustained and promiscuous DNA binding. Cell. Microbiol. 20, e12887. Nichols, B.A., Chiappino, M.L., O’connor, G.R., 1983. Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J. Ultrastruct. Res. 83, 85 98. Niedelman, W., Gold, D.A., Rosowski, E.E., Sprokholt, J.K., Lim, D., Farid arenas, A., et al., 2012. The rhoptry

805

proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferongamma response. PLoS Pathog. 8, e1002784. Niehrs, C., 2012. The complex world of WNT receptor signalling. Nat. Rev. Mol. Cell Biol. 13, 767 779. Olias, P., Etheridge, R.D., Zhang, Y., Holtzman, M.J., Sibley, L.D., 2016. Toxoplasma effector recruits the Mi-2/ NuRD complex to repress STAT1 transcription and block IFN-gamma-dependent gene expression. Cell Host Microbe 20, 72 82. Ong, Y.C., Reese, M.L., Boothroyd, J.C., 2010. Toxoplasma rhoptry protein 16 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6. J. Biol. Chem. 285, 28731 28740. Ong, Y.C., Boyle, J.P., Boothroyd, J.C., 2011. Straindependent host transcriptional responses to Toxoplasma infection are largely conserved in Mammalian and avian hosts. PLoS One 6, e26369. Ossorio, P.N., Schwartzman, J.D., Boothroyd, J.C., 1992. A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry. Mol. Biochem. Parasitol. 50, 1 15. Panas, M.W., Naor, A., Cygan, A.M., Boothroyd, J.C., 2019. Toxoplasma controls host cyclin E expression through the use of a novel MYR1-dependent effector protein, HCE1. mBio 10, 1 18. Peixoto, L., Chen, F., Harb, O.S., Davis, P.H., Beiting, D.P., Brownback, C.S., et al., 2010. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses. Cell Host Microbe 8, 208 218. Pellegrini, E., Palencia, A., Braun, L., Kapp, U., Bougdour, A., Belrhali, H., et al., 2017. Structural basis for the subversion of MAP kinase signaling by an intrinsically disordered parasite secreted agonist. Structure 25, 16 26. Pernas, L., Boothroyd, J.C., 2010. Association of host mitochondria with the parasitophorous vacuole during Toxoplasma infection is not dependent on rhoptry proteins ROP2/8. Int. J. Parasitol. 40, 1367 1371. Pernas, L., Adomako-Ankomah, Y., Shastri, A.J., Ewald, S. E., Treeck, M., Boyle, J.P., et al., 2014. Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS Biol. 12, e1001845. Qiu, W., Wernimont, A., Tang, K., Taylor, S., Lunin, V., Schapira, M., et al., 2009. Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii. EMBO J. 28, 969 979. Rabenau, K.E., Sohrabi, A., Tripathy, A., Reitter, C., Ajioka, J.W., Tomley, F.M., et al., 2001. TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2. Mol. Microbiol. 41, 537 547.

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Reese, M.L., Boothroyd, J.C., 2009. A helical membranebinding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole. Traffic. 10, 1458 1470. Reese, M.L., Zeiner, G.M., Saeij, J.P., Boothroyd, J.C., Boyle, J.P., 2011. Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence. Proc. Natl. Acad. Sci. U.S.A. 108, 9625 9630. Reese, M.L., Shah, N., Boothroyd, J.C., 2014. The Toxoplasma pseudokinase ROP5 is an allosteric inhibitor of the immunity-related GTPases. J. Biol. Chem. 289, 27849 27858. Reid, M.A., Wang, W.I., Rosales, K.R., Welliver, M.X., Pan, M., Kong, M., 2013. The B55alpha subunit of PP2A drives a p53-dependent metabolic adaptation to glutamine deprivation. Mol. Cell 50, 200 211. Riscal, R., Schrepfer, E., Arena, G., Cisse, M.Y., Bellvert, F., Heuillet, M., et al., 2016. Chromatin-bound MDM2 regulates serine metabolism and redox homeostasis independently of p53. Mol. Cell 62, 890-902. Roiko, M.S., Carruthers, V.B., 2013. Functional dissection of Toxoplasma gondii perforin-like protein 1 reveals a dual domain mode of membrane binding for cytolysis and parasite egress. J. Biol. Chem. 288, 8712 8725. Rosowski, E.E., Saeij, J.P., 2012. Toxoplasma gondii clonal strains all inhibit STAT1 transcriptional activity but polymorphic effectors differentially modulate IFNgamma induced gene expression and STAT1 phosphorylation. PLoS One 7, e51448. Rosowski, E.E., Lu, D., Julien, L., Rodda, L., Gaiser, R.A., Jensen, K.D., et al., 2011. Strain-specific activation of the NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208, 195 212. Rosowski, E.E., Nguyen, Q.P., Camejo, A., Spooner, E., Saeij, J.P., 2014. Toxoplasma gondii Inhibits gamma interferon (IFN-gamma)- and IFN-beta-induced host cell STAT1 transcriptional activity by increasing the association of STAT1 with DNA. Infect. Immun. 82, 706 719. Sadak, A., Taghy, Z., Fortier, B., Dubremetz, J.F., 1988. Characterization of a family of rhoptry proteins of Toxoplasma gondii. Mol. Biochem. Parasitol. 29, 203 211. Saeij, J.P., Boyle, J.P., Coller, S., Taylor, S., Sibley, L.D., Brooke-Powell, E.T., et al., 2006. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314, 1780 1783. Saridakis, V., Sheng, Y., Sarkari, F., Holowaty, M.N., Shire, K., Nguyen, T., et al., 2005. Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr nuclear antigen 1 implications for EBV-mediated immortalization. Mol. Cell 18, 25 36. Shastri, A.J., Marino, N.D., Franco, M., Lodoen, M.B., Boothroyd, J.C., 2014. GRA25 is a novel virulence factor of Toxoplasma gondii and influences the host immune response. Infect. Immun. 82, 2595 2605.

Sibley, L.D., 1993. Interactions between Toxoplasma gondii and its mammalian host cells. Semin. Cell Biol. 4, 335 344. Sibley, L.D., Krahenbuhl, J.L., 1986. Toxoplasma modifies macrophage phagosomes by secretion of a vesicular network rich in surface proteins. J. Cell Biol. 103, 867 874. Sinai, A.P., Joiner, K.A., 2001. The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane. J. Cell Biol. 154, 95 108. Soldati, D., Kim, K., Kampmeier, J., Dubremetz, J.F., Boothroyd, J.C., 1995. Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection. Mol. Biochem. Parasitol. 74, 87 97. Speer, C.A., Clark, S., Dubey, J.P., 1998. Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii. J. Parasitol. 84, 505 512. Speer, C.A., Dubey, J.P., Mcallister, M.M., Blixt, J.A., 1999. Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii [In Process Citation]. Int. J. Parasitol. 29, 1509 1519. Staal, F.J., Luis, T.C., Tiemessen, M.M., 2008. WNT signalling in the immune system: WNT is spreading its wings. Nat. Rev. Immunol. 8, 581 593. Suzuki, Y., Orellana, M.A., Schreiber, R.D., Remington, J.S., 1988. Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science 240, 516 518. Talevich, E., Kannan, N., 2013. Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors. BMC Evol. Biol. 13, 117. Tyler, J.S., Boothroyd, J.C., 2011. The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex. PLoS Pathog. 7, e1001282. Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., et al., 2006. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314, 1776 1780. Vinkemeier, U., 2004. Getting the message across, STAT! Design principles of a molecular signaling circuit. J. Cell Biol. 167, 197 201. Wei, F., Wang, W., Liu, Q., 2013. Protein kinases of Toxoplasma gondii: functions and drug targets. Parasitol. Res. 112, 2121 2129. Yamamoto, M., Standley, D.M., Takashima, S., Saiga, H., Okuyama, M., Kayama, H., et al., 2009. A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3. J. Exp. Med. 206, 2747 2760. Yamamoto, M., Ma, J.S., Mueller, C., Kamiyama, N., Saiga, H., Kubo, E., et al., 2011. ATF6beta is a host cellular target of the Toxoplasma gondii virulence factor ROP18. J. Exp. Med. 208, 1533 1546.

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18 Bradyzoite and sexual stage development Anthony P. Sinai1, Laura J. Knoll2 and Louis M. Weiss3,4 1

Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky, College of Medicine, Lexington, KY, United States 2Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, United States 3Department of Pathology, Albert Einstein College of Medicine, New York, NY, United States 4Department of Medicine, Albert Einstein College of Medicine, New York, NY, United States

18.1 Introduction The asexual cycle of Toxoplasma gondii can occur in every warm-blooded animal, including humans. The asexual cycle has two developmental stages: a rapidly replicating form called the tachyzoite and a slow growing form called the bradyzoite. The sexual cycle of T. gondii occurs naturally only within the feline intestine, after which infected cats excrete between 2 and 20 million oocysts per day in their feces. T. gondii is acquired orally either by ingestion of oocyst-contaminated food or water or by eating bradyzoite cyst-harboring meat products. After ingestion, sporozoites or bradyzoites will invade the intestinal epithelium, differentiate into the rapidly growing tachyzoite form, and disseminate throughout the body. Epidemiology suggests that the ingestion of bradyzoite cysts in undercooked meat is an important source of T. gondii infection for humans (Kimball et al., 1974; Mcauley et al., 1994).

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00018-9

The sexual cycle of T. gondii occurs in the domestic cat and other Felidae, where oocysts are produced in the feline intestinal track. Infection in the cat can occur due to ingestion of bradyzoites, tachyzoites, or oocysts; however, the prepatent period (i.e., the time to the first shedding of oocysts after infection) varies according to the life cycle stage ingested. The shortest prepatent period follows ingestion of tissue cysts (3
10 days) then tachyzoites (13 days), and the longest follows oocyst ingestion ($18 days) (Dubey, 1998a; Dubey et al., 1998). The feline enteroepithelial cycle typically starts upon the ingestion of bradyzoites in tissue cysts, and after several intermediate stages the resulting merozoite stages culminate in the production of gametes. Gamete fusion produces a zygote, which is surrounded by a rigid oocyst wall that can be seen to have blue autofluorescence that can be observed using ultraviolet (UV) light at 330
385 nm. After 2
3 days in the environment, unsporulated oocysts shed in feces sporulate to produce eight

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sporozoites within each oocyst (Dubey, 1998a; Dubey et al., 1998). Sporulated oocysts ingested by an intermediate host, for example, humans or mice, release sporozoites that infect host cells and differentiate into tachyzoites that disseminate in the host and cause acute toxoplasmosis. Sporulated oocysts, which are environmentally hardy and very infectious, are capable of contaminating food, for example produce and water, leading to disease transmission and outbreaks (Aramini et al., 1999; Fritz et al., 2012a). In both the definitive feline and intermediate hosts, tachyzoites disseminate throughout the body, and then differentiate into bradyzoites that remain for the lifetime of the host. The question of whether lifetime infection truly occurs has been the subject of recent debate (Rougier et al., 2017). Tachyzoites can invade virtually any nucleated cell during dissemination. Bradyzoite-containing cysts are found predominately in the host central nervous system and striated muscle (Swierzy et al., 2014; Luder and Rahman, 2017; Cabral et al., 2016), developing in cells that are conventionally viewed as postmitotic (De falco et al., 2009; Sharma et al., 2017) (Fig. 18.1). The number of tissue cysts formed in mouse brain appears to be under immune control and regulated by the class I gene Ld (Brown et al., 1995). More tissue cysts are produced in mice that become mildly ill from infection than in those that become highly symptomatic. Tissue cyst persistence may vary with regard to with both the strain of T. gondii and the strain of murine host (Ferguson et al., 1994a). Mice that are resistant to acute infection can be susceptible to chronic infection with encephalitis suggesting that control of chronic infection is not linked to the loci that control susceptibility to acute infection (Brown and Mcleod, 1990; Brown et al., 1994; Mcleod et al., 1993; Mcleod et al., 1989). Bradyzoites contained in cysts are refractory to most chemotherapeutic agents used for treatment of toxoplasmosis, and tissue cysts are

FIGURE 18.1

Ultrastructure of a Toxoplasma gondii tissue cyst in vitro. (A) This electron micrograph demonstrates multiple bradyzoites within a cyst. The cyst is within a murine astrocyte grown in vitro tissue culture. The clear vacuoles are consistent with amylopectin granules seen in bradyzoites. The cyst wall is formed from the parasitophorous vacuole membrane. Bar length is 25 μm. (B) The organization of the tissue cyst wall and the amorphous matrix surrounding the encysted bradyzoites, which likely has its origins in the tachyzoite intravacuolar network. Source: Courtesy (A) Dr. S. Halonen, MSU and (B) C. Vommaro.

produced in any animal capable of being infected with T. gondii. The persistence and reactivation of bradyzoite forms is a major cause of disease in humans. Symptomatic toxoplasmosis is believed to occur in immunocompromised patients, typically due to recrudescence of chronic infection mediated by bradyzoite reactivation as cellular immune

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18.2 Bradyzoite and tissue cyst morphology and biology

surveillance is lost (Luft and Remington, 1992). This potentially suggests that reactivation may occur spontaneously with the released and growing parasites being efficiently cleared by the intact immune system, something that unchecked in the context of immune suppression. As such the bradyzoite within a tissue cyst, while largely innocuous in a healthy host, represents a ticking time bomb in the immunocompromised host. This is not likely to be a desirable outcome for the parasite, as the tissue cyst is designed as a transmission form during the act of carnivory that completes the life cycle when the carnivore is a felid. Despite its critical role in clinical disease and the progression of the life cycle, our understanding of bradyzoites and the tissue cysts within which they reside remains limited. In gaining physiological insights into bradyzoites, which themselves are highly functionally heterogenous, we may better address the acute paucity of therapeutic approaches targeting this enigmatic though clinically central life cycle stage.

18.2 Bradyzoite and tissue cyst morphology and biology Tachyzoites (tachos 5 fast) refer to the rapidly growing life stage of T. gondii that have also been called endozoites or trophozoites. Bradyzoites (brady 5 slow), also called cystozoites, are the life stage found in the tissue cyst and are believed to replicate slowly. Both stages replicate by endodyogeny within a parasitophorous vacuole within the host cell, which is modified by the particular life stage into either a tachyzoite- or bradyzoite-specific vacuole. T. gondii tissue cysts are the modified bradyzoite parasitophorous vacuole found within a host cell. Evidence of endodyogeny within encysted organisms, which was found in early ultrastructural studies (Dubey et al., 1998; Ferguson, 2004; Ferguson et al., 1994b; Huskinson-Mark et al., 1991; Fortier et al.,

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1996), was confirmed in purified ex vivo tissue cysts wherein each stage of the process was captured morphologically (Watts et al., 2015). This was made possible by staining for the inner membrane complex protein TgIMC3 (Anderson-White et al., 2011), which possesses some unique feature. First, TgIMC3 is a marker of developing daughters, with daughter scaffolds considerably brighter than the gravid mother (Anderson-White et al., 2011). This intensity is maintained in the released progeny but fades over time (Anderson-White et al., 2011). As a result, TgIMC3 intensity can serve as an indicator of the recency of replication (Watts et al., 2015). The overall TgIMC3 signal and its distribution within a given tissue cyst provides a crucial insight into the previously unrecognized dynamism of bradyzoites within tissue cysts in vivo (Watts et al., 2015) (Fig. 18.2). This analysis revealed that tissue cysts are not the uniform dormant entities that they had long been viewed as (Weiss and Kim, 2000; Sinai et al., 2017); rather, the bradyzoites within tissue cysts display a remarkable diversity with evidence for sporadic, clustered, and even synchronized replication (Watts et al., 2015). This dynamism exposed by TgIMC3 labeling has a distinct temporal component that suggests that replication of bradyzoites within tissue cysts likely occurs in an opportunistic, though cyclical, fashion (Watts et al., 2015) that can potentially be modeled mathematically (Patwardhan and Sinai, unpublished). The definitive evidence for bradyzoite replication within tissue cysts and overall tissue cyst heterogeneity reveals that the tissue cyst represents a “colony” that comprises physiologically variable bradyzoites (Sinai et al., 2017). This heterogeneity is likely to underpin the ineffectiveness of drugs as the population of tissue cysts comprises a subpopulation of variable bradyzoites (Sinai et al., 2017; Alday and Doggett, 2017). Thus a true understanding of the chronic infection needs to address

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FIGURE 18.2 Evidence for active replication within an in vivo derived tissue cyst. The purified tissue cyst was labeled with an antibody specific for the inner membrane complex protein TgIMC3 and Hoechst dye, a nuclear stain (DNA). Parasites undergoing active endodyogeny captured at different stages of cytokinesis are evident with the intense TgIMC3 labeling, while bradyzoites that are not actively replicating at the time of fixation stain with varying TgIMC3 intensity, a function of the recency of replication. Two bradyzoites in the later stages of endodyogeny are highlighted in the red and yellow box to the bottom and right of the main image. Here the nuclear profiles, including the mitotic nuclei, are evident. These images have been rendered on the computer to highlight the distinct morphological features. Source: Courtesy E. Watts and A.P Sinai.

specific physiological states at the level of the individual encysted organisms. Toward this end, the Sinai laboratory has initiated a program to identify imaging-based physiological markers and the relationships between them (Watts et al., 2015). At the heart of the effort to exploit imaging based physiological markers the the expansion of the capabilities of BradyCount 1.0 from merely enumerating bradyzoite numbers within cysts (Watts et al., 2015) to quantifying replication status, amylopectin reserves and mitochondrial function (see next). This expanded capability is being incorporated into BradyCount 2.0. The ability of BradyCount 1.0 to streamline the quantification of encysted bradyzoite burdens by recording the Hoechst dye labeled nuclear profiles (1 to 1 ratio to bradyzoites) revealed previously unknown facts regarding the relationship between bradyzoites and the tissue cysts that house them (Watts et al., 2015). Most notably, the mean bradyzoite number within tissue cysts increased over time

before plateauing (Watts et al., 2015). Their relative occupancy, the packing density (bradyzoites per imaged cyst volume), followed an oscillatory pattern (Watts et al., 2015). This strongly suggests that tissue cyst size is not governed by bradyzoite occupancy or growth, but rather tissue cyst expansion likely provides the resident bradyzoites room to replicate and occupy the space, thereby increasing the packing density (Watts et al., 2015; Sinai et al., 2017). Cycles of tissue cyst expansion followed by rounds of replication likely contribute to the oscillatory profile of the packing density. Traditionally, bradyzoites have been viewed as nonreplicative terminally differentiated organisms (Sinai et al., 2017; Weiss and Kim, 2000). This view was supported by the finding that the vast majority of organisms were in a G0 with a DNA content of 1N (Radke et al., 2003). The true picture may actually be more complex, as one follows the replication potential of bradyzoites over time. Early tissue cysts (3 weeks postinfection) contain a large

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proportion of actively replicating organisms as defined by the overall high distribution of TgIMC3 intensities (Watts et al., 2015). Consistent with bradyzoites entering a “dormant” state, tissue cysts harvested at week 5 exhibited low levels of TgIMC3 expression, an indication not only of little active replication but also a significant time interval since the last replication event (Watts et al., 2015). This is followed by a surprising burst of replication between weeks 5 and 8 resulting in TgIMC3 levels intermediate to the week 3 and week 5 levels (Watts et al., 2015). This finding points to the “evolution” of the chronic infection not being entirely unidirectional toward dormancy, as was previously considered to be the case (Weiss and Kim, 2000). This ability to “reset” bradyzoite growth in tissue cysts suggests that bradyzoites within a cyst likely represent a continuum of physiological states covering the spectrum from transitional bradyzoites maintaining a high replication potential, intermediate bradyzoites capable of entering a replicative cycle but not committed to that fate, to the terminal bradyzoite that is differentiated to a state where the parasite is geared for transmission alone (Watts et al., 2017). An intriguing possibility, given the clonality of each tissue cyst, is that by balancing the relative proportions of these states, the parasite may have evolved a bethedging mechanism with organisms tailored for the maintenance of the bradyzoite burden, reactivation, and transmission (Sinai et al., 2017). Thus essentially each tissue cyst is its own “ecological” entity that is influenced by the environment of the parasitized host cell and by extension the physiological and immune status of the host. It has been suggested that in the G0 stage, some of these parasites may act like hypnozoites and represent a reservoir of parasites that are resistant to conventional anti
Toxoplasma therapy (Alday and Doggett, 2017). It is possible that the different

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populations of bradyzoites seen in cysts can undergo different differentiation pathways upon ingestion by a host, for example, the parasites in G0 may be committed to development in feline intestines and may not be capable of differentiation back into tachyzoites directly, and the dividing bradyzoites may be capable of going directly to tachyzoites without first differentiating into sporozoites. These population subsets would be consistent with other Coccidia that developmentally only proceed in forward direction from tachyzoites to bradyzoites to sporozoites, but cannot go from bradyzoites to tachyzoites. These subsets may explain the phenomenon that when a cyst is ruptured and the released bradyzoites are used for a plaque assay, only a fraction of the counted cysts are able to initiate tachyzoite vacuoles in tissue culture (Soete et al., 1993; Soete and Dubremetz, 1996). Immature tissue cysts may be as small as 5 μm containing only two organisms, suggesting that the commitment to bradyzoite differentiation is an early event in the establishment of a parasitophorous vacuole. Such immature cysts increase in size, and thus it is clear that T. gondii expressing bradyzoite-specific markers can replicate during the maturation of tissue cyst (van der Waaij, 1959), with evidence of bradyzoite replication occurring even in 8 week tissue cysts (Watts et al., 2015). The size of a tissue cyst is variable, but on average a mature cyst in the brain is spherical and 50
70 μm in diameter containing approximately 1000 crescent-shaped 7 by 1.5 μm bradyzoites. In muscle, such tissue cysts are more elongated and may be up to 100 μm in length (Dubey, 1998a). The development of the imaging application BradyCount 1.0 (Watts et al., 2015) revealed that these numbers are likely to underrepresent true bradyzoite burdens as imaging volumes of optical sections in medium and larger brain
derived tissue cysts (each cyst was typically optically sectioned into eight slices, in which the widest section was

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quantified) routinely had between 250 and 1500 bradyzoites (Watts et al., 2015). Notably, two cysts with exactly the same imaged volume could have 1.7
2.4-fold differences in the bradyzoite occupancy (packing density) (Watts et al., 2015). These differences highlight the fact that tissue cyst size is not an accurate measure of bradyzoite burden. It further highlights the inherent heterogeneity of tissue cysts necessitating the analysis of the chronic infection at the level of the bradyzoite. The size of brain-derived tissue cysts was found to be similarly distributed based on early or a more progressed infection (Watts et al., 2015). Tissue cyst size trends to be larger at later time points but not in a statistically reliable manner (Watts et al., 2015). Here, however, one needs to present a note of caution as smaller cysts at later time points could have their origins in the reactivation and reestablishment of cysts that originated earlier in the course of the infection. Evidence for such events is seen histologically with clusters of smaller cysts that are more frequent as the infection progresses (Watts et al., 2015). Perhaps not surprisingly, there is a correlation between small cyst size and a decreased oral transmission with some of the exotic strains of T. gondii (Fux et al., 2007). Young and old cysts can be distinguished readily by their ultrastructural features (Dubey, 1998a; Fortier et al., 1996; Sims et al., 1989; Scholytyseck et al., 1974). While tissue cysts can develop in any visceral organ (e.g., lungs, liver, and kidneys), they are more common in neural (e.g., brain and eyes) or muscle (e.g., skeletal and cardiac) tissue (Dubey, 1998a). In the central nervous system, cysts have been reported in neurons, astrocytes, and microglia (Ferguson et al., 1989; Ferguson and Hutchison, 1987a, 1987b; Melzer et al., 2010). Experimental data in mice has indicated that, in the central nervous system, neurons are the predominant cell in which cysts are found (Cabral et al., 2016; Koshy and Cabral, 2014). In

tissue culture, both astrocytes and neurons have been demonstrated to support the cyst formation (Halonen et al., 1996, 1998a; Fagard et al., 1999). Host cells have varying permissiveness for the development of tissue cysts with cells that are more resistant to bradyzoite formation having elevated levels of lactate, glycolysis, and Akt expression (Weilhammer et al., 2012). In vitro bradyzoites have been demonstrated to exit a cyst and establish new cysts in the same cell or adjacent cells (Dzierszinski et al., 2004). This observation, in addition to reactivation of latent infection with spread to adjacent foci, may account for the observation of clusters of cysts seen in vivo in the central nervous system, in addition to foci formed due to reactivation (Odaert et al., 1996). The tissue cyst wall or bradyzoite parasitophorous vacuole membrane is elastic, ,0.5 μm thick (on transmission electron microscopy (TEM) the cyst wall averaged 240 nm), faintly periodic acid
Schiff (PAS) positive, and argyrophilic, although this depends on the silver staining method used (Sims et al., 1988). Proteomic data indicates that many cyst wall proteins are, in fact, glycoproteins (Tomita et al., 2013, 2017; Weiss LM, unpublished data). In addition, experiments suggest that the proteins in the cyst wall, which are glycosylated, may be modified by host cell enzymes, resulting in host cell
specific glycosylation of the cyst wall (Tomita et al., 2013, 2017). This potential adaptation may contribute to the broad host range of T. gondii with host glycans effectively shielding parasite proteins from immune detection and clearance. The tissue cyst wall is phase lucent by phase-contrast microscopy, and the vacuole often contains an odd number of club-shaped parasites, highlighting that asynchronous division occurs (Weiss et al., 1995; Dzierszinski et al., 2004), a finding confirmed with in vivo tissue cysts. Pale blue autofluorescence of the tissue cyst wall can be observed using UV light at 330
385 nm (Lei et al., 2005), this signal is markedly

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18.2 Bradyzoite and tissue cyst morphology and biology

lower than that observed in the oocyst wall (Lindquist et al., 2003). On electron microscopy the membrane of the bradyzoite parasitophorous vacuole has a ruffled appearance and is associated with a precipitation of underlying material creating the cyst wall (Lemgruber et al., 2011b; Zhang et al., 2001; Tomita et al., 2013). Both tubules (65 nm) and vesicles can be seen in this underlying material and the matrix (Lemgruber et al., 2011b). The cyst wall appears to be composed of both host- (outside the parasitophorous membrane) and parasitederived (underlying the parasitophorous membrane) materials and lined by granular material, the matrix, which fills up the space between the bradyzoites, particularly in mature cysts (Sims et al., 1988; Ferguson and Hutchison, 1987a, 1987b; Lemgruber et al., 2011b) (Fig. 18.1). It has been reported that pores (30
40 nm) are present in the tissue cyst wall and that Lucifer yellow (457 Da), trypan blue (960 Da), and dextran
rhodamine (10 kDa) can cross the cyst wall (Lemgruber et al., 2011b). During development in astrocytes, the bradyzoite parasitophorous vacuole is surrounded by a layer of host cell intermediate filaments (glial fibrillary acidic protein) that is not incorporated into the cyst wall. This intermediate filament wrapping causes an increase in the distance between the bradyzoite vacuole with host mitochondria, but not the endoplasmic reticulum (ER), compared with that seen in tachyzoite vacuoles; although in both cases, ER and mitochondria are found to associate with the vacuole (Halonen et al., 1998b). Wrapping of the cyst wall with intermediate filaments and the present of a microtubule cage around the vacuole has also been observed other cell types, for example, epithelial kidney cells from Macaca mullata (LC-MK2) and human foreskin fibroblasts (Paredes-Santos et al., 2018). Tissue cysts were reported to have a specific gravity of 1.056 and can be purified from brain tissue using isopycnic centrifugation in a

813

Percoll gradient (Cornelissen et al., 1981), discontinuous Percoll step gradients (Pettersen, 1988; Blewett et al., 1983; Watts et al., 2017), and 20% dextran (Freyre, 1995) (see Chapter 20: Genetic manipulation of Toxoplasma gondii, for protocol). The heterogeneity of tissue cysts becomes apparent using a refinement of the tissue cyst purification protocol. With this threestep Percoll gradient, the majority of cysts have a specific gravity between 1.055 and 1.040, and significant numbers of higher density ( . 1.055) and lower density (,1.040) cysts are observed (Watts et al., 2015, 2017). Notably, the proportions of lower density cysts increase over time (Watts et al., 2015). This change may be reflective of larger tissue cysts being less occupied and possessing a lower packing density (Watts et al., 2015). The distribution of tissue cysts in Percoll gradients could serve as a potential marker for mutant phenotypes, affecting intracyst growth and occupancy or the effect of drug treatments where overall cyst burden is unaffected but the nature of the cysts and resident bradyzoites altered. Bradyzoites differ ultrastructurally from tachyzoites in that they have a posteriorly located nucleus, solid rhoptries that often loop back on themselves, numerous micronemes, and polysaccharide (amylopectin) granules (AG) (Ferguson and Hutchison, 1987b; Dubey, 1997; Lemgruber et al., 2011a). There is at least one bradyzoite rhoptry protein (BRP1) (TgME49_314250) that is absent in tachyzoites; knockout of BRP1 did not affect development (Schwarz et al., 2005). Lipid bodies are not seen in bradyzoites but are numerous in sporozoites and occasionally seen in tachyzoites. The contents of rhoptries in mature bradyzoites are electron dense in contrast to the labyrinthine rhoptries seen in tachyzoites and in immature bradyzoites (Dubey, 1998a; Lemgruber et al., 2011a). Bradyzoites stain red with PAS due to the presence of AG, whereas tachyzoites are usually PAS negative (Dubey et al., 1998; Dubey, 1998a). A closer examination of the

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18. Bradyzoite and sexual stage development

patterns for amylopectin accumulation reveals that it is not uniform. In fact, clusters of parasites with high levels of AG can be found adjacent to groups lacking the storage polysaccharide (Fig. 18.3). While the contribution of amylopectin synthesis to the progression of the chronic infection is not known, mutants resulting in amylopectin accumulation in tachyzoites are defective in the maintenance of the chronic infection (Sugi et al., 2017; Uboldi et al., 2015). In general, bradyzoites are more slender than tachyzoites. Bradyzoites are more resistant to acid pepsin (1
2 hour survival in pepsin
HCl) than tachyzoites (10 minutes survival); however, this cannot be relied on to absolutely distinguish these two life stages (Jacobs and Remington, 1960; Popiel et al., 1996; Dubey, 1998b). The time to oocyst shedding, called the prepatent period, in cats, following feeding of bradyzoites is shorter (3
7 days) than that following feeding of tachyzoites (more than 14 days). The length of the prepatent period is the most sensitive biologic marker of mature functional tissue cysts (Dubey et al., 1997; Dubey, 1997). In addition, oral feeding with oocysts generally results in higher tissue cyst burdens relative to i.p. injection of oocysts, tachyzoites, or bradyzoites (Fritz et al., 2012b). This is consistent with the natural course of the life cycle (Dubey, 1998a).

18.3 The development of tissue cysts and bradyzoites in vitro The development of tissue cysts in vitro was reported more than 40 years ago (Matsukayashi and Akao, 1963; Hogan et al., 1960); however, the morphologic similarity of bradyzoites and tachyzoites by light microscopy made it difficult to study these differentiation events until the development of antibodies to bradyzoite-specific antigens. In vitro produced bradyzoite cysts led to oocyte excretion in cats with a prepatent period consistent with that of tissue cysts (Hoff et al., 1977). Using TEM, it has been demonstrated that while cyst-like structures were present within 3 days of infecting host cells in tissue culture, in vitro cysts are not mature until 6 days postinfection according to the cat bioassay (Dubey, 1997). Prolonged passage of T. gondii or other Apicomplexa in vitro may lead to the loss of their ability to differentiate into other stages. For example, prolonged passage of Besnoitia jellisoni in vitro leads to a loss of its ability to form tissue cysts in mice, and many type II isolates (e.g., PLK, a clone of ME49) of T. gondii cannot form oocysts in cats (Frenkel et al., 1976). Bradyzoite-specific monoclonal antibodies (see Table 18.1) have greatly facilitated studies of bradyzoite development in vitro and the FIGURE 18.3 The distribution of amylopectin granules (AG) in a tissue cyst is nonuniform. (A) PAS reagent labeled accumulation of AG overlaid on the corresponding differential interference contrast (DIC) image of a purified tissue cyst reveals areas of high accumulation (green outline) and low accumulation (yellow outline) within the same cyst. (A*) A computer rendering of the overlaid micrograph reveals the distribution of AG and its relative levels. AG, Amylopectin granules; PAS, periodic acid
Schiff. Source: Courtesy E. Watts and A.P. Sinai.

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18.3 The development of tissue cysts and bradyzoites in vitro

TABLE 18.1 Common bradyzoite markers.

Monoclonal antibody

Size on immunoblot (kDa)

Location by IFA

Comments

Cloned ToxodB number

BAG1 (hsp30, BAG5)

7E5 74.1.8

28

Cytoplasm

small heat shock protein

TGME49_259020

BSR4 (p36, SRS16C)

T84A12

36

Surface

SRS family antigen, also in sporozoites

TGME49_320180

SAG4A (p18, SRS35A)

T83B1

18

Surface

SRS family

TGME49_280570

None

DC11

Not reactive

Surface

Surface antigen

Noa

p21

T84G11

21

Surface

Surface antigen

Noa

p34

T82C2

34

Surface

surface antigen

Noa

SRS9 (SRS16B)

Murine polyclonal only

43

Surface

SRS family

TGME49_320190

MAG1

None

65

Matrix

Studies indicate also expressed in tachyzoites

TGME49_270240

None

E7B2

29

Matrix

No

None

93.2 (Weiss, unpublished)

Not reactive

Matrix

No

None

1.23.29 (Weiss, unpublished)

19

Matrix

No

CST1

73.18, SalmonE; also recognized by DBA lectin

250 (116)

Cyst wall

CST1?

CC2

115 Cyst wall (bradyzoite) 40 (tachyzoite)

LDH2

Polyclonal sera weakly cross- 35 reacts to LDH1

ENO1

Polyclonal sera to ENO2 and ENO1 do not cross react

Name of antigen

48

structural protein

TGME49_064660

Same as CST1?

No

Cytosolic

Tachyzoite isoform LDH1

TGME49_291040

Nuclear and cytosolic

Tachyzoite isoform ENO2

TGME49_268860

a

- These may be members of the SRS family. BSR4, Bradyzoite-specific recombinant 4; IFA, immunofluorescence analysis.

recognition of techniques for the induction of differentiation. Parasite lines that use various fluorescent protein constructs (Ds Red, GFP, YFP, etc.) under the control of various stagespecific promoters have also been developed as useful tools to study bradyzoite development (Singh et al., 2002; Ma et al., 2004); this has included lines that express both bradyzoite(usually BAG1: TgME49_259020, or lactate

dehydrogenase 2 (LDH2): TgME49_291040 promoter driven) and tachyzoite- (usually SAG1: TgME49_233460 promoter driven) specific GFP constructs in the same cell (Unno et al., 2009). Using parasites-containing chloramphenicol acetyltransferase (CAT) expressed constitutively from the α-tubulin promoter (TUB1) and β-galactosidase, expressed from a bradyzoitespecific promoter (BAG1), one can measure

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18. Bradyzoite and sexual stage development

both growth rate and degree of bradyzoite differentiation (Eaton et al., 2005). Studies of the in vitro development of bradyzoites using transcriptomic [serial analysis of gene expression (SAGE) and RNA-seq] as well as proteomic techniques have confirmed that there are numerous stage-specific genes/proteins in these organisms (data available at www. ToxodB.org). A list of genes that had been functionally characterized can be found in Jeffers et al. (2018). In tissue culture studies, it is evident that bradyzoites spontaneously convert to tachyzoites and that tachyzoites spontaneously convert to bradyzoites (Bohne et al., 1993, 1994; De champs et al., 1997; Jones et al., 1986; Lane et al., 1996; Lindsay et al., 1991, 1993a, 1993b; Mchugh et al., 1993; Popiel et al., 1994, 1996; Soete et al., 1993, 1994; Odbergferragut et al., 1996; Weiss et al., 1994; Parmley et al., 1995). As noted by Matsubayashi in 1963 and confirmed by several groups using bradyzoitespecific monoclonal antibodies, T. gondii strains with a slower rate of replication are more likely to develop cysts in vitro, and slowing the replication of virulent strains allow tissue cysts of virulent strains to develop in vitro (Matsukayashi and Akao, 1963). Low-virulence strains are high cyst forming strains in mice, for example, type II and III strains such as ME49 or Pru or 76K and have a higher spontaneous rate of cyst formation in culture than do virulent type I strains such as RH (Soete et al., 1994). In addition, we have observed that for avirulent T. gondii isolates, both an increased growth rate and decreased efficiency of cyst production correlate with prolonged tissue culture passage. These observations probably reflect alterations in T. gondii epigenetic state associated with prolonged tissue culture passage. Many of these changes may be reset by passage of a strain in an animal model to produce cysts and using these cysts to infect host cells in vitro to reestablish the tissue culture line.

Stress conditions are associated with induction of bradyzoite development, that is, there are more bradyzoites under these conditions than would be expected from the simple inhibition of tachyzoite replication. Conditions that induce the formation of bradyzoite within host cells are temperature stress [43 C (Soete et al., 1994)], pH stress [pH 6.6
6.8 or 8.0
8.2 (Weiss et al., 1995; Soete et al., 1994; Odbergferragut et al., 1996)], chemical stress from Na arsenite (Soete et al., 1994), and nutrient stress from arginine starvation (Fox et al., 2004). In murine macrophage lines derived from bone marrow, interferon-γ increases bradyzoite antigen expression (Bohne et al., 1994). This induction appears to be due to the production of nitric oxide (NO) as bradyzoite differentiation was inhibited by treating macrophages with an inducible nitric oxide synthase inhibitor NGmonomethyl-L-arginine (Bohne et al., 1994). Bradyzoite differentiation is also enhanced by sodium nitroprusside (SNP), an exogenous NO donor (Bohne et al., 1994; Kirkman et al., 2001; Weiss et al., 1996). Similarly, both oligomycin, an inhibitor of mitochondrial ATP synthetase function, and antimycin A, an inhibitor of the electron transport of the respiratory chain, increase bradyzoite antigen expression (Bohne et al., 1994; Tomavo and Boothroyd, 1995). The use of pH stress (pH 8 media) often accompanied by low CO2 (0.5%) is the most commonly used method to induce bradyzoite differentiation in the laboratory, and we have found that this combination is very effective in vitro with more than 90% of all vacuoles displaying bradyzoite-specific markers (Fig. 18.4). It has been suggested that the T. gondii stresssensitive response leading to differentiation, which is not seen is the closely related apicomplexan Hammondia hammondi, increased the flexibility of the T. gondii life cycle and was a critical evolutionary step for its widespread distribution in nature (Sokol et al., 2018). The contribution of the host cell to stage conversion remains to be fully elucidated.

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18.3 The development of tissue cysts and bradyzoites in vitro

817

FIGURE 18.4 Development of Toxoplasma gondii life stages in vitro. Primary murine astrocytes (A, B) or human foreskin fibroblasts (C, D) infected with T. gondii (ME49/PLK strain). Human fibroblasts infected with T. gondii (ME49/ PLK strain) were maintained at pH 8.0 (C) or pH 7.2 (D) for 3 days. (A) Phase micrograph demonstrating an in vitro tissue cyst; (B) immunofluorescence analysis (IFA) using anti-CST1 (mAb 73.18) demonstrating staining of the cyst wall of the bradyzoite parasitophorous vacuole (the corresponding phase microscopy is shown in panel A); (C) IFA using anti-BAG1 (mAb 74.1.8) demonstrating bradyzoite development in several parasitophorous vacuoles, staining occurs throughout the cytoplasm of the bradyzoites; (D) IFA using anti-SAG1 (mAb DG52) demonstrating tachyzoite development and rosette formation, staining is localized to the surface of each tachyzoite. Source: Courtesy S. Halonen, T. Tomita and L. M. Weiss.

Exposure of extracellular tachyzoites to stress conditions (pH 8.1) will result in an increase in bradyzoite differentiation (Weiss et al., 1998; Yahiaoui et al., 1999), consistent with a direct effect of stress on the parasite. Nonetheless, most of the agents that induce differentiation have profound effects upon host cells, and it is likely that alterations in host cell signaling also have significant impact upon bradyzoite differentiation. For example, the kinase inhibitor compound 1 (Gurnett et al., 2002; Merck Pharmaceuticals Inc) requires host cell protein synthesis in order to induce bradyzoite formation, suggesting that its effect on differentiation is mediated through a perturbation of the host cell rather than directly on the parasite (Radke et al., 2006). Human cell division autoantigen-1 (CDA1) was identified as a key host factor in this analysis, and small interfering RNA knockdown of CDA1 demonstrated that CDA1 expression causes the inhibition of parasite replication that leads subsequently to the induction of bradyzoite differentiation (Radke

et al., 2006). The effect of CDA1 may reflect a role for the host cell cycle in triggering T. gondii differentiation (Luder and Rahman, 2017). In addition, the metabolic status of the host cell can affect its ability to support bradyzoite differentiation (Weilhammer et al., 2012). Host cell CD73-generated adenosine can facilitate bradyzoite differentiation in the central nervous system (Mahamed et al., 2012). The specific host cell infected can influence differentiation (Luder and Rahman, 2017). Cysts appear to develop and persist preferentially in neurons and skeletal muscle (mature myotubes), and transcriptional analysis has identified specific gene clusters associated with cyst development in these cell types (Swierzy et al., 2017). Given that infection with tachyzoites promotes entry into the cell cycle followed by host cell arrest in S phase (Molestina et al., 2008), infection of terminally differentiated cells such as neurons and myotubes that have a high barrier to cell cycle entry (Sharma et al., 2017) triggers parasite

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18. Bradyzoite and sexual stage development

differentiation because critical signals for parasite growth are not sensed. When cells are infected by bradyzoites derived from tissue cysts and cultured in vitro under permissive conditions (pH7), differentiation to tachyzoites and the appearance of the tachyzoite-specific antigen SAG1 are seen within 15 hours, which is before cell division occurs (Soete and Dubremetz, 1996; Soete et al., 1993). Vacuoles containing organisms expressing only tachyzoite antigens are evident within 48 hours of infection (Soete and Dubremetz, 1996; Soete et al., 1993). When bradyzoite differentiation occurs in cell culture following infection with tachyzoites and culture under bradyzoite-inducing stress conditions, all of the current well-characterized bradyzoite markers, with the exception of p21 (mAb T84G10), can be detected within 24 hours of infection (Gross et al., 1996; Lane et al., 1996; Soete and Dubremetz, 1996). This includes bradyzoite surface antigens as well as those markers related to cyst wall formation. Conversion between these two stages is a rapid event, and commitment to differentiation may occur at the time of or shortly after invasion and the formation of the parasitophorous vacuole. By 3 days after exposure to conditions that induce bradyzoite development, vacuoles are present in tissue culture exhibiting electron microscopic characteristics of cysts. Reactivity to mAb T84G10 (p21) does not appear until 5 days (Gross et al., 1996; Lane et al., 1996; Soete and Dubremetz, 1996). As assessed by the cat bioassay, mature/functional cysts are not formed until at least 6 days in culture. There are probably other differences in antigen expression, replication potential, and as-yet unidentified markers to distinguish early from late bradyzoites. With the capacity of bradyzoites to replicate within tissue cysts at later stages of the infection (Watts et al., 2015), a model is emerging of the tissue cyst as a multigenerational mosaic whose distribution of replicative and mature forms defines the overall

character of the tissue cyst. Identification of additional markers of mature functional cysts and methods suppress tachyzoite growth is needed to facilitate in vitro studies on cyst biology and characterize the full spectrum of states observed in vivo. The development of singlecell RNA-seq analysis applied to in vitro tissue cysts, and individual parasites should reveal the extent of heterogeneity in this currently enigmatic population.

18.4 The cell cycle and bradyzoite development It is probable that bradyzoite differentiation from tachyzoites is a programmed response related to a slowing of replication and lengthening of the cell cycle (Jerome et al., 1998; Croken et al. 2014b), similar to the programmed expansion and differentiation reported in other Coccidia. The cell cycle in tachyzoites is characterized by major G1 and S phases, and a relatively short or absent G2 and mitosis (M) (Francia and Striepen, 2014). As T. gondii replication slows, there is an increase in duration of the G1 phase of the cell cycle. It is not known if the checkpoints within this cell cycle differ from those observed in yeast and mammalian cells (Francia and Striepen, 2014), but preliminary studies suggest differences (Radke et al., 2001; Khan et al., 2002). Concrete evidence of these differences has been exposed by the identification of the cyclin-related kinases and putative cyclins in T. gondii that define unique checkpoints inherent in the cell cycle architecture of Apicomplexa (Alvarez and Suvorova, 2017). The organization of the cell cycle is linked to differentiation events. It appears that the unique late S/G2 represents a premitotic cell cycle checkpoint at which bradyzoite differentiation occurs (Radke et al., 2003; White et al., 2014). This idea is supported by the peak expression of bradyzoite mRNAs in the late

Toxoplasma Gondii

18.4 The cell cycle and bradyzoite development

mitotic period (Behnke et al., 2010). The timing of bradyzoite induction in the cell cycle is consistent with a model of commitment to differentiation occurring in G1, but completion of the bradyzoite developmental program requiring remodeling of chromatin structure with modulation of the epigenetic state of the parasite (Kim, 2018). An outstanding question is the role epigenetic chromatin (re)modeling in dictating the progression of the chronic infection and whether specific chromatin states reflect if bradyzoites replicate, are arrested in growth, or are primed for reactivation (dedifferentiation back to tachyzoites). When T. gondii sporozoites from the VEG strain (type III) infect human fibroblasts in vitro, they transform to rapidly dividing tachyzoites with a half-life of 6 hours. After 20 divisions, approximately 5 days in culture, these tachyzoites shift to a slower growth rate with a half-life of 15 hours (Radke et al., 2001; Khan et al., 2002). Bradyzoite differentiation, as defined by the expression of bradyzoitespecific antigens, occurs spontaneously when the population shifts to a slower growth rate (t1/2 16 hours) but is not seen in the rapidly dividing (t1/2 6 hours) organisms. This finding is consistent with observations that spontaneous bradyzoite differentiation occurs less readily in rapidly dividing strains of T. gondii such as RH, and that stress conditions that slow growth induce bradyzoite differentiation (Bohne et al., 1994; Jerome et al., 1998; Weiss and Kim, 2000). Bradyzoites can undergo asynchronous division, resulting in vacuoles with odd numbers of organisms instead of the usual multiples of two seen in tachyzoite vacuoles (Dzierszinski et al., 2004). With the exception of Δpkac3 mutant that had normal cell cycle progression and increased cyst formation in vitro (Sugi et al., 2016), bradyzoite differentiation cannot be uncoupled from slowing of the cell cycle. Differentiation may be a stochastic event that occurs at a specific point in

819

the cell cycle when replication has slowed sufficiently. Conditions that slow the cell cycle result in bradyzoite differentiation, and progression through the cycle is required for full differentiation to occur (Fox and Bzik, 2002; Khan et al., 2002). In support this idea, it was found that a point mutation in the TgCactin gene causes a temperature-sensitive (ts) cell cycle arrest in G1 (Gubbels et al., 2008) that was confirmed by genome-wide expression profiling of this temperaturesensitive mutant (Szatanek et al., 2012). This genome-wide expression profiling also demonstrated the induction of several extracellular parasites and bradyzoite genes, including AP2 transcription factors associated with extracellular and bradyzoite parasites (Szatanek et al., 2012). AP2 transcription factors TgAP2IX-9 [TgME49_306620 (Radke et al., 2013)], TgAP2XI-4 [TgME49_315769 (Walker et al., 2013)] appear to be key regulatory factors in bradyzoite development (Gubbels et al., 2008). Interestingly, TgAP2IX9 appears to function as repressor of differentiation (Radke et al., 2013). Conditional knockdown of a ribosomal protein small subunit 13 (RPS13: TgME49_270380) gene, using a tetracycline regulated promoter, caused T. gondii to arrest in G1, lead to depletion of ribosomes and an increase in BAG1 expression during in vitro culture (Hutson et al., 2010). Transcriptional analysis of these parasites demonstrated an early stress response; however, the full repertoire of bradyzoite genes was not seen, suggesting that some progression through G1 is likely needed for full differentiation (Hutson et al., 2010). Bradyzoite differentiation is associated with eIF2α phosphorylation (Sullivan et al., 2004; Narasimhan et al., 2008), which dampens global translation initiation favoring the preferential of translation of genes (mRNA) that encode proteins responding to a stress conditions including transcription factors (e.g., stage associated AP2s) associated with

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18. Bradyzoite and sexual stage development

differentiation (Wek et al., 2006; Sullivan and Jeffers, 2012). It is possible the effect seen with the RPS13 knockdown occurs through a similar common pathway. The rps13 promoter region contains putative AP2-binding regions, and pull-down studies suggest a complex of GCN5: TgME49_254555, TgME49: 243440, AP2, and Swi2/Snf2 ATPases bind to this region of rps13 [(Hutson et al., 2010); see Chapter 21 for a detailed discussion on gene regulation in T. gondii]. When bradyzoites liberated from tissue cysts are used to infect host cells, the tachyzoite-specific antigen SAG1 is expressed within 15 hours of infection prior to any significant cell division by the infecting bradyzoite (Soete and Dubremetz, 1996; Soete et al., 1993). By 48 hours after infection, vacuoles containing organisms expressing only tachyzoite antigens are evident. In a similar fashion, tachyzoites used for infection with stress exposure (pH 8.1) express bradyzoite antigens at 24 hours, and vacuoles containing 1 or 2 organisms can be found that have bradyzoite antigens. Although a small proportion of replicating parasites (,10%) have 1.8 to 2N DNA content (i.e., are in a G2 premitotic state), parasites that coexpress bradyzoite marker BAG1 and tachyzoite marker SAG1 are much more likely (approximately 50% of these parasites) to have a G2 premitotic DNA content (Radke et al., 2003). Flow cytometry measurements of DNA content of mature bradyzoites isolated from tissue cysts demonstrate that this stage has a 1N DNA content consistent with their being in a quiescent G0 state (Radke et al., 2003), although a G1 state cannot be ruled out as DNA content alone is not sufficient to distinguish between a G0 or G1 state. Here, additionally establishing the relative levels of suppressive epigenetic marks and/or the expression of genes associated with metabolic flux in the population will help one distinguish parasite in G0 (exited from the cell cycle) as opposed to G1(not necessarily actively cycling—but retaining the

capacity to enter a cell cycle). Overall, it appears that commitment to bradyzoite differentiation probably occurs at a particular point in the cell cycle, and that transit through the cell cycle is required for differentiation. Early “prebradyzoites” can continue to replicate, but at some point in the development and maturation of the tissue cyst, the fully mature bradyzoites enter a quiescent G0 state, or an intermediate state where the barrier to enter a new cell cycle is higher but still present.

18.5 The stress response and signaling pathways for bradyzoite formation While differentiation is a programmed response, bradyzoite differentiation is also a stress-related response of T. gondii to environmental conditions, including the inflammatory response of the host (Sullivan and Jeffers, 2012; Jeffers et al., 2018; Weiss et al., 1996). Many different classical stress response inducing conditions such as temperature, pH, and mitochondrial inhibitors are associated with bradyzoite development in vitro. Bradyzoite differentiation probably shares features common to other stress-induced differentiation systems such as glucose starvation and hyphae formation in fungi or spore formation in Dicyostelium (Thomason et al., 1999; Soderbom and Loomis, 1998). These systems have demonstrated unique proteins related to specific differentiation structures in each organism as well as the utilization of phylogenetically conserved pathways. Many of these signaling pathways involve cyclic nucleotides and kinases as part of the regulatory system in differentiation. It is also interesting to note that abscisic acid responses that result in the production of cyclic ADP ribose, such as those seen in plant stress responses, have been demonstrated in T. gondii, and that inhibition of abscisic acid synthesis by fluridone triggered bradyzoite differentiation (Nagamune et al., 2008).

Toxoplasma Gondii

18.5 The stress response and signaling pathways for bradyzoite formation

Studies of microorganisms including fungi and other protozoa suggest that differentiation involves conserved signaling pathways, such as cyclic nucleotides, that are also involved in the response to stress or nutrient starvation. The effect of cyclic nucleotides on bradyzoite differentiation has been assessed using nonmetabolized analogues of cAMP and cGMP as well as forskolin, which stimulates a short pulse of cAMP. In Dictyostelium, cAMP in the environment is a trigger for differentiation. It has been demonstrated that extracellular adenosine correlates with the number of cysts of T. gondii formed in vivo in the brain of mice as well as with the number of cysts formed in vitro in murine astrocytes (Mahamed et al., 2012). Bradyzoite induction in response to cyclic nucleotides was measured using either immunofluorescence analysis techniques monitoring bradyzoite markers such as the cyst wall lectin Dolichos biflorus (DBA) or a bradyzoite promoter reporter parasite (Kirkman et al., 2001; Eaton et al., 2005). These studies demonstrate that cGMP or forskolin can induce bradyzoite formation. Exposure of extracellular T. gondii tachyzoites to conditions such as pH 8.1, forskolin, or SNP induces bradyzoite formation and results in a transient three- to fourfold elevation in cAMP levels that within 30 minutes returned to the cAMP levels comparable to those seen in control parasites incubated in pH 7.1 media. No reproducible changes in cGMP were observed. Consistent with these inhibitor studies, using optogenetic modulation of cAMP, a minimum level of cAMP was required for the initiation of bradyzoite formation, but increased levels of cAMP impaired this process (Hartmann et al., 2013). Most of the effects of cAMP within cells can be attributed to regulation of cAMP-dependent protein kinase A activity (PKA). PKA catalytic subunit isoforms (PKAc) are typically involved in modulating the different cellular processes in other organisms such as response to nutrition starvation and respiratory functions

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through phosphorylation of its target substrates, modulating their activity. Effects of cGMP can be attributed to stimulation of a cGMPdependent kinase (PKG: TgMe49_311360), effects upon phosphodiesterases or other signaling molecules. Several kinases that are potentially involved in differentiation have been cloned. T. gondii has three PKAc: PKA1, TgME49_286470; PKA2: TgME49_228420; and PKA3, TgME49_226030 (Sugi et al., 2016). In addition, a glycogen synthase kinase (GSK-3: TgME49_288840) homolog (Qin et al., 1998) and a unique apicomplexan PKG (Gurnett et al., 2002; Nare et al., 2002; Donald et al., 2002; Donald and Liberator, 2002) may be involved in differentiation. Inhibitors of PKA or apicomplexan PKG inhibit replication and induce differentiation (Eaton et al., 2005; Nare et al., 2002), although as discussed earlier the PKG inhibitor compound 1 may have effects upon host signaling. PKA has effects upon metabolism, gene expression, and cell cycle. The dissection of exact signaling cascades is complicated by the presence of more than one PKA isoform and multiple phosphodiesterases that are likely to modulate signaling. Further, the inhibitors of PKA or PKG may have off target effects upon other kinases or effects upon host cell signaling. It is likely that there are both inhibitory and stimulatory pathways affected by cyclic nucleotide signaling. For example, TgPKAc3 was found to be associated with cAMPdependent tachyzoite maintenance, and a Δpkac3 mutant had increased cyst formation in vitro (Sugi et al., 2016). As protein phosphorylation has proven to be a major mechanism of regulation of gene expression and integration and amplification of extracellular signals, the presence of highly conserved signaling molecules suggests that many of the pathways identified in other eukaryotes are likely to be preserved in T. gondii. A novel mitogen-activated protein kinase (TgMAPK-1: TgME49_207820) related to p38 (a human MAPK involved in the stress response)

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is also increased during bradyzoite formation (Brumlik et al., 2004). At least two MAPK orthologs are present in the T. gondii genome (www. toxodb.org), and one of these MAPK may play a role in the signaling processes involved in bradyzoite differentiation. Inhibitors of bump kinases upregulate bradyzoite markers and affect parasite replication through interactions with TgCDPK1: TgME49_301440 and TgMAPKL1: TgME49_312570 (Winzer et al., 2015; Sugi et al., 2015). While transcription of TgMAPKL1 is increased during early bradyzoite differentiation, deletion of TgMAPKL1 leads to downregulation of known bradyzoite markers and affects parasite attachment and replication (Cao et al., 2016). The rhoptry kinases are a family found exclusively in Apicomplexa. While members of this family had been implicated in acute virulence, their role in the chronic infection had not garnered the same focus until a tour de force study by the Bzik laboratory (Fox et al., 2016). Here, they used a knock out (KO) approach to target 31 rhoptry kinases with the aim of identifying activities impacting the chronic infection. While several KO’s exhibited marked reductions in cyst burdens, they also were found to be less virulent during the preceding acute infection phase (ΔROP2: TgME49_215785, ΔROP17: TgME49_258580, and ΔROP18: TgME49_205250). One of these, ΔROP17 was found to have effects on both the acute and chronic phases. Remarkably, two deletion mutants ΔROP35: TgME49_304740 and a ΔROP38/29/19 mutant (the three genes, TgME49_242110, TgME49_ 242230, and TgME49_242240, are contiguous in the genome making the triple KO feasible) exhibited no acute virulence phenotype, but deletion had a selective effect during the chronic phase. Given that rhoptry contents are secreted only at the time of invasion, this presents a potential paradox as the rhoptry product effect must manifest at the time of the establishment of the tissue cyst, or during bradyzoite reinvasion, assuming in vivo events is similar to what is reported in

tissue culture models (Dzierszinski et al., 2004). Overall, these data are consistent with the idea that the commitment to establish a bradyzoite vacuole (i.e., tissue cyst) occurs at the time, or shortly after, invasion. In eukaryotes the cellular stress response is associated with phosphorylation of the alpha subunit of eIF2 (eukaryotic initiation factor-2) enhancing the translational expression of bZIP proteins such as GCN4 in yeast and ATF4 in mammals (Hinnebusch and Natarajan, 2002). A novel eIF2 protein kinase, designated TgIF2K-A (T. gondii initiation factor-2kinase: TgME49_229630), has been shown to phosphorylate the T. gondii translation initiation factor TgIF2α: TgME49_214270, and that phosphorylation is enhanced by conditions known to induce bradyzoite differentiation (Sullivan et al., 2004). Inhibiting TgIF2α dephosphorylation induces bradyzoite development, and TgIF2α is maintained in a phosphorylated state in latent cysts (Narasimhan et al., 2008). T. gondii has an additional initiation factor-2kinase (TgIF2K-B: TgME40_311510) that is localized in the cytoplasm and likely responds to cytoplasmic stresses, whereas TgIF2K-A is localized in the ER (Narasimhan et al., 2008). Control at the level of translation may not only manifest with regard to whether or not to initiate translation of specific transcripts, but additionally as to where on the transcript to initiate translation. TgBCP1 [TgME49_203450 (brain colonization protein 1)] is made as a 25 kD protein in tachyzoites and a 51 kD protein in bradyzoites due to the stage-specific initiation of translation from distinct in frame initiator methionine residues (Milligan-Myhre et al., 2016). Not only are the proteins different but also their localization is altered as the short tachyzoite-specific form is cytoplasmic while the larger form synthesized in bradyzoites is secreted and delivered to the cyst wall (Milligan-Myhre et al., 2016). The use of translation initiation as a switch in developmental

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18.6 Heat shock proteins

regulation points to an entirely novel mechanism of control. Epigenetic events, such as histone modification, are probably key factors in the differentiation process resulting in bradyzoite formation (Kim, 2018) (see Chapter 21: Regulation of gene expression in Toxoplasma gondii for a detailed discussion of histone modifications in T. gondii). As in other organisms, histone acetylation and methylation patterns correlate with gene activation or repression in T. gondii (Saksouk et al., 2005). As bradyzoite differentiation proceeds, gene activation markers are associated with region upstream of transcriptionally regulated bradyzoite genes (Saksouk et al., 2005). Epigenetically regulated changes in gene expression and changes in chromatin modifications typically require transit through S phase as has been observed for induction of expression of bradyzoite markers (Radke et al, 2003). T. gondii has three H2A histones that are differentially regulated during in vitro and in vivo bradyzoite conditions (Dalmasso et al., 2009). One of the H2A histone variants, H2AX, TgME49_261580, is localized to silent areas of the genome during bradyzoite development (Dalmasso et al., 2009). Inhibition of histone deacetylase activity with the compound FR235222 induced bradyzoite differentiation and derepressed stage-specific genes (Bougdour et al., 2009). Derivatives of FR235222 may be useful therapeutics for both acute and chronic infections (Maubon et al., 2010). T. gondi contains two distinct histone acetyltransferase GCN5 proteins (Hettmann and Soldati, 1999; Sullivan and Smith, 2000; Rodrigues-Pousada et al., 2004). TgGCN5-A (TgME49_254555) is enriched at the upstream regions of transcriptionally controlled bradyzoite genes under alkaline pH (Naguleswaran et al., 2010). TgGCN5A knockout parasites are unable to induce bradyzoite-associated genes in response to alkaline stress and have different patterns of cell cycle and bradyzoite gene expression from stressed wild type parasites

(Naguleswaran et al., 2010). TgGCN5-A knockout parasites are unable to induce bradyzoiteassociated genes in response to alkaline stress have different patterns of cell cycle and bradyzoite gene expression from stressed wild-type parasites (Croken et al., 2014a, 2014b). TgGCN5b (TgME49_243440) interacts with AP2 factors and is required for T. gondii proliferation (Wang et al., 2014). TgSRCAP [TgME49_280800 (T. gondii Snf2-related CBP activator protein)] is a SWI2/SNF2 family chromatin remodeler whose expression increases during cyst development (Nallani and Sullivan, 2005). A SRCAP homolog, which in other eukaryotes, is involved in the regulation of CREB (cAMP response element binding protein) and has been identified in T. gondii (Sullivan et al., 2003). Using yeast two-hybrid analysis, TgSRCAP was found to interact with several proteins that could be transcription regulators including TgLZTR: TGME49_298600 (Nallani and Sullivan, 2005). Insertion into TgRSC8 (TGME49_286920), a T. gondii homolog of Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p [switch/sucrose nonfermentable (SWI/SNF)], caused parasites to display a bradyzoite development defect (Craver et al., 2010). Further analysis of this TgRSC8 mutant showed that while steady-state transcript levels of several known bradyzoite genes were significantly reduced in the mutant, other known bradyzoite genes were not affected, highlighting the complexity of bradyzoite development (Rooney et al., 2011). A detailed analysis of these signaling pathways will be required in order to understand the regulatory network triggered during bradyzoite formation.

18.6 Heat shock proteins There is a significant body of evidence linking heat shock proteins with differentiation in various phyla (Heikkila, 1993a, 1993b). BAG1

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(also known as BAG5) has homology to small heat shock proteins and has also been called hsp30 (Bohne et al., 1995; Parmley et al., 1995). Both BAG1 mRNA and protein (a 28 kDa cytoplasmic antigen) are upregulated during bradyzoite formation, suggesting that transcriptional regulation of its expression. BAG1 is one of the most abundant bradyzoite-specific genes found in the T. gondii bradyzoite expressed sequence tags (ESTs) representing about 3% of all bradyzoite-specific clones. T. gondii expressing BAG1 is seen within 24 hours of exposure to pH 8.0 or other stress conditions. BAG1 antibody cross-reacts with the corresponding gene in Neospora caninum and is a marker for differentiation in this related apicomplexan (Weiss et al., 1999). The N. caninum BAG1 homolog has been cloned and characterized (Kobayashi et al., 2012). The carboxyl-terminal region of BAG1 has a small heat shock motif, most similar to the small heat shock proteins of plants, and the near the N-terminus is a synapsin Ia-like domain that may be involved in the association of this small heat shock protein with proteins during development. Four other small heat shock proteins are present in T. gondii; of these, hsp20 (TgME49_232940), hsp21(TgME49_ 312600), and hsp29 (TgME49_289600) are expressed in both tachyzoites and bradyzoites, and hsp 28 (TgME49_286720) is specific for tachyzoites (De miguel, 2005). None of these other small heat shock proteins are associated with bradyzoite differentiation, and all are present as multimeric forms in the cytosol of T. gondii. BAG1 appears not to form multimeric forms in T. gondii (Weiss LM, unpublished data). A homolog of heat shock protein 70 (hsp70, TgME49_273760) is induced during both the transition from tachyzoite to bradyzoite and from bradyzoite to tachyzoite (Weiss et al., 1998; Lyons and Johnson, 1995, 1998; Miller et al., 1999; Silva et al., 1998). Induction of hsp70 can be demonstrated at both the protein

and RNA level. Quercetin, an inhibitor of hsp synthesis, can suppress hsp70 and decrease the ability of pH shock to induce bradyzoite formation (Weiss et al., 1996, 1998). Extracellular T. gondii treated with a 1-hour exposure to pH 8.1 versus pH 7.1 express a 72 kDa inducible hsp70 (detected with mAb C92F3A-5; Stressgen) (Weiss et al., 1998), and this extracellular treatment induces bradyzoite formation. T. gondii-infected cultures treated with pH 8.1 show fourfold induction of the hsp70 levels compared to T. gondii grown in pH 7.1 treated cells (Weiss et al., 1996, 1998), which is similar to the magnitude of the hsp70 response demonstrated in Trypanosoma cruzi, Theileria annulata, and Plasmodium falciparum (Shiels et al., 1997). A similar increase in hsp70 is seen with in vivo cysts during reactivation in a murine model induced by anti-γ-interferon (Silva et al., 1998). The relative level of expression of hsp70 by T. gondii is also associated with virulence, and RH strain has four copies of a sevenamino acid repeat unit (GGMPGGM) at the C-terminus of its hsp70 compared with five copies in the ME49 strain (Lyons and Johnson, 1998). Other heat shock proteins have been associated with bradyzoite differentiation. TgHsp60 (TgME49_247550) transcripts from the PLK strain are upregulated within in vivo bradyzoites compared to in vitro tachyzoites, though Hsp60 proteins levels do not seem to differ between these two stages (Murata et al., 2017). Three Hsp40/DnaJ family members (TGME49_ 11590, TGME49_010430, and TGME49_023950) are also upregulated in tachyzoites exposed to pH8 stress (Sullivan and Jeffers, 2012). The 3D structures of Tghsp60 and 70 have been predicted based on homology modeling (Ashwinder et al., 2016). Hsp90 (TgME49_2883800) mRNA and protein levels also increase during bradyzoite differentiation (Echeverria et al., 2005). Fluorescence microscopy demonstrated that in tachyzoites, hsp90 is in the cytosol, whereas in

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18.6 Heat shock proteins

mature bradyzoites, hsp90 is present in both the nucleus and cytosol. In T. gondii mutants that are unable to differentiate, hsp90 is only found in the cytoplasm. Geldanamycin, a benzoquinone ansamycin antibiotic capable of binding and disrupting the function of hsp90, blocks conversion both from the tachyzoite to bradyzoite and the bradyzoite to tachyzoite (Echeverria et al., 2005). Deletion of TgHsp90 reduces the expression of BAG1 and MAG1 (TgME49_270240) in a type I strain, which typically do not differentiate well to bradyzoites (Sun et al., 2017). Hsp90 forms a complex with p23 (a smHSP) with a nuclear and cytosolic distribution in bradyzoites, but no nuclear localization is seen in tachyzoites (Echeverria et al., 2010). In contrast, a complex of Hip
Hsp70
Hsp90 was found in the cytoplasm of both tachyzoites and bradyzoites (Echeverria et al., 2010). DnaK-tetratricopeptide repeat (DnaK-TPR; TgME49_202020) is also associated with bradyzoite differentiation, and on yeast two-hybrid screening was also found to interact with p23 (Ueno et al., 2011). Three hsp40/DnaJ family members (TgME49_115690, TgME49_010430, and TgME49_023950) were also found to be unregulated in parasites exposed to alkaline stress-induced bradyzoite differentiation (Sullivan and Jeffers, 2012). Suppressive subtractive hybridization has been used to find differentially regulated genes and identified a protein with ankyrin domains (TgANK1: TgME49_216140) and TgDnaK-TPR as bradyzoite-specific genes. Both TgANK1 and TgDnaK-TPR contain TPRs (Friesen et al., 2008). TPR domains have been reported to play roles in protein
protein interactions, transcription, protein folding, and cell cycle processes. Using an ectopic promoter, TgANK1 was shown to localize mainly to the parasite cytoplasm with some staining in the nucleus (Friesen et al., 2008). TgANK1 was found to be upregulated in the type I strain RH-ERP over GT1 (Yang et al., 2013). TgDnaK-TPR is

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upregulated during in vitro bradyzoite conditions and localizes to the cytoplasm of in vitro and in vivo bradyzoites. Yeast two-hybrid screens demonstrated that TgDnaK-TPR interacts with cochaperone p23 (Ueno et al., 2011). A T. gondii-specific deoxyribose-phosphate aldolase-like gene (TgDPA) has been described as another protein that is upregulated in bradyzoites (examined in type II strains) (Ueno et al., 2009; Cleary et al., 2002). TgDPA: TgME49_318750 antibodies demonstrate that this is a cytosolic protein. Although the catalytic domain of TgDPA is not conserved, TgDPA has been characterized to bind to actindepolymerizing factor (Ueno et al., 2010). The expression of reporter genes driven by the hsp70 promoter is responsive to conditions that induce bradyzoite formation (Ma et al., 2004). The pH regulated cis-element of the hsp70 promoter maps to the region 2420 through 2340 from the initial ATG of the hsp70 gene (Ma et al., 2004). At 2650 bp from the initial ATG is the sequence AGAGACG, which has been described as a cis-acting element that acts as an enhancer in the transcription of several T. gondii genes (Mercier et al., 1996). There are a series of nGAAn repeats 2385 from the initial ATG, which have similarity to the heat shock element (HSE) described in other eukaryotes (Morimot et al., 1994). A CCGGGG located next to this HSE is similar to the sp1-hsp70 site in the human hsp70 promoter (Morgan, 1989). The hsp70 promoter also contains several AGGGG or CCCCT regions that are similar to the core region of the STRE (stress response element) described in many eukaryotic genes (Estruch, 2000). Similar STRE and HSE elements are seen in the promoter region of enolase 1 (TgENO1, TgME49_268860), a bradyzoite-specific isoform of enolase (Kibe et al., 2005). In yeast, enolase is also known as hsp48, as it is a stress-related heat shock protein (Iida and Yahara, 1985). The STRE-binding activity detected in nuclear extract from stress-induced bradyzoites is

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significantly higher than that from nonstressed tachyzoites (Kibe et al., 2005). Transcription factors responsible for regulation of hsp70 and enolase 1 have not yet been identified, although electromobility shift assays (EMSA) suggest that there are specific proteins that recognize the STRE (or perhaps HSE) elements of these genes (Kibe et al., 2005; Ma et al., 2004). Although there is an area of similarity between the BAG1 promoter region and that of hsp70 promoter region, oligonucleotides from this BAG1 upstream region do not compete in EMSA (Ma et al., 2004).

18.7 Transcriptional control of bradyzoite genes Expression of most bradyzoite-specific proteins is controlled at the level of transcription, and therefore numerous techniques that measure steady-state mRNA levels have been used to identify bradyzoite-specific genes. Sequencing of cDNA libraries, called ESTs, SAGE, microarray analysis, and nextgeneration sequencing projects of bradyzoites and tachyzoites, have all been used to identify stage specific genes, and the data from these methods is organized and available at http:// www.toxodb.org/. Analysis of ESTs was the first large-scale method used to identify genes that are induced during bradyzoite differentiation; the majority of these genes encode proteins of unknown function (Ajioka, 1998; Ajioka et al., 1998; Manger et al., 1998a). Microarrays have been used to analyze bradyzoite development mutants and to identify groups of genes that are coordinately regulated during bradyzoite differentiation (Manger et al., 1998a; Cleary et al., 2002). We and others have also used RNA-seq (data on RNA-seq are available on ToxodB.org) to examine the bradyzoite transcriptome during in vitro stressinduced bradyzoite development as well as cysts from chronic infection (Croken et al.,

2014a, 2014b; Knoll LJ, unpublished data; Weiss LM, unpublished data). These analyses have confirmed the induction of previously described bradyzoite genes, that is, BAG1, LDH2, and SAG4A:TgME49_280570, TgME49_280580 and identified other potential bradyzoite-specific genes. In addition, genes not known to be regulated during differentiation were shown to have altered mRNA expression (Cleary et al., 2002). SAGE was used to understand the progression of gene expression during bradyzoite development (Radke et al., 2005). This SAGE data suggest that T. gondii undergoes a programmed differentiation response similar to P. falciparum with the coordinate regulation of groups of genes during this developmental program. Bradyzoite-specific genes have also been identified by using a subtractive cDNA library approach (Yahiaoui et al., 1999). Sixty-five cDNA clones were analyzed from a bradyzoite subtractive cDNA library; of these, many were identified that were exclusively or preferentially transcribed in bradyzoites. This included homologs of chaperones (mitochondria heat shock protein 60 and T-complex protein 1), nitrogen fixation protein, DNA damage repair protein, KE2 protein, phosphatidylinosoitol synthase, glucose-6-phosphate isomerase, and enolase. Another study has used suppression subtractive hybridization to identify novel bradyzoite-specific genes (Friesen et al., 2008). The data from these expression analysis and genetic studies confirm that there are significant numbers of bradyzoite-specific genes involving many complex pathways. While it is believed that most of the control for the stage specificity for bradyzoite proteins is at transcription, only one bradyzoite-specific promoter element has so far been characterized. Mapping of cis-acting elements upstream of several bradyzoite genes identified a 6
8 bp sequence that controls bradyzoite gene expression under several bradyzoite induction conditions (Behnke et al., 2008). Gel-shift

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18.7 Transcriptional control of bradyzoite genes

experiments show that this promoter element is bound by parasite proteins to maintain in a “poised” chromatin state throughout the intermediate host life cycle in low-passage strains. T. gondii expresses transcription factors containing the plant-related DNA-binding domain Apetala2 (AP2). Apicomplexan AP2 transcription factors have been found to be involved in malaria development (Balaji et al., 2005). The T. gondii genome contains B67 genes that contain an AP2 domain, and several of these AP2 transcription factors have been found to be regulated in tachyzoites in a cell cycle manner (Behnke et al., 2010). The binding motifs of some of these AP2 transcription factors have been demonstrated to bind to the promoter regions of bradyzoite markers BAG1 and BNTPase, providing evidence that these AP2 proteins are trans-regulators of bradyzoite genes (Radke et al., 2013, 2018; Hong et al., 2017; Huang et al., 2017; Walker et al., 2013). Radke et al. (2013) identified 15 AP2 that were upregulated during bradyzoite induction, of which some transcriptionally oscillate in a cell cycle manner. AP2IV-3: TgME49_318610, AP2VI-3: TgME49_244510, AP2VIIa-1: TgME49_280470, AP2Viii-4: TgME49_272710, AP2Ib-1: TgME49_208020, and AP2IX-9: TgME49_306620 are associated with early bradyzoite development (Radke et al., 2018). The AP2 proteins AP2XI-4: TgME49_315760 and AP2IV-3: TgME49_318610 appear to be bradyzoite activators. Deletion of AP2XI-4 results in the downregulation of bradyzoite markers and impairment of cyst formation (Walker et al., 2013). AP2XI-4 binds to a “CACACAC” cis motif that appears on the promoter of many bradyzoite-upregulated genes (Walker et al., 2013). Deletion of AP2IV-3 results in a downregulation of bradyzoite genes and a modest decrease in cyst numbers during pH 7.8 in vitro induction, whereas overexpression of AP2IV-3 upregulates bradyzoite formation (the expression of BAG1 and LHD2) and increased cyst formation (Hong et al., 2017).

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In contrast, the AP2 proteins AP2IX-9, AP2IX-4, and AP2IV-4 (TgME49_318470) function as bradyzoite repressors (Hong et al., 2017; Huang et al., 2017; Radke et al., 2013, 2018). Overexpression of AP2IX-9 suppressed the rate of cyst formation, whereas deletion of this protein increased this rate (Radke et al., 2013). AP2IX-4 deletion affected cyst formation negatively, even though there was upregulation of bradyzoite markers BAG1, LDH2 (TgME49_291040), and microneme adhesive repeat domain
containing protein 4 (MCP4) (TgME49_208730) (Huang et al., 2017). AP2IV-4 acts as a repressor of bradyzoite differentiation. Deletion of AP2IV-4 results in upregulation of bradyzoite markers such as BAG1, B-NTPase, ENO1, LDH2, cyst wall proteins bradyzoite pseudokinase 1 (BPK1) (TgME49_253330) and MCP4, and an increase in cyst formation (Radke et al., 2018). Overall, AP2 factors play a key role in bradyzoite development most likely through interactions with epigenetic markers and binding cis elements of diverse sequence motifs. Uncovering the upstream regulators of these transcription factors would further elucidate the genetic pathway that regulates bradyzoite differentiation and increases our understanding of the molecular pathways involved in bradyzoite differentiation. In addition to transcriptional regulation of bradyzoite differentiation, T. gondii utilizes posttranslational modifications of proteins to exert control over its differentiation process. Eukaryotic initiation factor 2 (IF2) kinases are associated with stress responses, and data suggests that are important in T. gondii bradyzoite differentiation. Generally, eIF2α phosphorylation leads to a decrease in global protein synthesis in favor of translating transcription factor mRNAs related to the stress response (Hinnebusch, 1997), and this phosphorylation is seen in T. gondii exposed to pH8 media and other stresses that lead to bradyzoite differentiation (Sullivan et al., 2004; Narasimhan et al., 2008; Konrad et al., 2011). There is an

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association of TgeIF2α phosphorylation with translation of AP2 factors in response to ER stress with tunicamycin treatment (Joyce et al., 2013). TgIF2α remains phosphorylated after the parasite has differentiated into bradyzoites (Narasimhan et al., 2008). Inhibition of TgIF2α dephosphorylation in vitro leads to decreased tachyzoite replication, increased cyst formation, and lower reactivation rates (Konrad et al., 2011, 2013). Other translational control mechanisms in T. gondii include cis-acting regulatory RNA hairpins (Holmes et al., 2014), silencing by microRNA (Braun et al., 2010), tRNA cleavage (Galizi et al., 2013), and modulatory RNA-binding proteins (Galizi et al., 2013; Liu et al., 2014; Gissot et al., 2013). Mechanisms of translational control in apicomplexans are reviewed by Holmes et al. (2017) (see Chapter 21: Regulation of gene expression in Toxoplasma gondii). Tachyzoite-associated LDH1 mRNA contains a cis-acting regulatory element in the form of an RNA hairpin structure that represses translation (Holmes et al., 2014) of this tachyzoite specific LDH isoform. In Plasmodium, Puf RNA-binding proteins, which have been characterized to bind to the 30 UTR regions of select mRNAs to control their translation, modulate the differentiation of sporozoites (Mueller et al., 2011; Gomes-Santos et al., 2011). In T. gondii, Puf (TgME49_260680, TgME49_318350) homologs that have RNA-binding capacity are upregulated in bradyzoites (Liu et al., 2014). TgAlba (TgME49_221380, TgME49_218820) proteins bind to RNA and interacts with proteins that regulate translation. A T. gondii Alba knockout strain had reduced cyst formations in vitro and in vivo (Gissot et al., 2013). Other RNA-binding proteins that may play a role in controlling translation during bradyzoite development include Argonautes, DEAD-box helicases, and KH-type splicing regulatory proteins (Braun et al., 2010; Cherry and Ananvoranich, 2014). It is possible that upon

differentiation, tachyzoite transcripts are translationally repressed within bradyzoites and sequestered by ribonucleoproteins into granules awaiting translation upon reversion back to tachyzoites. The transcriptional expression of several rhoptry kinases (ROPK) has been found to be increased during chronic infection compared to acute infection (Jones et al., 2016; Pittman et al., 2014). While rhoptry kinases have been characterized for their roles in parasite virulence and protection of the parasite against host immunity-related GTPases (Behnke et al., 2016), the rhoptry kinome has also been shown to play a role in chronic infection (Jones et al., 2016; Fox et al., 2016). Of 32 deleted ROPKs in the Pru type II strain, 11 ROPK-KOs showed a modest decrease in cyst burden, while 5 ROPKKOs showed a significant reduction in cyst numbers (Fox et al., 2016). Characterization of the substrate and functional activities of these rhoptry kinases during bradyzoite differentiation will provide additional insights into the mechanisms of cyst formation in vivo. Despite the identification of many bradyzoite-specific genes, a promoter, and T. gondii mutants that are unable to differentiate, a unified model for bradyzoite differentiation has not yet been developed. However, several themes have emerged from the available data: 1. Tachyzoites and bradyzoites express related genes encoding structural homologs in a mutually exclusive way. 2. Metabolic genes that are stage specific exist suggesting that each stage is metabolically distinct. 3. Stress-related differentiation pathways and stress proteins are associated with these stage transitions. 4. Multiple mechanisms to control bradyzoite development likely exist and may be organized in a hierarchy of gene induction. 5. Chromatin remodeling is an important mechanism used to coordinate bradyzoite

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development (see Chapter 21: Regulation of gene expression in Toxoplasma gondii for a detailed discussion of gene regulation).

18.8 Cyst wall and matrix antigens The development of the tissue cyst wall and matrix are early events in the process of bradyzoite differentiation. The cyst wall is a modified parasitophorous vacuole membrane that surrounds the bradyzoites within the host cell. Under electron microscopy the cyst wall can be seen as an up to 250 nm thick layer of electron dense material containing vesicles and tubular structures underneath the parasitophorous vacuole membrane (Lemgruber et al., 2011b; Dubey et al., 1998). The lumen of the cyst contains the matrix, which consists of tubules, vesicles, and amorphous proteinaceous material. The cyst wall consists of a compact outer layer and a looser sponge-like layer that extends into the cyst matrix (Lemgruber et al., 2011b). Proteins have been described which localize to both the matrix and cyst wall, the cyst wall alone, or the matrix alone. Some of these proteins are found in both tachyzoites

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and bradyzoites but display stage-specific localization patterns. Cyst wall proteins are detected at the same time as other bradyzoite-specific antigens such as BAG1 (Gross et al., 1996; Zhang et al., 2001). An important function of the cyst wall and matrix is to protect bradyzoites from harsh environmental conditions during transmission. In addition, these structures provide a physical barrier to host immune defenses. Much of this may be due to carbohydrates present in the cyst wall. The cyst wall is a modification of the bradyzoite parasitophorous vacuole membrane formed by the parasite that is enclosed in host cell membrane, that is, tissue cysts are intracellular (Ferguson and Hutchison, 1987b; Scholytyseck et al., 1974). On electron microscopy the membrane of the bradyzoite parasitophorous vacuole has a ruffled appearance and is associated with a precipitation of underlying material creating the cyst wall (Fig. 18.5). The cyst wall is PAS positive, marks with some silver stains, and binds the lectins Dolichos biflorus (DBA) and succinylated-wheat germ agglutinin (S-WGA) suggesting that polysaccharides are present in this structure (Boothroyd et al., 1997; Guimaraes et al., 2003; FIGURE 18.5 Electron microscopy of the cyst wall of Toxoplasma gondii isolated from murine brain. (A) Transmission electron microscopy of cyst wall, (B) Immunoelectron microscopy of cyst stained with mAB 73.18 showing labeling of the cyst wall matrix (20 nm gold, arrows), and (C) Immunoelectron microscopy of cysts stained with Dolichos biflorans lectin demonstrating labeling of the cyst wall matrix (10 nm gold, arrows). Bar 5 1 μm. Source: Reproduced with permission from Zhang, Y.W., Halonen, S.K., Ma, Y. F., Wittner, M., Weiss, L.M., 2001. Initial characterization of CST1, a Toxoplasma gondii cyst wall glycoprotein. Infect. Immun., 69, 501
507.

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Sims et al., 1988; Dubey et al., 1998). The binding of these lectins to tissue cysts can be inhibited by competition by their specific sugar haptens including N-acetylgalactosamine (GalNAc) for DBA and N-acetylglucosamine (GlcNAc) for S-WGA (Boothroyd et al., 1997). This posttranslational glycosylation plays an important role in the biology of the cyst as disruption of glycosylation alters cyst biology resulting in a reduction in in vivo cyst numbers and/ or fragile cysts (Tomita et al., 2013, 2017; Caffaro et al., 2013). Deletion of the T. gondii nucleotidesugar transporter (TgNST1: TgME49_367380) results in the loss of α-N-acetylgalactosamine and N-acetyl-D-glucosamine in the cyst wall, and this ΔTgNST1 T. gondii strain produces fewer cysts during murine infection (Caffaro et al., 2013). Using a proteomic approach, SUMOylation was also demonstrated on the cyst wall formed in vitro under alkaline stress (Braun et al., 2009). Treatment with chitinase disrupts the cyst wall and eliminates S-WGA binding, suggesting that chitin or a similar polysaccharide may be present in this structure (Boothroyd et al., 1997). Alternatively activated macrophages in the central nervous system detect chitin on the brain cyst wall and are necessary for the clearance of cysts during chronic infection (Nance et al., 2012). Binding of the lectin DBA is a marker of cyst wall formation in vitro. CST1 (TgME40_ 264660) is a 250 kDa protein originally recognized as a 116 kDa antigen using twodimensional electrophoresis (2D-PAGE) (Weiss et al., 1992; Zhang et al., 2001; Tomita et al., 2013). CST1 is an SRS protein (SRS44, containing 13 SRS domains) with a large mucin-like domain recognized by DBA as well as the monoclonal antibodies 73.18 and SalmonE (Weiss et al., 1992; Zhang et al., 2001) (Figs. 18.5 and 18.6). Lectin overlay experiments of 2D-PAGE gels suggest that the lectins DBA and S-WGA recognize different antigens, with S-WGA recognizing a 48 kDa antigen (Zhang et al., 2001). CST1 localizes to the

granular material in the cyst wall under the limiting membrane of the bradyzoite parasitophorous vacuole (Ferguson, 2004; Zhang et al., 2001). Knockout of cst1 in the PruΔKU80 T. gondii background (Fox et al., 2011) eliminates DBA staining of the cyst wall. Δcst1 parasites are able to form brain cysts in mice, but the cysts are much more fragile, rupturing after gentle homogenization of brain. Ultrastructure studies of the cyst wall by electron microscopy revealed that a lack of CST1 results in disruption of the cyst wall granular layer. Complementation with full length CST1 rescues this phenotype, but a CST1 without the mucin domain did not fully complement the defect, suggesting a role for glycosylation of the mucin domain in cyst wall stability (Tomita et al., 2013) (Fig. 18.6). Several glycosyl transferase genes, including a polypeptide N-acetylgalactosaminyltransferase that may be involved in cyst wall formation, have been identified, expressed, and characterized in T. gondii (Stwora-Wojczyk et al., 2004a, 2004b, 2004c; Wojczyk et al., 2003). T. gondii ppGalNAc-T2 (TgME49_258770) and -T3 (TgME49_318730) deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3 (Tomita et al., 2017). Both T2 and T3 were found to glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall (Tomita et al., 2017). T. gondii ppGalNAc-T2 is required for the initial glycosylation of the mucin-like domain, and T. gondii ppGalNAc-T3 is responsible for the sequential glycosylation on neighboring acceptor sites (Tomita et al., 2017). Protocols for purification of the cyst wall have now been developed and have facilitated identification of component proteins using proteomic approaches (Zhang et al., 2001, 2010; Tu et al., 2018, 2019a). BioID techniques are defining the cyst wall protein interactome and identifying new cyst wall proteins (Tu et al., 2020).

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18.8 Cyst wall and matrix antigens

(A)

(B)

(C)

(D)

(E)

(F)

(G)

FIGURE 18.6 Functional characteristic of cyst wall protein CST1. (A
C) Immunofluorescent assay of HFF infected with EGS strain (Paredes-Santos et al., 2015) of Toxoplasma gondii probed with anti-SalmonE monoclonal antibody (red) DBA lectin (green). Panel A is taken with a rhodamine filter set, Panel B with a fluorescein filter set, and Panel C with a filter set that allows simultaneous viewing of red and green fluorescent labels. This confirms that the SalmonE antibody and DBA both label the cyst wall (bradyzoite parasitophorous vacuole membrane). SalmonE recognizes cyst wall protein 1 (CST1) (Tomita et al., 2013). (D, E) Fluorescent microscopy (D) and electron micrograph (E) of purified mouse brain cysts from wild-type PruΔku80 T. gondii. The PruΔku80 strain expresses GFP using the bradyzoite-specific LDH2 promoter. These cysts do not rupture with the standard purification technique and have a normal appearing cyst wall with amorphous material lining the cyst wall membrane. (F, G) Fluorescent microscopy (F) and electron micrograph (G) of purified mouse brain cysts from Δcst1 PruΔku80 T. gondii. This demonstrates the “fragile cyst” phenotype of the cst1 knockout and that this knockout has an abnormal cyst wall morphology on TEM, lacking the amorphous material that is usually present under the cyst wall membrane. (C) Representative brain cysts after gentle homogenization of WT Pru strain and Δcst1 strain. HFF, Human foreskin fibroblasts. Source: Figures D
G are adapted with permission from Tomita, T., Bzik, D.J., Ma, Y.F., Fox, B.A., Markillie, L.M., Taylor, R.C., et al., 2013. The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence. PLoS Pathog. 9, e1003823.

In addition to CST1, 11 other cyst wall proteins (CST2 to 11, and MCP3) have been identified using these approaches (Tu et al., 2019a; Tu et al., 2020), including CST2 (GRA50, TgME49_203600), CST3 (GRA51, TgME49_230705), CST4 (TgME49_

261650), CST5 (GRA52, TgME49_319340), CST6 (GRA53, TgME49_260520), CST7 (TgME49_ 258870), CST8 (TgME49_204340), CST9 (TgME49_ 310790), CST10 (TgME49_312330), CST11 (TgME49_239752), and MCP3 (TgME49_208740).

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(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

(J)

FIGURE 18.7 Distribution of dense granule proteins (GRA) in bradyzoite parasitophorous vacuoles. Representative sections from the lung of an acutely infected mouse containing tachyzoites (A) and the brain of a chronically infected mouse containing tissue cysts (B
J). All of the sections were stained for dense granules proteins using the peroxidase technique and corresponding antisera specific to each GRA protein. Bars represent 5 μm. (A) Section stained with anti-GRA8 showing strong labeling of the parasitophorous vacuole containing tachyzoites. (B
J) Tissue cysts demonstrate relatively uniform staining of the dense granules within the bradyzoites (B
I). There was variable staining of the cyst wall with positive staining for GRA1 (B), GRA3 (D), GRA5 (F), GRA6 (G), and GRA7 (H) but little staining with GRA2 (C), GRA4 (E), GRA8 (I), or NTPase (J). Source: Courtesy DJP Ferguson. Reprinted with permission from Ferguson, D.J. 2004. Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. Int. J. Parasitol. 34, 347
360.

Several dense granule proteins (GRA1 though 8: GRA2: TgME49_270250, GRA2: TgME49_227620, GRA3: TgME49_227280; GRA4: TgME49_310780, GRA5: TgME49286450, GRA6: TGME49_275440, GRA7: TgME49_ 203310, GRA8: TgME49_254720) localize to the parasitophorous vacuolar membrane, the matrix of the vacuole and the tubular structures within the parasitophorous vacuole of T. gondii (Ferguson, 2004) (see Figs. 18.7 and 18.8). GRA proteins play a role in the formation of the intravacuolar network within the parasitophorous vacuole (Mercier et al., 2002). It is possible the tubular structures seen within the cyst wall are derived from the intravacuolar nanotubules since GRA proteins that form this structure are also found within the cyst wall layer and membrane (Torpier et al., 1993; Lane et al., 1996; Cesbron-Delauw, 1994). GRA5 (Lecordier et al., 1993) is found in both tachyzoites and bradyzoites. By immunohistochemistry, GRA5 is localized primarily to the cyst wall membrane and not to the granular material under this

membrane (Ferguson, 2004; Lane et al., 1996). Less intense staining of the cyst wall membrane is demonstrated by antibodies to GRA1, GRA3, and GRA6 (Ferguson, 2004; Torpier et al., 1993). Knockouts of either GRA4 or GRA6 and especially the dual GRA4/GRA6 knockout have been shown have reduced brain cyst burdens in C57BL/b mice, (Fox et al., 2011). Interestingly, deletion of GRA24 (TgME49_230180) leads to enhanced cyst wall formation in vitro (Odell et al., 2015). Deletion of aspartyl protease 5 (ASP5, TgME49_242720), a protein involved in the processing of several GRA proteins and the formation of the intravacuolar network, resulted in parasites that had irregular cyst wall staining (Hammoudi et al., 2015). Deletion of a serine protease inhibitor (TgPl1, TgME49_217430) that is secreted to the parasitophorous vacuole via the dense granules causes increased frequency of bradyzoite switching and an increased parasite burden during acute infection (Pszenny et al., 2012).

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18.8 Cyst wall and matrix antigens

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FIGURE 18.8 Toxoplasma gondii tissue cyst and the localization of bradyzoite-specific markers. (A) T. gondii cyst isolated from mouse brain under phase microscopy (10 3 objective) and (B) location of various antigens that have been identified in T. gondii cysts. Source: (A) Courtesy Dr. L. Weiss, Albert Einstein College of Medicine and (B) Adapted with permission from Tu, V., Yakubu, R., Weiss, L.M., 2018. Observations on bradyzoite biology. Microbes Infect. 20, 466
476.

Given the localization of most of the dense granule proteins to the parasitophorous vacuole, it seems likely that many components of the cyst wall will be dense granule proteins, perhaps with new carbohydrate modifications or bradyzoite-specific glycoproteins secreted from dense granules. In support of this idea is that rat monoclonal antibody CC2 reacts with an 115 kDa antigen in bradyzoites (perhaps CST1) but recognizes a 40 kDa protein in tachyzoites (Gross et al., 1995). Other proteins discovered to be within the cyst wall include BPK1 (TgME49_253330) (Buchholz et al., 2011, 2013), MCP4 (TgME49_ 208730) (Buchholz et al., 2011), matrix antigen 1 (MAG1, TgME49_270240) (Parmley et al., 1994), proteophosphoglycan 1 (PPG1,

TgME49_297520) a member of a family of proteins with homology to proteophosphoglycans (Craver et al., 2010), and brain colonization protein 1 (BCP1) (Milligan-Myhre et al., 2016). To search for new bradyzoite-specific secreted proteins, transcriptome analysis of in vitro and in vivo bradyzoites, combined with programs to identify signal peptides, led to the prediction of more than 100 bradyzoite-secreted proteins (Buchholz et al., 2011). The first two proteins identified by this screen, BPK1 and MCP4, localized to the cyst wall by electron microscopy, but the authors were unable to determine if the proteins originated in the dense granules. Subsequent study showed that deletion of BPK1 did not influence in vivo and in vitro bradyzoite differentiation but

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significantly reduced mouse oral infectivity and pepsin
acid resistance (Buchholz et al., 2013). Similar transcriptome analysis of the T. gondii sexual stages has identified several putative oocyst wall proteins (Fritz et al., 2012b). PPG1 knockout downregulated BAG1 expression (Craver et al., 2010). Interestingly, BCP1 localizes to the cyst wall under immunofluorescence within in vivo cysts but not in vitro derived cysts (Milligan-Myhre et al., 2016). MAG1 was originally identified as a 65 kDa protein expressed in the cyst matrix that was not expressed in tachyzoites (Parmley et al., 1994). Reverse transcriptase polymerase chain reaction (RT-PCR) data indicates that mRNA for MAG1 is present in both tachyzoites and bradyzoites (Parmley et al., 1994). It has now been demonstrated that MAG1 is also expressed in tachyzoites and secreted into the parasitophorous vacuole, albeit less abundantly than in bradyzoites (Ferguson and Parmley, 2002). Antibodies (monoclonal and rabbit polyclonal) to MAG1 react with extracellular material in the cyst matrix and to a lesser extent with the cyst wall, but not with the surface or cytoplasm of bradyzoites (Han et al., 2019). A second matrix antigen, MAG2: TgME49_209755, has been identified using monoclonal antibodies and proteomic techniques that localizes primarily to the cyst matrix and has no cyst wall staining (Tu et al., 2019b). The dense granule proteins GRA1, GRA2, GRA3, GRA5, GRA6, and GRA7 are present in the matrix of both tachyzoites and bradyzoites, but GRA4 and GRA8 appear to be expressed higher in tachyzoites. In addition to dense granule proteins that are found in the intravesicular membrane network (i.e., matrix), tachyzoites have also been shown to secrete nucleoside triphosphate hydrolases (TgNTPases: TgME49_277270, TgME49_277240) and rhoptry proteins into the lumen of the parasitophorous vacuole (Bermudes et al., 1994; Ferguson et al., 1999).

While a bradyzoite-specific NTPase (B-NTPase) has been identified (Behnke et al., 2008; Radke et al., 2005), its localization within the bradyzoite stage remains unknown. Monoclonal antibodies E7B2 (Lane et al., 1996), 93.2 (Weiss LM, unpublished), and 1.23.29 (Weiss LM, unpublished) also recognize matrix antigens, but the corresponding genes have not been identified (see Table 18.1 for a list of these antibodies).

18.9 Surface antigens Most T. gondii surface antigens are members of family of 182 genes with similarity to SAG1 (Boothroyd et al., 1998; Manger et al., 1998b; Lekutis et al., 2001; Wasmuth et al., 2012). It is not clear why so many family members exist (although some are pseudogenes) (Wasmuth et al., 2012) because antigenic variation as described for Trypanosomes or Plasmodium has not been described. All of these SAGs appear to be attached to the plasma membrane by a similar glycolipid anchor. While SAG3 (TgME49_308020) is found in all life stages, several of these surface antigens appear to be stage specific. SAG1 (now SRS29B: TgME49_233460), SRS1
3 (TgME49_233450, TgME49_233480, TGME49_308840), SAG2A (TgME49_271050), and SAG2B (TgME49_208850) are expressed in tachyzoites (Burg et al., 1988; Lekutis et al., 2000; Lekutis et al., 2001), and SAG2C (TgME48_207160), SAG2D (TgME49_207150) (Lekutis et al., 2000), SAG2X (TgME49_207140), SAG2Y (TgME49_207130) (Saeij et al., 2008), SAG4A (TgME49_264660) (Odbergferragut et al., 1996; Tomavo et al., 1991), SAG5A/5.1 (now SRS36C: TGME49_292270), and SRS9 (TgME49320190) (Cleary et al., 2002) in bradyzoites (Kim and Boothroyd, 2005; Jung et al., 2004). See Wasmuth et al. (2012) for details of the proposed renaming and stage expression of the SRS family. These antigens may be involved in persistence of tissue cysts in their hosts and the relative lack of an immune response to

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18.9 Surface antigens

tissue cysts (Kim and Boothroyd, 2005). CST1 (SRS44) is an SRS domain family member that has 13 SRS domains and a mucin domain and localizes to the cyst wall but is not a surface antigen (Tomita et al., 2013). SRS13 (TgME49_222370), which has 2 SRS domains, was the only other SRS protein found to have a mucin domain, and it also localizes to the cyst wall and is not a surface antigen (Tomita et al., 2018). Both SAG3 (p43) and SAG1 (p30) have been implicated in adhesion to host cells. Disruption of SAG3 leads to twofold decreased adhesion of parasites (Dzierszinski et al., 2000). Disruption of SAG1 in the RH strain, but not in PLK, results in parasites that are more invasive (Mineo et al., 1993). Either the SAG1 or SAG3 disruption in RH strain results in a decrease in virulence (Dzierszinski et al., 2000). More recently, dual disruption of SAG1 (p30, SRS29B) and SAG2 (SRS34A, SAG2A, p22) showed upregulation of SRS29C (SRS2, p35) and decreased virulence, with overexpression of SRS29C in the virulent RH strain also associated with decreased virulence (Wasmuth et al., 2012). Bradyzoite-specific recombinant (BSR) 4 (TgME49_320180) was discovered in a promoter trap to isolate developmentally regulated genes and found to be a surface antigen that interacts with the p36 mAb T84A12 (Knoll and Boothroyd, 1998). It was later found that SRS9, a highly similar gene immediately downstream of BSR4, is also a bradyzoite-specific surface antigen that reacts with mAb T84A12 (Van et al., 2007). These genes demonstrate a restriction fragment length polymorphism between ME49 (PLK; type II) and CEP (type III) strains, which correlates with the lack of mAb T84A12 binding to CEP strain. Antibodies that recognize both BSR4 and SRS9 show that one or both of these proteins are expressed on the merozoite as well as the bradyzoite surface (Ferguson, 2004). While SRS9 is transcriptionally upregulated in bradyzoites (Cleary et al., 2002), RNA levels of BSR4 are similar in tachyzoites and

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bradyzoites, suggesting that posttranscriptional regulation of BSR4 occurs via some unknown mechanism. Disruption of BSR4 and SRS9 did not result in a tissue culture bradyzoite development phenotype (Knoll and Boothroyd, 1998). Deletion of SRS9 created parasites that were similar to wild type in size and abundance during early chronic infection (3
4 weeks) but were not able to persist in the brains of mice (Kim et al., 2007). Surface antigens similar to SAG1 with a conserved placement of 12 cysteines include SAG1, SAG3, BSR4, and SRS1
4 (SAG-related sequences), SAG5, SAG5.1, and SAG 5.2. A second group of SAG1 members has a less consistent conservation of cysteine spacing. This group includes SAG2A (SAG2 or p22), SAG2B, SAG2C, SAG2D, and SRS28 [TgME49_258550 (SporoSAG)]. SAG2C and SAG2D are only detected on in vivo bradyzoites not in vitro bradyzoites (Lekutis et al., 2001). Similar to SRS9, parasites deleted in the four-gene surface antigen cluster SAG2C/D/X/Y have a chronic infection persistence defect and a reduction in cyst numbers with time (Saeij et al., 2008). SAG4A (p18) is an 18 kDa surface protein transcriptionally regulated during bradyzoite development (Odbergferragut et al., 1996). Crystal structures of SAG1 family members from different life cycle stages, SAG1 [tachyzoite; (He et al., 2002)], BSR4 [bradyzoite (Crawford et al., 2009)], and SRS28 [sporozoite, aka SporoSAG (Crawford et al., 2009)], show overall similarly, but potentially biologically relevant structural diversity. A new family of 31 GPI-anchored surface antigens was described that are completely unrelated to the SAG1 proteins (Pollard et al., 2008). This new family of proteins was named SAG-unrelated surface antigens (SUSAa) (TgME49_278430, TgME49_278080). Analysis of the single-nucleotide polymorphism density shows that the SUSA genes are the most polymorphic within the T. gondii genome, highlighting that they are likely under immune

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pressure. One family member so far, SUSA1, has been shown to be bradyzoite specific (Pollard et al., 2008). Like the SAG1 superfamily, the function(s) of the SUSA family remain to be determined. A P-type ATPase (TgPMA1: TgME49_ 252640) has also been described on the surface of T. gondii (Holpert et al., 2001, 2006). TgPMA1 is upregulated in bradyzoites and shows punctate staining on the parasite plasma membrane. T. gondii ΔTgPMA1 parasites have a downregulation of bradyzoite genes and produce fewer bradyzoite-positive vacuoles in vitro; however, these parasites are still able to form mature cysts in vivo (Holpert et al., 2001, 2006).

18.10 Metabolic differences between bradyzoites and tachyzoites It is probable that energy metabolism of bradyzoites is different from tachyzoites given the location of bradyzoites in a thick-walled vacuole and their slower growth rate. An unusual and unexplained feature of T. gondii differentiation is the presence of stage-specific differences in the activity and isoforms of several

glycolytic enzymes. It is known that tachyzoites utilize the glycolytic pathway with the production of lactate as their major source of energy (Wastling et al., 2009). Functional mitochondria with a TCA cycle exist in tachyzoites and are thought to contribute to energy production. While both tachyzoites and bradyzoites utilize the glycolytic pathway for energy, data suggests that bradyzoites lack a functional TCA cycle and respiratory chain suggesting a predominate role for anaerobic glycolysis in bradyzoites (Denton et al., 1996). Many enzymes that function in the metabolism of oxygen radicals are also upregulated in bradyzoites, perhaps to deal with exposure to these compounds during latent infection (Manger et al., 1998a). Tissue cysts purified from mouse brains were labeled with the membrane potential sensitive dye MitoTracker revealed the mitochondrial activity is likely to complex; some cysts had the majority of resident bradyzoites taking up the dye, while other cysts had virtually no labeled bradyzoites (Watts and Sinai, unpublished), and the vast majority of cysts exhibited a heterogenous uptake in the bradyzoites within the cyst (Fig. 18.9). This heterogeneity covered both a spectrum of

FIGURE 18.9 Heterogenous presentation of active mitochondria within a tissue cyst. (A) A purified brain derived tissue cyst labeled with the fixable membrane potential sensitive dye MitoTracker reveals nonuniform staining ranging from the absence of labeling to intense punctate staining. Labeled mitochondria display a diversity of morphologies, a feature linked in tachyzoites to distinct physiological states. (A*) A gray0-scale computer rendering of the adjacent image highlights the inherent morphological and likely functional heterogeneity of the mitochondrion in the encysted bradyzoites. Source: Courtesy E. Watts and A. Sinai.

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18.10 Metabolic differences between bradyzoites and tachyzoites

MitoTracker levels as well as morphological differences in the labeled organelles, reflecting differences noted in tachyzoites (Ovciarikova et al., 2017). Recent work in tachyzoites has addressed the functional significance of such morphological heterogeneity suggesting such variations in encysted bradyzoites further cement that physiological heterogeneity is in fact the norm for bradyzoites (Ovciarikova et al., 2017). How this heterogeneity correlates with replication potential and amylopectin levels is not known. Overall, the regulation and activation of glycolysis appear to be different in T. gondii than many other eukaryotes (Saito et al., 2002; Maeda et al., 2003; Denton et al., 1996). LDH and pyruvate kinase activity are higher in bradyzoites than in tachyzoites while PPi-phosphofructokinase activity is higher in tachyzoites than bradyzoites (Denton et al., 1996). These data suggest that bradyzoite energy metabolism may be dependent on the catabolism of amylopectin, which is present in bradyzoites and essentially absent in tachyzoites, to lactate. The bradyzoite-specific glycolytic isoenzymes are resistant to acidic pH suggesting that bradyzoites are resistant to the acidification resulting from the accumulation of the glycolytic products from amylopectin catabolism to lactate. These metabolic differences may be involved in the observation that neurons and mature muscle cells are more likely to support development of tissue cysts (Luder and Rahman, 2017). During anaerobic growth, LDH is important for supplying energy to the parasite by converting pyruvate to lactate in the glycolytic pathway. T. gondii LDH occurs in two forms: TgLDH1 (tachyzoite expressed) and TgLDH2 (bradyzoite expressed) (Yang and Parmley, 1997; Yang and Parmley, 1995). TgLDH2 expression appears to be transcriptionally regulated because LDH2 mRNA is detectable by RT-PCR only in bradyzoites. TgLDH2 is highly upregulated within bradyzoites (Radke et al.,

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2005; Buchholz et al., 2011) and is able to function efficiently under acidic and pyruvate-rich conditions (Yang and Parmley, 1995; Yang and Parmley, 1997; Dando et al., 2001). Knockout of TgLDH2 (ΔTgLDH2) in the PruΔKu80 strain does not affect bradyzoite differentiation in vitro, but does reduce cyst formation in vivo. Interestingly, while TgLDH1 expression is higher in tachyzoites, deletion of this protein in the PruΔKu80 strain reduces bradyzoite development in vitro and also impairs cyst formation in vivo. When both TgLDH1 and TgLDH2 are deleted, a phenotype similar to the ΔTgLDH1 is seen (Abdelbaset et al., 2017). These results confirmed earlier studies that knockdown of TgLDH2 and TgLDH1 reduced cyst numbers in vivo (Al-Anouti et al., 2004). Knockdown of LDH2 has been achieved using dsRNA (Al-Anouti et al., 2004). When LDH2 expression was downregulated, bradyzoite differentiation and growth are impaired (Al-Anouti et al., 2004). Interestingly, when TgLDH1 and TgLDH2 are overexpressed in type I RH strain, an increase in in vitro cyst formation occurs under alkaline conditions (Liwak and Ananvoranich, 2009). Two stage-specific enolases have also been cloned and characterized; consistent with the hypothesis that utilization of the glycolytic pathway is different in tachyzoites compared to bradyzoites (Yahiaoui et al., 1999; Manger et al., 1998a). It has been shown that intron retention of TgENO1/2 and TgLDH1/2 transcripts plays a role in the stage-specific expression of these genes (Lunghi et al., 2015). Unspliced transcripts of TgENO and TgLDH are produced by the parasite and await a differentiation signal before splicing to mature transcripts (Lunghi et al., 2015). Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. In yeast, enolase is known to be a stress response protein, that is, hsp48 (Iida and Yahara, 1985). ENO2, the tachyzoite form, has threefold higher specific activity than ENO1, the bradyzoite enzyme,

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but both have similar Michaelis constants (Km) (Dzierszinski et al., 2001). Surprisingly, polyclonal antisera to each isoform do not cross react despite the similarity of these two isoforms (Dzierszinski et al., 1999, 2001). Both isoforms are found localized to the nucleus in dividing cells, but in late bradyzoites, which are quiescent, ENO1 is cytoplasmic (Ferguson and Parmley, 2002; Ferguson et al., 2002a). In the TgENO1 knockout, there is a reduction in cyst numbers in infected mice (Mouveaux et al., 2014). As TgENO1 and TgENO2 localize predominately to the nucleus of bradyzoites and tachyzoites, respectively (Ferguson et al., 2002b), the stage-specific expression and localization of these enolases suggest a role for them in transcriptional stage interconversion. Indeed, these enolases have the ability to bind to chromatin, regulate the expression of many genes, interact with the promoter of MAG1, and bind to “TTTTCT” DNA motifs (Mouveaux et al., 2014; Ruan et al., 2015). In addition, the promoters of TgENO1 and 2 have been characterized to bind nuclear extracts from tachyzoites or bradyzoites (Kibe et al., 2005). One of these binding proteins was an FK506BP homolog called TgNF3 (TgME49_260440), which is believed to silence TgENO1. TgNF3 is found in the nucleus of tachyzoites and in the cytoplasm of bradyzoites (Olguin-Lamas et al., 2011). The significance of these observations is unknown, but it is possible that some glycolytic enzymes may have alternate regulatory functions that are not yet fully understood. An obvious difference between tachyzoites and bradyzoites is the presence of cytosolic granules of amylopectin that are composed of glucose polymers (Guerardel et al., 2005). Structural studies of amylopectin have revealed it to be a plant-like amylopectin with predominantly (α1
4) linkages, which is most similar to the semicrystalline floridean starch accumulated by red algae (Coppin et al., 2005). AG disappear from bradyzoites when they

transform into tachyzoites during cell culture (Coppin et al., 2003). T. gondii calcium
dependent protein kinase (TgCDPK2, TgME49_255490) plays a role in the regulation of amylopectin (Uboldi et al., 2015). TgCDPK2 regulates the activity of pyruvate phosphate dikinase, an enzyme involved in amylopectin generation. Deletion of TgCDPK2 in RH, a type I strain, and PRU, a type II strain, results in abnormal parasite morphology and aberrant amylopectin accumulation. Tachyzoites that are ΔTgCDPK2 accumulate starch in their residual body, and bradyzoites have abnormally large AG in their cytoplasm and were unable to form cysts in vivo, although the cyst burdens in animals infected with the parental line were extremely low (Uboldi et al., 2015) raising concerns about the sensitivity of the in vivo study. Glycogen phosphorylase (TgGP, TgMe49_310670) has been demonstrated to be a critical enzyme for amylopectin storage dynamics, given its role in amylopectin catabolism. TgGP disruption, which like the ΔCDPK2-parasites results in the excessive accumulation amylopectin, was found to disrupt cyst formation in vivo (Sugi et al., 2017). TgGP1 mutants were created that substituted glutamate for Serine25 to mimic phosphorylation and to create a hyperactive TgGP1 or substituted alanine for Serine25 to produce a phospho-null mutant that has ablated activity. The hyperactive state of TgGP1S25E was not able to retain amylopectin, likely on account of increased catabolism, while the phospho-null mutant TgGP1S25A had abnormal accumulation of starch, similar to the TgCDPK2 and TgGP1 knockouts. While it has been shown that TgCDPK2 phosphorylates the catabolic enzymes of amylopectin upstream of TgGP1 (Uboldi et al., 2015), it does not appear to regulate TgGP1 directly (Sugi et al., 2017). Further studies on the kinases (including the TgGWD: TgME49_214260, the glucose water dikinase needed to phosphorylate the starch chain to promote unwinding and access to amylases)

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18.11 Genetic studies on bradyzoite biology

and phosphatases (such as TgLaforin: TgME49_205290, a glucan-specific phosphatase that is needed to reset the starch degradation cycle by removing phosphate allowing amylases to proceed with degradation) as well as the enzymes responsible for regulating TgGP1 are required to fully elucidate amylopectin catabolism. These studies demonstrate that regulation of amylopectin metabolism is required for a successful chronic infection. While not seen in tachyzoites, amylopectin is present in the sexual cycle in the cat intestine in macrogametes, persists during oocyst formation and in sporozoites. Merozoites on the other hand lack amylopectin. Amylopectin is believed to be a carbohydrate store for the bradyzoite or sporozoite during long periods of quiescence and nutrient deprivation. Candidate genes for enzymes involved in amylopectin breakdown and synthesis have been identified (Coppin et al., 2003; Sugi et al., 2017). With the confirmation of the replicative potential of encysted bradyzoites (Watts et al., 2015), an additional function in providing the energy and biosynthetic potential of releasing stored glucose presents an additional function during the course of the chronic infection. Examination of electron micrographs and PAS-stained tissue cysts reveal that encysted bradyzoites vary considerably with regard to the levels of detectable amylopectin. This heterogeneity may reflect the different replicative status of individual parasites within the tissue cyst and serve as a potential physiological marker. A bradyzoite-specific P type ATPase whose mRNA and protein are preferentially expressed in bradyzoites has been characterized and localizes in punctate pattern to the region of the plasma membrane (Holpert et al., 2001). A second P-type ATPase also may be preferentially expressed in bradyzoites as judged by RT-PCR of steady-state mRNA, although mRNA can also be detected in tachyzoites. It is not known if the metabolic changes between bradyzoites and tachyzoites cause

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differentiation or are a consequence of the differentiation process. One hypothesis is that monitoring for nutrient deprivation, a type of stress response, might serve as the sensor for differentiation in T. gondii. However, no sensors that respond to any environmental change have yet been identified in T. gondii.

18.11 Genetic studies on bradyzoite biology One would expect that the knockout of a bradyzoite-specific gene would be feasible as growth should occur in the tachyzoite stage even if bradyzoite development is prevented. This strategy has been applied to the bradyzoite-specific gene BAG1/hsp30 (BAG5) (Bohne et al., 1998; Zhang et al., 1999). A bag1 knockout was created using hypoxanthinexanthine-guanine phosphoribosyltransferase (HGXPRT) as a selectable marker in an HGXPRTneg PLK strain of T. gondii. Another bag1 knockout was performed using CAT as a selectable marker in a clone of PLK strain that had been passaged through mice to ensure it made cysts at the start of the study. Cyst formation in vitro and in vivo occurred in both knockouts, indicating that BAG1 is not essential for cyst formation. Zhang et al. (1999), however, found that the number of cysts formed in vivo in CD1 mice was reduced in the bag1 knockouts. Complementation resulted in the production of similar numbers of cysts in vivo as the wild-type PLK strain (Zhang et al., 1999). When parasites were grown in SNP, the bag1 knockout grew faster than PLK. This result may be a difference in transition rate from the rapidly growing tachyzoite to the slowly growing bradyzoite stage in this bag1 knockout. The decrease in cyst formation is a relatively subtle phenotype, which was not observed when BAG1 was disrupted in the HGXPRTneg PLK strain background (not passaged in mice prior to the knockout) and cyst

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formation was tested in highly susceptible C57BL/6 mice (Bohne et al., 1998). The capacity to convert from tachyzoite to bradyzoite is a key feature for T. gondii persistence in the host, and thus it is likely that multiple genes with redundant functions are involved in this process. Small heat shock proteins, such as BAG1, have been postulated to act as specialized chaperones for enzymes such as glutathione reductase during differentiation, but the exact function of such small heat shock proteins is unknown. It is possible that the other small heat shock proteins in T. gondii can partially compensate for a lack of BAG1. Promoter trapping has been an effective technique for the identification of genes induced during bradyzoite differentiation. These studies used a promoterless HGXPRT gene with 6-thoxanthine (6-TX) or 8azaguanine (8-AzaH) as negative selection and mycophenolic acid with xanthine (MPA-X) as a positive selection (Bohne et al., 1997; Knoll and Boothroyd, 1998). Selection works by growing transfected parasites at pH 7.0 in the presence of 6-TX that inhibits the growth of all organisms that have the HGXPRT gene on a constitutive or tachyzoite promoter. To confirm bradyzoite specificity, this population of organisms is then exposed to pH 8.0 and MPA-X, and only parasites that express HGXPRT (i.e., those with a bradyzoite or constitutive promoter in front of the HGXPRT gene) will survive. It should be noted that this approach can be “leaky,” depending on the concentrations of 6-TX and MPA-X used. Nonetheless, when the 6-TX and MPA-X selections are repeated several times, one will enrich the population for organisms with HGXPRT under the control of bradyzoite-specific gene promoters. Using this approach, additional bradyzoite-specific promoters (Donald and Roos, unpublished; cited in Matrajt et al., 2002) and 8 BSR strains were obtained (Knoll and Boothroyd, 1998). Additional genetic strategies have been developed to identify mutants unable to

undergo bradyzoite differentiation (Matrajt et al., 2002; Singh et al., 2002). Singh et al. (2002) generated point mutants in a LDH2-GFP Prugnaiud (type II) background, which expresses GFP under the control of the bradyzoite-specific promoter LDH2, to obtain mutants with an altered ability to transform into bradyzoites. Parasites unable to differentiate were identified by fluorescence activated cell sorting (FACS) enrichment of GFP-negative parasites when these organisms were exposed to bradyzoite inducing conditions. Matrajt et al. (2002) also utilized insertional mutagenesis of an engineered stable line expressing a bradyzoite-specific pT7-HGXPRT cassette in UPRT-deficient RH (type I) parasites. Earlier studies had demonstrated that RH UPRT disruptants differentiate into bradyzoites under conditions of CO2 starvation (Bohne and Roos, 1997). The pT7-HGXPRT stable line was obtained by rounds of negative and positive selection, alternating 6-TX (tachyzoite conditions) with MPA-X (bradyzoite conditions) selection. The result was a cell line where HGXPRT was highly regulated by differentiation conditions. Insertional mutagenesis was then performed in this pT7-HGXPRT line using DHFR cassettes that earlier were shown to have a high frequency of nonhomologous insertion (Donald and Roos, 1993). An inability to differentiate into bradyzoites was detected by the inability of the disruptant to express HGXPRT. Both groups were able to demonstrate that these differentiation mutants were unable to make bradyzoites at the same efficiency as the parental strains and both groups demonstrated global defects in expression of previously characterized markers as determined by immunofluorescence and microarray analysis. Due to technical difficulties the exact mutations could not be identified. The insertional mutants identified by Matrajt et al. (2002) were similar to the bag1 knockout (Zhang et al., 1999) and had more rapid growth under bradyzoite inducing

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conditions than seen in wild-type parasites (Matrajt et al., 2002). Microarray analysis of these mutants identified classes of genes, including a 14-3-3: TgME49_262090 homolog, a PISTLRE kinase, and a probable vacuolar ATPase, whose expression was decreased in the differentiation mutants (Singh et al., 2002; Matrajt et al., 2002). Other genes of interest identified included an AP2 factor (AP2XII-6, TgME49_249190) and an oocyst wall protein (Lescault et al., 2010). Microarray studies of these mutants also suggest that there was a hierocracy of gene expression during bradyzoite differentiation (Singh et al., 2002), which is consistent with the developmental program seen in Plasmodia. Further microarray analysis of bradyzoite development mutants has also highlighted an extracellular tachyzoite state that is distinct from intracellular tachyzoites and bradyzoites (Lescault et al., 2010). A FACS strategy, similar to that of (Singh et al., 2002) and Matrajt et al. (2002), combined with insertional mutagenesis has been useful to identify genes necessary for bradyzoite development. Mutant TBD-6 switched to bradyzoites with about half the efficiency of wildtype parasites (Vanchinathan et al., 2005). TBD6 had an insertion 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1), which causes an upregulation of the mRNA. The phenotype of decreased bradyzoite development could be replicated by directed integration into the same upstream region. ZFP1 is targeted to the parasite nucleolus by CCHC motifs, downregulated in wild-type bradyzoites and appears to be essential as multiple attempts yielded no knockouts (Vanchinathan et al., 2005). Mutants TBD-5 and TBD-8 had independent insertions into a pseudouridine synthetase homolog (PUS1: TgME49_202640) (Anderson et al., 2009). Knockout of PUS1 had a bradyzoite development phenotype similar to the TBD-8 mutant and could be complemented with the genomic PUS1 allele. During animal infections,

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PUS1 deletions had increased mortality during acute infection and higher cyst burdens during chronic infection (Anderson et al., 2009). Insertional mutagenesis of signature-tag containing parasites was used to isolate mutants defective in the establishment of a chronic infection (Frankel et al., 2007; Knoll et al., 2001). Immunofluorescence microscopy screening of this library for mutants parasites defective in their ability to form an intact cyst wall led to the identification of nine additional bradyzoite development mutants (Craver et al., 2010). In this study, a proteophosphoglycan was found to localize to the parasitophorous vacuole space enhance cyst wall formation. Two independently identified differentiation deficient mutants (Singh et al., 2002; Frankel et al., 2007) had disruption on a same locus that contains noncoding RNA (Tg-ncRNA-1), which encodes a REP-derived small RNA. Complementation of these mutants with WT Tg-ncRNA-1 locus rescued the differentiation defect (Patil et al., 2012). This suggests that noncoding RNAs probably have a role in the process of differentiation. Overall, these insertional mutagenesis strategies have discovered proteins involved in bradyzoite development that would not have been predicted through other strategies.

18.12 Sexual stage morphology, biology, and antigens As described in Chapter 1, The history and life cycle of Toxoplasma gondii, the sexual cycle of T. gondii is restricted to the feline intestinal tract. Ingested bradyzoites are released from the cyst in the stomach, then invade the enteroepithelial cells of the feline small intestine. Within these enteroepithelial cells, bradyzoites differentiate into five morphologically distinct types of schizonts (Dubey and Frenkel, 1972a, 1972b). Within 2 days in the feline intestine, parasites progress through all five stages of

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schizonts and then develop into merozoites. The merozoites undergo a limited proliferation of two-to-four doublings before they differentiate into macrogametes and microgametes. The macro- and microgametes fuse to produce diploid oocysts that develop thick impermeable walls and are shed in the feces (see Chapter 2: The ultrastructure of Toxoplasma gondii, for ultrastructural images of these life cycle stages). Peak oocyst shedding in cats usually occurs from days 5 to 15 postinfection. While some cats can excrete up to tens of millions of oocysts throughout the course of infection, other cats shed considerably fewer oocysts, even when infected with the same T. gondii strains. In ambient air and temperature, some but not all of the oocysts in the cat feces mature by sporulation. If an oocyst sporulates, it undergoes mitosis and meiosis to form two sporocysts, each containing four haploid sporozoites. The differences in oocysts shedding and sporulation suggest that biological factors of the cat influence T. gondii sexual reproduction. While beautiful microscopy has characterized the sexual stages in the cat intestine, as illustrated by the electron micrographs in Chapter 2, The ultrastructure of Toxoplasma gondii, few molecular markers and cat stagespecific antibodies have been developed. Bioinformatics analysis of the bradyzoiteenriched expressed sequence tags uncovered a novel rhoptry protein of the bradyzoite stage, BRP1, that is also expressed in the merozoite stages (Schwarz et al., 2005). Deletion of BRP1 appears to have no consequences to bradyzoite development or maintenance, but sexual development has not yet been investigated. For a global analysis of the sexual development genes, merozoites were harvested from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip (Behnke et al., 2014). Subsequently, the Hehl lab performed RNAseq on the enteroepithelial cells of cats infected with T. gondii for 3, 5, or 7 days (18, ToxoDB) (Hehl et al., 2015). Harvest

times were chosen to correlate with parasite life stages: schizonts and merozoites day 3, merozoite and gametes day 5, and gametes and oocysts at day 7. Analysis of this data revealed two gra genes with highest transcript levels in merozoites but significantly downregulated in the tachyzoite stage, gra11a (TGME49_212410) and gra11b (TGME49_237800) (Ramakrishnan et al., 2017). Antisera to GRA11B have been useful as both a marker of sexual development and to follow the localization changes of GRA11B through developmental progression. Two monoclonal antibodies, 3G4 and 4B6, have been generated against the T. gondii oocyst wall (Dumetre and Darde, 2005). By immunofluorescence, 3G4 and 4B6 recognize the wall of unsporulated and sporulated oocysts, respectively. Finally, sporulation can be confirmed by expression of sporozoite-specific surface antigen (SporoSAG, TGME49_258550). SporoSAG is not expressed in the asexual stages, or the sexual stages in the cat intestine, but it is transcriptionally upregulated during oocyst maturation in the environment (Crawford et al., 2010).

18.13 Sexual stage development in cell culture Determining conditions for T. gondii sexual development in cell culture would advance the field by creating a model to easily examine the sexual stages and making classical genetic crosses readily available. Early attempts to perform sexual crosses of T. gondii in cat small intestinal cells in tissue culture were unsuccessful, most likely because the rapidly replicating tachyzoite form outgrew the slower growing sexual forms, and lysed the cells (Elmer Pfefferkorn, personal communications). To combat this problem, T. gondii was engineered to express a negative selectable marker from an asexual stage-specific promoter. Cidal inhibition is caused when the T. gondii enzyme uracil

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18.13 Sexual stage development in cell culture

phosphoribosyltransferase (UPT) phosphoribosylates the uracil analog 5-fluorodeoxyuridine (FUDR). The UPT gene was deleted, then reintroduced it under the control of the tachyzoitespecific promoter surface antigen 1 (SAG1). When bradyzoites of this ΔUPT::SAG1-UPT strain are grown in cat intestinal epithelial cells with FUDR, induction of sexual development genes could be seen by RT-PCR and some oocysts were formed, but the system was not robust (Knoll lab, unpublished results). It was hypothesized that some metabolites were missing from the cell culture system for T. gondii sexual development to readily occur. It is well known that cats are auxotrophic for the sulfated amino acid taurine; however, supplementation of the tissue culture cat enteroepithelial cells with varying concentration of taurine did not enhance T. gondii sexual development (Knoll lab, unpublished results). While production of a metabolite from a cat-specific bacteria was a possibility, a wide variety of feline species can act as the definitive host for T. gondii, highlighting that the microbiome of the house cat was unlikely to be essential.

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Switching to cat serum brought success in cat enteroepithelial cells in tissue culture with abundant T. gondii oocysts production. One of the major differences between commercial feline and bovine sera is that bovine serum is delipidated. The T. gondii asexual stages scavenge fatty acids from the host, particularly oleic acid (Nolan et al., 2018). Similarly, sexual reproduction in fungi is dependent on linoleic acid (C18:2), but not on oleic acid (C18:1) (Brown et al., 2008). Supplementation of bovine serum with 200 μM, but not 20 μM linoleic acid, caused sexual differentiation (Fig. 18.10) and abundant oocyst production (Knoll lab, unpublished results; Martorelli Di Genova et al., 2019). Similar to fungi, 200 μM oleic acid did not enhance oocyst production, highlighting that the number of double bonds is critical in the biologic activity of the lipid used for supplementation. The dramatic difference between one and two double bonds supports the hypothesis that linoleic acid is used as a signaling molecule and not merely to meet basic nutritional needs.

FIGURE 18.10 Toxoplasma gondii sexual stage development in vitro. (A) Photomicrograph of cat intestinal cells in sexual stage differentiation media (containing bovine serum with 200 μM linoleic acid). Cells were infected with bradyzoites isolated from mouse brain derived EGS strain (Paredes-Santos et al., 2015). T. gondii tissue cysts digested with pepsin and acid. Cells were imaged at 40 3 magnification five days after infection using an EVOS cell imaging station. (Knoll L, manuscript in preparation). (B) Corresponding fluorescence image demonstrating expression of MSF-BFP in EGS strain T. gondii (arrows). This EGS strain expresses three distinct stage specific differentiation markers: SAG1-mCherry, LDH2sfGFP, and MSF-BFP (Weiss L., unpublished data). Source: Courtesy L. Knoll and Bruno Martorelli Di Genova.

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Quorum-sensing for sexual reproduction in fungi is dependent on oxygenation of linoleic acid (Brown et al., 2008). It is possible that T. gondii cyclooxygenases and lipoxygenases oxygenate linoleic acid to an oxylipin signaling molecule for T. gondii sexual development to proceed. The T. gondii genome contains four lipoxygenases in the T. gondii genome, with TGME49_315970 called TgLOX1 600-fold upregulated in the cat intestine compared to asexual mouse infection stages (Hehl et al., 2015). Future studies will analyze the oxylipins produced during T. gondii sexual development as well as addressing which oxygenase is responsible for their production.

18.14 Sexual stage development in a mouse model Addition of 20 μM linoleic acid did not enhance oocyst production, indicating that the concentration of linoleic acid was critical for proper signaling. The dependence of T. gondii sexual development on high levels of linoleic acid is key to understanding the species boundary because cats are the only mammal known to lack delta-6-desaturase (D6D) activity in their small intestines (Rivers et al., 1975; Sinclair et al., 1979). D6D is the first and ratelimiting step for the conversion of linoleic acid to arachidonic acid (Das, 2006). It was hypothesized that the lack of D6D activity in the cat small intestine allows for a buildup of linoleic acid, which T. gondii subsequently uses as a signal for sexual development. To test this hypothesis, sexual development markers were analyzed in linoleic acid supplemented mouse intestinal monolayers with or without the drug SC26196, a specific inhibitor of the D6D enzyme (Obukowicz et al., 1998). Five days after infection with T. gondii, abundant expression of the sexual development markers GRA11B and BRP1 was found only in linoleic

acid supplemented mouse cells where D6D was inhibited. As the D6D inhibitor SC26196 was effective as an antiinflammatory agent in whole animal experiments (Obukowicz et al., 1998; He et al., 2012), it was hypothesized that a mouse model of T. gondii sexual development could be created (Martorelli Di Genova et al., 2019). When mice were fed a linoleic acid rich diet and SC26196 throughout infection, qPCR and immunofluorescence on ileum demonstrated a high expression of the sexual stage marker GRA11B and a low expression of the asexual tachyzoite stage marker SAG1 (Burg et al., 1988) in mice supplemented with linoleic acid and 100 mg/kg SC26196 PO every 12 hours but not in control mice (Martorelli Di Genova et al., 2019). Starting as early as 5 days postinfection, oocysts with 3G4 antibody positive and blue autofluorescent walls (Belli et al., 2003) were present in the mouse feces, and there was increased shedding through day 7 when the mice were sacrificed. Genomic DNA extracted from murine fecal samples when examined by qPCR demonstrated T. gondii genomic DNA only in mice treated with the D6D inhibitor SC26196 but not in control animals (Martorelli Di Genova et al., 2019). Furthermore, the oocysts shed from mice were infectious to new mice as seen by anti-Toxoplasma antibody production and bradyzoite cysts in their brains 28 days following infection with the oocysts. This murine animal model will significantly advance studies on the sexual stages of T. gondii and facilitate the use of sexual crosses (forward genetic) approaches to examine the biology of this pathogen.

18.15 Summary The advances made in our understanding of bradyzoite biology and the regulation of the tachyzoite to bradyzoite transition have been facilitated by the identification of markers and

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References

the application of genetic approaches and imaging based tools to this life cycle stage. This work has fundamentally altered our understanding of chronic T. gondii infection. Chronic infection in vivo was previously viewed as a black box, defined by the tissue cyst, and it is now being investigated at the level of individual bradyzoites within the cyst. The evidence suggests that the bradyzoites within tissue cysts are physiologically heterogenous and capable of replication, the complexity, and dynamism of a life cycle stage that was viewed as being largely inert offers new areas for investigation. These studies will no doubt have a practical benefit in guiding the development of new drugs and treatment modalities to address what has been a refractory state. This new perspective on bradyzoite biology opens the path to addressing what are still elusive questions regarding their invisibility to the immune system and the critical events leading to the clinically relevant reactivation and recrudescence, which are the dominant features of this chronic infection in its hosts. Studies of the mechanism(s) by which development is triggered and coordinated should eventually result in the identification of new therapeutic strategies for the control toxoplasmosis. These advances may ultimately result in the radical cure of infection by eliminating the latent T. gondii cyst stage in tissue. Furthermore, genetic strategies that prevent cyst formation may also prove useful in the development of vaccine strains of this pathogenic apicomplexan. The significant recent advances in our understanding of the sexual cycle defined by in depth transcriptomic analyses and the development of both cell culture-based and nonfeline (mouse) animal-based systems to recapitulate the sexual cycle addresses one of the primary impediments to the study of sexual cycle of this parasite. The murine sexual cycle model not only established the key signals that defined the host range restriction but also has more broadly opened the gates to a

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broader dissection of this critical life cycle stage. The implications of this ground breaking work will be felt not only for T. gondii, but for all oocyst-forming Coccidia where sophisticated molecular genetic approaches may not be as well developed. This is an exciting time to be working on aspects of parasite biology that were largely experimentally intractable, though clinically important. Understanding the biology of these life cycle stages offers opportunities for discoveries that will impact the development of novel therapeutic approaches for chronic toxoplasmosis.

Acknowledgments This work was supported by NIH grants AI134753 and AI123495 (LMW), AI122894 (APS), AI144016 (LJK). We thank the members of our laboratories for their dedication and our colleagues in the Toxoplasma community for stimulating discussions and sharing unpublished data.

References Abdelbaset, E.A., Fox, B.A., Karram, M.H., Abd ellah, M.R., Bzik, D.J., Igarashi, M., 2017. Lactate dehydrogenase in Toxoplasma gondii controls virulence, bradyzoite differentiation, and chronic infection. PLoS One 12. Available from: https://doi.org/10.1371/journal.pone.0173745. Ajioka, J.W., 1998. Toxoplasma gondii: ESTs and gene discovery. Int. J. Parasitol. 28, 1025
1031. Ajioka, J.W., Boothroyd, J.C., Brunk, B.P., Hehl, A., Hillier, L., Manger, I.D., et al., 1998. Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa. Genome Res. 8, 18
28. Al-Anouti, F., Tomavo, S., Parmley, S., Ananvoranich, S., 2004. The expression of lactate dehydrogenase is important for the cell cycle of Toxoplasma gondii. J. Biol. Chem. 279, 52300
52311. Alday, P.H., Doggett, J.S., 2017. Drugs in development for toxoplasmosis: advances, challenges, and current status. Drug Des. Dev. Ther. 11, 273
293. Alvarez, C.A., Suvorova, E.S., 2017. Checkpoints of apicomplexan cell division identified in Toxoplasma gondii. PLoS Pathog. 13, e1006483. Anderson, M.Z., Brewer, J., Singh, U., Boothroyd, J.C., 2009. A pseudouridine synthase homologue is critical to

Toxoplasma Gondii

846

18. Bradyzoite and sexual stage development

cellular differentiation in Toxoplasma gondii. Eukaryot. Cell 8, 398
409. Anderson-White, B.R., Ivey, F.D., Cheng, K., Szatanek, T., Lorestani, A., Beckers, C.J., et al., 2011. A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii. Cell. Microbiol. 13, 18
31. Aramini, J.J., Stephen, C., Dubey, J.P., Engelstoft, C., Schwantje, H., Ribble, C.S., 1999. Potential contamination of drinking water with Toxoplasma gondii oocysts. Epidemiol. Infect. 122, 305
315. Ashwinder, K., Kho, M.T., Chee, P.M., Lim, W.Z., Yap, I.K.S., Choi, S.B., et al., 2016. Targeting heat shock proteins 60 and 70 of Toxoplasma gondii as a potential drug target: in silico approach. Interdiscip. Sci.—Comput. Life Sci. 8, 374
387. Balaji, S., Babu, M.M., Iyer, L.M., Aravind, L., 2005. Discovery of the principal specific transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains. Nucleic Acids Res. 33, 3994
4006. Behnke, M.S., Radke, J.B., Smith, A.T., Sullivan JR., W.J., White, M.W., 2008. The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements. Mol. Microbiol. 68, 1502
1518. Behnke, M.S., Wootton, J.C., Lehmann, M.M., Radke, J.B., Lucas, O., Nawas, J., et al., 2010. Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5, e12354. Behnke, M.S., Zhang, T.P., Dubey, J.P., Sibley, L.D., 2014. Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development. BMC Genomics 15, 350. Behnke, M.S., Dubey, J.P., Sibley, L.D., 2016. Genetic mapping of pathogenesis determinants in Toxoplasma gondii. Ann. Rev. Microbiol. 70, 63
81. Belli, S.I., Wallach, M.G., Luxford, C., Davies, M.J., Smith, N.C., 2003. Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalaninemediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima. Eukaryot. Cell 2, 456
464. Bermudes, D., Peck, K.R., Afifi, M.A., Beckers, C.J.M., Joiner, K.A., 1994. Tandemly repeated genes encode nucleoside triphosphate hydrolase isoforms secreted into the parasitophorous vacuole of Toxoplasma gondii. J. Biol. Chem. 269, 29252
29260. Blewett, D.A., Miller, J.K., Harding, J., 1983. Simple technique for the direct isolation of toxoplasma tissue cysts from fetal ovine brain. Vet. Rec. 112, 98
100.

Bohne, W., Roos, D.S., 1997. Stage-specific expression of a selectable marker in Toxoplasma gondii permits selective inhibition of either tachyzoites of bradyzoites. Mol. Biochem. Parasitol. 88, 115
126. Bohne, W., Heesemann, J., Gross, U., 1993. Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages. Infect. Immun. 61, 1141
1145. Bohne, W., Heesemann, J., Gross, U., 1994. Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion. Infect. Immun. 62, 1761
1767. Bohne, W., Gross, U., Ferguson, D.J., Heesemann, J., 1995. Cloning and characterization of a bradyzoitespecifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants. Mol. Microbiol. 16, 1221
1230. Bohne, W., Wirsing, A., Gross, U., 1997. Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements. Mol. Biochem. Parasitol. 85, 89
98. Bohne, W., Hunter, C.A., White, M.W., Ferguson, D.J., Gross, U., Roos, D.S., 1998. Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii. Mol. Biochem. Parasitol. 92, 291
301. Boothroyd, J.C., Black, M., Bonnefoy, S., Hehl, A., Knoll, L. J., Manger, I.D., et al., 1997. Genetic and biochemical analysis of development in Toxoplasma gondii. Philos. Trans. R. Soc. London, Ser. B: Biol. Sci. 352, 1347
1354. Boothroyd, J.C., Hehl, A., Knoll, L.J., Manger, I.D., 1998. The surface of Toxoplasma: more and less. Int. J. Parasitol. 28, 3
9. Bougdour, A., Maubon, D., Baldacci, P., Ortet, P., Bastien, O., Bouillon, A., et al., 2009. Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites. J. Exp. Med. 206, 953
966. Braun, L., Cannella, D., Pinheiro, A.M., Kieffer, S., Belrhali, H., Garin, J., et al., 2009. The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii. Int. J. Parasitol. 39, 81
90. Braun, L., Cannella, D., Ortet, P., Barakat, M., Sautel, C.F., Kieffer, S., et al., 2010. A complex small RNA repertoire is generated by a plant/fungal-like machinery and effected by a metazoan-like argonaute in the single-cell human parasite Toxoplasma gondii. PLoS Pathog. 6, e1000920. Brown, C.R., Mcleod, R., 1990. Class I MHC genes and CD8 1 T cells determine cyst number in Toxoplasma gondii infection. J. Immunol. 145, 3438
3441. Brown, C.R., David, C.S., Khare, S.J., Mcleod, R., 1994. Effects of human class I transgenes on Toxoplasma gondii cyst formation. J. Immunol. 152, 4537
4541.

Toxoplasma Gondii

References

Brown, C.R., Hunter, C.A., Estes, R.G., Beckmann, E., Forman, J., David, C., et al., 1995. Definitive identification of a gene that confers resistance against Toxoplasma cyst burden and encephalitis. Immunology 85, 419
428. Brown, S.H., Zarnowski, R., Sharpee, W.C., Keller, N.P., 2008. Morphological transitions governed by density dependence and lipoxygenase activity in Aspergillus flavus. Appl. Environ. Microbiol 74, 5674
5685. Brumlik, M.J., Wei, S., Finstad, K., Nesbit, J., Hyman, L.E., Lacey, M., et al., 2004. Identification of a novel mitogenactivated protein kinase in Toxoplasma gondii. Int. J. Parasitol. 34, 1245
1254. Buchholz, K.R., Fritz, H.M., Chen, X., Durbin-Johnson, B., Rocke, D.M., Ferguson, D.J., et al., 2011. Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii bradyzoites. Eukaryot. Cell 10, 1637
1647. Buchholz, K.R., Bowyer, P.W., Boothroyd, J.C., 2013. Bradyzoite pseudokinase 1 is crucial for efficient oral infectivity of the Toxoplasma gondii tissue cyst. Eukaryot. Cell. 12 (3), 399
410. Available from: https://doi.org/ 10.1128/EC.00343-12. Burg, J.L., Perelman, D., Kasper, L.H., Ware, P.L., Boothroyd, J.C., 1988. Molecular analysis of the gene encoding the major surface-antigen of Toxoplasma gondii. J. Immunol. 141, 3584
3591. Cabral, C.M., Tuladhar, S., Dietrich, H.K., Nguyen, E., Macdonald, W.R., Trivedi, T., et al., 2016. Neurons are the primary target cell for the brain-tropic intracellular parasite Toxoplasma gondii. PLoS Pathog. 12, e1005447. Caffaro, C.E., Koshy, A.A., Liu, L., Zeiner, G.M., Hirschberg, C.B., Boothroyd, J.C., 2013. A nucleotide sugar transporter involved in glycosylation of the toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites. PLoS Pathog. 9, e1003331. Cao, L., Wang, Z., Wang, S., Li, J., Wang, X., Wei, F., et al., 2016. Deletion of mitogen-activated protein kinase 1 inhibits development and growth of Toxoplasma gondii. Parasitol. Res. 115, 797
805. Cesbron-Delauw, M.F., 1994. Dense-granule organelles of Toxoplasma gondii—their role in the host-parasite relationship. Parasitol. Today 10, 293
296. Cherry, A.A., Ananvoranich, S., 2014. Characterization of a homolog of DEAD-box RNA helicases in Toxoplasma gondii as a marker of cytoplasmic mRNP stress granules. Gene 543, 34
44. Cleary, M.D., Singh, U., Blader, I.J., Brewer, J.L., Boothroyd, J.C., 2002. Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryot. Cell 1, 329
340.

847

Coppin, A., Dzierszinski, F., Legrand, S., Mortuaire, M., Ferguson, D., Tomavo, S., 2003. Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii. Biochimie 85, 353
361. Coppin, A., Varre, J.S., Lienard, L., Dauvillee, D., Guerardel, Y., Soyer-Gobillard, M.O., et al., 2005. Evolution of plant-like crystalline storage polysaccharide in the protozoan parasite Toxoplasma gondii argues for a red alga ancestry. J. Mol. Evol. 60, 257
267. Cornelissen, A.W., Overdulve, J.P., Hoenderboom, J.M., 1981. Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density-gradient centrifugation. Parasitology 83, 103
108. Craver, M.P.J., Rooney, P.J., Knoll, L.J., 2010. Isolation of Toxoplasma gondii development mutants identifies a potential proteophosphogylcan that enhances cyst wall formation. Mol. Biochem. Parasitol. 169, 120
123. Crawford, J., Grujic, O., Bruic, E., Czjzek, M., Grigg, M.E., Boulanger, M.J., 2009. Structural characterization of the bradyzoite surface antigen (BSR4) from Toxoplasma gondii, a unique addition to the surface antigen glycoprotein 1-related superfamily. J. Biol. Chem. 284, 9192
9198. Crawford, J., Lamb, E., Wasmuth, J., Grujic, O., Grigg, M.E., Boulanger, M.J., 2010. Structural and functional characterization of SporoSAG: a SAG2-related surface antigen from Toxoplasma gondii. J. Biol. Chem. 285, 12063
12070. Croken, M.M., Ma, Y., Markillie, L.M., Taylor, R.C., Orr, G., Weiss, L.M., et al., 2014a. Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage. PLoS One 9, e111297. Croken, M.M., Qiu, W., White, M.W., Kim, K., 2014b. Gene Set Enrichment Analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program. BMC Genomics 15, 515. Dalmasso, M.C., Onyango, D.O., Naguleswaran, A., Sullivan JR., W.J., Angel, S.O., 2009. Toxoplasma H2A variants reveal novel insights into nucleosome composition and functions for this histone family. J. Mol. Biol. 392, 33
47. Dando, C., Schroeder, E.R., Hunsaker, L.A., Deck, L.M., Royer, R.E., Zhou, X.L., et al., 2001. The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum. Mol. Biochem. Parasitol. 118, 23
32. Das, U.N., 2006. Essential fatty acids: biochemistry, physiology and pathology. Biotechnol. J. 1, 420
439.

Toxoplasma Gondii

848

18. Bradyzoite and sexual stage development

De champs, C., Imbert-Bernard, C., Belmeguenai, A., Ricard, J., Pelloux, H., Brambilla, E., et al., 1997. Toxoplasma gondii: in vivo and in vitro cystogenesis of the virulent RH strain. J. Parasitol. 83, 152
155. De falco, M., Cobellis, G., De luca, A., 2009. Proliferation of cardiomyocytes: a question unresolved. Front. Biosci. (Elite Ed) 1, 528
536. De miguel, N., Echeverria, P.C., Angel, S.O. Characterization and stage-specific expression analysis of members of Toxoplasma gondii alpha-crystallin/sHsp family. In: Eight International Congress on Toxoplasmosis, 2005, p. 85. Denton, H., Roberts, C.W., Alexander, J., Thong, K.W., Coombs, G.H., 1996. Enzymes of energy metabolism in the bradyzoites and tachyzoites of Toxoplasma gondii. FEMS Microbiol. Lett. 137, 103
108. Donald, R.G., Roos, D.S., 1993. Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drugresistance mutations in malaria. Proc. Natl. Acad. Sci. U.S.A. 90, 11703
11707. Donald, R.G., Liberator, P.A., 2002. Molecular characterization of a coccidian parasite cGMP dependent protein kinase. Mol. Biochem. Parasitol. 120, 165
175. Donald, R.G., Allocco, J., Singh, S.B., Nare, B., Salowe, S.P., Wiltsie, J., et al., 2002. Toxoplasma gondii cyclic GMPdependent kinase: chemotherapeutic targeting of an essential parasite protein kinase. Eukaryot. Cell 1, 317
328. Dubey, J.P., 1997. Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii [published erratum appears in 1998. J. Eukaryot. Microbiol. 45 (3), 367] J. Eukaryot. Microbiol. 44, 592
602. Dubey, J.P., 1998a. Advances in the life cycle of Toxoplasma gondii. Int. J. Parasitol. 28, 1019
1024. Dubey, J.P., 1998b. Re-examination of resistance of Toxoplasma gondii tachyzoites and bradyzoites to pepsin and trypsin digestion. Parasitology 116 (Pt 1), 43
50. Dubey, J.P., Frenkel, J.K., 1972a. Cyst-induced toxoplasmosis in cats. J. Protozool. 19, 155
177. Dubey, J.P., Frenkel, J.K., 1972b. Extra-intestinal stages of Isospora felis and I. rivolta (Protozoa: Eimeriidae) in cats. J. Protozool. 19, 89
92. Dubey, J.P., Speer, C.A., Shen, S.K., Kwok, O.C., Blixt, J.A., 1997. Oocyst-induced murine toxoplasmosis: life cycle, pathogenicity, and stage conversion in mice fed Toxoplasma gondii oocysts. J. Parasitol. 83, 870
882. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267
299.

Dumetre, A., Darde, M.L., 2005. Immunomagnetic separation of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall. J. Microbiol. Methods 61, 209
217. Dzierszinski, F., Popescu, O., Toursel, C., Slomianny, C., Yahiaoui, B., Tomavo, S., 1999. The protozoan parasite Toxoplasma gondii expresses two functional plant-like glycolytic enzymes. Implications for evolutionary origin of apicomplexans. J. Biol. Chem. 274, 24888
24895. Dzierszinski, F., Mortuaire, M., Cesbron-Delauw, M.F., Tomavo, S., 2000. Targeted disruption of the glycosylphosphatidylinositol-anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice. Mol. Microbiol. 37, 574
582. Dzierszinski, F., Mortuaire, M., Dendouga, N., Popescu, O., Tomavo, S., 2001. Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii. J. Mol. Biol. 309, 1017
1027. Dzierszinski, F., Nishi, M., Ouko, L., Roos, D.S., 2004. Dynamics of Toxoplasma gondii differentiation. Eukaryot. Cell 3, 992
1003. Eaton, M.S., Weiss, L.M., Kim, K., 2005. Cyclic nucleotide kinases and tachyzoite-bradyzoite transition in Toxoplasma gondii. Int. J. Parasitol. Available from: https://doi.org/10.1016/j.ijpara.2005.08.014. Echeverria, P.C., Matrajt, M., Harb, O.S., Zappia, M.P., Costas, M.A., Roos, D.S., et al., 2005. Toxoplasma gondii Hsp90 is a potential drug target whose expression and subcellular localization are developmentally regulated. J. Mol. Biol. 350, 723
734. Echeverria, P.C., Figueras, M.J., Vogler, M., Kriehuber, T., De miguel, N., Deng, B., et al., 2010. The Hsp90 cochaperone p23 of Toxoplasma gondii: Identification, functional analysis and dynamic interactome determination. Mol. Biochem. Parasitol. 172, 129
140. Estruch, F., 2000. Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol. Rev. 24, 469
486. Fagard, R., Van tan, H., Creuzet, C., Pelloux, H., 1999. Differential development of toxoplasma gondii in neural cells. Parasitol. Today 15, 504
507. Ferguson, D.J., 2004. Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. Int. J. Parasitol. 34, 347
360. Ferguson, D.J.P., Hutchison, W.M., 1987a. The host-parasite relationship of Toxoplasma gondii in the brains of chronically infected mice. Virchows Arch. A 411, 39
43. Ferguson, D.J.P., Hutchison, W.M., 1987b. An ultrastructural study of the early development and tissue cyst

Toxoplasma Gondii

References

formation of Toxoplasma gondii in the brains of mice. Parasitol. Res. 73, 483
491. Ferguson, D.J., Parmley, S.F., 2002. Toxoplasma gondii MAG1 protein expression. Trends Parasitol. 18, 482. Ferguson, D.J.P., Huchiso, W.M., Pettersen, E., 1989. Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study. Parasitol. Res. 73, 599
603. Ferguson, D.J., Huskinson-Mark, J., Araujo, F.G., Remington, J.S., 1994a. A morphological study of chronic cerebral toxoplasmosis in mice: comparison of four different strains of Toxoplasma gondii. Parasitol. Res. 80, 493
501. Ferguson, D.J., Huskinson-Mark, J., Araujo, F.G., Remington, J.S., 1994b. An ultrastructural study of the effect of treatment with atovaquone in brains of mice chronically infected with the ME49 strain of Toxoplasma gondii. Int. J. Exp. Pathol. 75, 111
116. Ferguson, D.J.P., Cesbron-Delauw, M.F., Dubremetz, J.E., Sibley, L.D., Joiner, K.A., Wright, S., 1999. The expression and distribution of dense granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Exp. Parasitol. 91, 203
211. Ferguson, D.J., Parmley, S.F., Tomavo, S., 2002a. Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication. Int. J. Parasitol. 32, 1399
1410. Ferguson, D.J.P., Parmley, S.F., Tomavo, S., 2002b. Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication. Int. J. Parasitol. 32, 1399
1410. Fortier, B., Coignard-Chatain, C., Soete, M., Dubremetz, J.F., 1996. [Structure and biology of Toxoplasma gondii bradyzoites]. C.R. Seances Soc. Biol. Fil. 190, 385
394. Fox, B.A., Bzik, D.J., 2002. De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii. Nature 415, 926
929. Fox, B.A., Gigley, J.P., Bzik, D.J., 2004. Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation. Int. J. Parasitol. 34, 323
331. Fox, B.A., Falla, A., Rommereim, L.M., Tomita, T., Gigley, J.P., Mercier, C., et al., 2011. Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryot. Cell 10, 1193
1206. Fox, B.A., Rommereim, L.M., Guevara, R.B., Falla, A., Triana, M.A.H., Sun, Y., et al., 2016. The Toxoplasma gondii rhoptry kinome is essential for chronic infection. mBio 7. Available from: https://doi.org/10.1128/ mBio.00193-16.

849

Francia, M.E., Striepen, B., 2014. Cell division in apicomplexan parasites. Nat. Rev. Microbiol. 12, 125
136. Frankel, M.B., Mordue, D.G., Knoll, L.J., 2007. Discovery of parasite virulence genes reveals a unique regulator of chromosome condensation 1 ortholog critical for efficient nuclear trafficking. Proc. Natl. Acad. Sci. U.S.A. 104, 10181
10186. Frenkel, J.K., Dubey, J.P., Hoff, R.L., 1976. Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni. J. Protozool. 23, 421
424. Freyre, A., 1995. Separation of toxoplasma cysts from brain tissue and liberation of viable bradyzoites. J. Parasitol. 81, 1008
1010. Friesen, J., Fleige, T., Gross, U., Bohne, W., 2008. Identification of novel bradyzoite-specific Toxoplasma gondii genes with domains for protein-protein interactions by suppression subtractive hybridization. Mol. Biochem. Parasitol. 157, 228
232. Fritz, H., Barr, B., Packham, A., Melli, A., Conrad, P.A., 2012a. Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts. J. Microbiol. Methods 88, 47
52. Fritz, H.M., Buchholz, K.R., Chen, X., Durbin-Johnson, B., Rocke, D.M., Conrad, P.A., et al., 2012b. Transcriptomic analysis of toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts. PLoS One 7, e29998. Fux, B., Nawas, J., Khan, A., Gill, D.B., Su, C., Sibley, L.D., 2007. Toxoplasma gondii strains defective in oral transmission are also defective in developmental stage differentiation. Infect. Immun. 75, 2580
2590. Galizi, R., Spano, F., Giubilei, M.A., Capuccini, B., Magini, A., Urbanelli, L., et al., 2013. Evidence of tRNA cleavage in apicomplexan parasites: half-tRNAs as new potential regulatory molecules of Toxoplasma gondii and Plasmodium berghei. Mol. Biochem. Parasitol. 188, 99
108. Gissot, M., Walker, R., Delhaye, S., Alayi, T.D., Huot, L., Hot, D., et al., 2013. Toxoplasma gondii alba proteins are involved in translational control of gene expression. J. Mol. Biol. 425, 1287
1301. Gomes-Santos, C.S.S., Braks, J., Prudencio, M., Carret, C., Gomes, A.R., Pain, A., et al., 2011. Transition of plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio. PLoS Pathog. 7, e1002046. Gross, U., Bormuth, H., Gaissmaier, C., Dittrich, C., Krenn, V., Bohne, W., et al., 1995. Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo. Clin. Diagn. Lab. Immunol. 2, 542
548. Gross, U., Bohne, W., Luder, C.G., Lugert, R., Seeber, F., Dittrich, C., et al., 1996. Regulation of developmental

Toxoplasma Gondii

850

18. Bradyzoite and sexual stage development

differentiation in the protozoan parasite Toxoplasma gondii. J. Eukaryot. Microbiol. 43, 114S
116S. Gubbels, M.J., Lehmann, M., Muthalagi, M., Jerome, M.E., Brooks, C.F., Szatanek, T., et al., 2008. Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii. PLoS Pathog. 4, e36. Guerardel, Y., Leleu, D., Coppin, A., Lienard, L., Slomianny, C., Strecker, G., et al., 2005. Amylopectin biogenesis and characterization in the protozoan parasite Toxoplasma gondii, the intracellular development of which is restricted in the HepG2 cell line. Microbes Infect. 7, 41
48. Guimaraes, E.V., De carvalho, L., Barbosa, H.S., 2003. An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts. Mem. Inst. Oswaldo Cruz 98, 915
917. Gurnett, A.M., Liberator, P.A., Dulski, P.M., Salowe, S.P., Donald, R.G., Anderson, J.W., et al., 2002. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J. Biol. Chem. 277, 15913
15922. Halonen, S.K., Lyman, W.D., Chiu, F.C., 1996. Growth and development of Toxoplasma gondii in human neurons and astrocytes. J. Neuropathol. Exp. Neurol. 55, 1150
1156. Halonen, S.K., Chiu, F., Weiss, L.M., 1998a. Effect of cytokines on growth of Toxoplasma gondii in murine astrocytes. Infect. Immun. 66, 4989
4993. Halonen, S.K., Weiss, L.M., Chiu, F.C., 1998b. Association of host cell intermediate filaments with Toxoplasma gondii cysts in murine astrocytes in vitro. Int. J. Parasitol. 28, 815
823. Hammoudi, P.-M., Jacot, D., Mueller, C., Di cristina, M., Dogga, S.K., Marq, J.-B., et al., 2015. Fundamental roles of the golgi-associated toxoplasma aspartyl protease, ASP5, at the host-parasite interface. PLoS Pathog. 11, e1005211. Han, B., Tomita, T., Yakubu, R., Tu, V., Mayoral, J., Sugi, T., et al., 2019. MAG1, a Toxoplasma gondii matrix protein involved in host cell responses. mSphere submitted. Hartmann, A., Arroyo-Olarte, R.D., Imkeller, K., Hegemann, P., Lucius, R., Gupta, N., 2013. Optogenetic modulation of an adenylate cyclase in Toxoplasma gondii demonstrates a requirement of the parasite cAMP for host-cell invasion and stage differentiation. J. Biol. Chem. 288, 13705
13717. He, X.L., Grigg, M.E., Boothroyd, J.C., Garcia, K.C., 2002. Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily. Nat. Struct. Biol. 9, 606
611. He, C., Qu, X., Wan, J., Rong, R., Huang, L., Cai, C., et al., 2012. Inhibiting delta-6 desaturase activity suppresses tumor growth in mice. PLoS One 7, e47567.

Hehl, A.B., Basso, W.U., Lippuner, C., Ramakrishnan, C., Okoniewski, M., Walker, R.A., et al., 2015. Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes. BMC Genomics 16, 66. Heikkila, J.J., 1993a. Heat shock gene expression and development. I. An overview of fungal, plant, and poikilothermic animal developmental systems. Dev. Genet. 14, 1
5. Heikkila, J.J., 1993b. Heat shock gene expression and development. II. An overview of mammalian and avian developmental systems. Dev. Genet. 14, 87
91. Hettmann, C., Soldati, D., 1999. Cloning and analysis of a Toxoplasma gondii histone acetyltransferase: a novel chromatin remodelling factor in Apicomplexan parasites. Nucleic Acids Res. 27, 4344
4352. Hinnebusch, A.G., 1997. Translational regulation of yeast GCN4
a window on factors that control initiatortRNA binding to the ribosome. J. Biol. Chem. 272, 21661
21664. Hinnebusch, A.G., Natarajan, K., 2002. Gcn4p, a master regulator of gene expression, is controlled at multiple levels by diverse signals of starvation and stress. Eukaryot. Cell 1, 22
32. Hoff, R.L., Dubey, J.P., Behbehani, A.M., Frenkel, J.K., 1977. Toxoplasma gondii cysts in cell culture: new biologic evidence. J. Parasitol. 63, 1121
1124. Hogan, M.J., Yoneda, C., Feeney, L., Zweigart, P., Lewis, A., 1960. Morphology and culture of Toxoplasma. Arch Ophthalmol 64, 655
667. Holmes, M., Itaas, V., Ananvoranich, S., 2014. Sustained translational repression of lactate dehydrogenase 1 in Toxoplasma gondii bradyzoites is conferred by a small regulatory RNA hairpin. Febs Journal 281, 5077
5091. Holmes, M.J., Augusto, L.D.S., Zhang, M., Wek, R.C., Sullivan JR., W.J., 2017. Translational control in the latency of apicomplexan parasites. Trends Parasitol. 33, 947
960. Holpert, M., Luder, C.G., Gross, U., Bohne, W., 2001. Bradyzoite-specific expression of a P-type ATPase in Toxoplasma gondii. Mol. Biochem. Parasitol. 112, 293
296. Holpert, M., Gross, U., Bohne, W., 2006. Disruption of the bradyzoite-specific P-type (H 1 )-ATPase PMA1 in Toxoplasma gondii leads to decreased bradyzoite differentiation after stress stimuli but does not interfere with mature tissue cyst formation. Mol. Biochem. Parasitol. 146, 129
133. Hong, D.-P., Radke, J.B., White, M.W., 2017. Opposing transcriptional mechanisms regulate toxoplasma development. Msphere 2, e00347-16.

Toxoplasma Gondii

References

Huang, S., Holmes, M.J., Radke, J.B., Hong, D.-P., Liu, T.K., White, M.W., et al., 2017. Toxoplasma gondii AP2IX-4 regulates gene expression during bradyzoite development. Msphere 2, e00054-17. Huskinson-Mark, J., Araujo, F.G., Remington, J.S., 1991. Evaluation of the effect of drugs on the cyst form of Toxoplasma gondii. J. Infect. Dis. 164, 170
171. Hutson, S.L., Mui, E., Kinsley, K., Witola, W.H., Behnke, M. S., El bissati, K., et al., 2010. T. gondii RP promoters & knockdown reveal molecular pathways associated with proliferation and cell-cycle arrest. PLoS One 5, e14057. Iida, H., Yahara, I., 1985. Yeast heat shock protein of Mr 48,000 is an isomer of enolase. Nature 315, 688
690. Jacobs, L., Remington, J., 1960. The resistance of the encysted form of Toxoplasma gondii. J. Parasitol. 46, 11
21. Jeffers, V., Tampaki, Z., Kim, K., Sullivan JR., W.J., 2018. A latent ability to persist: differentiation in Toxoplasma gondii. Cell Mol. Life Sci. 75, 2355
2373. Jerome, M.E., Radke, J.R., Bohne, W., Roos, D.S., White, M. W., 1998. Toxoplasma gondii bradyzoites form spontaneously during sporozoite-initiated development. Infect. Immun. 66, 4838
4844. Jones, T.C., Bienz, K.A., Erb, P., 1986. In vitro cultivation of Toxoplasma gondii cysts in astrocytes in the presence of gamma interferon. Infect. Immun. 51, 147
156. Jones, N.G., Wang, Q., Sibley, L.D., 2016. Secreted protein kinases regulate cyst burden during chronic toxoplasmosis. Cell. Microbiol. 19. Available from: https://doi. org/10.1111/cmi.12651. Joyce, B.R., Tampaki, Z., Kim, K., Wek, R.C., Sullivan JR., W.J., 2013. The unfolded protein response in the protozoan parasite Toxoplasma gondii features translational and transcriptional control. Eukaryot. Cell 12, 979
989. Jung, C., Lee, C.Y., Grigg, M.E., 2004. The SRS superfamily of Toxoplasma surface proteins. Int. J. Parasitol. 34, 285
296. Khan, F., Tang, J., Qin, C.L., Kim, K., 2002. Cyclindependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii. Mol. Microbiol. 45, 321
332. Kibe, M.K., Coppin, A., Dendouga, N., Oria, G., Meurice, E., Mortuaire, M., et al., 2005. Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii. Nucleic Acids Res. 33, 1722
1736. Kim, K., 2018. The epigenome, cell cycle, and development in Toxoplasma. Annu Rev Microbiol 72, 479
499. Kim, S.K., Boothroyd, J.C., 2005. Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence. J. Immunol. 174, 8038
8048. Kim, S.K., Karasov, A., Boothroyd, J.C., 2007. Bradyzoitespecific surface antigen SRS9 plays a role in maintaining

851

Toxoplasma gondii persistence in the brain and in host control of parasite replication in the intestine. Infect. Immun. 75, 1626
1634. Kimball, A.C., Kean, B.H., Fuchs, F., 1974. Toxoplasmosis: risk variations in New York city obstetric patients. Am. J. Obstet. Gynecol 119, 208
214. Kirkman, L.A., Weiss, L.M., Kim, K., 2001. Cyclic nucleotide signaling in Toxoplasma gondii bradyzoite differentiation. Infect. Immun. 69, 148
153. Knoll, L.J., Boothroyd, J.C., 1998. Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase. Mol. Cell. Biol. 18, 807
814. Knoll, L.J., Furie, G.L., Boothroyd, J.C., 2001. Adaptation of signature-tagged mutagenesis for Toxoplasma gondii: a negative screening strategy to isolate genes that are essential in restrictive growth conditions. Mol. Biochem. Parasitol. 116, 11
16. Kobayashi, T., Narabu, S., Yanai, Y., Hatano, Y., Ito, A., Imai, S., et al., 2012. Gene cloning and characterization of the protein encoded by the Neospora Caninum bradyzoite-specific antigen gene Bag1. J. Parasitol. Available from: https://doi.org/10.1645/12-65.1. Konrad, C., Wek, R.C., Sullivan JR., W.J., 2011. A GCN2like eukaryotic initiation factor 2 kinase increases the viability of extracellular toxoplasma gondii parasites. Eukaryot. Cell 10, 1403
1412. Konrad, C., Queener, S.F., Wek, R.C., Sullivan JR., W.J., 2013. Inhibitors of eIF2 alpha dephosphorylation slow replication and stabilize latency in Toxoplasma gondii. Antimicrob. Agents Chemother. 57, 1815
1822. Koshy, A.A., Cabral, C.M., 2014. 3-D imaging and analysis of neurons infected in vivo with Toxoplasma gondii. J Vis Exp . Available from: https://doi.org/10.3791/52237. Lane, A., Soete, M., Dubremetz, J.F., Smith, J.E., 1996. Toxoplasma gondii: appearance of specific markers during the development of tissue cysts in vitro. Parasitol. Res. 82, 340
346. Lecordier, L., Mercier, C., Torpier, G., Tourvieille, B., Darcy, F., Liu, J.L., et al., 1993. Molecular structure of a Toxoplasma gondii dense granule antigen (GRA 5) associated with the parasitophorous vacuole membrane. Mol. Biochem. Parasitol. 59, 143
153. Lei, Y., Davey, M., Ellis, J., 2005. Autofluorescence of Toxoplasma gondii and Neospora caninum cysts in vitro. J. Parasitol. 91, 17
23. Lekutis, C., Ferguson, D.J.P., Boothroyd, J.C., 2000. Toxoplasma gondii: identification of a developmentally regulated family of genes related to SAG2. Exp. Parasitol. 96, 89
96. Lekutis, C., Ferguson, D.J.P., Grigg, M.E., Camps, M., Boothroyd, J.C., 2001. Surface antigens of Toxoplasma

Toxoplasma Gondii

852

18. Bradyzoite and sexual stage development

gondii: variations on a theme. Int. J. Parasitol. 31, 1285
1292. Lemgruber, L., Lupetti, P., De souza, W., Vommaro, R.C., 2011a. New details on the fine structure of the rhoptry of Toxoplasma gondii. Microsc. Res. Tech. 74, 812
818. Lemgruber, L., Lupetti, P., Martins-Duarte, E.S., De souza, W., Vommaro, R.C., 2011b. The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form. Cell. Microbiol. 13, 1920
1932. Lescault, P.J., Thompson, A.B., Patil, V., Lirussi, D., Burton, A., Margarit, J., et al., 2010. Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state. PLoS One 5, e14463. Lindquist, H.D., Bennett, J.W., Hester, J.D., Ware, M.W., Dubey, J.P., Everson, W.V., 2003. Autofluorescence of Toxoplasma gondii and related coccidian oocysts. J. Parasitol. 89, 865
867. Lindsay, D.S., Dubey, J.P., Blagburn, B.L., Toivio-Kinnucan, M., 1991. Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites. J. Parasitol. 77, 126
132. Lindsay, D.S., Mitschler, R.R., Toivio-Kinnucan, M.A., Upton, S.J., Dubey, J.P., Blagburn, B.L., 1993a. Association of host cell mitochondria with developing Toxoplasma gondii tissue cysts. Am. J. Vet. Res. 54, 1663
1667. Lindsay, D.S., Toivio-Kinnucan, M.A., Blagburn, B.L., 1993b. Ultrastructural determination of cystogenesis by various Toxoplasma gondii isolates in cell culture. J. Parasitol. 79, 289
292. Liu, M., Miao, J., Liu, T., Sullivan JR., W.J., Cui, L., Chen, X., 2014. Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii. Parasites Vectors 7, 141. Liwak, U., Ananvoranich, S., 2009. Toxoplasma gondii: overexpression of lactate dehydrogenase enhances differentiation under alkaline conditions. Exp. Parasitol. 122, 155
161. Luder, C.G.K., Rahman, T., 2017. Impact of the host on Toxoplasma stage differentiation. Microb. Cell 4, 203
211. Luft, B.J., Remington, J.S., 1992. Toxoplasmic encephalitis in AIDS. Clin. Infect. Dis. 15, 211
222. Lunghi, M., Galizi, R., Magini, A., Carruthers, V.B., Di cristina, M., 2015. Expression of the glycolytic enzymes enolase and lactate dehydrogenase during the early phase of Toxoplasma differentiation is regulated by an intron retention mechanism. Mol. Microbiol. 96, 1159
1175. Lyons, R.E., Johnson, A.M., 1995. Heat shock proteins of Toxoplasma gondii. Parasite Immunol. 17, 353
359.

Lyons, R.E., Johnson, A.M., 1998. Gene sequence and transcription differences in 70 kDa heat shock protein correlate with murine virulence of Toxoplasma gondii. Int. J. Parasitol. 28, 1041
1051. Ma, Y.F., Zhang, Y., Kim, K., Weiss, L.M., 2004. Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus. Int. J. Parasitol. 34, 333
346. Maeda, T., Saito, T., Oguchi, Y., Nakazawa, M., Takeuchi, T., Asai, T., 2003. Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites. Parasitol. Res. 89, 259
265. Mahamed, D.A., Mills, J.H., Egan, C.E., Denkers, E.Y., Bynoe, M.S., 2012. CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system. Proc. Natl. Acad. Sci. U.S.A. 109, 16312
16317. Manger, I.D., Hehl, A., Parmley, S., Sibley, L.D., Marra, M., Hillier, L., et al., 1998a. Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: identification of developmentally regulated genes. Infect. Immun. 66, 1632
1637. Manger, I.D., Hehl, A.B., Boothroyd, J.C., 1998b. The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1. Infect. Immun. 66, 2237
2244. Martorelli Di Genova, B., Wilson, S.K., Dubey, J.P. & Knoll, L.J. 2019. Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction. PLOS Biology 17(8), e3000364. https://doi.org/10.1371/ journal.pbio.3000364. Matrajt, M., Donald, R.G., Singh, U., Roos, D.S., 2002. Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii. Mol. Microbiol. 44, 735
747. Matsukayashi, H., Akao, S., 1963. Morphological studies on the development of the toxoplasma cyst. Am. J. Trop. Med. Hyg 12, 321
333. Maubon, D., Bougdour, A., Wong, Y.S., Brenier-Pinchart, M.P., Curt, A., Hakimi, M.A., et al., 2010. Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form and study of new derivative compounds. Antimicrob. Agents Chemother. 54, 4843
4850. Mcauley, J., Boyer, K.M., Patel, D., Mets, M., Swisher, C., Roizen, N., et al., 1994. Early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis: the Chicago Collaborative Treatment Trial. Clin. Infect. Dis. 18, 38
72. Mchugh, T.D., Gbewonyo, A., Johnson, J.D., Holliman, R. E., Butcher, P.D., 1993. Development of an in vitro

Toxoplasma Gondii

References

model of Toxoplasma gondii cyst formation. FEMS Microbiol. Lett. 114, 325
332. Mcleod, R., Skamene, E., Brown, C.R., Eisenhauer, P.B., Mack, D.G., 1989. Genetic regulation of early survival and cyst number after peroral Toxoplasma gondii infection of A x B/B x A recombinant inbred and B10 congenic mice. J. Immunol. 143, 3031
3034. Mcleod, R., Brown, C., Mack, D., 1993. Immunogenetics influence outcome of Toxoplasma gondii infection. Res. Immunol. 144, 61
65. Melzer, T.C., Cranston, H.J., Weiss, L.M., Halonen, S.K., 2010. Host cell preference of Toxoplasma gondii cysts in murine brain: a confocal study. J Neuroparasitology 1, N100505. Available from: https://doi.org/10.4303/jnp/N100505. Mercier, C., Lefebvre-Van hende, S., Garber, G.E., Lecordier, L., Capron, A., Cesbron-Delauw, M.F., 1996. Common cis-acting elements critical for the expression of several genes of Toxoplasma gondii. Mol. Microbiol. 21, 421
428. Mercier, C., Dubremetz, J.F., Rauscher, B., Lecordier, L., Sibley, L.D., Cesbron-Delauw, M.F., 2002. Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. Mol. Biol. Cell 13, 2397
2409. Miller, C.M., Smith, N.C., Johnson, A.M., 1999. Cytokines, nitric oxide, heat shock proteins and virulence in Toxoplasma. Parasitol. Today 15, 418
422. Milligan-Myhre, K., Wilson, S.K., Knoll, L.J., 2016. Developmental change in translation initiation alters the localization of a common microbial protein necessary for Toxoplasma chronic infection. Mol. Microbiol. 102, 1086
1098. Mineo, J.R., Mcleod, R., Mack, D., Smith, J., Khan, I.A., Ely, K.H., et al., 1993. Antibodies to Toxoplasma gondii major surface protein (SAG-1, P30) inhibit infection of host cells and are produced in murine intestine after peroral infection. J. Immunol. 150, 3951
3964. Molestina, R.E., El-Guendy, N., Sinai, A.P., 2008. Infection with Toxoplasma gondii results in dysregulation of the host cell cycle. Cell. Microbiol. 10, 1153
1165. Morgan, W.D., 1989. Transcription factor Sp1 binds to and activates a human hsp70 gene promoter. Mol. Cell. Biol. 9, 4099
4104. Morimot, R.I., Tissieres, A., Georgopoulos, C. (Eds.), 1994. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Press, New York. Mouveaux, T., Oria, G., Werkmeister, E., Slomianny, C., Fox, B.A., Bzik, D.J., et al., 2014. Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator. PLoS One 9, e105820. Mueller, K., Matuschewski, K., Silvie, O., 2011. The Puffamily RNA-binding protein Puf2 controls sporozoite

853

conversion to liver stages in the malaria parasite. PLoS One 6, e19860. Murata, Y., Sugi, T., Weiss, L.M., Kato, K., 2017. Identification of compounds that suppress Toxoplasma gondii tachyzoites and bradyzoites. PLoS One 12, e0178203. Nagamune, K., Hicks, L.M., Fux, B., Brossier, F., Chini, E.N., Sibley, L.D., 2008. Abscisic acid controls calciumdependent egress and development in Toxoplasma gondii. Nature 451, 207
210. Naguleswaran, A., Elias, E.V., Mcclintick, J., Edenberg, H.J., Sullivan JR., W.J., 2010. Toxoplasma gondii lysine acetyltransferase GCN5-A functions in the cellular response to alkaline stress and expression of cyst genes. PLoS Pathog. 6, e1001232. Nallani, K.C., Sullivan JR., W.J., 2005. Identification of proteins interacting with Toxoplasma SRCAP by yeast twohybrid screening. Parasitol. Res. 95, 236
242. Nance, J.P., Vannella, K.M., Worth, D., David, C., Carter, D., Noor, S., et al., 2012. Chitinase dependent control of protozoan cyst burden in the brain. PLoS Pathog. 8, e1002990. Narasimhan, J., Joyce, B.R., Naguleswaran, A., Smith, A.T., Livingston, M.R., Dixon, S.E., et al., 2008. Translation regulation by eukaryotic initiation factor-2 kinases in the development of latent cysts in Toxoplasma gondii. J. Biol. Chem. 283, 16591
16601. Nare, B., Allocco, J.J., Liberator, P.A., Donald, R.G., 2002. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine toxoplasmosis: gamma interferon is required for efficacy. Antimicrob. Agents Chemother. 46, 300
307. Nolan, S.J., Romano, J.D., Kline, J.T., Coppens, I., 2018. Novel approaches to kill Toxoplasma gondii by exploiting the uncontrolled uptake of unsaturated fatty acids and vulnerability to lipid storage inhibition of the parasite. Antimicrob. Agents Chemother. 62, e00347-18. Obukowicz, M.G., Welsch, D.J., Salsgiver, W.J., MartinBerger, C.L., Chinn, K.S., Duffin, K.L., et al., 1998. Novel, selective delta6 or delta5 fatty acid desaturase inhibitors as antiinflammatory agents in mice. J. Pharmacol. Exp. Ther. 287, 157
166. Odaert, H., Soete, M., Fortier, B., Camus, D., Dubremetz, J.F., 1996. Stage conversion of Toxoplasma gondii in mouse brain during infection and immunodepression. Parasitol. Res. 82, 28
31. Odbergferragut, C., Soete, M., Engels, A., Samyn, B., Loyens, A., Vanbeeumen, J., et al., 1996. Molecular cloning of the Toxoplasma gondii sag4 gene encoding an 18 kDa bradyzoite specific surface protein. Mol. Biochem. Parasitol. 82, 237
244. Odell, A.V., Tran, F., Foderaro, J.E., Poupart, S., Pathak, R., Westwood, N.J., et al., 2015. Yeast three-hybrid screen

Toxoplasma Gondii

854

18. Bradyzoite and sexual stage development

identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation. PLoS One 10, e0120331. Olguin-Lamas, A., Madec, E., Hovasse, A., Werkmeister, E., Callebaut, I., Slomianny, C., et al., 2011. A novel Toxoplasma gondii nuclear factor TgNF3 is a dynamic chromatin-associated component, modulator of nucleolar architecture and parasite virulence. PLoS Pathog. 7, e1001328. Ovciarikova, J., Lemgruber, L., Stilger, K.L., Sullivan, W.J., Sheiner, L., 2017. Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii. Sci. Rep. 7, 42746. Paredes-Santos, T.C., Tomita, T., Yan Fen, M., de Souza, W., Attias, M., Vommaro, R.C., et al., 2015. Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo. Microbes Infect. 18, 39
47. Paredes-Santos, T.C., Martins-Duarte, E.S., De souza, W., Attias, M., Vommaro, R.C., 2018. Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro. Parasitology 145, 1027
1038. Parmley, S.F., Yang, S., Harth, G., Sibley, L.D., Sucharczuk, A., Remington, J.S., 1994. Molecular characterization of a 65-kilodalton Toxoplasma gondii antigen expressed abundantly in the matrix of tissue cysts. Mol. Biochem. Parasitol. 66, 283
296. Parmley, S.F., Weiss, L.M., Yang, S., 1995. Cloning of a bradyzoite-specific gene of Toxoplasma gondii encoding a cytoplasmic antigen. Mol. Biochem. Parasitol. 73, 253
257. Patil, V., Lescault, P.J., Lirussi, D., Thompson, A.B., Matrajt, M., 2012. Disruption of the expression of a non-coding RNA significantly impairs cellular differentiation in Toxoplasma gondii. Int. J. Mol. Sci. 14, 611
624. Pettersen, E.K., 1988. Resistance to avirulent Toxoplasma gondii in normal and vaccinated rats. APMIS 96, 820
824. Pittman, K.J., Aliota, M.T., Knoll, L.J., 2014. Dual transcriptional profiling of mice and Toxoplasma gondii during acute and chronic infection. BMC Genomics 15, 806. Pollard, A.M., Onatolu, K.N., Hiller, L., Haldar, K., Knoll, L.J., 2008. Highly polymorphic family of glycosylphosphatidylinositol-anchored surface antigens with evidence of developmental regulation in Toxoplasma gondii. Infect. Immun. 76, 103
110. Popiel, I., Gold, M., Choromanski, L., 1994. Tissue cyst formation of Toxoplasma gondii T-263 in cell culture. J. Eukaryot. Microbiol. 41, 17S. Popiel, I., Gold, M.C., Booth, K.S., 1996. Quantification of Toxoplasma gondii bradyzoites. J. Parasitol. 82, 330
332. Pszenny, V., Davis, P.H., Zhou, X.W., Hunter, C.A., Carruthers, V.B., Roos, D.S., 2012. Targeted disruption

of Toxoplasma gondii serine protease inhibitor 1 increases bradyzoite cyst formation in vitro and parasite tissue burden in mice. Infect. Immun. 80, 1156
1165. Qin, C.L., Tang, J., Kim, K., 1998. Cloning and in vitro expression of TPK3, a Toxoplasma gondii homologue of shaggy/glycogen synthase kinase-3 kinases. Mol. Biochem. Parasitol. 93, 273
283. Radke, J.R., Striepen, B., Guerini, M.N., Jerome, M.E., Roos, D.S., White, M.W., 2001. Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii. Mol. Biochem. Parasitol. 115, 165
175. Radke, J.R., Guerini, M.N., Jerome, M., White, M.W., 2003. A change in the premitotic period of the cell cycle is associated with bradyzoite differentiation in Toxoplasma gondii. Mol. Biochem. Parasitol. 131, 119
127. Radke, J.R., Behnke, M.S., Mackey, A.J., Radke, J.B., Roos, D.S., White, M.W., 2005. The transcriptome of Toxoplasma gondii. BMC Biol. 3, 26. Radke, J.R., Donald, R.G., Eibs, A., Jerome, M.E., Behnke, M.S., Liberator, P., et al., 2006. Changes in the expression of human cell division autoantigen-1 influence Toxoplasma gondii growth and development. PLoS Pathog. 2, e105. Radke, J.B., Lucas, O., De silva, E.K., Ma, Y., Sullivan JR., W.J., Weiss, L.M., et al., 2013. ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst. Proc. Natl. Acad. Sci. U.S.A. 110, 6871
6876. Radke, J.B., Worth, D., Honng, D.P., Huang, S., Sullivan, W.J., Wilson, E.H., 2018. Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis. PLoS Pathog. 14, e1007035. Ramakrishnan, C., Walker, R.A., Eichenberger, R.M., Hehl, A.B., Smith, N.C., 2017. The merozoite-specific protein, TgGRA11B, identified as a component of the Toxoplasma gondii parasitophorous vacuole in a tachyzoite expression model. Int. J. Parasitol. 47, 597
600. Rivers, J.P., Sinclair, A.J., Craqford, M.A., 1975. Inability of the cat to desaturate essential fatty acids. Nature 258, 171
173. Rodrigues-Pousada, C.A., Nevitt, T., Menezes, R., Azevedo, D., Pereira, J., Amaral, C., 2004. Yeast activator proteins and stress response: an overview. FEBS Lett. 567, 80
85. Rooney, P.J., Neal, L.M., Knoll, L.J., 2011. Involvement of a Toxoplasma gondii chromatin remodeling complex ortholog in developmental regulation. PLoS One 6, e19570. Rougier, S., Montoya, J.G., Peyron, F., 2017. Lifelong persistence of toxoplasma cysts: a questionable dogma?: (Trends in Parasitology 33, 93-101; 2017). Trends Parasitol. 33, 414. Ruan, J., Mouveaux, T., Light, S.H., Minasov, G., Anderson, W.F., Tomavo, S., et al., 2015. The structure of

Toxoplasma Gondii

References

bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions. Acta Crystallogr., Sect. D: Struct. Biol. 71, 417
426. Saeij, J.P., Arrizabalaga, G., Boothroyd, J.C., 2008. A cluster of four surface antigen genes specifically expressed in bradyzoites, SAG2CDXY, plays an important role in Toxoplasma gondii persistence. Infect. Immun. 76, 2402
2410. Saito, T., Maeda, T., Nakazawa, M., Takeuchi, T., Nozaki, T., Asai, T., 2002. Characterisation of hexokinase in Toxoplasma gondii tachyzoites. Int. J. Parasitol. 32, 961
967. Saksouk, N., Bhatti, M.M., Kieffer, S., Smith, A.T., Musset, K., Garin, J., et al., 2005. Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii. Mol. Cell. Biol. 25, 10301
10314. Scholytyseck, E., Mehlhorn, H., Muller, B.E., 1974. [Fine structure of cyst and cyst wall of Sarcocystis tenella, Besnoitia jellisoni, Frenkelia sp. and Toxoplasma gondii]. J. Protozool. 21, 284
294. Schwarz, J.A., Fouts, A.E., Cummings, C.A., Ferguson, D.J., Boothroyd, J.C., 2005. A novel rhoptry protein in Toxoplasma gondii bradyzoites and merozoites. Mol. Biochem. Parasitol. 144, 159
166. Sharma, R., Kumar, D., Jha, N.K., Jha, S.K., Ambasta, R.K., Kumar, P., 2017. Re-expression of cell cycle markers in aged neurons and muscles: Whether cells should divide or die? Biochim. Biophys. Acta Mol. Basis Dis. 1863, 324
336. Shiels, B., Aslam, N., Mckellar, S., Smyth, A., Kinnaird, J., 1997. Modulation of protein synthesis relative to DNA synthesis alters the timing of differentiation in the protozoan parasite Theileria annulata. J. Cell. Sci. 110, 1441
1451. Silva, N.M., Gazzinelli, R.T., Silva, D.A., Ferro, E.A., Kasper, L.H., Mineo, J.R., 1998. Expression of Toxoplasma gondii-specific heat shock protein 70 during In vivo conversion of bradyzoites to tachyzoites. Infect. Immun. 66, 3959
3963. Sims, T.A., Hay, J., Talbot, I.C., 1988. Host-parasite relationship in the brains of mice with congenital toxoplasmosis. J. Pathol. 156, 255
261. Sims, T.A., Hay, J., Talbot, I.C., 1989. An electron microscope and immunohistochemical study of the intracellular location of Toxoplasma tissue cysts within the brains of mice with congenital toxoplasmosis. Br. J. Exp. Pathol. 70, 317
325. Sinai, A.P., Watts, E.A., Dhara, A., Murphy, R.D., Gentry, M.S., Patwardhan, A., 2017. Reexamining chronic Toxoplasma gondii infection: surprising activity for a “dormant” parasite. Curr. Clin. Microbiol. Rep. 3, 175
185.

855

Sinclair, A.J., Mclean, J.G., Monger, E.A., 1979. Metabolism of linoleic acid in the cat. Lipids 14, 932
936. Singh, U., Brewer, J.L., Boothroyd, J.C., 2002. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Mol. Microbiol. 44, 721
733. Soderbom, F., Loomis, W.F., 1998. Cell-cell signaling during Dictyostelium development. Trends Microbiol 6, 402
406. Soete, M., Dubremetz, J.F., 1996. Toxoplasma gondii: kinetics of stage-specific protein expression during tachyzoitebradyzoite conversion in vitro. Curr. Top. Microbiol. Immunol. 219, 76
80. Soete, M., Fortier, B., Camus, D., Dubremetz, J.F., 1993. Toxoplasma gondii: kinetics of bradyzoite-tachyzoite interconversion in vitro. Exp. Parasitol. 76, 259
264. Soete, M., Camus, D., Dubremetz, J.F., 1994. Experimental induction of bradyzoite-specific antigen expression and cyst formation by the RH strain of Toxoplasma gondii in vitro. Exp. Parasitol. 78, 361
370. Sokol, S.L., Primack, A.S., Nair, S.C., Wong, Z.S., Tembo, M., Verma, S.K., et al., 2018. Dissection of the in vitro developmental program of Hammondia hammondi reveals a link between stress sensitivity and life cycle flexibility in Toxoplasma gondii. Elife 7, e36491. Stwora-Wojczyk, M.M., Dzierszinski, F., Roos, D.S., Spitalnik, S.L., Wojczyk, B.S., 2004a. Functional characterization of a novel Toxoplasma gondii glycosyltransferase: UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T3. Arch. Biochem. Biophys. 426, 231
240. Stwora-Wojczyk, M.M., Kissinger, J.C., Spitalnik, S.L., Wojczyk, B.S., 2004b. O-glycosylation in Toxoplasma gondii: identification and analysis of a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Int. J. Parasitol. 34, 309. Sugi, T., Kawazu, S.-I., Horimoto, T., Kato, K., 2015. A single mutation in the gatekeeper residue in TgMAPKL-1 restores the inhibitory effect of a bumped kinase inhibitor on the cell cycle. Int. J. Parasitol.: Drugs Drug Resist 5, 1
8. Sugi, T., Ma, Y.F., Tomita, T., Murakoshi, F., Eaton, M.S., Yakubu, R., et al., 2016. Toxoplasma gondii cyclic ampdependent protein kinase subunit 3 is involved in the switch from tachyzoite to bradyzoite development. mBio 7, e00755-16. Sugi, T., Tu, V., Ma, Y., Tomita, T., Weiss, L.M., 2017. Toxoplasma gondii requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of brain cysts. mBio 8, e01289-17. Sullivan JR., W.J., Smith 2ND, C.K., 2000. Cloning and characterization of a novel histone acetyltransferase homologue from the protozoan parasite Toxoplasma gondii reveals a distinct GCN5 family member. Gene 242, 193
200.

Toxoplasma Gondii

856

18. Bradyzoite and sexual stage development

Sullivan JR., W.J., Jeffers, V., 2012. Mechanisms of Toxoplasma gondii persistence and latency. FEMS Microbiol. Rev. 36, 717
733. Sullivan JR., W.J., Monroy, M.A., Bohne, W., Nallani, K.C., Chrivia, J., Yaciuk, P., et al., 2003. Molecular cloning and characterization of an SRCAP chromatin remodeling homologue in Toxoplasma gondii. Parasitol. Res. 90, 1
8. Sullivan JR., W.J., Narasimhan, J., Bhatti, M.M., Wek, R.C., 2004. Parasite-specific eIF2 (eukaryotic initiation factor2) kinase required for stress-induced translation control. Biochem. J. 380, 523
531. Sun, H., Zhuo, X., Zhao, X., Yang, Y., Chen, X., Yao, C., et al., 2017. The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth. Parasite 24, 22. Swierzy, I.J., Muhammad, M., Kroll, J., Abelmann, A., Tenter, A.M., Luder, C.G., 2014. Toxoplasma gondii within skeletal muscle cells: a critical interplay for food-borne parasite transmission. Int. J. Parasitol. 44, 91
98. Swierzy, I.J., Handel, U., Kaever, A., Jarek, M., Scharfe, M., Schluter, D., et al., 2017. Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions. Sci. Rep. 7, 7229. Szatanek, T., Anderson-White, B.R., Faugno-Fusci, D.M., White, M., Saeij, J.P., Gubbels, M.J., 2012. Cactin is essential for G1 progression in Toxoplasma gondii. Mol. Microbiol. 84, 566
577. Thomason, P., Traynor, D., Kay, R., 1999. Taking the plunge. Terminal differentiation in Dictyostelium. Trends Genet. 15, 15
19. Tomavo, S., Boothroyd, J.C., 1995. Interconnection between organellar functions, development and drug resistance in the protozoan parasite, Toxoplasma gondii. Int. J. Parasitol. 25, 1293
1299. Tomavo, S., Fortier, B., Soete, M., Ansel, C., Camus, D., Dubremetz, J.F., 1991. Characterization of bradyzoitespecific antigens of Toxoplasma gondii. Infect. Immun. 59, 3750
3753. Tomita, T., Bzik, D.J., Ma, Y.F., Fox, B.A., Markillie, L.M., Taylor, R.C., et al., 2013. The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence. PLoS Pathog. 9, e1003823. Tomita, T., Sugi, T., Yakubu, R., Tu, V., Ma, Y., Weiss, L.M., 2017. Making home sweet and sturdy: Toxoplasma gondii ppGalNAc-ts glycosylate in hierarchical order and confer cyst wall rigidity. mBio 8, e02048-16. Tomita, T., Ma, Y., Weiss, L., 2018. Characterization of a SRS13: a new cyst wall mucin-like domain containing protein. Parasitol. Res. 117, 2457
2466. Available from: https://doi.org/10.1007/s00436-018-5934-3.

Torpier, G., Charif, H., Darcy, F., Liu, J., Darde, M.L., Capron, A., 1993. Toxoplasma gondii: differential location of antigens secreted from encysted bradyzoites. Exp. Parasitol. 77, 13
22. Tu, V., Yakubu, R., Weiss, L.M., 2018. Observations on bradyzoite biology. Microbes Infect. 20, 466
476. Tu, V., Mayoral, J., Sugi, T., Tomita, T., Han, B., Ma, Y., et al., 2019a. Enrichment and proteomic characterization of the cyst wall from in vitro Toxoplasma gondii cysts. mBio 10, e00469-19. Tu, V., Mayoral, J., Yakubu, R., Sugi, T., Tomita, T., Han, B., et al., 2019b. Characterization of MAG2: a Toxoplasma gondii bradyzoite cyst matrix protein. mSphere in submission. Tu, V., Tomita, T., Sugi, T., Mayoral, J., Han, B., Yakubu, R.R., et al., 2020. The Toxoplasma gondii cyst wall interactome. mBio, 11, e02699
19. Uboldi, A.D., Mccoy, J.M., Blume, M., Gerlic, M., Ferguson, D.J.P., Dagley, L.F., et al., 2015. Regulation of starch stores by a Ca2 1 -dependent protein kinase is essential for viable cyst development in Toxoplasma gondii. Cell Host Microbe 18, 670
681. Ueno, A., Dautu, G., Munyaka, B., Carmen, G., Kobayashi, Y., Igarashi, M., 2009. Toxoplasma gondii: identification and characterization of bradyzoite-specific deoxyribose phosphate aldolase-like gene (TgDPA). Exp. Parasitol. 121, 55
63. Ueno, A., Dautu, G., Saiki, E., Haga, K., Igarashi, M., 2010. Toxoplasma gondii deoxyribose phosphate aldolase-like protein (TgDPA) interacts with actin depolymerizing factor (TgADF) to enhance the actin filament dynamics in the bradyzoite stage. Mol. Biochem. Parasitol. 173, 39
42. Ueno, A., Dautu, G., Haga, K., Munyaka, B., Carmen, G., Kobayashi, Y., et al., 2011. Toxoplasma gondii: a bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein. Exp. Parasitol. 127, 795
803. Unno, A., Suzuki, K., Batanova, T., Cha, S.Y., Jang, H.K., Kitoh, K., et al., 2009. Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins. Parasitology 136, 579
588. van der Waaij, D., 1959. Formation, growth and multiplication of Toxoplasma gondii cysts in mouse brain. Trop. Geogr. Med 11, 345
360. Van, T.T., Kim, S.K., Camps, M., Boothroyd, J.C., Knoll, L.J., 2007. The BSR4 protein is up-regulated in Toxoplasma gondii bradyzoites, however the dominant surface antigen recognised by the P36 monoclonal antibody is SRS9. Int. J. Parasitol. 37, 877
885. Vanchinathan, P., Brewer, J.L., Harb, O.S., Boothroyd, J.C., Singh, U., 2005. Disruption of a locus encoding a nucleolar zinc finger protein decreases tachyzoite-tobradyzoite differentiation in Toxoplasma gondii. Infect. Immun. 73, 6680
6688.

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Further reading

Walker, R., Gissot, M., Croken, M.M., Huot, L., Hot, D., Kim, K., et al., 2013. The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation. Mol. Microbiol. 87, 641
655. Wang, J., Dixon, S.E., Ting, L.M., Liu, T.K., Jeffers, V., Croken, M.M., et al., 2014. Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation. PLoS Pathog. 10, e1003830. Wasmuth, J.D., Pszenny, V., Haile, S., Jansen, E.M., Gast, A.T., Sher, A., et al., 2012. Integrated bioinformatic and targeted deletion analyses of the SRS gene superfamily identify SRS29C as a negative regulator of Toxoplasma virulence. mBio 3, e00321-12. Wastling, J.M., Xia, D., Sohal, A., Chaussepied, M., Pain, A., Langsley, G., 2009. Proteomes and transcriptomes of the Apicomplexa - Where’s the message? Int. J. Parasitol. 39, 135
143. Watts, E., Zhao, Y., Dhara, A., Eller, B., Patwardhan, A., Sinai, A.P., 2015. Novel approaches reveal that Toxoplasma gondii Bradyzoites within tissue cysts are dynamic and replicating entities in vivo. mBio 6, e01155-15. Watts, E.A., Dhara, A., Sinai, A.P., 2017. Purification Toxoplasma gondii tissue cysts using percoll gradients. Curr. Protoc. Microbiol. 45, 20C.2.1
20C.2.19. Weilhammer, D.R., Iavarone, A.T., Villegas, E.N., Brooks, G.A., Sinai, A.P., Sha, W.C., 2012. Host metabolism regulates growth and differentiation of Toxoplasma gondii. Int. J. Parasitol. 42, 947
959. Weiss, L.M., Kim, K., 2000. The development and biology of bradyzoites of Toxoplasma gondii. Front. Biosci. 5, D391
D405. Weiss, L.M., Laplace, D., Tanowitz, H.B., Wittner, M., 1992. Identification of Toxoplasma gondii bradyzoitespecific monoclonal antibodies. J. Infect. Dis. 166, 213
215. Weiss, L.M., Laplace, D., Takvorian, P.M., Cali, A., Tanowitz, H.B., Wittner, M., 1994. Development of bradyzoites of Toxoplasma gondii in vitro. J. Eukaryot. Microbiol. 41, 18S. Weiss, L.M., Laplace, D., Takvorian, P.M., Tanowitz, H.B., Cali, A., Wittner, M., 1995. A cell culture system for study of the development of Toxoplasma gondii bradyzoites. J. Eukaryot. Microbiol. 42, 150
157. Weiss, L.M., Laplace, D., Takvorian, P., Tanowitz, H.B., Wittner, M., 1996. The association of the stress response and Toxoplasma gondii bradyzoite development. J. Eukaryot. Microbiol. 43, 120S. Weiss, L.M., Ma, Y.F., Takvorian, P.M., Tanowitz, H.B., Wittner, M., 1998. Bradyzoite development in Toxoplasma gondii and the hsp70 stress response. Infect. Immun. 66, 3295
3302.

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Weiss, L.M., Ma, Y.F., Halonen, S., Mcallister, M.M., Zhang, Y.W., 1999. The in vitro development of Neospora caninum bradyzoites. Int. J. Parasitol. 29, 1713
1723. Wek, R.C., Jiang, H.Y., Anthony, T.G., 2006. Coping with stress: eIF2 kinases and translational control. Biochem. Soc. Trans. 34, 7
11. White, M.W., Radke, J.R., Radke, J.B., 2014. Toxoplasma development—turn the switch on or off? Cell. Microbiol. 16, 466
472. Winzer, P., Mueller, J., Aguado-Martinez, A., Rahman, M., Balmer, V., Manser, V., et al., 2015. In vitro and in vivo effects of the bumped kinase inhibitor 1294 in the related cyst-forming apicomplexans Toxoplasma gondii and Neospora caninum, Antimicrob. Agents Chemother., 59. pp. 6361
6374. Wojczyk, B.S., Hagen, F.K., Striepen, B., Hang, H.C., Bertozzi, C.R., Roos, D.S., et al., 2003. cDNA Cloning and expression of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase T1 from Toxoplasma gondii. Mol. Biochem. Parasitol. 131, 93
107. Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., Tomavo, S., 1999. Isolation and characterization of a subtractive library enriched for developmentally regulated transcripts expressed during encystation of Toxoplasma gondii. Mol. Biochem. Parasitol. 99, 223
235. Yang, S., Parmley, S.F., 1995. A bradyzoite stagespecifically expressed gene of Toxoplasma gondii encodes a polypeptide homologous to lactate dehydrogenase. Mol. Biochem. Parasitol. 73, 291
294. Yang, S., Parmley, S.F., 1997. Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts. Gene 184, 1
12. Yang, N., Farrell, A., Niedelman, W., Melo, M., Lu, D., Julien, L., et al., 2013. Genetic basis for phenotypic differences between different Toxoplasma gondii type I strains. BMC Genomics 14. Zhang, Y.W., Kim, K., Ma, Y.F., Wittner, M., Tanowitz, H. B., Weiss, L.M., 1999. Disruption of the Toxoplasma gondii bradyzoite-specific gene BAG1 decreases in vivo cyst formation. Mol. Microbiol. 31, 691
701. Zhang, Y.W., Halonen, S.K., Ma, Y.F., Wittner, M., Weiss, L. M., 2001. Initial characterization of CST1, a Toxoplasma gondii cyst wall glycoprotein. Infect. Immun. 69, 501
507. Zhang, Y.W., Halonen, S.K., Ma, Y.F., Tanowtiz, H.B., Weiss, L.M., 2010. A purification method for enrichment of the Toxoplasma gondii cyst wall. J. Neuroparasitol. 1, N101001. Available from: https:// doi.org/10.4303/jnp/N101001.

Further reading Gross, U., 1996. Toxoplasma Gondii. Springer, Berlin.

Toxoplasma Gondii

C H A P T E R

19 Development and application of classical genetics in Toxoplasma gondii Michael S. Behnke1, Jeroen P.J. Saeij2 and Jon P. Boyle3 1

Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States 2Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, United States 3Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, United States

19.1 Summary Apicomplexan parasites have complex life cycles involving sexual replication in a single definitive host and asexual replication in a variety of alternative hosts. Toxoplasma gondii undergoes its sexual cycle only in cats, yet it also infects a wide range of other vertebrates where it propagates asexually. In Europe and North America the bulk of T. gondii infections are due to one of three major clonal lines (types 1, 2, and 3) which only recently originated from a few closely related parental lines. In South America, T. gondii strains exhibit significantly more genotypic diversity. Despite being genetically quite similar, even the North American clonal types differ substantially in various biological traits including virulence in laboratory mice, induction of changes in host-cell

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00019-0

signaling, and transcription. The ability to perform experimental genetic crosses has been exploited extensively in the past 15 years, first to generate linkage maps to facilitate completion of the genome assembly, and then later to map traits differing in the parental lines to individual loci. This chapter will highlight the power of this approach to map both simple and complex biological traits, including drug resistance, replication rate, pathogenesis in laboratory mice, induction of host gene expression, and modulation of the immune response. The most exciting findings to emerge from this approach in T. gondii have been the unequivocal identification of families of secreted effectors that are capable of direct modulation of the host environment, including those capable of disrupting cell-autonomous immune responses and the recruitment of host organelles.

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19.2 Biology of Toxoplasma 19.2.1 Life cycle T. gondii has a heteroxenous life cycle, alternating between sexual replication in members of the cat family, which serve as the definitive host, and asexual replication in a wide range of warm-blooded vertebrates that serve as intermediate hosts (Dubey, 1977) (see Chapter 1: The history and life cycle of Toxoplasma gondii). During the initial infection, T. gondii grows rapidly as a haploid form called the tachyzoite, which is capable of invading and replicating within any nucleated cell in its many warmblooded hosts. In response to stress brought on by the immune response, the parasite converts to a slow growing form called a bradyzoite that is encased in a thick-walled cyst. Ingestion of tissue cysts by cats leads to sexual differentiation within intestinal epithelial cells and eventually the fusion of male and female gametes to form a diploid zygote (Dubey and Frenkel, 1972). Following the development of an impervious wall, oocyst stages are shed in the feces, and they undergo meiosis in the environment to yield eight haploid progeny (Frenkel et al., 1970). Oocysts are long-lived, resistant to environmental conditions, and responsible for dissemination due to the contamination of food or water (de Moura et al., 2006; Dubey, 2004; Mead et al., 1999). Among many unique T. gondii features is the fact that the T. gondii life cycle is facultatively heteroxenous, in that the parasite is capable of indefinite asexual propagation and therefore does not require the sexual phase of the life cycle (Dubey and Sreekumar, 2003). This has likely played a prominent role in the dominance of a small number of clonal strains that are separated by a relatively small number of genetic crosses (Boyle et al., 2006).

19.2.2 Defining the sexual phase The sexual cycle of T. gondii only takes place in the enterocytes of the small intestine of

members of the cat family (Felidae) (Dubey, 2010). Factors that restrict the development to this host and tissue location are uncertain but presumably result from coadaptation of the parasite and host. By comparison, Hammondia hammondi, which is closely related to T. gondii, also undergoes the sexual cycle in cats, while more distant relatives Hammondia heydorni and Neospora caninum undergo sexual development only in canines (Dubey, 1977). The relative divergence of Toxoplasma and Neospora coincides with that of their respective carnivore hosts (Reid et al., 2012), suggesting these parasites speciated following separation of their definitive hosts. Initiation of the sexual cycle in T. gondii occurs when bradyzoites infect epithelial cells in the ileum of the cat intestine, although the reasons why this niche is preferred remain mysterious. For T. gondii, infection of cats with oocysts or tachyzoites can also lead to oocyst shedding, although this mode of infection is accompanied by considerable delays in oocyst shedding (Dubey, 2005, 2006), suggesting that the parasite must first differentiate into bradyzoites prior to commencing the sexual phase. This facultatively heteroxenous life cycle is yet another trait unique to T. gondii, since for H. hammondi the sexual cycle can only be initiated via infection with bradyzoites (Dubey and Sreekumar, 2003; Frenkel and Dubey, 1975a,b, 2000). Sexual development proceeds through several rounds of merogony, leading to the development of morphologically distinct male and female gametocytes, which fuse to form a zygote (Dubey and Frenkel, 1972). The diploid oocyst is shed in the feces in an unsporulated state, and it then undergoes meiosis in the environment (Ferguson et al., 1979; Speer et al., 1998). The development of these sexual stages has been extensively studied in histopathology sections by both light and electron microscopy, and their transcriptomes have been directly queried using microarrays and RNA sequencing (Behnke et al., 2014; Hehl et al., 2015; Ramakrishnan et al., 2019) (also see Gene Expression Omnibus GSE108740). However, the

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cat has been required for oocyst production, as there are no means as of yet to generate oocysts in vitro or in more tractable animal models like mice. Development of such an approach would be a huge advancement for the field given the extensive labor and cost associated with oocyst production in cats (see Chapter 18: Bradyzoite and Sexual Stage Development). Following the development of in vitro cell culture systems to isolate clones of T. gondii (Pfefferkorn and Pfefferkorn, 1976), cats fed with cysts that had developed from a cloned parasite line shed oocysts (Pfefferkorn et al., 1977). Similar findings by another group (Cornelissen and Overdulve, 1985) confirmed that T. gondii has the capability to differentiate into both micro- and macrogametocytes starting from a single progenitor parasite cell. A strict genetic basis for gametocyte differentiation (e.g., sex chromosome) can thus be discounted, although the cellular mechanisms that lead to differentiation into male (micro-) or female (macro-) gametocytes are as yet unknown. The developmental program necessary for completion of transmission through the cat is easily disrupted. Passage of tachyzoites through mice resulted in the loss of ability to form oocysts after B30 passages (likely fewer than 103 cell divisions) (Frenkel et al., 1976), and while the number of passages varies from study to study and most likely based on culture conditions and even strain type, the loss of cat competency either implies that any single mutation in large number of essential genes can disrupt the process or by the emergence of laboratory adapted parasite lines harboring effectively irreversible epigenetic changes. This interesting observation raises the question as to how common it is for T. gondii to propagate asexually in the wild, since this may eventually result in loss of competence to enter the sexual phase in the definitive host. This would eliminate the phase of the life cycle most responsible for the expansion of the parasite population (infection of a cat with a single cyst containing tens to hundreds of bradyzoites

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can produce up to a billion oocysts within 7 10 days) (Dubey and Frenkel, 1972; Miller et al., 1972). While anecdotal, most isolates obtained from human patients or nonfeline wild animals have “oocyst competence” at the time of their isolation, suggesting that most parasite lineages participate in the sexual cycle at some minimal frequency. Recent work showing an association with low seroprevalence rates in humans on islands that have always been, or were recently made, cat free provides further support for the sexual cycle in human infection and maintenance of the parasite population (e.g., de Wit et al., 2019). Regardless this predictable loss of an entire phase of the life cycle during laboratory passage requires further investigation and may shed light on T. gondii life cycle evolution. Classic studies by Cornelissen et al. (1984a, b) examined the ploidy of stages during gametogenesis and meiosis in the sexual cycle by analyzing the DNA content of gametes, zygote/oocyst, and sporozoites. These studies concluded that mature macrogametes (presumably fertilized) contain twice the amount of DNA found in all other stages, suggesting that most stages are haploid, and that meiosis takes place during sporogony. The absolute content of DNA in one haploid stage predicted a genome size of B80 Mbp, which is on the same order of magnitude as the assembled genome size of 65 Mbp (see later). Morphological studies of sporulation in T. gondii indicate that the initial division into two sporocysts is followed by reduction into four haploid sporozoites each (Speer et al., 1998). Presumably, the first division represents the meiotic division, although this has not been confirmed experimentally. Classical tetrad analysis using microdissection and single-cell polymerase chain reaction (PCR) amplification might resolve this and also provide a rapid means of evaluating the fraction of recombination in future crosses. Other advantages of T. gondii as a classical genetic system include the following: (1) techniques for harvesting and sporulating oocysts

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(Dubey et al., 1972) allow for infections to be initiated in vitro from material collected as the result of naturally or experimentally infected cats; (2) oocysts can be stored for long periods of time at 4 C (Dubey, 1998), which facilitates the isolation of progeny from experimental crosses as described later; and (3) progeny clones can be genotyped and cryopreserved, such that they can be used for multiple rounds of phenotyping and genetic mapping as new strain specific phenotypes are identified or new technologies allow for more refined analyses of parasite biology in vitro or in vivo.

19.2.3 Population structure and major strain types T. gondii has an unusual population structure comprising three clonal lineages, referred to as 1, 2, and 3, which are wide spread in North America and Europe (Sibley and Ajioka, 2008). Recently a fourth clonal lineage has been described, and strains of this genotype (referred to as haplotype 12) appear to be more common in wild versus domestic animals in North America (Khan et al., 2011). Strains of T. gondii are much more diverse in other regions, especially in South America where distinct nonclonal lineages show much greater evidence of out crossing (Khan et al., 2007; Lehmann et al., 2006; Minot et al., 2012; Pena et al., 2008). The global population structure of T. gondii has recently been compared using a variety of different typing methods to analyze .950 independent isolates grouping them into 16 haplogroups that comprise 6 major clades (Su et al., 2012). Studies on the population genetic structure of T. gondii from different regions are further discussed in Chapter 3, Molecular epidemiology and population structure of Toxoplasma gondii. To date all of the genetic crosses performed have had a member of the type 1, 2, or 3 lineage as at least one parent.

The three clonal lineages are themselves quite closely related and derived from a few closely related ancestral strains that underwent limited genetic recombination in the wild (Boyle et al., 2006; Grigg et al., 2001a; Su et al., 2003). Mapping of strain-specific single nucleotide polymorphisms (SNPs) identified in expressed sequence tags (ESTs) to the whole genome assemblies confirmed that the three clonal lineages arose through a small number of recombination events between separate ancestral strains of type 1 and 3 lineages that each crossed with a type 2 like parental strain (Boyle et al., 2006). This inheritance pattern is evident across the genome, where for certain genomic segments, 2 strains share the same parental chromosome and are therefore nearly identical (Boyle et al., 2006; Minot et al., 2012). For example, on most of chromosome Ia types 1, 2, and 3 are very similar, indicating that all three lineages received much of that chromosome from their shared parent (a type 2 ancestor; Boyle et al., 2006). For chromosome IV, types 1 and 2 have nearly identical sequence, while on much of chromosome XI types 2 and 3 have nearly identical sequence. Effectively for these types of chromosomal regions T. gondii is biallelic. In contrast, for other chromosomal segments (such as most of chromosome X and chromosomes II and III), the SNP pattern is tri-allelic, with type 1 and 3 strains having unique polymorphisms that distinguish them from their shared type 2 ancestral parent (Boyle et al., 2006; Minot et al., 2012). As discussed later, this ancestry pattern had an important impact on the T. gondii linkage map, particularly in terms of identifying polymorphisms capable of distinguishing the parental lines in some of these SNP-poor regions where the parents have shared ancestry. Regardless, these data clearly indicate that only a small number of strains interbred and gave rise to the three major clonal lineages that came to dominate in Europe and North America. Whether this occurred via a population bottleneck or a

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selective sweep (or a combination of both) is not currently clear. While the genetic similarity between the three major clonotypes has been an advantage for mapping lineage-specific traits, there will also be traits that are specific to other T. gondii lineages that are missed using these approaches. Analyses of divergent (so-called exotic) strains are lacking compared to those performed in the prototypical North American/European strains, but they are critical for unraveling some of the more dramatic phenotypic differences exhibited by divergent strains from South America.

19.3 Establishment of transmission genetics 19.3.1 Intra-strain crosses and meiosis Although morphological observations of T. gondii infection in the cat provided a framework for the sexual cycle, key mechanistic details such as sexual differentiation/determination, and recombination required the establishment of transmission genetics. Early work by Pfefferkorn et al. laid the foundation for T. gondii as a genetic model organism by generating necessary tools and carrying out the first genetic crosses. Performing genetic crosses in T. gondii is relatively complex and not easily undertaken in most laboratories, in part due to the biological hazard posed by oocysts, which are highly infectious. Oocysts are resistant to chemical treatments and disinfectants (i.e., chlorine, aldehyde fixation, strong acids or bases, and UV irradiation). Heat inactivation is the only reliable means of disinfecting surfaces that come in contact with T. gondii oocysts (Dubey, 1998). Other stages of T. gondii (tachyzoites and bradyzoites) can be used more widely with standard BSL-2 level containment, making these relatively easy to work with the laboratory. Successful completion of a genetic cross requires the production of tissue cysts of

FIGURE 19.1 Diagram of genetic crosses of Toxoplasma gondii. Tachyzoites of different genetic types are propagated in vitro in host cell monolayers and used to generate drug-resistant parental lines. Inoculation of mice leads to chronic infections characterized by bradyzoites found in tissue cysts in the brain. Cofeeding of tissue cysts to cats initiates the sexual cycle, which takes place in enterocytes of the small intestine. Oocysts are shed into the environment where they undergo meiosis. Oocysts are hatched and inoculated onto host cell monolayers to isolate haploid progeny clones that correspond to F1 hybrids. Source: Modified with permission from Sibley, L.D., Ajioka, J.W., 2008. Population structure of Toxoplasma gondii: clonal expansion driven by infrequent recombination and selective sweeps. Ann. Rev. Microbiol. 62, 329 351.

two compatible parental strains, obtained by chronically infecting mice or rats (although in vitro-derived cysts and even tachyzoites are infectious to cats and lead to oocyst production) (Dubey, 2005; Lindsay et al., 1991), cofeeding of tissue cysts to specific pathogen free cat(s), collection of oocysts shed in the feces, sporulation, reinitiation of in vitro cultures, cloning, and phenotyping of progeny (Fig. 19.1). Given the complexity of this process, it is not surprising that only a handful of crosses have been conducted to date (Table 19.1), yet the results of these experimental crosses have proven highly informative. As with other genetic model organisms, engineering of drug-resistant lines of T. gondii proved useful for tracking recombination in

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TABLE 19.1 Summary of genetic crosses among clonal lineages of Toxoplasma gondii. Parental strains

Parental strains

Individual crossesa

Unique progeny

Frequency of Markers recombination

Maternal inheritance

233

ME49 B7 3 CTG ARA-Ar/SNFr

S

10b

135d

ND

No bias

b

CL

133

9

PTG FUDR /ANO 3 CTG ARA- C96 Ar/SNFr/DCLr

21b

135d

ND

Type 3 predominates

GT-1 FUDRr 3 CTG ARA-Ar/ SNFr

C285

11c

175e

B100%g

Slight type 1 bias

C295

20b

1603f

B20-35%g

No bias

r

r

3d 132

2 3 10

GT-1 SNFr 3 ME49 B7 FUDRr

ME49 B7 FUDRr 3 VAND SNFr

37b

Ct3

b c

Ct7

3 1

Ct10

4b

TX405

11b c

13

499,470h 12.8% 544

VAND bias

i

a Separate crosses were done in parallel cats fed similar mixtures of bradyzoites from the two parental strains for Cl and S clones in the 2 3 3 cross (Sibley et al., 1992), for c285, c295 clones in the 1 3 3 cross (Su et al., 2002) and for the Ct3, Ct7, Ct10 clones in the 1 3 2 cross (Behnke et al., 2011). b Clones isolated by drug section. c Clones selected at random and genotyped by PCR. d PCR screened for recombination on chromosome VIIa. e PCR-RFLP markers (Khan et al., 2005b). f Affymetrix array hybridizations (Behnke et al., 2011). g Determined by isolating clones and then genotyping with 10 unlinked PCR-RFLP markers. h SNPs from Illumina WGS (Khan et al., 2014). i The number of useful markers used in the genetic map. ND, Not determined.

progeny from genetic crosses. These studies exploited the fact that T. gondii tachyzoites are haploid and can be maintained indefinitely in vitro and cryopreserved, thus aiding in isolation of defined clones (Pfefferkorn, 1988). Alkylating agents such as ethyl-nitrosourea (ENU) proved effective for inducing mutations and isolation of mutant parasite lines (Pfefferkorn and Pfefferkorn, 1979). Among the most useful were lines resistant to 5-fluorodeoxyuridine (FUDR), which is an antimetabolite that is incorporated by the enzyme uracil phsophoribosyltransferase (Pfefferkorn

and Pfefferkorn, 1977), and adenosine arabinoside (ARA-A), which is likewise incorporated by adenosine kinase (Pfefferkorn and Pfefferkorn, 1978). The results from these early studies showed that mutants with selectable phenotypes could be easily recovered, opening up the possibility to use genetic crosses to study the genetic basis for phenotypes such as drug resistance. For the first T. gondii cross, two mutant lines resistant to ARA-A and FUDR were separately selected from the parental C T. gondii strain (a.k.a. CTG, or C Elmer Pfefferkorn; CEP) after ENU mutagenesis (Pfefferkorn and

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19.3 Establishment of transmission genetics

Pfefferkorn, 1980). The CTG strain was isolated from a naturally infected cat in New Hampshire by Pfefferkorn (Pfefferkorn and Pfefferkorn, 1977), and it was later found to be a member of the type 3 lineage (Howe and Sibley, 1995). All of the oocyst-derived clones recovered from the ARA-Ar line were drug resistant, confirming the haploid nature of the parasite. The ability of this clonal line to produce oocysts also demonstrated the potential to make both micro- and macrogametocytes and to undergo self-fertilization. When roughly equal numbers of ARA-Ar and FUDRr bradyzoites were fed to a cat, approximately 12% of the clones recovered were resistant to both drugs (Pfefferkorn and Pfefferkorn, 1980). This rate of doubly resistant clones was observed in two separate experiments and is consistent with the expected meiotic recombination yield, given that half of all fertilizations will be self-fertilizations and of the remaining cross-fertilizations; 25% of the progeny from the latter category are expected to be doubly resistant when observed for two independent markers, hence an overall frequency of 12.5%. This calculation assumes these markers to be unlinked, something that was later confirmed by linkage mapping studies. These data argue that mating types are not predetermined, a finding confirmed by others (Cornelissen and Overdulve, 1985), and that parthenogenesis does not contribute substantially to the output of oocysts during experimental crosses. By examining the inheritance of drug resistance in self-crosses versus outcrosses, it was also shown that only a haploid model can explain the inheritance of drug resistance in T. gondii.

19.3.2 Genetic crosses between different lineages The first interstrain cross was conducted between the CTG strain (type 3) described above and the ME49 strain (type 2), originally

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isolated from a sheep in California (Lunde and Jacobs, 1983) and later shown to be representative of the type 2 lineage (Howe and Sibley, 1995). Prior to being used in this cross, the ME49 line was passaged through a cat to obtain sporulated oocysts, and a clone called B7 was isolated by limiting dilution in vitro for use as the parental line. The other parental line used in this cross was a doubly resistant clone of CTG bearing the drug resistance markers AraAr and sinefungin (SNFr), previously isolated as part of the intra-strain cross among singly drug-resistant clones of CTG (Pfefferkorn and Kasper, 1983). Each of the parental strains were used to infect mice, brain homogenates containing tissue cysts were cofed to two different naı¨ve cats, and oocysts were collected after shedding (Fig. 19.1). Out of 20 recombinant clones isolated in vitro, 17 were singly resistant to either SNF or ARA-A, two were doubly resistant, and one was doubly susceptible (Table 19.1). A second cross between type 2 and 3 lineages was performed several years later (c.1996) using a similar strategy (Table 19.1). In this cross the parental strains were carrying the following drug-resistance markers: the type 2 P T. gondii strain [a separate clone of ME49 (Ware and Kasper, 1987)] was resistant to aprinocideN-oxide (ANO) and FUDR and the type 3 strain CTG was resistant to SNF, ARA-A, and diclazuril (DCL) (Khan et al., 2005b) (Table 19.1). A total of 21 recombinants from this cross were selected from a pool of parasites that was doubly selected with SNF and ANO and then phenotyped for the other drug resistance markers (Table 19.1). In both of the crosses between types 2 and 3, all of the progeny were isolated based on drug resistance, hence the natural frequency of outcrossing versus selfing is unknown (Table 19.1). Expanding on the success of these early interstrain crosses, a genetic cross between the type 1 strain GT-1 (Dubey, 1980) and the type 3 strain CTG (Pfefferkorn et al., 1977) was conducted to investigate the genetic basis of acute

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19. Development and application of classical genetics in Toxoplasma gondii

virulence since these lineages differ dramatically in this phenotype (Su et al., 2002) (Table 19.1). A drug-resistant clone of the GT-1 strain was isolated following chemical mutagenesis and selection with FUDR as described previously (Pfefferkorn and Pfefferkorn, 1977). GT-1 was originally isolated from a goat (Dubey, 1980), and unlike the T. gondii type 1 strain RH is fully capable of undergoing the entire life cycle. The GT-1 FUDRr line was crossed with the CTG strain that was doubly resistant to ARA-A and SNF, described above. Progeny from this cross were initially selected for segregation of these resistance markers to obtain FUDRR-Ara-AR and FUDRR-SNFR clones (Table 19.1). Subsequent to this, clones were isolated randomly and typed by PCR analysis using polymorphic DNA markers (Su et al., 2002). In the absence of any drug selection during the cloning of progeny, 24 of 25 clones had recombinant genotypes, while one clone had the type 3 genotype at all queried markers (Table 19.1). The very high frequency of outcrossing cannot be explained simply by inefficient gamete formation as each strain readily produced oocysts when fed to cats alone (unpublished data). In addition, examination of the inheritance of apicoplast markers revealed that both parental types were capable for forming microgametes and macrogametes, although there was a slight bias for type 1 to form macrogametes (C. Su unpublished data) (Table 19.1). The final pair-wise combination of crosses between the three clonal lineages was completed with the successful crossing of type 1 and 2 strains (Behnke et al., 2011). A sinefungin resistant (SNFr) clone of the type 1 GT-1 strain was crossed with a FUDR resistant (FUDRr) B7 clone of ME49. Following coinfection of cats with a mixture of bradyzoites of the GT-1-SNFr and ME49-FUDRr strains, recombinant progeny were isolated from sporulated oocysts either by selection with the combination of both drugs, or by random cloning and genotyping using

polymorphic DNA markers (Table 19.1). Among the nondrug selected progeny, recombinants were isolated at the expected frequency (i.e., B20% 35% of randomly selected clones were recombinants), and in contrast to the 1 3 3 cross (described earlier), there was no maternal bias in this cross (Table 19.1, Behnke et al., 2011). To increase the diversity represented in genetic mapping a cross using a sinefungin resistant type 10 South American VAND strain was generated (Khan et al., 2014). Interestingly, initial attempts to self the 2 3 10 parental strains in cats resulted in recovered oocysts for the ME49-FUDRr strain but not for VANDSNFr. The failure of VAND to produce oocysts was rescued when both ME49-FUDRr and VAND-SNFr were cofed to a cat, resulting in viable oocysts for both self-mated parental strains, as well as recombinant progeny. Initial genotyping of drug-selected progeny revealed a recombination frequency (12.8%), very close to the expected of 12.5%. Cloned progeny were selected with either 10 PCR-based markers or double drug selection, resulting in 24 informative progeny. Both parents were capable of producing macrogametes, with a slight maternal bias for type 10 VAND (Table 19.1).

19.3.3 Implications of selfing versus outcrossing for population structure Population genetic studies of T. gondii indicate a high level of clonality in North America and Europe, which could be maintained by asexual transmission or by a high proportion of self-fertilization (Sibley and Ajioka, 2008). Occasional hybrids between the clonal types are observed in nature (Su et al., 2012); however, the relative frequency of selfing versus outcrossing is uncertain. In experimental crosses where the frequency of selfing has been monitored, it is typically B50%, with the exception of the 1 3 3 cross (Table 19.1).

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19.4 Development of genetic mapping

Selfing has been linked to outbreaks of toxoplasmosis spread by a waterborne route due to contamination with oocysts as a single point source (Wendte et al., 2010). Despite having a highly clonal population structure (Ajzenberg et al., 2002b; Darde´ et al., 1992; Howe and Sibley, 1995; Sibley and Boothroyd, 1992b), pockets of high genetic diversity also occur in the wild (Ajzenberg et al., 2002a, 2004) suggesting that locally high rates of genetic crossing are important in the population structure of T. gondii. Studies in South America have emphasized a much more diverse genetic structure, suggesting that outcrossing is much more frequent (Khan et al., 2007; Lehmann et al., 2006; Minot et al., 2012; Pena et al., 2008). The observation that some experimental genetic crosses yield a high proportion of outcrossing (i.e., 1 3 3 cross) suggests that this phenomenon contributes to genetic diversity of natural populations, particularly in some regions.

19.4 Development of genetic mapping 19.4.1 Advances in molecular genetic tools A number of technical advances coincided with the development of classical genetic mapping, and these led to synergism in advancing genetic approaches in T. gondii. Establishment of linkage groups was aided by the separation of T. gondii chromosomes by pulsed-field gel electrophoresis (PFGE) and mapping of DNA probes to these gel-separated bands by Southern blot hybridization (Sibley and Boothroyd, 1992a). Large-scale sequencing of ESTs from different strains (Ajioka et al., 1998; Li et al., 2003; Manger et al., 1998), originally undertaken for gene discovery, also allowed the identification of SNPs that facilitated the development of polymorphic genetic markers. The use of PCR-based methods to amplify regions, containing polymorphisms that define

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restriction fragment length polymorphisms (RFLPs), allowed higher throughput genotyping (Su et al., 2002). The development of an Affymetrix array for T. gondii provided an alternative means for genotyping using single feature polymorphisms based on differences in the ESTs, as well as a set of previously derived PCR-RFLP genetic markers (Bahl et al., 2010; Behnke et al., 2015b). Eventually, costs dropped enough to genotype the progeny using whole genome sequencing (WGS; Khan et al., 2014). Completion of whole genome sequences of representatives of the three major lineages T. gondii and establishment of a database for housing genomic data (www.ToxoDB. org; Gajria et al., 2007) provided a framework for localizing genes once initial linkages were established. The assembly of the genome went hand-in-hand with the generation of linkage maps, which were used to identify contigs and scaffolds that belonged to individual chromosomes (Khan et al., 2005b). Without these efforts, along with the various iterations of the Toxoplasma genome database, mapping loci responsible for phenotypic differences would not have led to the identification of the responsible genes.

19.4.2 Development of linkage maps for forward genetic analysis The generation of genetic linkage maps for T. gondii proceeded through several phases. Initially, a set of 64 polymorphic markers that recognized RFLPs detected by Southern blot was used to analyze segregation from a genetic cross between types 2 and 3 (Sibley et al., 1992). Segregation of these markers in 19 recombinant progeny led to the first generation linkage map. At this early stage, 11 chromosomes were recognized and the total genetic map distance was less than 150 cMs. Although low resolution, this map was sufficient to link several drug resistance markers to specific

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19. Development and application of classical genetics in Toxoplasma gondii

chromosomes (i.e., SNF and ARA-A) and to establish the basic parameters of meiotic recombination in T. gondii. The next advance in genetic mapping in T. gondii came with the successful analysis of a cross between the virulent type 1 (GT-1) and the nonvirulent type 3 (CTG) lineages. The RFLP markers were converted to PCR-based typing, and 112 markers were used to generate a linkage map from two parallel crosses (Su et al., 2002). The number of chromosomes remained the same, but the linkage map expanded to B400 cM. Finally, in phase three, an expanded number of PCRRFLP based genetic markers (B250 in all) was used to analyze 71 progeny from genetic crosses between two of the three pair-wise groupings (2 3 3 and 1 3 3) (Khan et al., 2005b). MapMaker EXP 3.0 (Lander et al., 1987) was used to generate linkage maps with a LOD score of .3.0. The precise order and distance between markers was determined based on physical maps that came from assembly of the whole genome sequence using the linkage map. The combined genetic linkage map of T. gondii, derived by combining the results from 2 3 3 and 1 3 3 crosses, consists of 14 linkage groups that collectively comprise 590 cM (Khan et al., 2005b). The chromosomes are largely the same as those identified in earlier studies, with the addition of several new groups that had not been resolved by PFGE and/or had been missed due to low density of markers in the earlier studies. The combined genetic map is based on markers that are spaced approximately every 300 kb across the genome. Individual linkage maps for each putative chromosome and their corresponding markers are shown in Fig. 19.2. As expected, the relative size of each chromosome, based on PFG separation and physical maps from the genome assembly, shows a roughly linear relationship with genetic size in map units (Khan et al., 2005b). The rate of crossover in T. gondii averages about 100 Kb/cM although marked

differences are seen from a low of 42 Kb/cM on chromosome Ia to a high of B150 Kb/cM on chromosome VIIb (Khan et al., 2005b). The average crossover frequency for T. gondii is substantially lower than for Plasmodium falciparum (17 Kb/cM) (Su et al., 1999) making it more difficult to fine map genetic traits in T. gondii. This combined linkage map was also used to assemble contigs from whole genome sequencing into genomic scaffolds that was initially performed on the same B7 clone of ME49. Scaffolds were assembled and ordered into putative chromosomes using the linkage maps and based on the position of genetic markers, along with bacterial artificial chromosomeend and cosmid-end sequences from several large insert libraries (Khan et al., 2005b). More than 95% of the 65 Mb genome was assembled using this strategy, thus providing a framework for genetic mapping and for identify genes within regions of interest. A genetic linkage map was generated for the type 1 3 2 cross based on the segregation of markers among 45 recombinant progeny (Behnke et al., 2011). In this case the polymorphisms were scored based on SNPs detected following the hybridization of gDNA to an Affymetrix array for each of the parental and progeny clones. Because the array is based on the genomic sequence of the type 2 lineage, hybridizations were scored based on perfect match (type 2) versus mismatch (type 1). Using this approach greater than 85% of the genome is represented in this linkage map based on 1603 informative probes (Table 19.1). On average, there are 10 markers for each of the 151 genetic intervals defined by crossovers in this genetic map. Although this map provides a much higher resolution of markers, the precision for mapping is still limited by the number for crossovers, which depends more on the number of progeny than the density of the genetic map. Moreover there are still regions of the map that are invisible in this cross (such as chromosome IV where types 1 and 2 are nearly

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FIGURE 19.2 Genetic linkage maps for T. gondii and polymorphism map (inset) based on the 3 major clonal lineages from Europe and North America. Maps for each of the 14 linkage groups labeled as chromosomes (numbered at the top) were determined by a combination of genetic crosses between types 2 3 3 and 1 3 3. Markers are shown to the right of the vertical line, and genetic distances between each node are shown to the left in centimorgans. Total genetic distances are shown at the bottom of each chromosome. Color code: red indicates polymorphism unique to type 1, green unique to type 2, blue unique to type 3, brown indicates multiple polymorphisms. Inset: map of polymorphism types across the T. gondii genome based on alignments between GT1 (type 1), Me49 (type 2), and VEG (type 3) genome assemblies (ToxoDB.org; version 30), showing large chromosomal segments being dominated by a given SNP type (e.g., chromosomes XI and IV), as well as regions showing more type 2-specific polymorphisms amidst a background of type 1 and type 3 (e.g., chromosomes II and X). Data binned in 1 kb windows and colored by the dominant polymorphism type in that region. Source: Used with permission from Khan A., Taylor S., Su C., Mackey A.J., Boyle J., Cole R. et al., 2005b. Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii. Nucleic Acids Res. 33, 2980 2992.

identical and chromosome XI where types 2 and 3 are nearly identical; Fig. 19.2—inset; Boyle et al., 2006). Interestingly, both T. gondii (Khan et al., 2005b) and P. falciparum (Su et al., 1999) show a high frequency of closely spaced double crossover events. These occur at much closer distances than would be predicted by crossover frequency suggesting they are gene

conversions rather than reciprocal events. These closely spaced double crossovers appear more frequently in the array-based SNP map from the 1 3 2 cross (Behnke et al., 2011), reflecting the greater density of markers in this map. Most recently, the type 2 3 10 cross was mapped using WGS (Khan et al., 2014). The genomes of the two parental strains and

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19. Development and application of classical genetics in Toxoplasma gondii

24 informative recombinant progeny were sequenced using Illumina HiSeq 2000 technology. Alignment of the reads to the parental reference genomes resulted in the identification of 499,470 SNPs used to detect crossover points. The development of the software package REDHORSE allowed for the precise identification of 79 conventional crossovers and 59 double crossovers in the 24 progeny (Shaik et al., 2015). The double crossovers spanned less than 5 kb, and typically were less than 1 kb in length, whereas conventional crossovers occurred over much longer distances. The precise mechanism for how double crossovers are generated in T. gondii are not known, but they are likely to be important for increasing genetic diversity. A majority of the double crossovers occurred within genes, the annotations of which span a range of Kyoto Encyclopedia of Genes and Genomes pathway and GO ontological functions. The total genetic size of the 2 3 10 genetic map was 350 cM, slightly lower than the 590 cM for the combined genetic linkage of the clonal strains, which may have been due to the higher genetic divergence of the VAND parental strain. The 2 3 10 genetic map has 544 markers that specify the crossover points and span the chromosomes.

19.4.3 Limitation of the current linkage maps In the past the existing genetic map has had blind spots since strain-specific haplotypes are conserved across large regions of the genome (Boyle et al., 2006; Minot et al., 2012). This has led to a situation where most of the informative markers on a given chromosome are unique to a given lineage (Khan et al., 2005b). An extreme example of this is chromosome XI where the genetic linkage map is based almost exclusively on progeny from the 1 3 3 cross and based on the PCR-RFLP markers used in multiple crosses this chromosome is effectively

invisible in the 2 3 3 cross (Fig. 19.2—inset). The lack of useful genetic markers is no longer limiting given the existence of whole genome sequences for the parental lines used in all of the T. gondii genetic crosses performed to date. For example, a search on ToxoDB identifies 1189 SNPs distinguishing the type 2 strain Prugniaud from the type 3 strain VEG on chromosome XI, plenty for easy identification after even low-coverage sequencing of F1 progeny from a given cross. In the past the weight placed on one genetic cross compared to another (e.g., 1 3 3 compared to 2 3 3) has impacted the resulting linkage groups that are formed. A consistent theme across multiple iterations of the genetic map is inconsistent, but statistically significant, linkage between markers thought to be on distinct physical chromosomes (e.g., chromosomes VI and VIII and VIIb and VIII; Khan et al., 2005b). For example using PCR-RFLP markers, chromosomes VIIb and VIII are unlinked when analyzing type 2 3 3 cross progeny, but markers on the right arm of VIIb and left arm of VIII show significant evidence for linkage when analyzing the type 1 3 3 (Khan et al., 2005b) and type 2 3 10 cross (Khan et al., 2014). Differences in linkage for these particular chromosomes depending on the queried cross is complicated by the fact that the right arm of chromosome VIIb and the left arm of chromosome VIII is invisible to the PCR-RFLP markers for the 2 3 3 cross, since all of them only distinguish type 1 from strain types 2 and 3 (Fig. 19.2 and its inset). Interestingly, recently published Hi-C data for T. gondii [which examines inter- and intrachromosomal associations; (Rao et al., 2014)], shows strong evidence for Chromosomes VIIb and VIII being a part of the same physical chromosome, joining the right arm of VIIb to the left arm of VIII (Bunnik et al., 2019), a finding that should be investigated further. Importantly, unlike VIIb and VIII, this Hi-C study found no evidence for physical contacts

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19.5 Mapping phenotypic traits by classical genetics

between chromosomes VI and VIII which have shown linkage disequilibrium in quantitative trait locus (QTL) studies, suggesting that not all linkage disequilibrium is due to discrepancies between the genetic and physical maps (Khan et al., 2005b). Obtaining WGS sequence data from existing F1 progeny as well as long read sequencing technologies such as Oxford Nanopore (Jain et al., 2016) or PacBioRS (Rhoads and Au, 2015) should settle these discrepancies.

19.5 Mapping phenotypic traits by classical genetics 19.5.1 Mapping drug resistance The association between drug resistance and specific genetic markers has been evaluated for several phenotypic markers, including FUDR, SNF, and ARA-A (Khan et al., 2005b). Resistance to each of these compounds is primarily determined by a single genetic locus. Their association with specific genetic markers was evaluated using QTL mapping (Lander and Kruglyak, 1995; Lander and Botstein, 1989) to analyze both single-locus effects and secondary loci that influence resistance. QTL analysis showed a single strong association for each of these compounds with no detectable contribution from other loci. The molecular targets for two of these compounds, FUDR and ARA-A, were previously known, and they serve as controls for the precision of mapping by linkage analysis. Resistance to FUDR occurs due to loss of UPRT activity, the gene for which is located in the center of chromosome XI. Mapping FUDR resistance showed perfect correspondence to this locus. Resistance to ARA-A occurs due to disruption of adenosine kinase activity, the gene for which is located at the distal end of chromosome XII between markers AK165 and AK163 (Khan et al., 2005b). The relatively large genomic intervals defined by these mapping

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studies illustrate what continues to be a limitation the linkage mapping in T. gondii: the low rate of recombination limits resolution. The map unit (cM) in T. gondii is approximately 100 kb (Khan et al., 2005b), and therefore a 1-cM region contains 10 20 genes. As mentioned above, while some added level of precision can be gained from genotyping using microarray probe hybridization and/or WGS sequencing, the major limitation is the number of recombination events per chromosome. The target for SNF resistance in T. gondii was identified using genetic mapping and WGS of F1 progeny. In a prior type 1 3 3 cross the SNF resistance phenotype mapped to a region on chromosome IX associated with the markers AK123 to MIC1 (Khan et al., 2005b), although this region was too broad to identify the casual gene located within the locus. In the type 2 3 10 cross the VAND parent was made SNF resistant by chemical mutagenesis (Khan et al., 2014). Since the progeny of this cross were genotyped using WGS, mutations could be identified at the base pair level. As in the 1 3 3 cross, SNF resistance mapped to the middle of chromosome IX in the 2 3 10 cross. This region contained one SNP that was present in all SNF resistant type 2 3 10 progeny resulting in an early stop codon in a putative amino acid transporter, named SNR1 (TGVAND_290860). Several CRISPR/Cas9 gRNAs were used to target the candidate gene confirming SNF resistance results from the disruption of SNR1 (Behnke et al., 2015b).

19.5.2 Mapping quantitative traits Many phenotypic traits such as growth and virulence are not governed by a single locus but rather by the combination of genes at different loci. In addition, other regions of the genome can modulate traits that are strongly influenced by a single locus. Studies by Lander and Botstein (Lander and Kruglyak, 1995;

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19. Development and application of classical genetics in Toxoplasma gondii

Lander and Botstein, 1989; Lander et al., 1987) provided the foundation for genetic analysis of such complex traits. In particular, these studies demonstrated that interval mapping based on genetic markers can reliably map QTLs. The power of QTL mapping strategies for analyzing complex phenotypes has led to great interest in application of this technology in parasites (Su and Wootton, 2004). QTL analysis not only estimates the contributions of different loci but also provides statistical methods to account for the potential of false-positive associations that occur with large datasets (Lynch and Walsh, 1998). Typically, this is expressed as a likelihood ratio that relates the probability that a given association is real versus that it might occur by chance. The outcomes of these calculations are referred to as the log likelihood ratio statistic or log odds ratio (i.e., LOD score) (Lynch and Walsh, 1998). There has been considerable literature about the use of LOD score thresholds to determine the probability of linkage between two randomly segregating genetic markers. An acceptable LOD score threshold of $ 3.3 has been proposed as “significant” at P , .05 when using the human genome. However, QTL mapping tools such as R/qtl allow for more robust measures of genomewide statistical significance thresholds by permutation analyses, and for T. gondii LOD scores much lower than 3.3 have been found to indicate significant linkage between a given trait and underlying causal genes (see later).

19.5.3 Genetic approaches for defining virulence genes One of the main advantages of classical or forward genetics is that it provides an unbiased analysis of complex phenotypes. In other words, genetic linkage mapping has the potential to identify unknown genes that mediate important, naturally occurring biological phenotypes. The strength of this approach is that it

requires no a priori assumptions about the underlying molecular basis of the trait, so long as it differs between natural isolates and it can be reliably measured. Strains of T. gondii differ substantially in their ability to cause disease in laboratory mice. More specifically, strains of the type 1 genotype are acutely virulent in the mouse model. Type 1 strains are uniformly lethal in most inbred mice, and even at low inocula, infected animals do not survive challenge with tachyzoites given by i.p. inoculation (translates into an effective LD100 of a single organism) (Howe et al., 1996; Sibley and Boothroyd, 1992b). Although laboratory passage might affect this trait, the commonly used RH strain was reported to be virulent for laboratory mice on primary isolation (Sabin, 1941), and similar high virulence was reported among a collection of type 1 isolates (Sibley and Boothroyd, 1992b). In contrast, type 2 strains show intermediate virulence (i.e. LD50 103 105 depending on mouse strain), and lab mice can survive relatively high infections (105 106) with type 3 strains (Howe et al., 1996; Saeij et al., 2006; Sibley and Boothroyd, 1992b; Walzer et al., 2013). Although more susceptible to toxoplasmosis than natural hosts of transmission such as wild mice (Frenkel, 1953; Lilue et al., 2013), rats (Dubey and Frenkel, 1998; Wang et al., 2019) or chickens (Dubey, 2008), laboratory mice offer the potential to uncover the molecular basis of differences in acute virulence between natural isolates of T. gondii. 19.5.3.1 Mapping differences in the type 1 3 3 cross Quantitative trait mapping was initially used to evaluate the acute virulence of T. gondii in the mouse model using the progeny of a type 1 (virulent) 3 3 (avirulent) cross (Su et al., 2002). GT-1 has a virulent phenotype typical of type 1 strains, such that a single viable organism is lethal in outbred laboratory mice, while CTG has an LD50 of .106 in outbred mice.

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19.5 Mapping phenotypic traits by classical genetics

Twenty-six F1 progeny clones were initially tested to establish their virulence phenotype in mice following inoculation with 101, 102, or 103 tachyzoites and virulence was defined based on cumulative mortality. Virulent clones did not give rise to chronic infection (all infected animals died), while other clones showed intermediate or low levels of virulence (Su et al., 2002). Phenotypic analysis of recombinant progeny revealed a range of phenotypes from fully virulent to nonvirulent and including intermediate levels not seen in either parental strain (Su et al., 2002). When acute mortality was considered as single gene model, it was mapped to a single QTL in the central region of chromosome VIIa (Su et al., 2002). These results confirmed that virulence was largely heritable rather than epigenetic and that it was primarily controlled by a single locus (Su et al., 2002). This finding was an important conceptual advance, validating the use of forward genetics to analyze the molecular basis of virulence. However, at this early stage, the genome was not yet sequenced or assembled, hence it remained only a hypothetical possibility to map and identify individual genes that conferred enhanced virulence. Completion of the genome sequence of the type 2 strain ME49 and the assembly of the genome using the combined genetic map (Khan et al., 2005b) made it possible to map traits to defined genomic intervals and to identify genes that controlled important biological phenotypes. Differences between the virulent type 1 and avirulent type 3 lineages were reexamined using an expanded set of 34 unique clones typed with 175 different PCRRFLP based markers (Taylor et al., 2006). In addition to acute virulence, these strains also differ in their ability to migrate across polarized monolayers, to migrate under agarose in vitro (Barragan and Sibley, 2002) and in their intrinsic growth rate (Radke et al., 2001). Differences in migration in vitro exist between the type 1 (high) and 3 (low) parental strains

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(Taylor et al., 2006). The migration potential of the progeny and differences in acute virulence in the mouse varied continuously with some progeny showing phenotypes more extreme than one parent or the other (Taylor et al., 2006). Analysis of these traits by QTL mapping revealed that migration in vitro, transmigration across polarized monolayers, acute mortality (i.e., virulence), and the serum response of surviving mice all mapped to a single peak on chromosome VIIa (Taylor et al., 2006). The single QTL on chromosome VIIa was predicted to account for B65% of the variance in virulence between the parental types, leaving open the possibility that other loci contribute smaller effects that were not mapped due to the low number of progeny examined. Finer mapping of the peaks for acute virulence and serum response of surviving animals showed perfect correspondence on chromosome VIIa. Addition of new markers within this locus, and several additional recombinant clones with crossovers on chromosome VIIa, allowed finer mapping (Fig. 19.3A) (Taylor et al., 2006). To further refine the choice of candidate genes within this region, RNAs from the parental stains were hybridized to an Affymetrix array for T. gondii (Bahl et al., 2010), revealing a marked difference in one gene on the right end of the original QTL locus: expression levels for this gene in a type 3 strain were B100-fold lower than that in type 1 (Taylor et al., 2006) (Fig. 19.3B). This differentially expressed gene was annotated as rhoptry protein 18 (ROP18) based on a previous proteomic study of purified rhoptry contents examined by mass spectrometry (Bradley et al., 2005). ROP18 contains a signal peptide and a prodomain that is processed during secretion, giving rise to a mature protein that consists of an N-terminal amphipathic helical region and a C-terminal serine/threonine (S/T) protein kinase domain (Fig. 19.3C). Rhoptries are organelles that discharge their contents into the host cell during attachment and invasion

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FIGURE 19.3 Fine mapping of the acute virulence phenotype in the type 1 3 3 cross. (A) Plot of acute virulence and serum responsiveness showing the prominent peak on chromosome VIIa, which accounts for .65% of the variance in these traits. (B) Microarray showing hybridization of cRNA to multiple genes in the center of this region identifies a prominent difference in gene 20. This gene, annotated as rhoptry protein 18 (ROP18) is poorly expressed in the type 3 strain by B100 fold compared to type 1. (C) Model of the domain structure of ROP18 revealing a signal peptide, prodomain that is cleaved during maturation, N-terminal low complexity region involved in membrane targeting, and a S/T protein kinase domain. (D) Electron micrograph showing discharge of rhoptries during invasion, releasing ROP proteins into the host cell cytoplasm and the lumen of the parasitophorous vacuole. Image provided by Wandy Beatty. Scale bar 5 100 nm. serine/threonine (S/T) Source: Used with permission from Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., et al., 2006. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314, 1776 1780.

(Ha˚kansson et al., 2001; Nichols et al., 1983) (Fig. 19.3D). Confirmation that ROP18 was indeed responsible for the differences in acute virulence between types 1 and 3 relied on reverse genetic approaches, which have been extensively developed in T. gondii (see Chapter 17: Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages in this volume). Taking advantage of the fact that ROP18 gene expression is highly reduced in the type 3 background, transgenic parasites were generated to express the type 1 ROP18 allele at levels similar to that in the type 1 strain (Taylor et al.,

2006). Restored expression of a kinase-active form of ROP18 in a type 3 recipient conferred enhanced virulence that nearly matched the type 1 parental strain (Taylor et al., 2006). However, expression of a mutant form of ROP18, where the catalytic Asp had been changed to Ala, failed to enhance virulence (Taylor et al., 2006). These studies confirmed that ROP18 is largely responsible for the differences in acute virulence between types 1 and 3 in the laboratory mouse. The phenotypes of transmigration and enhanced in vitro migration also map to a region on chromosome VIIa; however, they are not mediated by ROP18, nor is the QTL for growth on chromosome VIIa

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related to ROP18 (Taylor et al., 2006). Rather these traits are potentially mediated by additional genes found in this relatively broad region of the genome. Clues to the molecular function of ROP18 came from its cellular location. Following injection during host cell invasion, ROP18 is targeted to the cytoplasmic surface of the parasitophorous vacuole (El Hajj et al., 2007; Taylor et al., 2006). The N-terminal amphipathic helical region is responsible for tethering ROP18 on the cytoplasmic surface of the parasitophorous vacuole membrane (PVM) (El Hajj et al., 2007; Reese and Boothroyd, 2009), and this localization is essential to its virulence enhancing potential (Fentress and Sibley, 2012). On the PVM, ROP18 targets host immunity related GTPases (IRGs), which are normally recruited to the vacuole causing its vesiculation and destruction (Howard et al., 2011). Destruction by IRGs is strain dependent, and type 1 strains avoid the IRG pathway, while types 2 and 3 undergo IRG recruitment,

leading to vesiculation and destruction in activated macrophages (Khaminets et al., 2010; Zhao et al., 2009). ROP18 was shown to directly phosphorylate a number of IRGs on key threonine residues in switch region 1, thus presumably inactivating the GTPase domain and preventing oligomerization (Fentress et al., 2010b; Steinfeldt et al., 2010) (Table 19.2). In separate studies based on a yeast 2-hybrid screen, it was shown that ROP18 also phosphorylates ATF6β (Table 19.2), an endoplasmic reticulum stress response transcription factor (Yamamoto et al., 2011). Mice lacking ATF6β show decreased antigen presentation by dendritic cells to CD81 T cells (Yamamoto et al., 2011), suggesting phosphorylation of this factor by ROP18 might alter adaptive immunity. These same type 1 3 3 cross progeny were used to genetically map a superinfection phenotype where chronically infected mice were susceptible to reinfection with a number of South American strains, as well the type 1 parent of the 1 3 3 cross (GT-1) (Jensen et al.,

TABLE 19.2 Summary of phenotypes mapped in genetic crosses between clonal strains of Toxoplasma gondii. Genetic cross

Phenotypes

Chromosomes with QTLs

Genes

Function

133

Acute virulence

VIIa, Ia

ROP18

Phosphorylates IRGs,

Growth

VIIa, XI, XII, Ia VIIa

Migration a

233

132

ATF6β

Superinfection

XII

ROP5

Activates ROP18, binds IRGs

Acute virulence

VIIa, VIIb, XII

ROP18

Targets IRGs

ROP5

Activates ROP18, Binds IRGs

Host gene expression

VIIb

ROP16

Phosphorylates STAT3/6

IL-12 induction

X

GRA15

Activates NF-κB

Acute virulence

XII

ROP5

Activates ROP18 Binds Irga6

2 3 10

Acute virulence

XII

ROP5

Drug resistance

IX

SNR1

a

Amino acid Transporter

From Jensen et al. (2015). IRGs, Immunity related GTPases; QTLs, quantitative trait locus; STAT, signal transducer and activator of transcription.

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2015). Using selected F1 progeny that harbored the type 1 allele for ROP18 (the major virulence determinant in that particular cross), differences between GT-1 and the type 3 parent in the ability to cause disease in previously infected mice mapped to the SAG3 marker on chromosome XII (LOD 4.1; genome-wide P-value , .05). SAG3 is the genetic marker with the closest proximity to another major T. gondii locus determining virulence differences between T. gondii strains, ROP5 (discussed in Sections 19.5.3.2 and 19.5.3.3), and indeed the type 1 ROP5 allele was found to be strongly associated with superinfection ability (Jensen et al., 2015). This particular study highlights the long-term utility of F1 cross-progeny to map new phenotypes as they emerge, and the use of selective phenotyping to more precisely hone in on phenotypes that might be masked by other more dominant loci (like ROP18). 19.5.3.2 Mapping differences in the type 2 3 3 cross Type 2 and 3 strains have been categorized as similarly “avirulent,” but depending on the context infection outcomes with the members of these two lineages can be quite different. For example, in BALB/c mice type 2 strains are 2 3 orders of magnitude more pathogenic compared to type 3 parasites when mouse morbidity is quantified (Saeij et al., 2006). Initial studies on the progeny of a type 2 3 3 cross showed that recombination of genes can lead to higher levels of virulence than is seen in either of the parental strains, albeit still not to the extreme level exhibited by type 1 strains (Grigg et al., 2001a). Further analysis of the progeny from this cross was used to identify loci determining multiple traits: time to death following low (100 parasites i.p.) or high (100,000 parasites i.p.) dose challenge, as well as a binary trait indicating the presence of mortality (or not) at any dose. During the primary QTL scan a locus named VIR1 was located near the SAG3 genetic marker on chromosome

XII (Saeij et al., 2006) (Fig. 19.4A). When VIR1 was treated as a covariant, it revealed four additional QTLs that showed varying levels of statistical support, with genome-wide logarithm of odds (LOD) scores ranging from 2.5 to 3.5) (Saeij et al., 2006) (Fig. 19.4B). Comparison of other features such as degree of polymorphism, differences in expression level, and likelihood of being secreted, and hence interacting with the host, led to the identification of ROP18 as the candidate locus for VIR3 on chromosome VIIa (Saeij et al., 2006). Quantitative PCR revealed that ROP18 was underexpressed by .1000 fold in type 3 relative to type 2, which expresses similar levels to type 1 (the fold differences reported here versus above are likely due to sensitivity of microarrays vs qPCR). Generation of transgenic type 3 parasites expressing the type 2 ROP18 allele also led to enhanced virulence, confirming the role of this kinase in pathogenesis of T. gondii (Saeij et al., 2006). In comparing the two genetic crosses, it seems evident that the differences in expression level of ROP18 are largely responsible for the extremely low virulence of type 3, since expression of either the type 1 or type 2 alleles is sufficient to rescue this phenotype (Table 19.2). The type 1 and 2 alleles are highly polymorphic, having 28 amino acid polymorphisms between them (Walzer et al., 2013), and this high polymorphism rate is accompanied by a high dN/dS ratio and strong evidence for positive selection at this locus (Boyle et al., 2006). However, when directly compared, their ability to alter in vivo proliferation profiles when expressed in the poor ROP18expressing type 3 genetic background is nearly identical (Walzer et al., 2013). To date, the impact of this high polymorphism rate is unknown. The major differences in ROP18 expression have been traced to a difference upstream of the gene where the type 1 and 2 alleles have indels that distinguish them from type 3 strains as well as the related parasite N. caninum (Boyle et al., 2008; Khan et al., 2009).

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FIGURE 19.4 Genetic mapping of virulence phenotypes of F1 progeny from type 2 3 3 crosses. BALB/c and CBA/J mice were infected with 100,000 or 100 tachyzoites from 40 different F1 recombinant progeny from 2 3 3 crosses, and mortality was recorded daily for 40 days. As there were no significant differences between the two different mouse strains, results were pooled. Three phenotypes are represented: (1) “high-dose survivability,” log10 survival time (in days) after injection of 100,000 parasites (black line); (2) “avirulence,” a binary trait defined as no mortality at any dose (red line); (3) “low-dose survivability,” log10 survival time (in days) after injection of 100 parasites (blue line). Plots indicate the loglikelihood association of phenotypes with markers aligned across the genome. Marker positions (in cM) are given by tick marks. Significance levels as determined by 1000 permutations are indicated by horizontal lines [upper lines are significant; lower line is suggestive (P 5 .1)]. Because the significance levels for all three of the phenotypes differed by less than 0.1 LOD unit, only one significance line is drawn for all three. (A) Primary genome scan. (B) Secondary genome scan after the effect of the major virulence peak on chromosome XII, evident in (A) and cosegregating with the SAG3 marker, is neutralized by making it a covariate. (C) Table summarizing virulence QTLs identified from the 2 3 3 cross and if known the genes responsible for the virulence phenotype. QTLs, quantitative trait locus. Source: Used with permission from Saeij, J.P.J., Boyle, J.P., Coller, S., Taylor, S., Sibley, L.D., Brooke-Powell, E.T., et al., 2006. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314, 1780 1783.

Multiple studies have shown that upstream sequences from types 1 and 2 are effective promoters while the type 3 sequence is hundreds to thousands of times less effective (Boyle et al., 2008; Khan et al., 2009). Therefore the most critical feature that determines the ability of a given ROP18 allele to confer heightened

virulence in the mouse model is the expression level determined by its upstream sequences. Interestingly, both the low-expression type 3 allele and the higher expression type 2 and 1 alleles show evidence of long-term stability in the wild, suggesting that they are adapted for different niches in nature (Khan et al., 2009).

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In addition to ROP18 (aka “VIR3” in Saeij et al., 2006), four additional virulence QTLs were identified using data from the type 2 3 3 genetic cross (named VIR1-5; Fig. 19.4C), and in some cases the genes underlying those QTLs are known. VIR1 was the virulence QTL with the highest LOD score and has since been shown to be driven by heightened virulence determined by genes within the type 1 and 3 ROP5 loci compared to type 2 (Behnke et al., 2011; Reese et al., 2011). For VIR2, on the left arm of chromosome X, two candidate genes were proposed at the time (42.m03493 and 42.m03409) which are now named TGME49_226380 and TGME49_225160. TGME49_226380 is annotated as GRA35 based on its localization to the dense granules (Nadipuram et al., 2016) and mediates inflammasome activation in rat cells (Wang et al., 2019). While this makes GRA35 a compelling candidate for the VIR2 QTL gene, allelic differences between type 2 and 3 strains and their impact on the rat inflammasome phenotype have not been investigated. VIR4 is due to ROP16 (Saeij et al., 2006, 2007) which operates by modulating STAT3 and STAT6 activity in the host cell (see Section 19.5.4.2), while VIR5 has not yet been confirmed but was proposed to be due to adenosine kinase (which was mutated in the type 3 parental background). As shown in Fig. 19.4B, the right arm of chromosome X had a slight QTL peak that was not statistically significant (black line), but this locus encompasses the gene product encoding GRA15 (Rosowski et al., 2011) which modulates the activity of host NF-κB. 19.5.3.3 Mapping differences in the type 1 3 2 cross Another cross was performed between the type 1 strain GT-1 and type 2 strain ME49 (Behnke et al., 2011). Analysis of virulence differences among progeny from this cross was based on mortality in groups of outbred CD-1 mice challenged by i.p. inoculation with 100

parasites. Using this dose, the parental GT-1 strain causes complete mortality, while ME49 shows no lethality. The phenotypes of 45 F1 progeny were analyzed from this cross, revealing a wide range of virulence in outbred mice (Behnke et al., 2011). QTL analysis of the mortality data revealed a single strong peak on the left arm of chromosome XII (Fig. 19.5A) (Table 19.2). This QTL accounted for B90% of the variance in virulence between the strains (Behnke et al., 2011). Neither ROP18 nor ROP16 showed QTL peaks (Fig. 19.5A), indicating that these genes either contribute similarly to acute virulence in the type 1 and 2 backgrounds or the low dose used and/or mouse genetic background was such that their impact was not revealed using a strict mortality-based phenotype. The major virulence QTL on chromosome XII spans B400 kb and includes 51 genes flanked by several single-feature polymorphism probes that were informative in this cross (Fig. 19.5B). To identify the responsible gene within the chromosome XII QTL, differences in copy number were estimated from hybridization of gDNA, while differences in expression level were determined from cDNA hybridization to the T. gondii Affymetrix array. Together these approaches identified a cluster of polymorphic genes, all of which encoded different paralogs of the pseudokinase ROP5. ROP5 had previously been identified as a pseudokinase related to ROP2 (El Hajj et al., 2006), and with some similarity to ROP18, although it had no known role in virulence. To confirm the importance of ROP5, deletion of the entire locus in the type 1 RH strain lead to highly attenuated parasites, which were rescued by reexpression of ROP5 from a type 1 cosmid (Behnke et al., 2011). Reassembly of the genomic locus using Sanger-based whole genome shotgun sequencing data from types 1, 2, and 3 revealed two different patterns of gene arrangement (Fig. 19.5C). In types 1 and 3 a cluster of six genes is found, where the

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FIGURE 19.5 Genetic analysis of acute virulence in a cross between type 1 and 2 strains. (A) Genome wide associate of genetic markers defined by allelic differences detected by hybridization to Affymetrix arrays. A single strong peak was mapped on chromosome XII, while previously mapped genes for ROP16 and ROP18 play no role in virulence differences between these strains. (B) Enlargement of the region on the left end of XII reveals B50 genes lie within this region, including a polymorphic cluster of pseudokinases known as ROP5 that shows copy number variation. (C) Diagram of the genetic loci encoding ROP5 variants in the three lineages. Types 1 and 3 contain almost identical loci consisting of a major allele that contains the catalytic triad KHD (marked H). In contrast, type 2 strains contain more copies, dominated by a major allele that has a catalytic triad of KRD (marked R). Alleles marked with * represent minor variants. ψ denotes pseudogene. Source: Used with permission from Behnke, M.S., Khan, A., Wootton, J.C., Dubey, J.P., Tang, K., Sibley, L.D., 2011. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc. Natl. Acad. Sci. U.S.A. 108, 9631 9636.

dominant alleles are the same and are characterized by an incomplete catalytic triad consisting of KHD (Fig. 19.5C). In contrast, type 2 contains B11 genes in tandem (two of which are pseudogenes), where the major allele has number of differences from type 1 or 3, including having an incomplete catalytic triad consisting of KRD (Fig. 19.5). Consistent with higher copy number, expression levels of ROP5 are actually higher in type 2 parasites, indicating that unlike ROP18, expression level

is unlikely to be the basis for its contribution to virulence. The functional significance of ROP5 allelic differences is becoming more clear. Genetic studies demonstrate that the type 1 allelic cluster is associated with enhanced virulence, while that in type 2 is associated with lower virulence (Behnke et al., 2012). A similar role for ROP5 was also found in reanalysis of the progeny of the 2 3 3 cross, leading to the identification of the major peak on chromosome XII

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as ROP5 (Reese et al., 2011). In this context the type 3 allele was again associated with higher levels of virulence, while the type 2 allele was associated with lower virulence. In this study the impact of divergent ROP5 paralogs was assayed in the ΔROP5 background, and complementation with a single or even a double copy of an individual paralog did not fully complement the parental virulence phenotype. Interestingly, parental virulence was only recovered after simultaneous complementation with two distinct ROP5 paralogs (denoted as “A” and “B”; Reese et al., 2011), suggesting slightly distinct functions of individual ROP5 paralogs. Although ROP5 is catalytically inactive (Reese and Boothroyd, 2011), it plays a major role in virulence, and studies indicate it does so in combination with ROP18 (Behnke et al., 2012; Etheridge et al., 2014). Pseudokinases in other systems have recently been recognized as playing important roles in the regulation of active kinases (Boudeau et al., 2006; Zeqiraj and van Aalten, 2010). Subsequent studies have shown that ROP5 and ROP18 act together to disrupt IRG accumulation on the PVM, in part by binding of ROP5 to IRGs, and the ability of ROP5 to directly enhance the enzymatic activity of ROP18 (Behnke et al., 2012; Fleckenstein et al., 2012; Niedelman et al., 2012). ROP5 and ROP18 can also collaborate to disrupt the binding of another host antiparasitic factor, the guanylate binding proteins (GBPs) (Virreira Winter et al., 2011) 19.5.3.4 Mapping differences in crosses to “exotic” lineages The aforementioned crosses all used strains representing the three clonal lineages found throughout Europe and North America, and each was crucial in identifying parasite virulence factors. Whether these or other unknown factors contributed to virulence in genetically divergent South American strains was unknown. A cross between the intermediately

virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain was used to map the genetic basis for differences in virulence in the mouse (Behnke et al., 2015a), as well as to identify the SNF-resistance gene. Virulence of the type 2 3 10 progeny was assessed in CD-1 mice. There were 7 progeny that were avirulent, 14 progeny that were virulent killing $ 75% of the mice, and 3 with an intermediate phenotype. A genome-wide primary scan of the virulence phenotype generated one significant peak on the left side of chromosome XII, a locus that spanned the previously identified ROP5 gene. This peak accounted for 71% of the variance in the virulence phenotype. A secondary scan failed to find additional peaks, although a two-locus scan found a significant interaction between the primary peak on XII and chromosome VIIa, which contains ROP18. Based on the results from previous crosses, ROP5 became the primary candidate gene in the peak on XII. The VAND strain is predicted to contain eight copies of ROP5, all likely expanded in tandem. To delete this large region, two CRISPR/CAS9 gRNAs were designed targeting both sides of the ROP5 locus. Two VANDΔrop5 clones were selected and shown to be avirulent in CD-1 mice. Complementation of VANDΔrop5 with the type 1 RH TOXOM52 cosmid restored virulence. These results confirm that the major difference in virulence between ME49 and VAND is due to ROP5. Because ROP5 was mapped in several crosses, its contribution to virulence in other South American strains was of interest. Indeed, ROP5 knockouts in the virulent South American type 4 TgCtBr18 and type 8 TgCtBr5 strains resulted in avirulent parasites, which was restored with complementation. Given ROP18 was mapped in two crosses and has a two-locus interaction in the 2 3 10 cross, its contribution to virulence in South American strains was also implicated. CRISPR knockouts

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of ROP18 in VAND, TgCtBr18, and TgCtBr5 all resulted in completely avirulent parasites. This was in contrast to ROP18 knockouts in type 1 strains that are still lethal to mice but with either a delayed time to death (RHΔrop18) (Fentress et al., 2010a) or a change in lethal dose (GT-1Δrop18) (Shen et al., 2014). This indicates that both ROP5 and ROP18 are major virulence factors in South American strains. The convergence onto the ROP5 locus using such a wide variety of genetic crosses indicates that types 1 and 3 share the “more virulent” form of ROP5, which on its surface might seem counterintuitive given the significant virulence differences between the type 1 and 3 lineages. However, this is consistent with the fact that T. gondii is a sexual organism and harbors multiple virulence loci (two that have a dramatic phenotypic impact but most certainly many others that will prove to have more moderate effects on infection outcome). Unlike bacterial pathogens, these loci are not clustered in pathogenicity islands but rather spread throughout the genome, and the presence or absence of the “virulent” allele for a given locus is not sufficient to make predictions about the level of virulence associated with a given strain.

19.5.4 Expression quantitative trait locus mapping Jansen and Nap were the first to propose the use of genome-wide expression profiles, at that time generated by microarrays, in a segregating population to better understand the molecular basis underlying QTLs (they proposed the term “genetical genomics” for this type of analysis) (Jansen and Nap, 2001). The principal idea behind genetical genomics is that gene expression itself is a quantitative trait that can be mapped; these QTLs are called expression QTLs or eQTLs. The integration of eQTLs and QTLs for complex traits can provide insights into the molecular basis of the

881

complex trait. For example, if the differences in expression of many genes map to the same physical location as the complex trait, analyses of functional overlap (e.g., regulation by the same transcription factor, belonging to same signaling pathway) among these genes could provide insights into the molecular pathways that determine the complex trait. This approach can be particularly powerful in a host parasite system like T. gondii, where RNA from both host and parasite can be harvested and analyzed using both host and parasite-derived microarrays or simultaneously using RNA sequencing. From the perspective of parasite transcriptional activity, this approach can leverage extant strain-specific differences in gene expression to identify regulatory motifs for individual genes (via identification and follow up of cis-mapping eQTLs) as well as strain-specific global regulators of transcription (via identification of trans-mapping eQTLs). This approach has also been used to identify T. gondii genes that directly modulate the host transcriptome in a strain-specific manner, where transcript abundance of thousands of host genes are measured as quantitative traits and linked back to the parasite genetic map. 19.5.4.1 Using eQTL mapping to characterize mechanisms of strain-specific gene regulation in Toxoplasma T. gondii-specific spotted cDNA microarrays were the first platform upon which T. gondii gene expression at thousands of loci was assessed as a quantitative trait (Boyle et al., 2008). In this study, labeled cRNA from 2 3 3 F1 progeny clones was hybridized to two different spotted cDNA arrays (one representing tachyzoite gene expression and another representing genes expressed by bradyzoite stages; Cleary et al., 2002; Hehl et al., 1997). Expression profiles were analyzed as a quantitative trait to identify gene expression changes that significantly segregated among the F1 progeny and

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significantly associated with a given genetic marker (or markers). Genome-wide significance was determined using permutation of each individual spot on the microarray. Overall these data identified 16 T. gondii genes with robust differential expression phenotypes which could be mapped to segregating markers in the genome. While again limited by the recombination rate, the assumption was that these represented “cis” eQTLs, where the sequences that drove the differences in hybridization intensity across the F1 cross progeny were within, or at least near, the gene itself. This turned out to highlight a rather interesting set of genes, some of which were found to play critical roles in mediating T. gondii strain-specific phenotypes. One of these was ROP18, which as mentioned above is an important determinant of pathogenesis in the mouse model and is also much more highly expressed in the type 2 parent compared to that of type 3. This difference in expression turned out to be driven by a key set of indels in the sequences just upstream of the ROP18 start codon that effectively rendered the type 3 ROP18 promoter null (Boyle et al., 2008; Saeij et al., 2006). Another eQTL controlling hybridization intensity for T. gondii gene TGME49_234380 (annotated as 46. m01601 at the time; Boyle et al., 2008) showed a similar dependence upon polymorphic upstream regulatory sequences, and 7 polymorphisms found within the upstream B600 bp of the transcriptional start site from the type 3 allele were found to increase luciferase reporter gene expression compared to the type 2 allele (Boyle et al., 2008). The role for this gene in mediating strain-specific biological differences is not yet known. Finally, there was a significant “cis” eQTL for gene TGME49_220950 (formerly 41.m01274; Boyle et al., 2008), again where the type 3 parent and all F1 progeny with a type 3 allele had higher transcript abundance compared to type 2. This gene has since been identified as

being responsible for host mitochondrial association (HMA) in T. gondii, a phenotype where host mitochondria are tethered to the parasitophorous vacuole, and has been named Mitochondrial Association Factor 1b (Fig. 19.6A; MAF1b; Adomako-Ankomah et al., 2016; Blank et al., 2018; Pernas et al., 2014). After it was discovered that HMA was strainspecific in T. gondii (where type 2 strains lack this phenotype compared to strain types 1 and 3), the HMA phenotype was genetically mapped to chromosome II (Fig. 19.6B), and MAF1 was identified as one of a number of candidate genes within this QTL for a number of reasons, including its differential transcript abundance between type 2 and 3 strains (Fig. 19.6C). Deletion of the MAF1 gene in type 1 T. gondii dramatically reduced HMA, while complementation of a type 2 strain (which is naturally HMA-deficient; Pernas et al., 2014) with the type 1 MAF1 allele restored the HMA phenotype (Pernas et al., 2014) (Fig. 19.6D). While it is clear that MAF1 gene expression itself is a heritable trait (Fig. 19.6C), the precise mechanism for this difference is unknown. This is complicated by the fact that the MAF1 locus does not harbor a single copy gene, but rather a gene cluster where extensive tandem duplication and diversification has occurred (Adomako-Ankomah et al., 2016; Pernas et al., 2014). The locus harbors two classes of MAF1 paralogs (“a” and “b”,) with only “b” paralogs being capable of mediating the HMA phenotype when used to complement type 2 strains (Adomako-Ankomah et al., 2016; Blank et al., 2018; Pernas et al., 2014). When analyzed separately, the “a” paralogs are well expressed in all strains, while transcript abundance for “b” paralogs is significantly lower in type 2 strains. However, despite the fact that transcript levels for “b” paralogs are still detectable by qPCR and microarray (Fig. 19.6C), MAF1b is undetectable at the protein level by both immunoblot (Fig. 19.6C) and immunofluorescence (Adomako-Ankomah et al., 2016; Blank et al., 2018; Pernas et al., 2014).

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FIGURE 19.6 Host mitochondrial association is strain-specific in Toxoplasma gondii and maps to a locus on chromosome II. (A) HFFs were labeled for 30 min at 37 C with 50 nM MitoTracker and then infected with type 1 (RH), type 2 (ME49), or type 3 (CEP) strains of T. gondii. Cells were fixed 4 hpi and visualized by fluorescence microscopy. Phase, fluorescence (MitoTracker), and merged images are shown for each type. Scale bar, 5 μm. Transmission electron micrographs depicting the PVM surrounding type 1, 2, and 3 parasites grown in HFFs. Cells were fixed and processed for electron microscopy at 4 hpi. Host mitochondria are indicated by M and parasites by P. Percentage of the PVM associated with mitochondria in type 1, 2, and 3 vacuoles as determined by ImageJ analysis of electron micrographs (n 5 20 for each). Values shown are mean 6 SEM. ****P , .0001 using an unpaired t test. (B) LOD Score plot indicates the log-likelihood of association of HMA with markers across the entire Toxoplasma genome in numerical chromosome order. The only significant peak spanned the entirety of chromosome II since there were very few progeny with recombination events on that chromosome. (C) MAF1 gene expression in types 1, 2, and 3 and multiple 2 3 3 F1 progeny as determined by Affymetrix microarrays (left) showing the reduced MAF1 transcript abundance in the type 2 strain compared to types 1 and 3 as well as in all F1 progeny with the type 2 MAF1 allele. Immunoblot (right) shows that what we now know is MAF1b is not detectable in type 2 strains. (D) The percentage of the PVM associated with mitochondria at 4 hpi with type 1 and type 1:Δmaf1 in HFFs, and BMMs at 17 hpi with type 2 and type 2:MAF1b (TGGT1_253770, AKA TGGT1_053770) vacuoles were determined by ImageJ analysis of electron micrographs (n 5 20 for each). Values shown are mean 6 SEM. ****P , .0001 using an unpaired t test. HFFs, Human foreskin fibroblasts; HMA, host mitochondrial association; PVM, parasitophorous vacuole membrane. Source: Used in accordance with editorial policies from Pernas, L., Adomako-Ankomah, Y., Shastri, A.J., Ewald, S.E., Treeck, M., Boyle, J.P., et al., 2014. Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS Biol. 12, e1001845.

The mechanism for selective transcriptional and/or translational control at this locus certainly bears further investigation, as it appears that multiple copies of the MAF1b gene are effectively silenced while their neighboring MAF1a genes are highly transcriptionally active.

To date the only well-characterized eQTLs in T. gondii are those that map very strongly in cis. While it is clear that cis-mapping eQTLs tend to have much higher LOD scores than trans eQTLs, it was somewhat surprising that no reliable trans eQTL loci have been identified that control the expression of a number of

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downstream target genes. All studies to date have used the tachyzoite stage, and it remains to be seen if bradyzoites, or even sporozoites, may be more amenable to the detection of trans eQTLs. 19.5.4.2 Cross-species eQTL mapping: identifying Toxoplasma loci that affect host gene expression One hypothesis to explain Toxoplasma strain differences in virulence is that strains differ in the modulation of host cell signaling pathways, which could eventually determine differences in virulence. Indeed, microarray analysis of human foreskin fibroblasts (HFFs) infected with a type 1, type 2, or a type 3 strains

demonstrated that these strains differed significantly in the modulation of host gene expression. To determine the genetic basis of this strain-dependent regulation of host gene expression, HFFs were infected with 19 unique F1 recombinant progeny derived from crosses between type 2 and type 3 strains (Sibley et al., 1992), and microarrays were used to determine human gene expression levels. QTL analyses showed that the differences in expression levels of 3188 human cDNAs after infection with the 19 F1 progeny could be mapped to a specific T. gondii genomic locus (Fig. 19.7A and B; Table 19.3). A large fraction of these (38%) mapped to T. gondii chromosome VIIb, 18% mapped to chromosome X, 14% to chromosome

FIGURE 19.7 (A) HFFs were infected with type 1, 2, or 3 Toxoplasma strains, or 19 F1 progeny from a type 2 and a type 3 parent. At 24 hours postinfection, expression profiles were obtained using human cDNA arrays. The averaged results (from at least three biological replicates) for median-centered expression levels for cDNAs are displayed using a log2 blue (low) to yellow (high) scale. Values log2 . 2 or log2 , 2 2 were assigned the values 2 and 22, respectively. Also displayed is the unsupervised clustering of experiments. (B) For each Toxoplasma chromosome, the number of cDNAs that mapped significantly (P , .05, permutation test) to a genetic marker on that chromosome is shown (left). Plots indicate the loglikelihood association of expression of the human cDNAs for RGS4 (solid line), JAK2 (dashed line), and SOCS1 (dotted line) with markers aligned across the entire Toxoplasma genome in chromosome order, center. Select chromosomes are indicated (middle). An enlargement of center plot focusing on chromosome VIIb with the names of genetic markers indicated (right). (C) Type 2 parasites expressing HA-tagged type 1 ROP16 (type 2:ROP161, left), type 2 ROP16 (type 2:ROP162, middle) or type 1 ROP16 with a mutated NLS (type 2:ROP161NLSmut, right) were added (MOI 10) to HFFs and cells were fixed after 4 h. ROP16 was visualized using anti-HA antibodies followed by incubation with secondary (anti-rat Alexa 488) antibodies, showing robust localization of ROP16 to the host nucleus except for the NLS mutant. HFFs, Human foreskin fibroblasts. Source: Reprinted with permission from Saeij, J.P., Coller, S., Boyle, J.P., Jerome, M.E., White, M.W., Boothroyd, J.C., 2007. Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445, 324 327.

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TABLE 19.3 Host genes mapping as expression quantitative trait locus to the Toxoplasma gondii genome across three different cell types, by linkage group. LGa

Human fibroblastsb

Chicken fibroblastsc

Murine macrophagesd

VIIbe

686 (0.40)

119 (0.24)

1003 (0.42)

X

302 (0.17)

116 (0.23)

206 (0.09)

257 (0.15)

70 (0.14)

395 (0.17)

VIII

96 (0.06)

47 (0.09)

31 (0.01)

Ia

85 (0.05)

93 (0.19)

88 (0.04)

Ib

75 (0.04)

0 (0.00)

8 (0.00)

II

61 (0.04)

19 (0.04)

10 (0.00)

XII

41 (0.02)

4 (0.01)

38 (0.02)

III

39 (0.02)

19 (0.04)

6 (0.00)

VI

37 (0.02)

0 (0.00)

9 (0.00)

V

29 (0.02)

1 (0.00)

45 (0.02)

IX

18 (0.01)

8 (0.02)

450 (0.19)

IV

10 (0.01)

6 (0.01)

77 (0.03)

Total

1736

502

2366

VIIa e

a

LG: Linkage group. From Saeij et al. (2007). c From Ong et al. (2011). d From Shastri et al. (2014). e Linkage groups VIIb and VIII are possibly merged into a single chromosome (Bunnik et al., 2019; Khan et al., 2005b). b

VIIa, and the rest to other chromosomes. Most human genes for which the difference in gene expression upon infection with the F1 progeny mapped to T. gondii chromosome VIIb had the highest LOD-score around genetic marker L339 (Fig. 19.7B). This suggested that in the vicinity of that marker, there was a T. gondii gene whose product had a major impact on the regulation of human gene expression. Analysis of enrichment in functional annotation of this group of genes identified that they were significantly enriched for human genes involved in the IL-4, IL-6, and Janus kinase (JAK)/STAT signaling pathways. Activation of the IL-4 and IL-6 signaling pathway culminates in the activation of the transcription factors STAT6 and STAT3, respectively (Ihle, 2001). Indeed, HFFs

infected with type 1 and 3 strains, but not type 2 strains, contained activated STAT3 and STAT6. To fine map the T. gondii genomic region involved in the activation of STAT3/6 new genetic markers, identified by using a genome-wide SNP map generated from ESTs (Boyle et al., 2006), were used to identify the point of recombination in progeny that were recombinant for Toxoplasma chromosome VIIb. To further limit the region involved a new genetic cross between a types 2 and 3 strain was made, and two new F1 progeny that had undergone informative recombinations on chromosome VIIb were isolated (Saeij et al., 2007). These progeny allowed the region involved in strain-specific activation of STAT3/ 6 to be narrowed down to B0.6 Mb. However,

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this region still contained B80 predicted proteins (ToxoDB.org). Therefore candidate genes were prioritized using the following criteria: (1) number of SNPs between types 2 and 3 that change an amino acid, (2) presence of secretory signal peptide, and (3) predicted ability to modify host proteins either by, phosphorylation, dephosphorylation, ubiquitinylation, SUMOylation, cleavage, or attachment. Only one protein had clearly evident SNPs, a 707 amino acid protein with high homology to protein kinases. This protein had previously been described to localize to the T. gondii rhoptries (Bradley et al., 2005), an organelle known to be able to secrete proteins into the host cell upon invasion (Ha˚kansson et al., 2001), and was named ROP16. Interestingly, ROP16 from all three strains was seen to localize to the host cell nucleus early after infection and was still apparent in the nucleus as late as 24 hours postinvasion (Fig. 19.7C). Although wild-type type 2 strains do not activate STAT3/6 significantly, the type 2 strains expressing the type 1 allele of ROP16 (Type 2 1 ROP16I) induced significant STAT3 phosphorylation. Furthermore, expression of an active ROP16 by the type 2 strain led to a significantly reduced secretion of IL-12 by macrophages (Saeij et al., 2007). Type 2 1 ROP16I and type 2 1 ROP16III strains were also significantly less virulent suggesting ROP16 is likely VIR4. ROP16 was subsequently shown to directly phosphorylate STAT3 and STAT6, leading to their prolonged activation (Ong et al., 2010; Yamamoto et al., 2009). ROP16 has also been implicated in phosphorylation of STAT1 and STAT5 (Jensen et al., 2013; Rosowski and Saeij, 2012). Surprisingly, just a single amino acid difference at amino acid 503 accounts for the difference in ROP16 activity between type 2 versus type 1/3 strains (Yamamoto et al., 2009). Using similar methods, it was shown that the promoters of the human genes of which the differential expression mapped to T. gondii chromosome X were enriched in NF-κB

binding sites. Indeed, HFFs infected with type 2 strains had high levels of NF-κB p65 in the host nucleus, while type 1 and 3 strains did not induce translocation of visible levels of NF-κB p65 to the nucleus (Rosowski et al., 2011). This confirmed earlier studies that also showed that type 2 but not type 1 strains induce translocation of NF-κB to the nucleus of mouse splenocytes (Dobbin et al., 2002) and mouse bone marrow derived macrophages (Robben et al., 2004). Using this assay and the F1 progeny from the type 2 3 3 cross, this phenotype was mapped to a region on the distal end of chromosome X. A candidate gene approach subsequently identified that the secretory dense granule protein GRA15 from the type 2 strain was responsible (Rosowski et al., 2011) (Table 19.2). The GRA15 found in type 2 strains is capable of activating NF-κB when expressed in HeLa cells, showing it acts directly (Rosowski et al., 2011). Expression of type 2 GRA15 in a type 1 strain also increases NF-κB translocation and leads to elevated IL-12 production (Rosowski et al., 2011). Because GRA15 is found on the distal end of chromosome X while the VIR2 locus identified in the 2 3 3 cross (Saeij et al., 2006) is at the proximal end of chromosome X, it is unlikely that GRA15 is VIR2. Indeed, deletion of GRA15 in the type 2 strain does not change lethality but does lead to increased parasite growth in vivo (Rosowski et al., 2011). Exactly how GRA15 activates NF-κB was unknown, but it was shown that its function was independent of Myd88/TRIF but dependent on TNF receptor-associated factors (TRAF)6, which is an upstream adapter protein in the NF-κB pathway. Indeed, it was recently shown that GRA15 interacts with TRAFs through multiple TRAF-binding sites present in GRA15 (Sangare´ et al., 2019). The exact combination of GRA15 and ROP16 in a particular Toxoplasma strain also dramatically affects the polarization of the infected macrophage toward either proinflammatory M1 macrophages, which are good at

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19.5 Mapping phenotypic traits by classical genetics

killing intracellular pathogens and are induced by GRA151, or antiinflammatory M2 macrophages which are induced by ROP161/3. (Butcher et al., 2011; Jensen et al., 2011) (see also Chapter 17: Effectors produced by rhoptries and dense granules: an intense conversations between parasite and host in many languages ). Gene expression profiling of chicken fibroblasts with the F1 progeny from the 2 3 3 cross showed very similar results as those obtained in HFFs with the majority of differentially regulated transcripts mapping to loci on T. gondii chromosome VIIb (ROP16) and X (GRA15), although loci on chromosome Ia and VIIa were also involved (Table 19.3; Ong et al., 2011). This showed that the ROP16 and GRA15 strain-differences in activation of STAT3/6 and NF-κB are likely conserved in chickens (Ong et al., 2011). A larger study using 30 F1 progeny from the 2 3 3 cross identified strainspecific modulation of gene expression in the murine RAW 264.7 macrophage cell line (Table 19.3; Shastri et al., 2014). As in previous studies, loci on T. gondii chromosome VIIb, X, and VIIa seemed to determine most of the differential regulation of host gene expression after infection of macrophages with the F1 progeny. However, unlike the studies in HFFs and chicken fibroblasts, many transcripts also mapped to chromosome IX (Table 19.3). GRA25 was proposed as the candidate gene for this phenotype. Although knockout of GRA25 in a type 2 strain made this parasite avirulent complementation of either a type 2 or type 3 GRA25 was able to rescue this reduced virulence (Shastri et al., 2014). Deletion of GRA25 affected the expression of CCL2, of which the expression level differences mapped to chromosome IX, but it was not investigated if either GRA252 or GRA253 were able to rescue the knockout (Shastri et al., 2014). Thus although GRA25 was established as a novel virulence gene, it is still unclear if it is the T. gondii gene that determines the differential host gene expression that mapped to chromosome IX.

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The F1 progeny from the type 2 3 3 cross were also used to determine that strain differences in the induction of CD40 expression on macrophages mapped to chromosome X and were due to GRA15 (Morgado et al., 2014). T. gondii strains also differ significantly in the regulation of host miRNA expression. Using F1 progeny from the 1 3 2 cross, it was determined that ROP161 suppresses the expression of miR-146a. Interestingly, miR-146a expression levels in the brain of chronically infected mice correlates with cyst burden (Cannella et al., 2014). Thus Toxoplasma strains can differ dramatically in the modulation of host gene expression that can impact the pathogenesis of these strains.

19.5.5 Summary of differences between lineages The natural differences seen in acute virulence in the mouse between the three lineages are likely due to the different assortment of alleles they acquired following the relatively few genetic crosses since their common origin (Grigg and Suzuki, 2003; Su et al., 2003). Comparison of the four genetic crosses reveals that the differences in acute virulence are due to the combination of particular alleles of ROP18 and ROP5. Type 1 strains express a highly active ROP18 allele and a “virulent” form of ROP5 that enhances virulence. Type 2 strains have intermediate virulence and contain a ROP5 locus that is genetically “avirulent,” while harboring a fully functional form of ROP18. Although type 2 strain parasites do not normally avoid IRG recruitment to the PVM, expression of type 2 ROP18 in a type 3 background reduces IRG recruitment, along with enhancing virulence (Niedelman et al., 2012). This finding is consistent with ROP18 working cooperatively with ROP5 to prevent IRG accumulation. Type 1 and 3 strains share a major allele of ROP5 that is able to enhance

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virulence, although this is only fully apparent in the presence of ROP18. In contrast the ROP5 form found in type 2 is unable to enhance virulence, although the exact mechanistic reason is presently unclear. It might seem somewhat surprising that so few genes contribute such strong effects to virulence in the mouse model. However, this result may reflect the inbred nature of laboratory mice, which are derived from a narrow genetic stock (Yang et al., 2011) and the relatively inbred state of the three clonal lineage of T. gondii (Boyle et al., 2006; Howe and Sibley, 1995; Su et al., 2003). Such limited genetic diversity may magnify the effects of a few genes, which otherwise would contribute less to pathogenesis in either genetically more diverse host or parasite lineages. Collectively, the rhoptry kinome includes some 40 1 members, about half of which are predicted to be active while the remaining ones are predicted to be pseudokinases (Peixoto et al., 2010). Although current genetic studies have only implicated a few of these, others may influence different aspect of the host pathogen interaction, including during other life cycle stages and/or in other hosts.

19.5.6 Relevance of the mouse model to other species The role of parasite virulence factors in nonrodent hosts is not well understood, in part because most relevant hosts are not experimentally tractable models. However, it can be argued that any evolutionary pressure on traits that influence pathogenesis would only occur in natural hosts, where potential effects on fitness might affect transmission. Hence, the interaction between ROP kinases and IRGs has likely been heavily influenced by the role of rodents in natural transmission. IRGs are greatly expanded in the rodents but much more restricted in other mammals and almost entirely absent in humans (Howard et al.,

2011). Consequently, it has been argued that ROP5 ROP18 may not contribute to evasion of innate immunity in human cells, although this has only been evaluated in fibroblasts activated in vitro with IFN-γ (Niedelman et al., 2012). In contrast to IRGs a second family of GTPases called the p65 or GBPs is much more widely expressed in humans (Shenoy et al., 2008). Recent genetic studies have shown that GBPs are important in innate immunity to T. gondii in the mouse (Kim et al., 2011; Yamamoto et al., 2012), and also in a growing number of human cell types including mesenchymal stromal cells (Qin et al., 2017) and lung fibroblasts (Johnston et al., 2016). T. gondii is known it infect a wide range of mammals, many of these such as goats, chickens, and pigs are relatively resistant to disease during acute infection, yet commonly become chronically infected. In contrast, other groups show high susceptibility, including marsupials and new world monkeys (Dubey, 2010). Rodents show a wide range of susceptibilities: laboratory mice are highly susceptible but deer mice are resistant to type 1 strains (Frenkel, 1953), and house mice may also harbor increased resistance not found in the lab strains (Lilue et al., 2013; Hassan et al., 2019). Rats are relatively resistant to infection (Dubey and Frenkel, 1998) and one factor mediating this has been mapped to a locus called toxo1 (Cavailles et al., 2006), which was later fine mapped to a 891 kb interval containing 29 genes (Cavailles et al., 2014). Among the genes contained in this region is NLRP1, a member of the nucleotide-binding oligomerization domain-like receptor family, a system of intracellular sensors involved in activating the inflammasome (Rathinam et al., 2012). It was later shown that rat sterile immunity to Toxoplasma is perfectly correlated with NLRP1 sequence, which determines macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation (Cirelli et al., 2014; Cavailles et al., 2014). Recent

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19.6 Future challenges

studies in human susceptibility to congenital toxoplasmosis also report an associated with NALP1 (Witola et al., 2011), suggesting they may share a similar pathway for resistance. Because humans are an accidental host for T. gondii, strain differences in pathogenicity seen in humans are not shaped by evolutionary pressure but rather are coincidental. In North America and Europe, human infections are most often caused by type 2 strains (Ajzenberg et al., 2002b; Howe et al., 1997; Howe and Sibley, 1995), yet several studies suggest that type 1 strains, or strains harboring alleles typically found in type 1 strains, are also more pathogenic in humans (Fuentes et al., 2001; Grigg et al., 2001b; Khan et al., 2005a). In addition, a number of South American lineages have been associated with severe ocular disease in otherwise healthy individuals (Jones et al., 2006; Khan et al., 2006; Vallochi et al., 2005).

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(2) Development of non-feline in vivo models for generation of gametocytes, such as an oocyst-competent mouse model via genetic engineering and/or surgical implantation like those that have been developed most recently for Cryptosporidium spp. (Pawlowic et al., 2017; Vinayak et al., 2015) and Plasmodium spp. (Soulard et al., 2015). Recent work by Martorelli Di Genova et al. (2019) producing T. gondii oocysts in mice suggests that mice lacking delta-6-desaturase may represent such a model for T. gondii. (3) Generation of new genetic crosses between divergent lineages, including those from South America, China, and Africa. (4) Development of higher throughout assays for monitoring meaningful biological phenotypes including host cell transcriptional differences, growth, and effects on host cell signaling.

19.6.2 Expanding phenotypic analyses

19.6.1 Overcoming current limitations Classical genetic analyses in T. gondii have been extremely useful for defining basic parameters of recombination, mapping drugresistance loci, assembling the genome, and probing the molecular basis of pathogenesis in laboratory mice. Despite this progress, classical genetics remains a relatively difficult process in T. gondii compared to model organisms such as yeast, Drosophila and Caenorhabditis elegans. Improvements that would accelerate progress include the following: (1) Development of in vitro methods for the generation of gametocyte formation. Such techniques have been reported for Eimeria (Hofmann and Raether, 1990) and Plasmodium (Al-Olayan et al., 2002). Successful completion of the life cycle in vitro would remove the single biggest obstacle to performing genetic crosses in T. gondii.

While crosses in T. gondii are challenging, once the F1 progeny are generated and genotyped, they can be used indefinitely as new parental phenotypes are discovered. There are many known strain-specific differences and F1 progeny have been deposited at ATCC and current genetic maps are freely available. Differences in the migration rate of lineages of T. gondii have been linked to virulence (Barragan and Sibley, 2002). Although mapped to a broad region of chromosome VIIa, the molecular basis for this trait has not been defined. Differences in growth rates have been described for strains of T. gondii (Radke et al., 2001), although it is not known to what extent these contribute to pathogenesis. Differences in the migratory responses of dendritic cells to chemokines (Diana et al., 2004) have been reported to be strain-dependent in the mouse model. The development of intestinal necrosis (Liesenfeld, 2002) and CNS pathology (Suzuki et al., 1989) in

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the mouse model are also influenced in part by the genotype of the parasite. As shown in Table 19.3, the causal parasite gene(s) for a large number of host eQTL phenotypes are unknown, and they may be driven by previously uncharacterized classes of T. gondii effectors and/or targets of the host innate immune response. Only a tiny fraction of the genetic variation that exists in the extant T. gondii population has been probed using these powerful forward genetic techniques, and the field can still benefit greatly from further work in this area. In the future, bulk segregant analyses may prove to be particularly useful to study certain phenotypes. In this method, F1 progeny are pooled (either after genotyping or even prior) and placed under some form of selection and then the input and output pools are queried using whole genome sequencing. In this manner, large populations of F1 progeny could be queried simultaneously and would not require the generation of formal haplotype maps.

Acknowledgments We would like to acknowledge the following individuals who have made important contributions to the advances summarized here. James Ajioka and David Sibley wrote this chapter for editions 1 and 2 and this provided a solid foundation upon which to build for the current edition. Others include John Boothroyd, J.P. Dubey, Michael Grigg, Asis Khan, Jessica Kissinger, Hernan Lorenzi, Loraine Pfefferkorn, Elmer Pfefferkorn, David Roos, Chunlei Su, Sonya Taylor, Michael White, and John Wootton. The National Institutes of Health (USA) has supported work in the authors’ laboratories.

References Adomako-Ankomah, Y., English, E.D., Danielson, J.J., Pernas, L.F., Parker, M.L., Boulanger, M.J., et al., 2016. Host mitochondrial association evolved in the human parasite Toxoplasma gondii via neofunctionalization of a gene duplicate. Genetics 203, 283 298. Ajioka, J.A., Boothroyd, J.C., Brunk, B.P., Hehl, A., Hillier, L., Manger, I.D., et al., 1998. Gene discovery by EST

sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa. Gen. Res 8, 18 28. Ajzenberg, D., Ban˜uls, A.L., Tibayrenc, M., Darde´, M.L., 2002a. Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups. Int. J. Parasitology 32, 27 38. Ajzenberg, D., Cogne´, N., Paris, L., Bessieres, M.H., Thulliez, P., Fillisetti, D., et al., 2002b. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis and correlation with clinical findings. J. Infect. Dis. 186, 684 689. Ajzenberg, D., Ban˜uls, A.L., Su, C., Dume`tre, A., Demar, M., Carme, B., et al., 2004. Genetic diversity, clonality and sexuality in Toxoplasma gondii. Intl. J. Parasitol. 34, 1185 1196. Al-Olayan, E.M., Beetsma, A.L., Butcher, G.A., Sinden, R. E., Hurd, H., 2002. Complete development of mosquito phases of the malaria parasite in vitro. Science 295, 677 679. Bahl, A., Davis, P.H., Behnke, M., Dzierszinski, F., Jagalur, M., Chen, F., et al., 2010. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii. BMC Genomics 11, 603. Barragan, A., Sibley, L.D., 2002. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. J. Exp. Med. 195, 1625 1633. Behnke, M.S., Khan, A., Wootton, J.C., Dubey, J.P., Tang, K., Sibley, L.D., 2011. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc. Natl. Acad. Sci. U.S. A. 108, 9631 9636. Behnke, M.S., Fentress, S.J., Mashayekhi, M., Li, L.L., Taylor, G.A., Sibley, L.D., 2012. The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18. PLoS Path. 8, e1002992. Behnke, M.S., Zhang, T.P., Dubey, J.P., Sibley, L.D., 2014. Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development. BMC Genomics 15, 350. Behnke, M.S., Khan, A., Lauron, E.J., Jimah, J.R., Wang, Q., Tolia, N.H., et al., 2015a. Rhoptry proteins ROP5 and ROP18 are major murine virulence factors in genetically divergent South American strains of Toxoplasma gondii. PLoS Genet 11, e1005434. Behnke, M.S., Khan, A., Sibley, L.D., 2015b. Genetic mapping reveals that sinefungin resistance in Toxoplasma gondii is controlled by a putative amino acid transporter locus that can be used as a negative selectable marker. Eukaryot. Cell 14, 140 148. Blank, M.L., Parker, M.L., Ramaswamy, R., Powell, C.J., English, E.D., Adomako-Ankomah, Y., et al., 2018.

Toxoplasma Gondii

References

A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions. Mol. Microbiol. 108, 519 535. Boudeau, J., Miranda-Saavedra, D., Barton, G.J., Alessi, D. R., 2006. Emerging roles of pseudokinases. Trends Cell Biol. 16, 443 452. Boyle, J.P., Rajasekar, B., Saeij, J.P., Ajioka, J.W., Berriman, M., Paulsen, I., et al., 2006. Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103, 10514 10519. Boyle, J.P., Saeij, J.P., Harada, S.Y., Ajioka, J.W., Boothroyd, J.C., 2008. Expression quantitative trait locus mapping of Toxoplasma genes reveals multiple mechanisms for strain-specific differences in gene expression. Eukaryot. Cell 7, 1403 1414. Bradley, P.J., Ward, C., Cheng, S.J., Alexander, D.L., Coller, S., Coombs, G.H., et al., 2005. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in T. gondii. J. Biol. Chem. 280, 34245 34258. Bunnik, E.M., Venkat, A., Shao, J., McGovern, K.E., Batugedara, G., Worth, D., et al., 2019. Comparative 3D genome organization in apicomplexan parasites. Proc. Natl. Acad. Sci. U.S.A. 116, 3183 3192. Butcher, B.A., Fox, B.A., Rommereim, L.M., Kim, S.G., Maurer, K.J., Yarovinsky, F., et al., 2011. Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1dependent growth control. PLoS Pathog. 7, e1002236. Cannella, D., Brenier-Pinchart, M.P., Braun, L., van Rooyen, J.M., Bougdour, A., Bastien, O., et al., 2014. miR-146a and miR-155 delineate a MicroRNA fingerprint associated with Toxoplasma persistence in the host brain. Cell Rep. 6, 928 937. Cavailles, P., Sergent, V., Bisanz, C., Papapietro, O., Colacios, C., Mas, M., et al., 2006. The rat Toxo1 locus directs toxoplasmosis outcome and controls parasite proliferation and spreading by macrophage-dependent mechanisms. Proc. Natl. Acad. Sci. U.S.A. 103, 744 749. Cavailles, P., Flori, P., Papapietro, O., Bisanz, C., Lagrange, D., Pilloux, L., et al., 2014. A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage. PLoS Pathog. 10, e1004005. Cirelli, K.M., Gorfu, G., Hassan, M.A., Printz, M., Crown, D., Leppla, S.H., et al., 2014. Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii. PLoS Pathog. 10, e1003927. Cleary, M.D., Singh, U., Blader, I.J., Brewer, J.L., Boothroyd, J.C., 2002. Toxoplasma gondii asexual development: identification of developmentally regulated

891

genes and distinct patterns of gene expression. Eukaryot. Cell 1, 329 340. Cornelissen, A.W.C.A., Overdulve, J.P., 1985. Sex determination and sex differentiation in coccidia: gametogony and oocyst production after monoclonal infection of cats with free-living and intermediate host stages of Isospora (Toxoplasma) gondii. Parasitology 90, 35 44. Cornelissen, A.W.C.A., Overdulve, J.P., Van der Ploeg, M., 1984a. Cytochemical studies on nuclear DNA of four eucoccidian parasites, Isospora (Toxoplasma) gondii, Eimeria tenella, Sarcocystis cruzi and Plasmodium berghei. Parasitology 88, 13 25. Cornelissen, A.W.C.A., Overdulve, J.P., Van der Ploeg, M., 1984b. Determination of nuclear DNA of five eucoccidian parasites, Isospora (Toxoplasma) gondii, Sarcocystis cruzi, Eimeria tenella, E. acervulina, and Plasmodium berghei, with special reference to gametogenesis and meiosis in I. (T.) gondii. Parasitology 88, 531 553. Darde´, M.L., Bouteille, B., Pestre-Alexandre, M., 1992. Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications. J. Parasitol. 78, 786 794. de Moura, L., Bahia-Oliveira, L.M., Wada, M.Y., Jones, J.L., Tuboi, S.H., Carmo, E.H., et al., 2006. Waterborne toxoplasmosis, Brazil, from field to gene. Emerg. Infect. Dis. 12, 326 329. de Wit, L.A., Croll, D.A., Tershy, B., Correa, D., LunaPasten, H., Quadri, P., et al., 2019. Potential public health benefits from cat eradications on islands. PLoS Negl. Trop. Dis. 13, e0007040. Diana, J., Persat, F., Staquet, M.J., Assossou, O., Ferrandiz, J., Gariazo, M.J., et al., 2004. Migration and maturation of human dendritic cells infected with Toxoplasma gondii depends on parasite strain type. FEMS Immunol. Med. Microbiol. 42, 321 331. Dobbin, C.A., Smith, N.C., Johnson, A.M., 2002. Heat shock protein 70 is a potential virulence factor in murine toxoplasma infection via immunomodulation of host NF-kappa B and nitric oxide. J. Immunol. 169, 958 965. Dubey, J., 1980. Mouse pathogenicity of Toxoplasma gondii isolated from a goat. Am. J. Vet. Res. 41, 427 429. Dubey, J.P., 1977. Toxoplasma, Hammondia, Besnoitia, Sarcocystis, and other tissue cyst-forming coccidia of man and animals. In: Kreier, J.P. (Ed.), Parasitic Protozoa. Academic Press, New York, pp. 101 237. Dubey, J.P., 1998. Toxoplasma gondii oocyst survival under defined temperatures. J. Parasitol. 84, 862 865. Dubey, J.P., 2004. Toxoplasmosis—a waterborne zoonosis. Vet. Parasitol. 126, 57 72. Dubey, J.P., 2005. Unexpected oocyst shedding by cats fed Toxoplasma gondii tachzoites: in vivo stage conversion and strain variation. Vet. Parasit. 133, 289 298.

Toxoplasma Gondii

892

19. Development and application of classical genetics in Toxoplasma gondii

Dubey, J.P., 2006. Comparative infectivity of oocysts and bradyzoites of Toxoplasma gondii for intermediate (mice) and definitive (cats) hosts. Vet. Parastiol. 140, 69 75. Dubey, J.P., 2008. Toxoplasma gondii infections in chickens (Gallus domesticus): prevalence, clinical disease, diagnosis, and public health significance. Zoonoses Public Health 57, 60 73. Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans. CRC Press, Boca Raton, FL. Dubey, J.P., Frenkel, J.F., 1972. Cyst-induced toxoplasmosis in cats. J. Protozool. 19, 155 177. Dubey, J.P., Frenkel, J.K., 1998. Toxoplasmosis of rats: a review, with considerations of their value as an animal model and their possible role in epidemiology. Vet. Parasitol. 77, 1 32. Dubey, J.P., Sreekumar, C., 2003. Redescription of Hammondia hammondi and its differentiation from Toxoplasma gondii. Int. J. Parasitol. 33, 1437 1453. Dubey, J.P., Swan, G.V., Frenkel, J.K., 1972. A simplified method for isolation of Toxoplasma gondii from the feces of cats. J. Parasitol. 58, 1005 1006. El Hajj, H., Lebrun, M., Fourmaux, M.N., Vial, H., Dubremetz, J.F., 2006. Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association with the parasitophorous vacuole membrane. Cell. Microbiol. 9, 54 64. El Hajj, H., Lebrun, M., Arold, S.T., Vial, H., Labesse, G., Dubremetz, J.F., 2007. ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii. PLoS Pathog. 3, e14. Etheridge, R.D., Alaganan, A., Tang, K., Lou, H.J., Turk, B. E., Sibley, L.D., 2014. The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice. Cell Host Microbe 15, 537 550. Fentress, S.J., Sibley, L.D., 2012. An arginine-rich domain of ROP18 is necessary for vacuole targeting and virulence in Toxoplasma gondii. Cell. Microbiol. 14, 1921 1933. Fentress, S.J., Behnke, M.S., Dunay, I.R., Mashayekhi, M., Rommereim, L.M., Fox, B.A., et al., 2010a. Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence. Cell Host Microbe 8, 484 495. Fentress, S.J., Behnke, M.S., Dunay, I.R., Moashayekhi, M., Rommereim, L.M., Fox, B.A., et al., 2010b. Phosphorylation of immunity-related GTPases by a parasite secretory kinase promotes macrophage survival and virulence. Cell Host Microbe 16, 484 495. Ferguson, D.J.P., Birch-Andersen, A., Siim, J.C., Hutchison, W.M., 1979. Ultrastructural studies of the sporulation of oocysts of Toxoplasma gondii. Acta Pathol. Microbiol. Scand. 87, 183 190.

Fleckenstein, M.C., Reese, M.L., Konen-Waisman, S., Boothroyd, J.C., Howard, J.C., Steinfeldt, T., 2012. A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins. PLoS Biol. 10, e1001358. Frenkel, J.K., 1953. Host, strain and treatment variation as factors in the pathogenesis of toxoplasmosis. Am. J. Trop. Med. Hyg. 2, 390 415. Frenkel, J.K., Dubey, J.P., 1975a. Hammondia hammondi gen. nov., sp.nov., from domestic cats, a new coccidian related to Toxoplasma and Sarcocystis. Z. Parasitenkd. 46, 3 12. Frenkel, J.K., Dubey, J.P., 1975b. Hammondia hammondi: a new coccidium of cats producing cysts in muscle of other mammals. Science 189, 222 224. Frenkel, J.K., Dubey, J.P., 2000. The taxonomic importance of obligate heteroxeny: distinction of Hammondia hammondi from Toxoplasma gondii—another opinion. Parasitol. Res. 86, 783 786. Frenkel, J.K., Dubey, J.P., Miller, N.L., 1970. Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science 167, 893 896. Frenkel, J.K., Dubey, J.P., Hoff, R.L., 1976. Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni. J. Protozool. 23, 421 424. Fuentes, I., Rubio, J.M., Ramı´rez, C., Alvar, J., 2001. Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples. J. Clin. Microbiol. 39, 1566 1570. Gajria, B., Bahl, A., Brestelli, J., Dommer, J., Fischer, S., Gao, X., et al., 2007. ToxoDB: an integrated Toxoplasma gondii database resource. Nucl. Acids Res. 36, D553 556. Grigg, M.E., Suzuki, Y., 2003. Sexual recombination and clonal evolution of virulence in Toxoplasma. Microbes Infect. 5, 685 690. Grigg, M.E., Bonnefoy, S., Hehl, A.B., Suzuki, Y., Boothroyd, J.C., 2001a. Success and virulence in Toxoplasma as the result of sexual recombination between two distinct ancestries. Science 294, 161 165. Grigg, M.E., Ganatra, J., Boothroyd, J.C., Margolis, T.P., 2001b. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J. Infect. Dis. 184, 633 639. Ha˚kansson, S., Charron, A.J., Sibley, L.D., 2001. Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole. EMBO J. 20, 3132 3144. Hassan, M.A., Olijnik, A.A., Frickel, E.M., Saeij, J.P., 2019. Clonal and atypical Toxoplasma strain differences in virulence vary with mouse sub-species. Int. J. Parasitol. 49, 63 70. Hehl, A., Manger, I.D., Boothroyd, J.C., 1997. Genetic analysis in Toxoplasma: gene discovery with expressed

Toxoplasma Gondii

References

sequence tags and rapid mapping of natural polymorphisms. Methods 13, 89 102. Hehl, A.B., Basso, W.U., Lippuner, C., Ramakrishnan, C., Okoniewski, M., Walker, R.A., et al., 2015. Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of nonoverlapping gene families to attach, invade, and replicate within feline enterocytes. BMC Genomics 16, 66. Hofmann, J., Raether, W., 1990. Improved techniques for the in vitro cultivation of Eimeria tenella in primary chick kidney cells. Parasitol. Res. 76, 479 486. Howard, J.C., Hunn, J.P., Steinfeldt, T., 2011. The IRG protein-based resistance mechanism in mice and its relation to virulence in Toxoplasma gondii. Curr. Opin. Microbiol. 14, 414 421. Howe, D.K., Sibley, L.D., 1995. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J. Infect. Dis. 172, 1561 1566. Howe, D.K., Summers, B.C., Sibley, L.D., 1996. Acute virulence in mice is associated with markers on chromosome VIII in Toxoplasma gondii. Infect. Immun. 64, 5193 5198. Howe, D.K., Honore´, S., Derouin, F., Sibley, L.D., 1997. Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis. J. Clin. Microbiol. 35, 1411 1414. Ihle, J.N., 2001. The Stat family in cytokine signaling. Curr. Opin. Cell Biol. 13 (2), 211 217. Jain, M., Olsen, H.E., Paten, B., Akeson, M., 2016. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239. Jansen, R.C., Nap, J.P., 2001. Genetical genomics: the added value from segregation. Trends Genet. 17, 388 391. Jensen, K.D., Wang, Y., Wojno, E.D., Shastri, A.J., Hu, K., Cornel, L., et al., 2011. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation. Cell Host Microbe 9, 472 483. Jensen, K.D., Hu, K., Whitmarsh, R.J., Hassan, M.A., Julien, L., Lu, D., et al., 2013. Toxoplasma gondii rhoptry 16 kinase promotes host resistance to oral infection and intestinal inflammation only in the context of the dense granule protein GRA15. Infect. Immun. 81, 2156 2167. Jensen, K.D., Camejo, A., Melo, M.B., Cordeiro, C., Julien, L., Grotenbreg, G.M., et al., 2015. Toxoplasma gondii superinfection and virulence during secondary infection correlate with the exact ROP5/ROP18 allelic combination. MBio 6, e02280. Johnston, A.C., Piro, A., Clough, B., Siew, M., Virreira Winter, S., Coers, J., et al., 2016. Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii. Cell Microbiol. 18, 1056 1064. Jones, L.A., Alexander, J., Roberts, C.W., 2006. Ocular toxoplasmosis: in the storm of the eye. Parasite Immunol. 28, 635 642.

893

Khaminets, A., Hunn, J.P., Konen-Waisman, S., Zhao, Y.O., Preukschat, D., Coers, J., et al., 2010. Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole. Cell Microbiol. 12, 939 961. Khan, A., Su, C., German, M., Storch, G.A., Clifford, D., Sibley, L.D., 2005a. Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J. Clin. Microl. 43, 5881 5887. Khan, A., Taylor, S., Su, C., Mackey, A.J., Boyle, J., Cole, R., et al., 2005b. Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii. Nucleic Acids Res. 33, 2980 2992. Khan, A., Jordan, C., Muccioli, C., Vallochi, A.L., Rizzo, L.V., Belfort Jr., R., et al., 2006. Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis Brazil. Emerg. Infect. Dis. 12, 942 949. Khan, A., Fux, B., Su, C., Dubey, J.P., Darde, M.L., Ajioka, J.W., et al., 2007. Recent transcontinental sweep of Toxoplasma gondii driven by a single monomorphic chromosome. Proc. Natl. Acad. Sci. U.S.A. 104, 14872 14877. Khan, A., Taylor, S., Ajioka, J.W., Rosenthal, B.M., Sibley, L.D., 2009. Selection at a single locus leads to widespread expansion of Toxoplasma gondii lineages that are virulent in mice. PLoS Genet 5, e1000404. Khan, A., Dubey, J.P., Su, C., Ajioka, J.W., Rosenthal, B.M., Sibley, L.D., 2011. Genetic analyses of atypical Toxoplasma gondii strains reveals a fourth clonal lineage in North America. Int. J. Parasitol. 41, 645 655. Khan, A., Shaik, J.S., Behnke, M., Wang, Q., Dubey, J.P., Lorenzi, H.A., et al., 2014. NextGen sequencing reveals short double crossovers contribute disproportionately to genetic diversity in Toxoplasma gondii. BMC Genomics 15, 1168. Kim, B.H., Shenoy, A.R., Kumar, P., Das, R., Tiwari, S., MacMicking, J.D., 2011. A family of IFN-gammainducible 65-kD GTPases protects against bacterial infection. Science 332, 717 721. Lander, E., Kruglyak, L., 1995. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat. Genet. 11, 241 247. Lander, E.S., Botstein, D., 1989. Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121, 185 199. Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E., et al., 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1, 174 181. Lehmann, T., Marcet, P.L., Graham, D.H., Dahl, E.R., Dubey, J.P., 2006. Globalization and the population structure of Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103, 11423 11428.

Toxoplasma Gondii

894

19. Development and application of classical genetics in Toxoplasma gondii

Li, L., Brunk, B.P., Kissinger, J.C., Pape, D., Tang, K., Cole, R.H., et al., 2003. Gene discovery in the Apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Gen. Res 13, 443 454. Liesenfeld, O., 2002. Oral infection of C57BL/6 mice with Toxoplasma gondii: a new model of inflammatory bowel disease? J. Infect. Dis. 185, S96 101. Lilue, J., Muller, U.B., Steinfeldt, T., Howard, J.C., 2013. Reciprocal virulence and resistance polymorphism in the relationship between Toxoplasma gondii and the house mouse. eLife 2, e01298. Lindsay, D.S., Dubey, J.P., Blagburn, B.L., Toivio-Kinnucan, M., 1991. Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites. J. Parasitol. 77, 126 132. Lunde, M.N., Jacobs, L., 1983. Antigenic differences between endozoites and cystozoites of Toxoplasma gondii. J. Parasitol. 65, 806 808. Lynch, M., Walsh, B., 1998. Genetics and Analysis of Quantitative Traits. Sinauer Associates, Inc., Sunderland. Manger, I.D., Adrian, H., Parmley, S., Sibley, L.D., Marra, M., Hillier, L., et al., 1998. Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: identification of developmentally regulated genes. Infect. Immun. 66, 1632 1637. Martorelli Di Genova, B., Wilson, S.K., Dubey, J.P., Knoll, L.J., 2019. Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction. PLoS Biol 17 (8), e3000364. Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., et al., 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5, 607 625. Miller, N.L., Frenkel, J.K., Dubey, J.P., 1972. Oral infections with Toxoplasma cysts and oocysts in felines, other mammals, and in birds. J. Parasitol. 58, 928 937. Minot, S., Melo, M.B., Li, F., Lu, D., Niedelman, W., Levine, S.S., et al., 2012. Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity. Proc. Natl. Acad. Sci. U.S.A. 109, 13458 13463. Morgado, P., Sudarshana, D.M., Gov, L., Harker, K.S., Lam, T., Casali, P., et al., 2014. Type II Toxoplasma gondii induction of CD40 on infected macrophages enhances interleukin-12 responses. Infect. Immun. 82, 4047 4055. Nadipuram, S.M., Kim, E.W., Vashisht, A.A., Lin, A.H., Bell, H.N., Coppens, I., et al., 2016. In vivo biotinylation of the Toxoplasma parasitophorous vacuole reveals novel dense granule proteins important for parasite growth and pathogenesis. MBio 7, e00808 16. Available from: https://doi.org/10.1128/mBio.00808-16. Nichols, B.A., Chiappino, M.L., O’Connor, G.R., 1983. Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J. Ultrastruct. Res. 83, 85 98.

Niedelman, W., Gold, D.A., Rosowski, E.E., Sprokholt, J.K., Lim, D., Farid Arenas, A., et al., 2012. The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferongamma response. PLoS Pathog. 8, e1002784. Ong, Y.C., Reese, M.L., Boothroyd, J.C., 2010. Toxoplasma rhoptry protein 16 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6. J. Biol. Chem. 285, 28731 28740. Ong, Y.C., Boyle, J.P., Boothroyd, J.C., 2011. Straindependent host transcriptional responses to Toxoplasma infection are largely conserved in mammalian and avian hosts. PLoS One 6, e26369. Pawlowic, M.C., Vinayak, S., Sateriale, A., Brooks, C.F., Striepen, B., 2017. Generating and maintaining transgenic cryptosporidium parvum parasites. Curr. Protoc. Microbiol. 46, 20B.22.21 20B.22.32. Peixoto, L., Chen, F., Harb, O.S., Davis, P.H., Beiting, D.P., Brownback, C.S., et al., 2010. Integrative genomics approaches highlight a family of parasite-specific kinases that regulate host responses. Cell Host Microbe 8, 208 218. Pena, H.F., Gennari, S.M., Dubey, J.P., Su, C., 2008. Population structure and mouse-virulence of Toxoplasma gondii in Brazil. Int. J. Parasitol. 38, 561 569. Pernas, L., Adomako-Ankomah, Y., Shastri, A.J., Ewald, S.E., Treeck, M., Boyle, J.P., et al., 2014. Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS Biol. 12, e1001845. Pfefferkorn, E.R., 1988. Toxoplasma gondii viewed from a virological perspective. In: Englund, P.T., Sher, A. (Eds.), The Biology of Parasitism. Alan R, Liss, Inc, New York, pp. 479 501. Pfefferkorn, E.R., Kasper, L.H., 1983. Toxoplasma gondii: genetic crosses reveal phenotypic suppression of hydroxyurea resistance by fluorodeoxyuridine resistance. Exp. Parasitol. 55, 207 218. Pfefferkorn, E.R., Pfefferkorn, L.C., 1976. Toxoplasma gondii: isolation and preliminary characterization of temperature sensitive mutants. Exp. Parasitol. 39, 365 376. Pfefferkorn, E.R., Pfefferkorn, L.C., 1977. Toxoplasma gondii: characterization of a mutant resistant to 5-fluorodeoxyuridine. Exp. Parasitol. 42, 44 55. Pfefferkorn, E.R., Pfefferkorn, L.C., 1978. The biochemical basis for resistance to adenine arabinoside in a mutant of Toxoplasma gondii. J. Parasitol. 64, 486 492. Pfefferkorn, E.R., Pfefferkorn, L.C., 1979. Quantitative studies of the mutagenesis of Toxoplasma gondii. J. Parasitol. 65, 363 370. Pfefferkorn, L.C., Pfefferkorn, E.R., 1980. Toxoplasma gondii: genetic recombination between drug resistant mutants. Exp. Parasitol. 50, 305 316.

Toxoplasma Gondii

References

Pfefferkorn, E.R., Pfefferkorn, L.C., Colby, E.D., 1977. Development of gametes and oocysts in cats fed cysts derived from cloned trophozoites of Toxoplasma gondii. J. Parasitol. 63, 158 159. Qin, A., Lai, D.H., Liu, Q., Huang, W., Wu, Y.P., Chen, X., et al., 2017. Guanylate-binding protein 1 (GBP1) contributes to the immunity of human mesenchymal stromal cells against Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 114, 1365 1370. Radke, J.R., Striepen, B., Guerini, M.N., Jerome, M.E., Roos, D.S., White, M.W., 2001. Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii. Molec. Biochem. Parasitol. 115, 165 175. Ramakrishnan, C., Maier, S., Walker, R.A., Rehrauer, H., Joekel, D.E., Winiger, R.R., et al., 2019. An experimental genetically attenuated live vaccine to prevent transmission of Toxoplasma gondii by cats. Sci. Rep. 9, 1474. Rao, S.S., Huntley, M.H., Durand, N.C., Stamenova, E.K., Bochkov, I.D., Robinson, J.T., et al., 2014. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665 1680. Rathinam, V.A., Vanaja, S.K., Fitzgerald, K.A., 2012. Regulation of inflammasome signaling. Nat Immunol 13, 333 342. Reese, M.L., Boothroyd, J.C., 2009. A helical membranebinding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole. Traffic 10, 1458 1470. Reese, M.L., Boothroyd, J.C., 2011. A conserved noncanonical motif in the pseudoactive site of the ROP5 pseudokinase domain mediates its effect on Toxoplasma virulence. J. Biol. Chem. 286, 29366 29375. Reese, M.L., Zeiner, G.M., Saeij, J.P., Boothroyd, J.C., Boyle, J.P., 2011. Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence. Proc. Natl. Acad. Sci. U.S.A. 108, 9625 9630. Reid, A.J., Vermont, S.J., Cotton, J.A., Harris, D., HillCawthorne, G.A., Konen-Waisman, S., et al., 2012. Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy. PLoS Pathog. 8, e1002567. Rhoads, A., Au, K.F., 2015. PacBio sequencing and its applications. Genomics Proteomics Bioinform. 13, 278 289. Robben, P.M., Mordue, D.G., Truscott, S.M., Takeda, K., Akira, S., Sibley, L.D., 2004. Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype. J. Immunol. 172 (6), 3686 3694. Rosowski, E.E., Saeij, J.P., 2012. Toxoplasma gondii clonal strains all inhibit STAT1 transcriptional activity but polymorphic effectors differentially modulate IFNgamma induced gene expression and STAT1 phosphorylation. PLoS One 7, e51448.

895

Rosowski, E.E., Lu, D., Julien, L., Rodda, L., Gaiser, R.A., Jensen, K.D., et al., 2011. Strain-specific activation of the NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208, 195 212. Sabin, A.B., 1941. Toxoplasmic encephalitis in children. J. Am. Med. Assoc. 116, 801 807. Saeij, J.P.J., Boyle, J.P., Coller, S., Taylor, S., Sibley, L.D., Brooke-Powell, E.T., et al., 2006. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314, 1780 1783. Saeij, J.P., Coller, S., Boyle, J.P., Jerome, M.E., White, M.W., Boothroyd, J.C., 2007. Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445, 324 327. Sangare´, L.O., Yang, N., Konstantinou, E.K., Lu, D., Mukhopadhyay, D., Young, L.H., et al., 2019. Toxoplasma GRA15 activates the NF-κB pathway through interactions with TNF receptor-associated factors. mBio 10, e00808 e00819. Available from: https:// doi.org/10.1128/mBio.00808-19. Shaik, J.S., Khan, A., Beverley, S.M., Sibley, L.D., 2015. REDHORSE-REcombination and double crossover detection in haploid organisms using next-geneRation SEquencing data. BMC Genomics 16, 133. Shastri, A.J., Marino, N.D., Franco, M., Lodoen, M.B., Boothroyd, J.C., 2014. GRA25 is a novel virulence factor of Toxoplasma gondii and influences the host immune response. Infect. Immun. 82, 2595 2605. Shen, B., Brown, K.M., Lee, T.D., Sibley, L.D., 2014. Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. MBio 5, e01114-14. Shenoy, A.R., Kim, B.H., Choi, H.P., Matsuzawa, T., Tiwari, S., MacMicking, J.D., 2008. Emerging themes in IFNgamma-induced macrophage immunity by the p47 and p65 GTPase families. Immunobiology 212, 771 784. Sibley, L.D., Ajioka, J.W., 2008. Population structure of Toxoplasma gondii: clonal expansion driven by infrequent recombination and selective sweeps. Ann. Rev. Microbiol. 62, 329 351. Sibley, L.D., Boothroyd, J.C., 1992a. Construction of a molecular karyotype for Toxoplasma gondii. Mol. Biochem. Parasitol. 51, 291 300. Sibley, L.D., Boothroyd, J.C., 1992b. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature (Lond.) 359, 82 85. Sibley, L.D., LeBlanc, A.J., Pfefferkorn, E.R., Boothroyd, J.C., 1992. Generation of a restriction fragment length polymorphism linkage map for Toxoplasma gondii. Genetics 132, 1003 1015. Soulard, V., Bosson-Vanga, H., Lorthiois, A., Roucher, C., Franetich, J.F., Zanghi, G., et al., 2015. Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice. Nat. Commun. 6, 7690.

Toxoplasma Gondii

896

19. Development and application of classical genetics in Toxoplasma gondii

Speer, C., Clark, S., Dubey, J., 1998. Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii. J. Parasitol. 84, 505 512. Steinfeldt, T., Konen-Waisman, S., Tong, L., Pawlowski, N., Lamkemeyer, T., Sibley, L.D., et al., 2010. Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol. 8, e1000576. Su, X.Z., Wootton, J.C., 2004. Genetic mapping in the human malaria parasite Plasmodium falciparum. Molec. Microbiol. 53, 1573 1582. Su, X., Ferdig, M.T., Huang, Y., Huynh, C.Q., Liu, A., You, J., et al., 1999. A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum. Science 286, 1351 1353. Su, C., Howe, D.K., Dubey, J.P., Ajioka, J.W., Sibley, L.D., 2002. Identification of quantitative trait loci controlling acute virulence in Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 99, 10753 10758. Su, C., Evans, D., Cole, R.H., Kissinger, J.C., Ajioka, J.W., Sibley, L.D., 2003. Recent expansion of Toxoplasma through enhanced oral transmission. Science 299, 414 416. Su, C.L., Khan, A., Zhou, P., Majumdar, D., Ajzenberg, D., Darde´, M.L., et al., 2012. Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proc. Natl. Acad. Sci. U.S.A. 109, 5844 5849. Suzuki, Y., Conley, F.K., Remington, J.S., 1989. Differences in virulence and development of encephalitis during chronic infection vary with the strain of Toxoplasma gondii. J. Infect. Dis. 159, 790 794. Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., et al., 2006. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314, 1776 1780. Vallochi, A.L., Muccioli, C., Martins, M.C., Silveira, C., Belfort Jr., R., Rizzo, L.V., 2005. The genotype of Toxoplasma gondii strains causing ocular toxoplasmosis in humans in Brazil. Am. J. Ophthalmol. 139, 350 351. Vinayak, S., Pawlowic, M.C., Sateriale, A., Brooks, C.F., Studstill, C.J., Bar-Peled, Y., et al., 2015. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523, 477 480. Virreira Winter, S., Niedelman, W., Jensen, K.D., Rosowski, E.E., Julien, L., Spooner, E., et al., 2011. Determinants of GBP recruitment to Toxoplasma gondii vacuoles and the parasitic factors that control it. PLoS One 6, e24434.

Walzer, K.A., Adomako-Ankomah, Y., Dam, R.A., Herrmann, D.C., Schares, G., Dubey, J.P., et al., 2013. Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes. Proc. Natl. Acad. Sci. U.S.A. 110, 7446 7451. Wang, Y., Cirelli, K.M., Barros, P.D.C., Sangare, L.O., Butty, V., Hassan, M.A., et al., 2019. Three Toxoplasma gondii dense granule proteins are required for induction of Lewis rat macrophage pyroptosis. MBio 10, e02388 18. Ware, P.L., Kasper, L.H., 1987. Strain-specific antigens of Toxoplasma gondii. Infect. Immun. 55, 778 783. Wendte, J.M., Miller, M.A., Lambourn, D.M., Magargal, S. L., Jessup, D.A., Grigg, M.E., 2010. Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii. PLoS Genet. 6, e1001261. Witola, W.H., Mui, E., Hargrave, A., Liu, S., Hypolite, M., Montpetit, A., et al., 2011. NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondiiinfected monocytic cells. Infect. Immun. 79, 756 766. Yamamoto, M., Standley, D.M., Takashima, S., Saiga, H., Okuyama, M., Kayama, H., et al., 2009. A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3. J. Exp. Med. 206, 2747 2760. Yamamoto, M., Ma, J.S., Mueller, C., Kamiyama, N., Saiga, H., Kubo, E., et al., 2011. ATF6-beta is a host cellular target of the Toxoplasma gondii virulence factor ROP18. J. Exp. Med. 208, 1533 1546. Yamamoto, M., Okuyama, M., Ma, J.S., Kimura, T., Kamiyama, N., Saiga, H., et al., 2012. A cluster of interferon-gamma-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii. Immunity 37, 302 313. Yang, H., Wang, J.R., Didion, J.P., Buus, R.J., Bell, T.A., Welsh, C.E., et al., 2011. Subspecific origin and haplotype diversity in the laboratory mouse. Nat. Genet. 43, 648 655. Zeqiraj, E., van Aalten, D.M., 2010. Pseudokinasesremnants of evolution or key allosteric regulators? Curr. Opin. Struct. Biol. 20, 772 781. Zhao, Y., Ferguson, D.J., Wilson, D.C., Howard, J.C., Sibley, L.D., Yap, G.S., 2009. Virulent Toxoplasma gondii evade immunity-related GTPas-mediated parasite vacuole disruption within primed macrophages. J. Immunol. 182, 3775 3781.

Toxoplasma Gondii

C H A P T E R

20 Genetic manipulation of Toxoplasma gondii Damien Jacot1, Sebastian Lourido2,3, Markus Meissner4, Lilach Sheiner5, Dominique Soldati-Favre1 and Boris Striepen6 1

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland 2Whitehead Institute for Biomedical Research, Cambridge, MA, United States 3 Biology Department, Massachusetts Institute of Technology, Cambridge, MA, United States 4 Experimental Parasitology, Department of Veterinary Sciences, Ludwig Maximilians University, Munich, Germany 5Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary & Life Sciences, Sir Graeme Davies Building, University of Glasgow, Glasgow, United Kingdom 6Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, United States

20.1 Introduction The first genetic manipulations applied to Toxoplasma gondii were performed by using chemical mutagenesis. These studies were pioneered in the 1970s by the Pfefferkorn laboratory (Pfefferkorn and Pfefferkorn, 1976; Pfefferkorn, 1988) who developed protocols to reproducibly cultivate tachyzoites in a tissue culture system and to mutagenize, select, and finally clone parasites by limiting dilution. Based on these protocols, a series of chemically induced mutants were used to map out the parasite’s nucleotide biosynthetic pathways. These studies were critical for the establishment

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00020-7

of protocols for genetic crosses in the cat (Pfefferkorn and Pfefferkorn, 1980). Crosses can be used to map a given phenotype to a single or multiple genome loci. This classical forward genetic approach has been instrumental to map virulence factors and to analyze Toxoplasma population structure and evolution. See Chapter 3, Molecular epidemiology and population structure of Toxoplasma gondii, and Chapter 19, Development and application of classical genetics in Toxoplasma gondii, for further discussion of these topics. The reverse genetics approach, which introduces foreign DNA into parasites, was achieved using electroporation (Soldati and

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Boothroyd, 1993). Initially, the transient transfection of plasmid DNA containing reporter genes flanked by T. gondii 50 and 30 flanking sequences allowed the expression of reporter genes used for the characterization of the elements controlling transcription. This methodology was rapidly utilized to identify and validate several selectable marker genes, which then opened an avenue for stable transformation and the development of an invaluable panoply of tools associated with DNA transfection. A wide range of positive and negative selectable markers have been tailored for homologous recombination (HR) leading to allelic replacement and gene knockouts (Messina et al., 1995; Soldati et al., 1995; Donald et al., 1996; Kim et al., 1993; Donald and Roos, 1993). In addition, nonhomologous random integration vectors have been designed as a strategy for random insertional mutagenesis. More recently, the CRISPR/Cas9 system, which utilizes the bacterial RNA-guided endonuclease Cas9, was successfully implemented in T. gondii (Shen et al., 2014; Sidik et al., 2016). The ease of redirecting the Cas9 nuclease by changing the 20 bases of homology that specify target recognition within the guide RNA has streamlined the functional analysis of parasite genes and enabled high-throughput loss-offunction screens (Sidik et al., 2016). The availability of several T. gondii genomes and those of other related apicomplexans (http://toxodb.org/toxo/ and https:// eupathdb.org/eupathdb/) constitutes a formidable source of information. In this postgenomics area the accessibility of T. gondii to multiple genetic manipulations approaches and to high throughput studies continue to rank it as a very attractive and powerful system to improve our understanding of the basic biology of the apicomplexan parasites. Fig. 20.1 summarizes the available sources of information and experimental approaches. There is no limitation to the identification of relevant genes

and little or no experimental barrier to unravel their biological functions. The purpose of this chapter is to recapitulate and describe the strategies associated with DNA transfection and genetic manipulations including the most recent technological developments and to provide a list of the most useful protocols, reagents, and strains available to the researchers.

20.2 The mechanics of making transgenic parasites 20.2.1 Transient transfection Successful manipulation of the Toxoplasma genome is critically dependent on the efficiency of DNA transfection. Electroporation was and remains the method of choice to introduce DNA into tachyzoites. Importantly, the combination of this method with media mimicking the cytosolic ion composition of the cells (cytomix) confers the best survival rate (Vandenhoff et al., 1992). The protocol, initially established using a BTX electroporator, led to an efficiency of transient expression that oscillated between 30% and 50% (Soldati and Boothroyd, 1993). The optimal settings chosen on the BTX electroporator were fixed for the RH strain (type I, virulent strain) and were slightly modified for the cyst forming strains (ME49 and Prugniaud; type II strains). It has been frequently observed that the cyst forming strains are less amenable to genetic manipulation probably due to several factors. As an alternative, AMAXA Nucleofector system is also offering a very high efficiency of transfection (Upadhya et al., 2011). To monitor transfection efficiency, chloramphenicol acetyltransferase (CAT) and β-galactosidase were originally used as reporter genes and subsequently the β-lactamase, alkaline phosphatase, and firefly luciferase (LUC). These enzymes are classically used as reporters because

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20.2 The mechanics of making transgenic parasites

Genetic cross Expression vectors Episomal expression and random integration Insertional mutagenesis

Micronemes Inner membrane complex Rhoptries

Homologous recombination

Apicoplast

Gene knock-outs

Dense granules

Endogenous epitope tagging

Parasitophorous vacuole

Conditional protein stabilization Genome sequence

Conditional protein degradation

Microarrays

Tet-inducible system Conditional U1 gene silencing

Proteomics

CRISPR/Cas9

Metabolomics

Complementation cloning Signature-tagged mutagenesis

FIGURE 20.1 Sources of information and manipulation strategies. Schematic drawing of an intracellular parasite with the subcellular structures and organelles and the list of the tools currently available for functional analysis. Source: Adapted from Soldati, D., Meissner, M., 2004. Toxoplasma gondii a model organism for the apicomplexans. In: Genomes and the Molecular Cell Biology of Malaria Parasites, vol. 5. Horizon Press, pp. 135167.

their activities can be monitored with great sensitivity and in a quantitative fashion. In addition, these enzymes are absent in eukaryotic cells, leading to virtually no background activity. In addition, the β-lactamase and alkaline phosphatase can be exploited to study the secretory pathway and quantify parasite secretion (Chaturvedi et al., 1999; Karsten et al., 1998). LacZ activity can be measured using a colorimetric assay that transforms yellow chlorophenol red-β-Dgalactopyranoside (CPRG) substrate into a red product using an absorbance spectrophotometer at 570 nm (Seeber and Boothroyd, 1996). This colorimetric readout assay can be monitored in live parasites using culture medium without phenol red and in multiwell plates allowing (at a high throughput level)

the screening of a drug efficacy against the parasite (McFadden et al., 1997). Faithful expression of a reporter gene requires adequate 50 and 30 flanking sequences that are derived from T. gondii genes. The flanking sequences must contain the control elements necessary to drive an optimal level of transcription. The monocistronic nature of transcription in T. gondii facilitated the identification of promoter elements that are usually in close proximity to the transcription start site. Numerous vectors suitable for transfection are currently available, and as they exhibit different range of promoter strength and stage specificities, they can be chosen appropriately according to the purpose of the experiment. A constitutive level of expression can be obtained by using vectors derived from the

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housekeeping gene TUB1 (α-tubulin) or other genes such as DHFR (dihydrofolate reductase), ROP1 (rhoptry protein 1), MIC2 (microneme protein 2), several GRA (dense granules proteins), and HXGPRT (hypoxanthine-guanine phosphoribosyl transferase) genes. The strength of these and other promoters have not been systematically compared; however the GRAs and MIC2 promoters are among the strongest, TUB1 and ROP1 promoters are intermediate while DHFR is a weak promoter (Soldati et al., 1995). In addition, the selfcleaving T2A sequence from picornavirus, which mediates ribosome-skipping events can be used to enable the generation of two or more separate peptide products from one mRNA (Tang et al., 2016; Brown and Sibley, 2018; Wang et al., 2015). Stage-specific expression can be achieved using the 50 -flanking sequences of stagespecific genes and so far, no stage-specific regulatory elements have been mapped in the 30 untranslated region (UTR) sequences. Tachyzoite-specific expression is conferred by vectors derived from SAG1 (surface antigen 1), ENO1 (enolase 1), and LDH1 (lactate dehydrogenase 1) genes. In contrast, vectors constructed from BAG1 (bradyzoite antigen 1), ENO2 (enolase 2), or SAG4 genes confer expression in the bradyzoite stage exclusively. Detailed promoter analysis and identification of cis-acting elements have only been undertaken for a limited number of genes (Bohne et al., 1997; Kibe et al., 2005; Mercier et al., 1996; Matrajt et al., 2004; Soldati and Boothroyd, 1995; Yang and Parmley, 1997). See Chapter 19, Development and application of classical genetics in Toxoplasma gondii, for a discussion of regulation of gene expression. Importantly, in numerous cases the considerable cell cycle-dependency of gene expression (Behnke et al., 2010) stresses the importance of using the endogenous promoter to control the expression of the gene of interest (GOI).

In addition to the promoter elements, sequence features carried on the mRNAs also contribute to the success of transfection. Sequence information derived from the 50 and 30 UTRs likely affects gene expression, but this level of regulation has not been rigorously investigated to date. The 30 UTR is an important element as transcription drops to less than 10% when such an element is not included. In Plasmodium partial deletion of 30 UTRs has been exploited to modulate the level of expression of essential genes offering a way to analyze their function (Thathy and Menard, 2002). At the start codon, a consensus sequence termed the “Kozak sequence” is recognized by the ribosome as a favorable sequence to initiate translation. A compilation of abundantly expressed genes in T. gondii was used to establish a consensus translational initiation sequence gNCAAaATGg, which is similar but not identical to the Kozak sequence found in higher eukaryotes (Seeber, 1997). Several genes including GFP were initially very difficult to express using their native sequence but the lack of expression was solved by the generation of fusions at the N-terminus (Striepen et al., 1998). These observations suggested a significant influence of the N-terminal amino acid sequences in recombinant protein expression. A systematic analysis aiming at the evaluation of the importance of the amino acid following the initiation methionine confirmed the existence of an N-end rule in T. gondii (Matrajt et al., 2002b). Typically, amino acids such as Ala, Glu, and Asp confer a high level of expression of the transgene. Like in other multicellular organism alternative splicing also occurs in T. gondii (Lunghi et al., 2016; Yeoh et al., 2019). Although the number of alternatively spiced genes is not exactly known (ranging from 0.8% to 22.6%) it is of biological significance (Yeoh et al., 2015; Hassan et al., 2012). In humans, a major hypothesized role of alternative splicing is to produce different proteins in different tissues.

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In T. gondii, the analog to tissue specificity could be stage specificity, with different protein isoforms in different life stages (Chen et al., 2018). However, only a small fraction of the alternative transcripts detected so far results in alternative proteins. Therefore proteome diversity does not appear to be the main outcome in apicomplexans. Alternative splicing may instead be involved in transcript turnover (Lunghi et al., 2016; Yeoh et al., 2019). Nevertheless, alternative splicing is essential to parasite survival (Suvorova et al., 2013; Yeoh et al., 2015). Alternative splicing should be considered when a complementation strategy is undertaken. Indeed, complementation with a cDNA or a gDNA could result with different outcomes (Hammoudi et al., 2015).

20.2.2 Stable transformation and positive and negative selectable markers Most of the selectable marker genes commonly used for eukaryotic cells are not suitable for the selection of stable transformants in T. gondii. As T. gondii is an obligate intracellular parasite, only drugs selectively affecting the parasite while keeping the host cells intact could be considered. In spite of this restriction, various selection protocols have been developed and are listed in Table 20.1. Chloramphenicol shows a potent but delayed parasiticidal effect, allowing the use of Escherichia coli CAT not only as reporter enzyme but also as a tight selectable marker (Kim et al., 1993). Parasite must complete up to three cycles of host cell lysis (up to 7 days) before an effect of the drug is evident. At this point, parasites are cloned in 96 well plates for about 5 days, in presence of drug selection. Another selection strategy based on the resistance to a drug can be achieved by exploiting the protective effect of the ble gene product of Streptoalloteichus or Tn5 against the DNA

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breaking activity of phleomycin (Messina et al., 1995; Soldati et al., 1995). Parasites expressing BLE become resistant to the drug, however, this selection needs to be applied on extracellular parasites to be effective as; otherwise, the host DNA would titrate down the drug. Phleomycin selection has been used successfully for the random insertion of transgenes (Soldati et al., 1995) and to disrupt genes by HRs (Mercier et al., 1998). As an alternative to drug resistance, stable selection can be achieved by complementation of the naturally occurring tryptophan auxotrophy of T. gondii by addition of indole to the culture medium (Sibley et al., 1994) following the introduction of the bacterial tryptophan synthase (trpB) gene. Two genes coding for nonessential nucleotide salvage pathway enzymes have been exploited as negative selectable markers. Loss of uracil phosphoribosyl transferase (UPRT) activity confers resistance to the pro-drug 50 fluo-20 -deoxyuridine (FUDR) (Donald and Roos, 1995), whereas in the absence of hypoxanthinexanthineguanine phosphoribosyl transferase (HXGPRT) activity, 6-thioxanthine (6-Tx) cannot be converted into an inhibitor of GMP synthase (Donald et al., 1996). Conversely, in HXGPRT deficient mutants, this gene is also used as a powerful positive selectable marker. Mycophenolic acid (MPA) efficiently kills parasites lacking the enzyme while supplementation with xanthine is required here for optimal growth. A high frequency of stable transformation is achievable using pyrimethamine resistance vectors derived from the parasite’s bifunctional dihydrofolate reductasethymidylate synthase DHFRTS. An artificially mutated dhfr-ts gene from T. gondii was used to design an expression vector pDHFR*TSc3 (no. 2854) that confers pyrimethamine resistance (Donald and Roos, 1993). The DHFRTS based selection is unique and shows an exceptional frequency of chromosomal integration of up to 5%

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TABLE 20.1 Selection strategies, gene markers, and conditions. Selectable marker genes

Recipient strain

Drug or selection procedure

Concentration range

CAT

Wild type

Chloramphenicol (CM)

20 μM CM

Escherichia coli

Drugs treatment during 7 days before cloning Wild type

Pyrimethamine (PYR); treatment during 2 days before cloning

1 μM PYR

Wild type

Phleomycin (PHLEO): 2 cycles of treatment during 510 h on extracellular parasites

5 μg/mL PHLEO

HXGPRT

RH hxgprt 2

Positive selection: MPA 1 xanthine: treatment during 3 days before cloning

25 μg/mL MPA

T. gondii

ME49 hxgprt 2

Negative selection: 6-Tx

50 μg/mL XAN

DHFRTS Toxoplasma gondii Ble Streptoalloteichus or Tn5

PRU hxgprt 2 UPRT

80 μg/mL 6-Tx

RH uprt 2

Negative selection: FUDR

5 μM FUDR

Wild type

FACS

TATi-1 conditional KO

ATc

Max 1 μM ATc

Transgenes flanked by loxP sites (recycling of markers)

Transient transfection with Cre expressing plasmid. Cloning immediately after electroporation

No selection

Wild type

Ganciclovir 24 h treatment

10 μM GCV

Wild type

5-Fluorocytosine

40 μM FLUC

T. gondii GFP/YFP Aequorea victoria Essential genes T. gondii Cre recombinase Enterobacteria phage P1 TK Herpes simplex CD E. coli 6-Tx, 6-Thioxanthine; FUDR, 50 -fluo-20 -deoxyuridine; MPA, mycophenolic acid.

(Donald and Roos, 1993). The flanking sequences of the DHFRTS genes are responsible for this unusual property, which can be partially conferred to other selectable marker genes such as the HXGPRT if this latter is controlled by the DHFRTS flanking sequences.

Fluorescence-activated cell sorting (FACS) is another way to select for stable transformation, when using a fluorescent tag as the selectable marker (Sheiner et al., 2011). To obtain clonal parasite lines stably expressing fluorescent proteins, two rounds of FACS and

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expansion of sorted parasites in culture (Gubbels et al., 2003) are routinely performed. Multiple fluorescent proteins can be used and sorted simultaneously, however, an instrument with multiple lasers might be required (see Section 20.5.5). The frequency of stable transformation significantly fluctuates depending on the type of selectable marker used. The conformation of the transfection plasmid (circular versus linearized by restriction digest) can also affect transfection efficiency. As a rule, circular plasmids are used for transient transfections while linearized plasmids are used to generate stable lines. Furthermore, restriction enzyme-mediated integration (REMI) can be used to further enhance the frequency of stable transformation up to 400-fold (Black et al., 1995) and enables cotransfection of several unselected constructs together with a single selectable marker. Any of the selectable marker genes listed earlier can, if needed, be efficiently recycled via excision by the site-specific Cre recombinase. The adaptation of the cre loxP system from bacteriophage P1 to T. gondii enables the specific in vivo excision of any introduced sequence, which is flanked by loxP sequences (Brecht et al., 1999).

20.2.3 Homologous recombination and random integration Unlike the situation in many protozoans, where integration into chromosomes occurs exclusively by HR and requires only a short segment of homology, HR is not favored in T. gondii. Vectors lacking long stretches of contiguous genomic DNA typically integrate into chromosomal DNA at random. The high frequency of transformation and random integration throughout the small genome size of haploid T. gondii tachyzoites was used as an efficient strategy to mutagenize

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the entire genome of T. gondii within one single electroporation cuvette (Roos et al., 1997). Such genomic scale tagging allows identification of any gene whose inactivation is not lethal to tachyzoites and for which a suitable functional selection or screen is available (Fig. 20.2). For example, positive/negative selection can be employed as selection schemes for mutants and promoter traps. The HXGPRT gene has been exploited to identify genes that are expressed in a stagespecific fashion (Knoll and Boothroyd, 1998). Parasites expressing HXGPRT under the control of a bradyzoite-specific promoter were mutagenized by random insertion of a plasmid and subjected to in vitro tachyzoite to bradyzoite conversion under 6-Tx selection to isolate mutants deficient in differentiation (Matrajt et al., 2002a). Signature-tagged mutagenesis has also been successfully applied to discover virulence genes in forward genetic screens, where the growth of insertional mutants has been compared in in vitro and in vivo (Frankel et al., 2007). HR leading to gene replacement (Fig. 20.2) is instrumental to study gene function and can be accomplished in T. gondii using different strategies. In wild-type parasites the efficiency of HR is favored if long contiguous stretches of homologous DNA are used to target the locus (Donald and Roos, 1994; Kim et al., 1993). Originally, the construction of vectors for HR was a relatively cumbersome approach mainly due to the cloning of long flanking regions. The multisite Gateway (Invitrogen) recombination technique is used to avoid restriction enzyme-mediated steps (Upadhya et al., 2011). Recombineering approaches using a cosmid library could also be followed to facilitate double HR (Brooks et al., 2010; Francia et al., 2012). A large insert DNA library was constructed in a copy-control fosmid backbone. Recombineering can thus be performed at single copy, which enhances stability and fidelity, followed by DNA production at higher copy number. The library was arrayed and end sequenced (Vinayak et al., 2014). Another way

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(A) Plasmid

Target...

Insertional

ori.

AmpR

DHFR-TS

...locus

Target... DHFR-TS

Promoter trap

(B)

...locus

(C) 1.5–2 kb

1.5–2 kb

1.5–2 kb A

CAT

B CAT

Target... YFP

YFP Target locus

CAT

A/B digest

Target locus Target... YFP

CAT

Target locus

Single homologous recombination CAT

Double homologous recombination

FIGURE 20.2 Exploiting nonhomologous insertion and homologous recombination to manipulate the Toxoplasma gondii genome. (A) Schematic representation of insertional genomic tagging using a DHFRTS plasmid (Roos et al., 1997). Plasmid DNA is indicated on top, genomic insertions below. For insertional mutagenesis expression of the DHFRTS pyrimethamine resistance gene is driven by its own promoter, the insertion is therefore not necessarily within the open reading frame but might also act through inactivating a regulatory region (e.g., promoter). In the case of promoter trapping DHFRTS does not carry its own promoter, and expression of the resistance gene depends on insertion close to an active promoter, or as an in-frame fusion into an expressed gene. Tandem insertions can complicate the identification of the tagged locus (Sullivan et al., 1999; Roos et al., 1997). (B) Schematic representation of gene knockout through double homologous recombination. The homologous regions destined for homologous recombination are represented by white boxes. Restriction enzymes A and B are used to generate fully homologous ends. In this case, YFP is used as a negative selectable marker to enrich for homologous recombination (YFP is lost and parasites are FACS negative) (Mazumdar et al., 2006). (C) Schematic representation of allelic replacement through single homologous recombination. In this strategy a circular plasmid inserts and tags the locus with a YFP fusion (which can be omitted, or replaced by a shortened open reading frame to create a functional knock-out). The gene-locus 30 of the plasmid backbone is functionally inactivated by the lack of a promoter. YFP, Yellow fluorescent protein.

to increase HR frequencies is by a combination of positive/negative selections (Fox et al., 1999, 2001; Mazumdar et al., 2006; Radke and White, 1998). Gene targeting was dramatically enhanced by the isolation of ΔKu80 strains. Ku80 is a

component of the nonhomologous end joining (NHEJ) pathway of DNA repair present in T. gondii yet lacking in many other apicomplexans including the Plasmodium species. Consequently, the usually high frequency of random integration events observed in wild-

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type parasites is almost completely abolished in ΔKu80, resulting in a parasite strain that allows efficient isolation of gene replacement and endogenous gene tagging events (Huynh and Carruthers, 2009; Fox et al., 2009).

20.2.4 Enhanced genetic manipulation through CRISPR/Cas9 The methods for the manipulation of specific loci discussed earlier, all rely on HR to site-specifically alter the genomic region of interest. Even when NHEJ is suppressed by the deletion of KU80, HR events are rare enough to require selection and large homology regions to enrich for parasites bearing the desired rearrangements. The efficiency of genetic engineering can be increased by generating doublestranded breaks at the locus of interest. In T. gondii, this has been achieved by adapting the CRISPR/Cas9 system, which directs the RNAguided Cas9 endonuclease from Streptococcus pyogenes to a specific locus through coexpression with a guide RNA. The guide RNA contains sequences necessary to bind Cas9 preceded by 20 bases of homology to a site in the parasite genome adjacent to an NGG or NAG motif. Redirecting the Cas9 nuclease is easily achieved by changing the sequence of the guide RNA. The impressive efficiency of this approach was demonstrated by transfecting parasites with plasmids expressing Cas9 under a T. gondii Pol II promoter, and a guide RNA under the T. gondii U6 promoter (Shen et al., 2014; Sidik et al., 2014). Large portions of parasites transfected with these constructs lost expression of the target gene, obviating the implementation of time-consuming selective strategies. The double-stranded breaks generated by CRISPR/Cas9 are repaired by the existing cellular machinery. In wild-type T. gondii strains, the double-stranded break in a target coding sequence is repaired by the NHEJ repair

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machinery leading to frequent indels resulting in premature stop codons and efficient gene knockout (Fig. 20.3A). Integration of CRISPR/ Cas9 plasmid fragments is also frequently observed and results in efficient disruption of the target GOI. Of relevance, knockouts generated via indels could revert to wild type by acquiring subsequent mutations resulting in the correction of the open reading frame. However, among numerous knockouts generated so far, only one such case was reported (Tosetti et al., 2019). To promote the complete deletion of the GOI, two sgRNAs can be used (Fig. 20.3B) (Tosetti et al., 2019). Of relevance, transfection of two CRISPR/Cas9 plasmids encoding different sgRNAs was found inefficient potentially due to the deleterious effect of the Cas9 overexpression (Jacot, unpublished). In contrast, two sgRNAs encoded on the same plasmid expressing Cas9 give excellent results and is recommended (Fig. 20.3B). Importantly, in ΔKu80 strains, doublestranded breaks are repaired through microhomology-mediated repair or through HR when an appropriate repair template is supplied. This offers the possibility to use PCR amplicons instead of the cumbersome cloning of large homology regions in plasmids, requiring merely forward and reverse primers with 3040 bp of homology to the target site and an appropriate plasmid as a template (Di Cristina and Carruthers, 2018; Shen et al., 2014; Sidik et al., 2014). The template plasmid can be a selection cassette and the appropriate selection used to enrich transgenic parasites (Fig. 20.3C). Remarkably, even without selection pressure, as many as 20% of the parasites that survive the operation may carry the desired rearrangement, enabling the generation of precise genetic changes like point mutations, difficult to achieve through previous methods (Sidik et al., 2014). To promote the complete deletion of the GOI, as presented earlier, two sgRNAs can be used to promote a large deletion or complete knockout of the GOI (Fig. 20.3D). The

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(A)

(B) Cas9

Cas9

+ sgRNA-1

v

v

sgRNA Transient transfection of a CRISPR/Cas9 plasmid

RH genomic locus

sgRNA-2

+

v v vv v

v v vv

v

sgRNA

sgRNA-2

sgRNA-1 Transient transfection of a CRISPR/Cas9 plasmid

RH genomic locus

STOP v v vv

v

v v vv

v

v

v

NNNN

Deletion of the first exon

Indels resulting in premature STOP codon

(C) Cas9

sgRNA +

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FIGURE 20.3 Toxoplasma gondii genetic manipulation with CRISPR/Cas9. (A) In wild-type parasites the doublestranded breaks generated by CRISPR/Cas9 are repaired by NHEJ leading to frequent indels resulting in premature stop codons. (B) Complete deletion of the GOI can be achieved using 2 sgRNAs encoded on the same plasmid expressing the Cas9. (C) In ΔKu80 strains, double-stranded breaks are repaired through HR when an appropriate repair template is supplied. PCR amplicons of an appropriate selection cassette with 30 bps homology regions are sufficient for efficient gene knockout. The 50 and 30 homology regions should not be contained within the sgRNA sequence. (D) As presented earlier, two sgRNAs can be used to generate large deletion. This is particularly suitable for the complete deletion of the GOI. The 50 and 30 homology regions must be 50 to the sgRNA-1 and 30 to the sgRNA-2, respectively. Note that in all strategies, the CRISPR/Cas9 machinery is only expressed transiently and will be lost after a few division cycles. GOI, Gene of interest; NHEJ, nonhomologous end joining.

20.3 Using transgenic parasites to study the function of parasite genes

CRISPR/Cas9 approach (combined with the use of a selectable marker) has also been used for the rapid construction of mutant lines with point mutations and for following the kinetics of gene manipulation in a population of organisms in culture allowing assessment of the fitness of a particular mutation (Sugi et al, 2016). The CRISPR/Cas9 approach was also adapted for the rapid generation of inducible knock-out strains (see next) (Brown and Sibley, 2018; Long et al., 2017; Jacot et al., 2019). A critical extension of the CRISPR/Cas9 system in T. gondii has been the development of pooled genome-wide screens (Sidik et al., 2016). Using massively parallel oligonucleotide synthesis, large libraries of guide RNAs have been constructed to target every protein-coding gene in the T. gondii genome with 10 different guide RNAs. By transfecting these libraries into a parasite strain constitutively expressing Cas9 and selecting for plasmid integration, large collections of mutants can be generated, barcoded by the guide RNA sequence each carries, such that their relative abundance in a population can be monitored by next-generation sequencing of the variable region from the guide RNA. This has enabled a complete assessment of the fitness contribution of each T. gondii gene to the infectious cycle in cultured fibroblasts. Changing the selective pressures in cell culture or deploying similar screens in vivo will be a valuable tool to understand parasite gene function in a wide variety of contexts.

20.3 Using transgenic parasites to study the function of parasite genes 20.3.1 Tagging subcellular compartments Visualizing and tracking the morphology and behavior of different subcellular compartments through the cells’ life cycle is an essential tool for cell biological analysis. Proteins localizing to almost any organelles of T. gondii have been

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described and a large number of constructs allowing expression of organelle-specific fluorescent proteins is now available (Fig. 20.4). Numerous versions of green fluorescent protein (GFP) and related fluorescent proteins have been successfully expressed in T. gondii (Kim et al., 2001; Striepen et al., 1998) and a range of colors is currently available for the simultaneous use of multiple markers. Cyan and yellow fluorescent protein (YFP) are a suitable pair for double labeling experiments and have been used in in vivo microscopic studies of T. gondii organelle biogenesis (Striepen et al., 2000; Pelletier et al., 2002; Joiner and Roos, 2002). A tandem repeat of the YFP gene yields exceptionally bright fluorescent transgenics that are now widely used to track parasites in tissue culture and in infected animals (Gubbels and Striepen, 2004; Gubbels et al., 2003, 2005; Egan et al., 2005). Red fluorescent proteins (RFP) further extend the options. DsRed produces brightly fluorescent parasites (Striepen et al., 2001); however, the requirement of tetramerization of this marker can be problematic if the tagged protein is part of a complex or structure. Monomeric variants of RFP [e.g., mRFP (Campbell et al., 2002)] can help overcome these problems but suffer from considerably weaker fluorescence. The mCherry and tomato variants (Shaner et al., 2004) provide a reasonable compromise and a tandem tomato marker produces exceptionally bright fluorescence when expressed in T. gondii (van Dooren et al., 2008). Parasites expressing fluorescent proteins can be analyzed and sorted by flow cytometry. In addition, fluorescent protein expression can be detected using a plate reader. This provides a convenient growth assay for drug screening and genetic selection (Gubbels et al., 2003).

20.3.2 Tagging of parasite proteins The cellular localization of a protein of interest is a first important step in order to characterize

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20. Genetic manipulation of Toxoplasma gondii

FIGURE 20.4 Tagging subcellular compartments with fluorescent protein markers in Toxoplasma gondii. This figure provides examples of single and dual fluorescent protein labeling T. gondii, all images were obtained by live-cell microscopy. (A) Dense granules and parasitophorous vacuole, P30GFP (Striepen et al., 1998); (B) centrocones (outermost dots) and posterior IMC rings of mother (innermost) daughter cells (lines), MORN1YFP (Gubbels et al., 2006); (C) nuclei, PCNAGFP (Radke et al., 2001); (D) plasma membrane, P30GFPGPI (Striepen, unpublished); (E) micronemes, MIC3GFP (Striepen et al., 2001); (F) cytoplasm, YFPYFP (Gubbels et al., 2003); (G) inner membrane complex, IMC3YFP (Gubbels et al., 2004); (H) microtubules, YFPTUB (Hu et al., 2002), (I) mitochondria, HSP60RFP (van Dooren, unpublished); (J) dividing tachyzoites IMC3YFP and H2bmRFP (Hu et al., 2004), (K) nuclear division and cytokinesis, H2bmRFP and MORN1YFP (Gubbels et al., 2006); (L) apicoplast division, FNRRFP and MORN1YFP (Striepen et al., 2000), (M) Golgi division, GRASPRFP and MORN1IMC; (N) apicoplast, ACPGFP (Waller et al., 1998); (O) rhoptries, ROP1GFP (Striepen et al., 1998); (P) endoplasmatic reticulum, P30GFPHDEL (Hager et al., 1999).

its function. Specific antibodies raised against subcellular fractions or individual proteins are widely used for this purpose for both light and electron microscopy techniques. This approach,

however, requires the production of antigen, either by the production of synthetic peptides or by recombinant expression and subsequent immunization, which is time-consuming and

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20.3 Using transgenic parasites to study the function of parasite genes

not always technically feasible. Through transfection experiments, proteins expressed as second copies from a heterologous promoter can be tagged by gene fusion using a generic epitope (for which antibodies are already available) or using fluorescent proteins. These tags can be placed at the N- or Cterminal end or even inserted internally. However, not all proteins can be investigated that way, as the tagging can affect targeting, maturation or function of its fusion partner. The bulky fluorescent proteins are sometimes problematic and short epitope tags with limited steric hindrance such as Myc (Delbac et al., 2001), HA (Karsten et al., 1997), FLAG (Sullivan et al., 2005), or Ty-1 (Herm-Gotz et al., 2002) offer suitable alternatives. Epitope tags require fixation and staining with a specific antibody before visualization. While not suitable for live-cell imaging, they can be used for subcellular and ultrastructural localization, immunoprecipitation experiments or to monitor protein processing during targeting or maturation. It has been frequently observed that the strength, and probably also the timing of expression with respect to the cell cycle, critically influence the outcome of an experiment and can lead to localization artifacts. For example, the overexpression of microneme proteins often results in accumulation in the early compartment of the secretory pathway or mistargeting to the parasitophorous vacuole (Soldati et al., 2001). To overcome this issue, localization of a protein of interest can also be achieved via endogenous tagging in ΔKu80 strain, this provides the advantage of preservation of strength and timing of expression as the native promoter element is used to drive transcription. Several C-terminal tagging constructs have been generated, taking advantage of ligation independent cloning (LIC) (Huynh and Carruthers, 2009). In combination with HR in a ΔKu80 strain the endogenous protein can be directly tagged (Huynh and Carruthers, 2009) and localized. Similar experiments can be performed using

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recombineered genomic cosmids and fosmids (Brooks et al., 2010). A CRISPR/Cas9 approach was also efficiently developed to insert epitope-tags at the endogenous C-terminus locus. Here, tagging is accomplished by electroporation of a plasmid expressing the Cas9 and a GOI specific sgRNA together with amplicons generated from plasmids containing epitope tags followed by a selectable marker. Note that the CRISPR/Cas9 machinery is only expressed transiently and while the amplicon will be selected for integration, the CRISPR/ Cas9 plasmid will be lost after a few division cycles. Such strategy was successfully applied to tag and localize calmodulin-like proteins to the conoid (Long et al., 2017). Multiple vectors with markers and selection cassette are available on https://www.addgene.org/ (Long et al., 2017). Importantly, in all cases a C-terminal tagging can interfere with the function of the proteins preventing the isolation of the tagged strain. Here, the versatility and power of the CRISPR/ Cas9 can be exploited to insert epitope tags anywhere in the endogenous locus as exemplified in Fig. 20.3C. The selection cassette can be replaced by an epitope tag in conjunction with an appropriate sgRNA to induce a doublestrand break at the appropriate location.

20.3.3 Genetic analysis of essential genes In order to study the function of essential genes in a haploid organism, several tools allowing the engineering of conditional knockout, knockdown or trans-dominant mutants were developed. Currently, multiple strategies operating at different levels, such as transcriptional control (Fig. 20.5A), control of protein stability (Fig. 20.5B), and gene excision by sitespecific recombination (Fig. 20.5C), have been implemented in T. gondii. Each of these technologies presents advantages and disadvantages to be considered for gene-function analysis.

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FIGURE 20.5 Inducible systems in T. gondii. (A) Schematic representation of the tetracycline-inducible systems. Here, a promoter containing 7 tetO replaces the endogenous promoter. The transactivator TATi-1 binds to the tetO sequences and promotes the transcription of GOI. The addition of ATc results in gene silencing. TATi-1 is either contained in the inducible plasmid or expressed constitutively in the recipient strain. (B) Schematic representation of the inducible systems targeting protein stability. (Upper panel) In absence of the small compound Shld1 the DD-fused protein is rapidly degraded. Addition of Shld1 will bind and fold the DD preventing protein degradation. The DD can be inserted either in the N- or C-terminal end of the protein. Although more cumbersome to engineer, N-terminal tagging is more tightly regulated. (Lower panel) The AID sequence is inserted at the C-terminus of the GOI. In the absence of IAA, the plant auxin receptor TIR1 is inactive (Apo state). Upon addition of IAA, auxin-bound TIR1 assembles into an active ubiquitin ligase complex. This complex now recognizes and polyubiquitinates the AID, targeting the fused protein to degradation. (C) Schematic representation of the inducible systems based on site-specific recombination. (Upper panel) The GOI is flanked by two loxP sites. Upon the addition of rapamycin, a functional DiCre recombinase is reconstituted. This will subsequently excise the GOI. (Lower panel) The endogenous 30 UTR of the GOI is first replaced by the SAG1 30 UTR that is flanked by two loxP sites and a 30 U1 snRNA recognition sequence. Addition of rapamycin leads to the transposition of the U1 element immediately after the STOP codon. This results in the recruitment of the T. gondii U1 snRNP (small nuclear ribonucleic particles) to the pre-mRNA, inhibiting polyadenylation and pre-mRNA maturation. Both systems are dependent on a recipient strain expressing constitutively the two inactive fragments of the Cre recombinase. AID, auxin-inducible degron; ATc, anhydrotetracycline; GOI, gene of interest; IAA, indole-3acetic acid; tetO, tet-Operators; UTR, untranslated region.

20.3 Using transgenic parasites to study the function of parasite genes

20.3.3.1 Tetracycline inducible systems One widely used approach to modulate expression is based on the E. coli tetracyclinerepressor system, which controls gene expression at the transcriptional level. The original tetracycline-repressor system interferes with transcription and has been optimized and coupled to T7 polymerase to tightly regulate gene expression in Trypanosoma brucei (Wirtz et al., 1999). The tet-repressor system has also been developed for other protozoan parasites including T. gondii (Meissner et al., 2001). Gene fusion of the tet-repressor (van Poppel et al., 2006) has led to higher transgene expression and tighter regulation. Although suitable for the expression of toxic genes and dominant negative mutants, this system proved not to be appropriate for the isolation of conditional knockouts in T. gondii. Indeed, the necessity to keep the parasites in presence of drug [anhydrotetracycline (ATc)] during a prolonged period in order to maintain the expression of an essential gene led to the generation of revertants that lost regulation. To improve the system, a genetic screen based on random insertion was designed to identify a functional transcriptional activating domain in T. gondii and to establish a tetracycline transactivator-based inducible system (Fig. 20.5A) (Meissner et al., 2002). This screen led to the isolation of two artificial transactivators that were not functional in HeLa cells, illustrating the differences between the transcription machinery in the parasite and its higher eukaryotic hosts. This system is suitable for the conditional disruption of Toxoplasma essential genes with no apparent reversion effect and operates on the parasites in the animal model. A line expressing one of the transactivators (TATi-1) was implemented to functionally analyze numerous genes including TgMyoA (Meissner et al., 2002), TgAMA-1 (Mital et al., 2005), TgMIC2 (Huynh and Carruthers, 2006), TgACP (Mazumdar

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et al., 2006), and profilin (Plattner et al., 2008), to name only the few pioneering studies. In its first design, the tet-inducible system is relatively laborious, requiring two steps of selection (Fig. 20.6A). The first step is the construction of a stable line expressing an inducible copy of the GOI via random integration in a TATi-1 expressing strain. The second step is the actual knockout of the target gene (see protocol section for details). More streamlined single-step strategies were established (Fig. 20.6B). In the first system the native promoter of the GOI is either replaced or distanced from the ATG by double HR with the DHFR selectable marker and the tetinducible promoter (Fig. 20.6B, upper panel). This can be performed in a ΔKu80 TATi-1 expressing line (Sheiner et al., 2011) to favor HR events. In the resulting mutant parasite the tet-inducible promoter, in its genomic context, directly controls the GOI, and addition of ATc induces the knockdown (Sheiner et al., 2011). Numerous studies used this strategy (Francia et al., 2012; Sampels et al., 2012; Biddau et al., 2018; Daher et al., 2015; Sheiner et al., 2015). An alternative single-step approach consists in using vectors that carry the coding sequence of TATi-1 under the control of a tubulin promoter (Tub8) downstream of the 50 UTR of the GOI. The 30 recombination results in the replacement of the endogenous promoter by the inducible tet-operator containing promoter. Simultaneously, a tag can be placed at the N-terminus of the GOI (Fig. 20.6B, lower panel). The introduction of CRISPR/Cas9 improved the efficiency of both strategies presented in Fig. 20.5B and C. Instead of cloning large homology regions, PCR-generated amplicons with 30 bps 50 and 30 homology regions to the targeted gene could be used. Double HR at the locus of interest is assisted by CRISPR/Cas9. Fig. 20.6C illustrates such a strategy where the sgRNA is designed close to the start codon. A plasmid containing a self-contained inducible system is used as template for PCR using primers with 30 bps 50 and 30 homology regions.

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FIGURE 20.6 Tetracycline inducible systems. (A) Schematic representation of the “two-step” process in the strain expressing constitutively TATi-1. The first step consists with the random insertion of the GOI cDNA under the control of the TetO7 promoter. The second step is the knockout of the GOI. (B) The disruption of Ku80 in TATi-1 expressing strain favored the recovery of double homologous recombination events where the promoter of the GOI is replaced by the TetO7 promoter. Alternatively, an “all in one vector” includes all the different elements for transactivation. (C) The self-contained approach can be efficiently used in combination with a CRISPR/Cas9 approach. There, a PCR amplicon with 30 bp homology regions is cotransfected with a sgRNA targeting the region just upstream to the ATG. Double homologous recombination will result in the generation of the inducible locus. GOI, Gene of interest.

20.3 Using transgenic parasites to study the function of parasite genes

This amplicon is then cotransfected with a plasmid expressing the Cas9 and the sgRNA. Note that the CRISPR/Cas9 machinery is only expressed transiently and while the amplicon will be selected for integration, the CRISPR/ Cas9 plasmid will be lost after a few division cycles. This strategy was well established in the RHΔKu80 parasite strain (Jacot et al., 2016; Frenal et al., 2017). However, CRISPR/Cas9 based genome editing using short homology regions (30 bps) was demonstrated to be successful in wild-type stain suggesting that this strategy can be applied broadly without being restricted to strains lacking Ku80 (Di Cristina and Carruthers, 2018; Shen et al., 2014; Sidik et al., 2018). A complete protocol for CRISPR/Cas9mediated generation of tetracycline inducible knockdown is available (Jacot et al., 2019). An additional example can be found here (Lacombe et al., 2019). The “two steps” strategy in TATi is a robust but laborious method to produce conditional knockout parasites (Fig. 20.6A). The first step results with different clones of various levels of expression (due to number of random integrations, integration in highly transcribed loci, etc.) and allows therefore the selection of the most suitable expression. The other strategies reduce this process to only one step but the transactivation only takes place in the GOI endogenous locus and in a single copy. The level and the proper timing of transactivation are critical for the establishment of conditional systems and therefore each method has to be considered for different genes. 20.3.3.2 Regulation of protein stability A major limitation of the earlier described conditional systems is their relatively slow kinetics Indeed, the proteins of interest are still present after removal or downregulation of the respective genes. In the case of very stable proteins, it can take up to 96 hours

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before a phenotype becomes fully evident. Such long incubation times can complicate the interpretation of the observed phenotypes in particular to discriminate primary from secondary effects. Two fast methods, based on conditional regulation of protein stability, have been successfully adapted to apicomplexans. 20.3.3.2.1 Destabilization domain (ddFKBP)

This system is based on mutated forms of the FKBP12-rapamycin binding protein that result in its fast degradation (Herm-Gotz et al., 2007; Armstrong and Goldberg, 2007). Fusion of this degradation domain (ddFKBP) to a protein of interest results in the degradation of the entire protein by the proteasome. Addition of the inducer shield (Shld-1) (a rapamycin analog) results in rapid stabilization of the protein (Fig. 20.5B, upper panel) (Banaszynski et al., 2006). Regulation can be achieved by fusing ddFKBP either at N- or C-terminus, however N-terminal fusion confers a vastly more performant destabilization in T. gondii (Herm-Gotz et al., 2007). In principle, this system should be suitable to construct conditional mutants by direct endogenous tagging. While direct allelic replacement was successful in some cases in Plasmodium falciparum (Dvorin et al., 2010; Farrell et al., 2011; Russo et al., 2009), most attempts using this strategy in T. gondii failed and resulted in the expression of ddFKBP-tagged proteins that remained stable in the absence of Shld-1. One exception is presented in the following paper (Heredero-Bermejo et al., 2019). Despite these obstacles the ddFKBP system is very well suited to generate overexpression or trans-dominant mutants (van Dooren et al., 2009; Santos et al., 2011; Daher et al., 2010; Breinich et al., 2009; Agop-Nersesian et al., 2010) however extreme caution is required in analyzing and interpreting the effects of dominant negative mutants as this strategy is prone to artifacts.

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20.3.3.2.2 Auxin-based degron system

20.3.3.3 Site-specific recombination

An auxin-inducible degron (AID) system was established in T. gondii to rapidly destabilize proteins and study essential genes (Fig. 20.5B, lower panel) (Brown and Sibley, 2018; Brown et al., 2017). This system is based on phytohormones called auxins that target certain proteins for proteasomal degradation in plants (Teale et al., 2006). Two transgenic components are required to implement this system. A recipient strain constitutively expressing the Oryza sativa transport inhibitor response 1 (TIR1) has to be expressed and the protein of interest has to be tagged with an AID. Addition of auxin [indole3-acetic acid (IAA)] results with the activation of a TIR1 mediated ubiquitin ligase activity, which targets exclusively the AID-tagged protein ultimately leading to rapid ubiquitin-dependent proteasomal degradation. Either tagging of the protein of interest can be achieved using a single HR strategy to insert the tag at the C-terminal endogenous locus or using a CRISPR/Cas9 approach as described previously. A detailed protocol for the generation of CRISPR/Cas9-mediated conditional knockdown of proteins using the AID system is available (Brown and Sibley, 2018). This system has recently been used successfully to generate several tightly regulated inducible knockdowns (Brown et al., 2017; Brown and Sibley, 2018; Bisio et al., 2019). The rapid kinetic of the both the ddFKBP and AID systems is of particular interest, when rapid processes are to be analyzed, such as components of trafficking systems or signaling cascades. However, these systems are not suited to destabilize proteins targeted to the secretory pathway except if the protein harbors N- or C-terminal domains facing the cytosol allowing access to the proteasome for degradation. This is notably the case for polytopic proteins (Bisio et al., 2019; Brown and Sibley, 2018; Santos et al., 2011).

20.3.3.3.1 Excision of LoxP flanked genes

The yeast recombinases Cre and Flp recognize DNA sequences, LOX and FRT sites, respectively, that are short enough for convenient cloning, but long enough to be specific and absent from even large genomes when not deliberately introduced. Both recombinases are highly efficient in excising DNA that lies in between the recognition sites and recombination requires only a minimal amount of recombinase activity. However, in order to generate conditional knockouts, temporal control of Cre is required. This can in principle be achieved via transient transfections with a Cre expression construct (Heaslip et al., 2010), however, transfection efficiencies can vary and Cre overexpression is toxic [most likely due to nonspecific recombination events (Xiao et al., 2012)]. A solution to this problem is provided by conditional Cre-systems, such as ligand controlled Cre-recombinases (Metzger et al., 1995) or dimerisable Cre (DiCre) (Jullien et al., 2003). While fusions of Cre to hormone binding domains have been shown to still be constitutively active in T. gondii (Brecht et al., 1999), the DiCre-system allows rapid, specific and efficient temporal control of Cre activity (Andenmatten et al., 2013). Here, the Cre recombinase is split into two inactive fragments that are fused to the rapamycin binding proteins FRB and FKBP, respectively. Addition of the ligand rapamycin results in reconstitution of the functional enzyme and excision of the GOI flanked by LoxP sites (Fig. 20.5C, upper panel). 20.3.3.3.2 U1 small nuclear ribonucleic particlesmediated gene silencing

Based on the same DiCre-mediated strategy, the U1 small nuclear ribonucleic particles mediated gene silencing was also established (Fig. 20.5C, lower panel) (Pieperhoff et al., 2015).

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20.3 Using transgenic parasites to study the function of parasite genes

Here, the rapamycin-induction resulted in the repositioning of the U1 recognition sites adjacent to the termination codon of the GOI, leading to pre-mRNA degradation and efficient gene knockdown. In contrast to the excision of the GOI flanked by LoxP sites, this strategy is more straightforward as it requires less genetic engineering. A successful use of this strategy is described here (Rugarabamu et al., 2015). A clear advantage of the DiCre approaches is that the GOI is under the control of its endogenous promoter, ensuring correct timing and levels of gene expression. In addition, future constructs can be easily modified to allow high-content cloning of knockout vectors, comparable to approaches applied in mice (Skarnes et al., 2011). Challenges of the DiCre system include a difficulty to obtain clonal knockout population, since induction of DiCre results in recombination rate between 20% and 90% leading to a mixed population of KO and wild-type parasites. Another disadvantage is the irreversibility of the recombination event. A major issue of the originally generated DiCre strains was the very low and unpredictable recombination rate (Andenmatten et al., 2013) This was caused by the original design of the plasmid, allowing the simultaneous expression of the two Cre-fragments. Since both expression cassettes shared the

Ku80 3′UTR

α-tub prom

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T2A

same promoter and 30 UTR, intrachromosomal recombinations within this cassette led to the loss of the expression cassette for one DiCre fragment and therefore in total loss of Creactivity over time. One strategy to minimize this problem is to constantly subclone the DiCre-expressing strain and to regularly test for DiCre-activity. However, several labs observed a loss of DiCre-activity during the selection process of conditional mutants. A new design by the Treeck laboratory for expression of the DiCre-fragments makes use of the T2A-self-cleaving peptide (Wang et al., 2015), allowing expression of the DiCrecassette from the same promoter and hence avoiding the risk of intrachromosomal recombinations. Furthermore, the selection marker CAT was placed between the two DiCrefragments to allow a constant selection for the intact expression cassette (Fig. 20.7) (Hunt et al., 2019). The inducible DiCre line can be used in a reverse way to create inducible overexpression. For this, the GOI is separated from a strong promoter with a “spacer” sequence (e.g., fluorescent marker) that is flanked with LoxP sites. Upon DiCre activation the spacer is spliced-out and the GOI is placed directly downstream of the strong promoter that in turn drives its overexpression. An example for this is found in Biddau’s 2018 paper (Biddau et al., 2018).

CAT

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FIGURE 20.7 Stable DiCre expressing strain. Schematic representation of the strategy used to generate a stable DiCre expressing strain. T2A-self-cleaving peptide allows the expression of the DiCre-cassette from the same promoter. A chloramphenicol selection cassette (CAT) is placed in between and could be used to select back the parasites. FRB_Cre2 and FKBP_Cre1 are the two split parts of the Cre recombinase. All elements are under the control of the strong tub8 promoter. The Ku80 30 and 50 UTR sequence target the integration of the inducible cassette in the Ku80 locus generation a ΔKu80 strain. CAT, Chloramphenicol acetyltransferase; DiCre, dimerisable Cre; UTR, untranslated region.

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20.3.4 Insertional mutagenesis and promoter trapping as tools of functional genetic analysis Random high-frequency integration of a genetic element into the parasite genome can be used to disrupt loci and produce pools of insertional mutants. The integrated sequence can subsequently be exploited to identify the targeted gene with modest effort (Fig. 20.2). The exceptionally high frequency of non-HR of transgenes in T. gondii allows the use of simple plasmid constructs similar to the way transposons are used in other organisms (Donald et al., 1996). Several nonessential genes have been identified using the random insertion of a DHFRTS or HXGPRT cassette (Sullivan et al., 1999; Donald and Roos, 1995; Chiang et al., 1999; Arrizabalaga et al., 2004). The genomic locus tagged by the insertion can be identified by plasmid rescue or inverse PCR strategies (Roos et al., 1997). The insertional strategy is not limited to gene disruption but can also be used to trap promoters and genes. Bradyzoite specific genes (Bohne et al., 1997; Knoll and Boothroyd, 1998) as well as genes controlling differentiation (Matrajt et al., 2002a; Vanchinathan et al., 2005) have been identified using differential HXGPRT selection under culture conditions that favor differentiation into bradyzoites followed by counterselection under “tachyzoite” culture conditions. Trapping of native T. gondii transcription factors might also be achievable. For this, a recipient strain harboring a YFPYFP marker under the control of a tet-regulated promoter would be randomly inserted. The tagging plasmid would harbor a tet-repressor gene lacking a stop codon and 30 UTR sequences. Translational fusion of this marker with a transcription factor should result in transactivation and hence green fluorescence as designed to isolate TATi-1 (Meissner et al., 2001, 2002). The fact that tachyzoites are haploid precludes the generation/identification of essential

genes by insertional mutagenesis. Nevertheless, it is possible to generate a library of parasite mutants for essential genes by coupling random insertion to the tet-inducible system (Jammallo et al., 2011). Signature-tagged mutagenesis is another strategy that has been used to identify essential genes by insertional tagging. In this case, screening is performed in a different life-cycle stage or under different growth conditions to permit the identification of “differentially essential” genes. This approach has recently been adapted to T. gondii (Knoll et al., 2001). Wild-type parasite clones are first tagged with unique oligonucleotide insertions (the signature-tag). These clones are then mutagenized (chemical or insertional) followed by another cloning step. Pools of mutants, which are distinguishable by their tag, are subsequently exposed to a selective condition, for example, infection into an animal. Tagging of genes that are essential in this condition will result in loss of the mutant. “Missing” mutants are then identified by comparing the tags present in pools before and after selection. Several candidate genes important for parasite persistence in the mouse have been identified using this approach (Craver et al., 2010; Frankel et al., 2007; Payne et al., 2011; Skariah et al., 2012).

20.3.5 Forward genetic analysis using chemical mutagenesis and complementation cloning Genetic analysis of pathways essential for growth in culture requires the generation of conditional mutants. Temperature sensitivity (ts) due to chemically induced point mutations can be exploited to obtain strains that are viable at the permissive temperature and display a mutant phenotype at the restrictive temperature. Heat-sensitive (Pfefferkorn and Pfefferkorn, 1976; Radke et al., 2000; Gubbels et al., 2008) and cold-sensitive (Uyetake et al.,

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20.3 Using transgenic parasites to study the function of parasite genes

2001) mutants have been isolated using ENU (N-ethyl-N-nitrosourea) as mutagen of choice in most T. gondii studies to induces random point mutations. Chemical mutagenesis has been successfully used to produce parasite mutants with defects in stage differentiation (Singh et al., 2002), invasion and egress (Black et al., 2000; Uyetake et al., 2001), and cell division and cell-cycle progression (Radke et al., 2000; White et al., 2005). While generating chemical mutants is straightforward, identifying the mutated gene responsible for the phenotype is not. The two avenues most commonly used to accomplish this goal are physical mapping through crosses, and phenotypic complementation by transfection with a wild-type DNA library. While crosses are feasible in T. gondii, the need of cats and their limited throughput makes them less practical as a general tool for mutant analysis (also the RH strain used as the molecular biology workhorse for T. gondii is unable to complete the sexual life cycle). The second approach to identify the gene affected in a given mutant is based on phenotypic complementation using a library of wild-type DNA. This strategy faces two technical challenges: full representation of the genome (or transcriptome) in the complementation library, and efficient recovery of the complementing sequence. Black and colleagues identified a genetic element that maintains stable episomes in T. gondii (Black and Boothroyd, 1998) allowing convenient rescue by heat lysis and transformation of bacteria. A library harboring an episomal maintenance sequence on the backbone successfully complemented the HXGPRT locus in the knockout mutant under MPA selection. Analysis of the recovered plasmids however suggested that they might undergo extensive recombination, potentially decreasing their stability and usefulness (Black and Boothroyd, 1998). The second effort to generate a complementation system was built on high-frequency

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integration of library plasmids (Striepen et al., 2002). Mutants are transfected with a plasmid library and subjected to selection. Subsequently complementing DNA sequences (carried as stable chromosomal insets) are rescued into plasmid using an in vitro recombination protocol (Invitrogen Gateway system) (Hartley et al., 2000). Rescued library inserts can be shuttled back into a parasite expression plasmid through a second recombination step to confirm their complementation capacity. A cDNA library builds on this model successfully complemented the Toxoplasma HXGPRT locus at high efficiency (Striepen et al., 2002) and was used to identify a phenotypic suppressor of the T. gondii ts cell cycle mutant C911 (Radke et al., 2000; White et al., 2005). An analogous library carrying Cryptosporidium parvum genomic DNA was used for heterologous complementation resulting in the identification of a Cryptosporidium gene encoding the purine salvage enzyme inosine-50 -monophosphate dehydrogenase (Striepen et al., 2002; Umejiego et al., 2004). Several ts mutants could not be complemented using the cDNA libraries described earlier (Gubbels, White, and BS unpublished). Genes encoding large mRNAs and/or transcribed at low levels are typically underrepresented in cDNA libraries. To overcome these problems, a large insert (4050 kb) genomic cosmid library, built on a DHFRTS containing supercos vector, was constructed. This library provides sufficient coverage and transformation efficiency to complement the lack of HXGPRT in every transfection reaction attempted. In addition, we complemented numerous mutants with a ts cell division defect (Gubbels et al., 2008). Note that the increase power of sequencing technology now also allows to sequence the complete genome of mutants, thus permitting to pinpoint the genetic basis of temperature-sensitive defects even in mutants that fail to complement efficiently (Gubbels et al., 2008).

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20.4 Perspectives T. gondii has proven itself as an excellent experimental model and reverse genetic approaches were key to build a detailed molecular picture of apicomplexan biology. The reverse genetic toolbox has seen constant extension and refinement. The potential and limits of the genetic approaches have been reviewed in light of the biological specificities that differ between T. gondii and rodent or human malaria parasites (Limenitakis and Soldati-Favre, 2011). The critical extension of the CRISPR/Cas9 system in T. gondii with the development of pooled genome-wide screens (Sidik et al., 2016) allowed a complete assessment of the fitness contribution of each 8000 individual genes contained in T. gondii genome. Using this approach and by changing the selective pressures in cell culture (drug pressure, metabolite starvation, and cyst conversion) represent powerful and high throughput tools to investigate parasites biology. Forward genetic approaches have seen considerable progress as well. These approaches could hold the key to mechanistic analysis of phenomena for which the genome does not immediately present an obvious list of candidate genes and proteins. While the tools to complement mutants have improved and may now be at a level to permit robust analysis, the ways to generate and select such mutants still lag. Robust screens that reduce a complex cell biological phenomenon to a phenotype that can be easily scored in thousands of mutants with limited effort are needed. The success of visual screens using automated microscopic detection (Carey et al., 2004) points to one avenue to reach this goal. The past decade has seen tremendous progress driven by the ability to transfect and genetically manipulate the parasites. Now, a set of tools based on the CRISPR/Cas9 technology took full advantage of the genome

sequence. With knockouts and conditional knockouts becoming standard and easy practice, the future of the genetic research of the Toxoplasma biology might be a community effort to harmonize and integrate different ways of selecting and phenotyping mutants into a unified understanding of parasite physiology, cell biology, and pathogenesis.

20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization 20.5.1 Propagation of Toxoplasma tachyzoites in tissue culture T. gondii is promiscuous in its choice of host cell and will infect almost any mammalian cell commonly used in tissue culture work. In general, large spread-out cells like fibroblasts or Vero cells are most suitable. Infection of these cells results in distinctive rosettes, which makes it easy to monitor parasite development by microscopy. Many laboratories use transformed cell lines like Vero or 3T3, which produce high parasite yields. Immortal lines grow fast, are easy to culture and can be obtained from many sources. Primary cell lines like human foreskin fibroblasts (HFFs) are also widely used. Their strong contact inhibition and slow growth make them the cell of choice for plaque assays, bradyzoite induction experiments, genetic selections, or any experiment in which cultures have to be maintained for longer periods of time. They also provide excellent microscopy for cell biological analysis. The disadvantage of primary lines is that they have to be managed more carefully as they will die at higher passage number due to senescence. A sufficient amount of early passage cells has to be cryopreserved to reinitiate the culture at that point. The human telomerase reverse transcriptase (hTERT)-immortalized cells (BD Biosciences) have emerged as a compromise;

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these cells are immortal but retain many characteristics of primary fibroblasts. We have found these cells to be equivalent to HFF cells in almost all applications. The protocol s below are based on HFF cells but can be used for HTERT cells as well (note the difference in glutamine concentration). Many companies supply reagents for tissue culture, the suppliers mentioned in the following are the ones we have used, products from other sources might work just as well. 20.5.1.1 Maintenance of human foreskin fibroblast cells • T25 flask tissue cultures typically yield 4107 parasites (yields are typically lower for the types II and III cyst-forming strains). The protocols below are based on this scale. If more material is needed, larger flasks (e.g., T175), roller bottles and cell factories have been used successfully with appropriately scaled protocols. • Warm media and trypsin solution in a 37 C water bath. • Aspirate medium from a confluent culture and add 2.5 mL of trypsin solution to the flask (0.25% trypsin and 0.2 g/L ethylenediaminetetraacetic acid in HBSS, Hyclone, store this solution in smaller 5 mL aliquots at 220 C for convenience). Carefully “wash” monolayer by tilting flask several times, aspirate most of the solution and leave enough to just cover the cells (B0.5 mL). Incubate at 37 C for 2 minutes. Inspect cells for rounding and detachment using an inverted microscope equipped with phase or interference contrast optics. If cells are still attached after 2 minutes, tap flask with flat hand and/or prolong incubation. HFFs are relatively fragile so take care and do not overtrypsinize. • Immediately take up detached cells in a defined volume of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% new born calf serum (NBCS,

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Hyclone, cosmic calf serum), penicillin and streptomycin (1:200 of a 10,000 U/mL of antibiotic stock, Hyclone), glutamine (1:100 of a 200 mM stock in water, note: for HTERT cells do not add glutamine to avoid overgrowing of cultures), and split 1:8 into new flasks. If fungal contaminations are a frequent problem use 1:100 Fungizone (250 μg/mL amphothericin B, Invitrogen). Move to incubators gassed to 5% CO2 at 37 C. Allow gas exchange by loosening caps. Confluent cultures can be kept for several weeks prior to T. gondii infection. 20.5.1.2 Maintenance of tachyzoites • Aspirate medium from a confluent HFF culture. • Add 10 mL of infection medium [DMEM supplemented with 1% fetal calf serum (FCS, Invitrogen. For experiments which require tight control over the small molecule composition use dialyzed FCS), penicillin and streptomycin as above]. • Infect a new flask with culture supernatant of a freshly lysed culture. As a rule of thumb, passing 0.51 mL into a T25 culture will result in complete lysis within 23 days for RH derived strains. A high inoculum is preferable if parasites are to be used, for example, in a transfection experiment as the majority of the tachyzoites will egress synchronously resulting in high overall parasite viability. To maintain strains, pass smaller number of parasites (e.g., 100 μL of a lysed culture). Transfection efficiency and invasion efficiency are greatly enhanced by using freshly lysed parasites. Host cells should not be overinfected. Ideally, every host cell should be infected with one parasite. 20.5.1.3 Cryopreservation of host cells and parasites • The aim is to freeze slowly and to thaw quickly. Wear a lab coat, face protection, and appropriately insulated gloves when handling liquid nitrogen. For best results

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have all tubes and reagents prepared and labeled, chill them on ice, and work quickly (if you have to freeze many vials at a time, divide them into smaller batches). Label 2 mL cryovials (fitted with silicone o-ring, Nalgene) using a pen dispensing ink that resists liquid nitrogen and chill on ice. Prepare an isopropanol/water containing freezing container (VWR, using this simple and inexpensive device will result in about 1 C/min cooling in a 280 C freezer, alternatively use a thin-walled foam container to slow cooling) Use “freshly” confluent (T175) HFF cultures for freezing. Trypsinize cells as described earlier and recover detached cells in DMEM 10% NBCS into a 15 mL sterile centrifuge tube. Pellet cells in a table-top centrifuge at 900 3 g for 10 minutes at 4 C using a swing bucket rotor. Discard the supernatant and resuspended cells in 1.8 mL chilled DMEM (no serum). Add 1.8 mL freezing medium (25% tissue culture grade dimethyl sulfoxide (DMSO) and 20% fetal bovine serum (FBS) in DMEM) and mix quickly. Immediately dispense 0.5 mL aliquots into chilled freezing tubes, tightly cap tubes, and move into chilled (ice) freezing container and place into a 280 C freezer. Thaw one vial the next day to ensure that your stocks are viable and move the remaining vials into a liquid nitrogen storage container. Solid bookkeeping which keeps track of rack, box, and vial position is essential as it is not easy to “search” for vials in liquid nitrogen stocks. Parasites are preserved as extracellular tachyzoites. Pellet a freshly lysed culture (1500 3 g, 20 minutes, 4 C) and then proceed as described for host cells above. Plan to freeze 2 3 108 per vial which means that you will produce 3 vials from a single T25 culture using 0.8 mL of DMEM and 0.8 mL

of freezing medium. Test for viability by thawing before you discontinue the culture of the given line. • Parasites can also be cryopreserved in host cells at the rosette stage in DMEM with 50% FBS/10% DMSO. • To thaw HFF cells, prepare a flask with medium warmed to 37 C. Remove one vial at a time from liquid nitrogen with thongs and immediately immerse into a beaker filled with water warmed to 37 C gently shaking the vial. Once the medium is thawed, transfer cells to the flask and incubate as described for standard culture. Replace medium after 12 hours. • To thaw parasites, use the above procedure and inoculate a confluent T25 culture. 20.5.1.4 Mycoplasma detection and removal • Mycoplasma contamination is a frequent plague of tissue cultures. Heavy infection can affect the growth of host cells, mycoplasma DNA can produce unwanted background in genetic experiments, and bacterial contamination is a severe problem for immunological experiments as mycoplasmaderived molecules potently stimulate a variety of immune cells and functions. • A simple test for contamination can be performed by DNA staining. Culture cells (and/or parasites) for two passages in the absence of antibiotics (which will lead to massive amplification of the bacteria) then transfer to six well plates with coverslips. • Stain coverslip cultures for bacterial DNA using DAPI using the standard IFA protocol provided below more sensitive staining can be obtained by acid/alcohol fixation and Hoechst staining (see Chen, 1977 for a detailed protocol). • In contaminated cultures, you will observe numerous small dots of DNA staining (about the size of the typical apicoplast

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genome staining) throughout the cytoplasm of the host cell. More sensitive PCR (ATCC, Stratagene) or LUC-based (MycoAlert, Cambrex) assays are also available. If you suspect a recent contamination, discard your cultures, thaw fresh vial from liquid nitrogen, and retest. Protocols to screen strains obtained from other laboratories should be routine. If you have to “rescue” your particular strain treat with Mycoplasma Removal Agent according to the manufacturer’s guidelines (an inhibitor of bacterial gyrase, e.g., MP Pharmaceuticals) for three passages and then retest (this antibiotic is reasonably tolerated by T. gondii at the suggested concentration). Other commercial agents kill T. gondii and should be screened prior to use as mycoplasma elimination agents. Alternatively, passage of the strain through a mouse and reisolation into tissue culture will remove mycoplasma.

20.5.1.5 Passaging Toxoplasma tachyzoites/ bradyzoite cysts in animal Tachyzoites of any strain can be maintained by passage in the peritoneal cavities of mice; 104 (type I strain, i.e., RH) or 105 (type II or III strain, i.e., ME49 or Prugniaud) are injected intraperitoneally into the mouse. • Replicating T. gondii can be harvested from the peritoneal cavity 3 (for type I strains) and 5 days later (type II or III strain) by peritoneal lavage with 4 mL of sterile saline or phosphate buffered saline (PBS). • This material can be used to serially passage the strain in the peritoneal cavities of mice or to infect tissue culture cells. Murine inflammatory cells (macrophages and neutrophils) will also be seen in this lavage material. • Passage through mice can be useful to remove microorganisms that have

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contaminated T. gondii tissue culture, provided that they cannot replicate in murine peritoneum. Anecdotal data indicate that periodic murine passage of a T. gondii strain passaged continuously in tissue culture helps to maintain the vigor and biologic characteristics of the strain.

20.5.2 Transfection and stable transformation protocols 20.5.2.1 Transient transfection • Cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, and 5 mM MgCl2) can be prepared in larger batches, filter sterilized, and stored in aliquots at 220 C or 4 C (Vandenhoff et al., 1992). • Weigh 12 mg ATP and 15.2 mg glutathione, add to 10 mL of cytomix and sterilize by passing through a 0.22 μm filter. • Sterilize DNA by ethanol precipitation. Adjust 50 μg of plasmid DNA (typically in B10 μL and purified using a commercial plasmid purification kit, e.g., Qiagen) to 100 μL with TE (pH 8.0). Add 11 μL 3 M NaOAc, and 250 μL ethanol. Precipitate DNA for 5 minutes at 220 C and spin at full speed in a microcentrifuge. • Wash the pellet with 1 mL cold 70% ethanol by gently inverting the tube and spin for 2 minutes in a microcentrifuge. • Move tubes into the laminar flow hood and discard the ethanol (keep an eye on the pellet). • Let ethanol evaporate for 510 minutes (be careful not to “overdry” as it can be hard to redissolve DNA). Resuspend DNA in 100 μL cytomix. • Filter parasites from a freshly lysed T175 flask into a 50 mL polypropylene tube and count t in a hemocytometer (dilute sample 1:10 in PBS for counting). Pellet parasites at

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1500 3 g, 20 minutes, 4 C and resuspend in complete cytomix to 3.3 3 107 parasites per mL (if required the parasite concentration can be increased up to eight times). • Mix 100 μL plasmid DNA and 300 μL parasites in a 2 mm gap electroporation cuvette (genetronix) and electroporate parasites with a single 1.5 kV pulse, a resistance setting of 25 Ω, and a capacitor setting of 25 μF using a BTX ECM 630. If you use a BioRad electroporator set to 1.5 kV, 25 μF and square wave. If you are employing an Amaxa system use the T-cell solution instead of cytomix and set the electroporation conditions to program U33. • Transfer parasites immediately into a confluent T25 HFF culture (for selection and biochemical experiments) or onto coverslips for microscopy (see next). • Expression of the transgene can be detected beginning 8 hours after transfection (depending on the transgene and the sensitivity of the assay employed) and peaks around 36 hours after electroporation. To measure transient transfection efficiency electroporate with a robust and easy to score visual marker [e.g., plasmid tubYFPYFPsagCAT (Gubbels et al., 2003)]. Inoculate coverslips and count total number of vacuoles and number of fluorescent vacuoles for several fields. All three electroporators yield transient efficiencies of 30%50% 24 hours after electroporation. 20.5.2.2 Selection of stable transformants CAT: Selection for CAT can start immediately after electroporation in presence of 20 μM chloramphenicol (34 mg/ mL stock in ethanol). Since the effect of the drug is delayed, it is important to passage the parasites every 2 days by inoculating at least 106 parasites to keep the pool of parasites as heterogeneous as possible. The minimal amount of plasmid required to generate stable transformants depends on

the vector used but 1050 μg of linearized plasmid will usually yield stable transformants. DHFRTS: Electroporate parasites with 50 μg of a plasmid encoding the drugresistant dihydrofolate reductasethymidylate synthase allele (Donald and Roos, 1993), for example, plasmid pDHFR*TScABP (Sullivan et al., 1999). After electroporation culture in the presence of 1 μM pyrimethamine (1 μL of a 10 mM stock in ethanol). This plasmid results in the highest frequency of transformation (up to 1%5%). Be careful handling transgenic strains as pyrimethamine is used in the treatment of human toxoplasmosis. HXGPRT: This selection requires a hypoxanthinexanthineguanine phosphoribosyltransferase null mutant (such mutants are available now for multiple strains, see, e.g., Donald et al., 1996 for RH). 24 hours after transfection add 25 μg/mL MPA (25 mg/mL stock in ethanol) and 50 μg/mL xanthine (50 mg/mL stock in 0.1 N KOH). MPA/xanthine should kill parasites within 23 days. BLE: For phleomycin selection electroporate parasites with an expression vector encoding the resistance marker BLE (Messina et al., 1995) transfer to HFF cells until complete lysis of the host culture occurred (1224 hours later). The lysed culture is forced three times through a 25-ga needle to assure that all the parasites are extracellular (see safety section for concerns about needle passing before using this protocol). The suspension of parasites is adjusted to 5 mg/mL of phleomycin (stock solution: 20 mg/mL in water and stored at 220 C) and incubated at 37 C for 10 hours. Parasites are transferred for recovery to HFF cultures in media containing 5 μg/mL of phleomycin. After a new cycle of lysis the extracellular parasites are treated again in

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20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization

presence of drug for 10 hours and cloned thereafter by limiting dilution in 96-well microtiter plates containing HFF cells in the presence of 5 μg/mL of phleomycin. All the selection processes described earlier could also be combined with a CRISPR/Cas9 targeted approach as described in Fig. 20.3. Here, 1020 μg of CRISPR/Cas9 plasmid along with 1015 μg of the selection cassette amplicon is recommended for efficient generation of a stable line (Shen et al., 2014; Sidik et al., 2014). Importantly, overexpression of the Cas9 is detrimental for parasites’ survival and a limited concentration of CRISPR/Cas9 plasmid must be transfected. 20.5.2.3 Restriction enzyme-mediated integration Stable random integration efficiency can be enhanced by adding 50100 μm of BamHI, NotI, or SacII to the cuvette immediately prior to electroporation [these three enzymes have worked in the past; choose one that does not cut an essential part of your plasmid(s)]. Note that REMI often results in multicopy integration of plasmid(s) (Black et al., 1995). 20.5.2.4 Cloning of transgenic lines by limiting dilution in 96 well plates • Seed tissue culture treated 96 well plates with HFF cells and grow to confluency. Remove medium and add 100 μL DMEM 1% FCS to each well. • Harvest freshly lysed parasites by filtration and centrifugation as described earlier. • Count using a hematocytometer and dilute to 250 parasites per mL. • Add 100 μL (25 tachyzoites) to each well in the first and seventh vertical column. • Using a multichannel pipetor perform a serial dilution from left to right transferring 100 μL at each step (mix each well by pipetting up and down three times).

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Discard medium after you reached column 6 and start over at row 7. • Incubate for 7 days without disturbing the culture. • Inspect each row from left to right using an inverted microscope and identify wells that contain a single plaque and mark those wells. Expand clonal lines by passage into a T25 flask.

20.5.3 Measuring parasite survival and growth 20.5.3.1 Plaque assay • Plaque assays are a reliable way to measure the number of viable and infectious parasites in a sample and are well suited to measure stable transfection efficiency. The following protocol will measure stable transformation using a DHFRTS resistance plasmid. • Electroporate tachyzoites as described earlier using 50 μg of pDHFR*TScABP (Sullivan et al., 1999). After electroporation, dilute 50 μL of the content of the cuvette into 950 μL cytomix or medium. • Infect T25 HFF cultures in drug-free medium with 3 and 6 μL diluted parasite suspension and two cultures with 6 and 60 μL to be cultured in the presence of 1 μM pyrimethamine. • Incubate for 7 days without disturbing the flasks (optimal time may depend on strain used, 23 mm plaques are best for scoring, a few extra flasks can be added in a larger experiment to be “developed” individually to test when the right plaque size is achieved). The period of selection takes longer with types II and III strains. • To stain the monolayer aspirate the medium, rinse with PBS, fix for 5 minutes with ethanol, and stain for 5 minutes with a crystal violet solution (dissolve 12.5 g crystal violet in 125 mL ethanol and mix with 500 mL 1% ammonium oxalate in water).

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• Remove crystal violet solution and rinse with PBS. • Air dry and count the number of plaques. • This assay can also be used to quantify parasite growth by measuring plaque area. To do this scan stained flasks with a standard flatbed scanner at 600 dpi and use image analysis for measurements. The area of plaques can be reasonably approximated using an ellipse. Measure the longest and shortest diameters of each plaque and use πab/4 to calculate the area. 20.5.3.2 Fluorescence assay • This assay will produce dynamic growth curves over the time of the experiment (usually a week). • Seed tissue culture treated black 384 or 96 well plates with special optical bottom (Beckton Dickinson) with HFF cells. For larger scale assays an automatic liquid dispenser (e.g., Genetix Q-Fill) will increase throughput and reproducibility. • Once plates are confluent replace medium with DMEM (without phenol red, Hyclone) 1% FCS and antibiotics as described earlier. • Infect each well with 2000 (384 well) or 5000 (96 well) tachyzoites [e.g., the YFPYFP strain (Gubbels et al., 2003)]. Plan to have quadruple wells for each experimental condition (e.g., drug concentration) and include negative (no parasites) and positive controls on each plate. Fill all wells with medium but do not use the outermost wells as they evaporate faster which affects parasite growth. • Measure fluorescence daily for each well for 58 days using a sensitive plate reader (BMG Fluostar, bottom excitation and emission 510/12 and 540/12 nm, respectively). • Plot the results (average of four wells and standard deviation) as percent positive in relation to the untreated positive control in each plate.

20.5.3.3 β-Galactosidase (LacZ) assay • This is an endpoint growth assay that can be used in multiwell formats (McFadden et al., 1997), a yellow substrate will be turned into a red product. • Seed HFF cells into standard tissue culture treated 384 well plates as described earlier. • Change medium of confluent cultures to DMEM 1% FCS without phenol red (50 μL/ well) and infect with 2000 β-galactosidase expressing tachyzoites (wash parasites in PBS before infection to eliminate phenol red). • At the desired read-out day (usually 5 days after infection, optimal staining has to be established empirically for each strain and condition), add 4.5 μL CPRG (Boehringer Mannheim, 4.5 mM stock in medium without phenol red). • Develop color to desired intensity (if you wait too long all wells will turn red, use your negative and positive controls as guide) and read absorbance at 570 nm. Plot endpoints as percent positivity as described earlier. 20.5.3.4 Uracil incorporation assay • In contrast to mammalian cells T. gondii can directly salvage uracil through UPRT. This can be exploited to measure parasite growth as a function of [3H]-uracil incorporation into parasite trichloroacetic acid (TCA) precipitable nucleic acids (Pfefferkorn and Guyre, 1984; Roos et al., 1994). The advantage of this assay is that it can be used in all strains and does not require a transgene. Recently a 96 well real-time format has been developed for this assay which is described in detail in Nare et al. (2002). • Infect 24 well cultures with parasites and incubate under test conditions (e.g., in presence of a drug).

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• Add 5 μCi of [5,6-3H]-uracil (3060 Ci/ mmol) to each well and incubate for 2 hours at 37 C. • Chill cultures and add an equal volume of ice-cold 0.6 N TCA to the medium of each well and incubate on ice for at least 1 hour. • Remove TCA and rinse plates under running water overnight (make sure to use a sink designated for radioactivity work). • Dry plates, add 500 μL of 0.1 N NaOH to each well, incubate for 1 hour, and measure radioactivity in half of the sample by liquid scintillation counting. Depending on the scintillation cocktail used neutralization of the base can help to avoid background. • Note: if the UPRT locus is used for cloning (e.g., FUDR is then used for selection of a recombinant clone) then this growth assay will no longer work as the UPRT locus will be disrupted and [3H]-uracil incorporation will not occur

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20.5.4 Live-cell and indirect immunofluorescence microscopy

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• Sterilize round 23 mm glass coverslips in 70% ethanol (or autoclave) and transfer to a six well plates. Seed coverslips with host cells and culture to confluency. Infect wells with tachyzoites 2436 hours before microscopic examination. • To observe parasites expressing fluorescent protein transgenes remove coverslip from dish with sterile forceps, wipe off medium from the bottom side and gently invert onto a microscope glass slide. If longer observation is required (e.g., for time-lapse microscopy), use spacer circles (e.g., Secure Seal, Invitrogen) to generate a small reservoir of medium. Alternatively use dishes that have a coverslip bottom (e.g., ΔT3 dishes, Bioptechs). • To use antibodies to stain cells remove medium, and fix cells in 2 mL of 3%

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paraformaldehyde in PBS for 1020 minutes. Remove fixative and permeabilize cells in 2 mL 0.25% Triton X100 (in PBS) for 10 minutes. Block in 2 mL 1% w/v BSA in PBS/0.25% Triton X100 for 30 minutes. React with primary antibody (diluted 1:1001:5000 in PBS/BSA/0.25% Triton X100 depending on titer) for 1 hour. This can be done with minimal reagent by inverting the coverslip onto 100 μL drops on parafilm in a moist chamber. Place back into six well dish (cell side up) and wash three times with 3 mL PBS (5 minutes each). React with secondary antibody diluted in BSA/PBS for 1 hour. Wash four times in 3 mL PBS (5 minutes each). To counter-stain DNA add 2 μL of a 2 mg/mL DAPI stock solution to the first wash. Apply a drop of mounting medium to a microscope slide. Briefly wash coverslip in dH2O (to prevent crystal formation after drying) and invert into mounting medium (cells down). Some epitopes are sensitive to aldehyde fixation. In that case use 2 mL of methanol for 20 minutes as fixative (methanol will also permeabilize the cells, and no Triton treatment is required). This protocol also works better to stain proteins secreted into the parasitophorous vacuole (these are often washed out by Triton permeabilization). A more elaborate protocol for secreted protein which preserves subcellular structures better than methanol can be found in Lecordier et al. (1999).

20.5.5 Cytometry of parasites and infected cells T. gondii tachyzoites can be efficiently sorted using a fluorescence-activated cell sorter (FACS)

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after labeling with specific antibodies to the surface of the parasite (Kim and Boothroyd, 1995; Radke et al., 2004) or based on the expression of autofluorescent protein (Striepen et al., 1998; Gubbels et al., 2004; Gubbels and Striepen, 2004). Parasites expressing fluorescent proteins can also be sorted within their host cells (Gubbels and Striepen, 2004; Egan et al., 2005). • For sorting autofluorescent parasites, harvest a freshly lysed culture and filter parasite through a 3 μm polycarbonate filter. Count parasites and take up in sterile PBS at 107/mL. • Use a high-speed sorter equipped with a 488 nm argon laser and the following filter and mirrors (GFP or YFP: DM: 555 SP, F: 530/40 BP; DRFP or Tomato DM: 555 SP, F: 570/40 BP). Note that for sorting the flow stream is broken into droplets, which carries the potential to produce aerosolized parasites. Extra safety can be provided by an evacuated and HEPA filtered enclosure of the sorting chamber. Discuss biosafety aspects with the FACS facility director and operator. • For enrichment sort into tubes preloaded with 0.5 mL of PBS or medium and transfer to a confluent T25 HFF culture. For cloning sort directly into seeded multiwell plates. Using a MOFLO sorter we found three events per well to result in the maximum number of single clones per plate. • To sort infected cells, inoculate parasites into a confluent HFF culture 124 hours prior to sorting. • Aspirate medium and wash twice with sterile PBS. • Trypsinize cells as described earlier and recover in 10 mL DMEM 1% FCS. • Filter through a 75 μm cell strainer (Becton Dickinson), spin down and resuspend in 0.5 mL PBS and sort as described earlier. • Detail on antibody staining for FACS of tachyzoites is provided in Radke et al. (2004).

20.5.6 Disruption of nonessential genes T. gondii is haploid and nonessential genes can be disrupted by HR using single or double crossover. As discussed in detail, the main challenge is to overcome the background of nonhomologous plasmid insertions. Indeed, T. gondii possesses an efficient NHEJ repair machinery limiting the generation of HR unless large homology regions are used. The isolation of ΔKu80 strains; the Ku80 gene is a key component of the NHEJ (Fox et al., 2009, 2011; Huynh and Carruthers, 2009), partly resolved this issue and allowed the efficient generation of transgenic parasites using homology regions of around 1000500 bp. The implementation of the CRISPR/Cas9 that generates double-stranded breaks at the locus of interest dramatically improved HR approaches and allowed fast and efficient gene targeting using homology regions of a few dozen base pairs (30 bp). PCR-generated amplicons with primers containing 30 bp homology regions are now routinely used (Di Cristina and Carruthers, 2018; Shen et al., 2014; Sidik et al., 2014). Here, we first describe a CAT/YFP positive/ negative selection for HR by double crossover and a protocol for the disruption of nonessential genes using the CRISPR/Cas9 system. 20.5.6.1 Disruption of nonessential genes using a CAT/YFP positive/negative selection • Construct a targeting plasmid that flanks a sagCATsag selectable marker cassette with homologous sequences from the target gene (typically the 50 and 30 genomic sequences flanking the actual coding sequence). If the recipient strain is wt, use 23 kb of homology sequence, if the recipient strain is ΔKu80 0.51 kb is enough. • Introduce a YFP expression cassette 30 adjacent to the 30 homologous flanking region. Be sure that your targeting plasmid contains a unique restriction site that will

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20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization

•

• •

• •

• •

allow you to linearize the construct without cutting into markers or flanking regions (e.g., in the multicloning site of the plasmid backbone). Test for YFP expression in a transient transfection experiment (B30% of the vacuole should show cytoplasmic fluorescence). Transfect with 10, 25, and 50 μg of linearized plasmid and select for stable transformation in the presence of 20 μM chloramphenicol. Subject the drug-resistant population (typically after 34 passages) to FACS. Clone nonfluorescent parasites by sorting into confluent 96 well plate cultures. Parasites can be labeled with Hoechst for better identification. Use the nontransfected parent strain and a YFP expressing strain as positive and negative controls. Leave plates undisturbed and check for single plaques after 7 days and mark clones. Suspend parasites by pipetting up and down and transfer 100 μL of each well into a well of a 24 well plate. Replenish medium in the 96 well plate and keep in the incubator. 24 well cultures will lyse within 34 days. Resuspend lysed parasites by pipetting and harvest by centrifugation. Extract genomic DNA using conventional DNA extraction kits and proceed to genotyping using appropriate primers.

20.5.6.2 Disruption of nonessential genes using CRISPR/Cas9 Several plasmid encoding the CRISPR/Cas9 machinery are available and two are described in details: • pSag1-Cas9-NLS-GFP/pU6-gRNA plasmid (Shen et al., 2014). • pTub1-FLAG-NLS-Cas9-NLS-pU6-SAG1 (Sidik et al., 2014). • The plasmid with 2sgRNAs is also available (Soldati-Favre, unpublished).

927

Several well-described protocols for CRISPR/Cas9 genome editing are available (Brown et al., 2018; Sugi, et al 2016; Jacot et al., 2019), but we will briefly describe here the key steps of a protocol to generate a knock-out using a selection cassette (Fig. 20.3C). • Extraction of the genomic sequence of your GOI: Genomic sequences can be downloaded on http://toxodb.org/toxo/. • Design of the gRNA: Several online tools are available for optimal sgRNA design. The Eukaryotic Pathogen CRISPR guide RNA/DNA Design Tool http://grna.ctegd.uga.edu/ is one example. Be careful to select the appropriate T. gondii strain. Your sgRNA must be designed within the GOI. Alternatively, 2 sgRNAs can be designed at both the 50 and 30 of the GOI. Importantly, both sgRNAs should be encoded in the same plasmid to avoid overexpression of the Cas9 (Fig. 20.3D). • Modification of the sgRNA: To modify the sequence of the sgRNA in the CRISPR/Cas9 plasmid the Q5 siteDirected Mutagenesis (New England Biolabs) is appropriate (Brown et al., 2018; Jacot et al., 2019, Methods Mol. Biol., in press). Directed mutagenesis is usually performed with a universal reverse primer and a forward primer containing the new sgRNA. The example next is designed for the pSag1-Cas9-NLS-GFP/pU6-gRNA plasmid (Shen et al., 2014). Follow the manufacturer protocol to perform the mutagenesis. • Reverse universal primer: aacttgacatccccatttac • Forward gRNA primer: NNNNNNNNNNNNNNNNNNNN gttttagagctagaaatagc The Ns correspond to the 20 nucleotides of the gRNA sequence.

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20. Genetic manipulation of Toxoplasma gondii

• Design of the gene-specific selection cassette: Design the 50 and 30 homology regions, respectively, directly 50 and 30 to the gRNA sequence for optimal efficiency. These regions should not include the gRNA sequence to avoid cleavage of the template vector. Both the 30 and 50 homology regions should contain around 30 bp. If a 2 sgRNA strategy is chosen, design the homology regions directly 50 and 30 to the sgRNAs. • PCR amplification of the selection cassette and clean-up: The Q5 DNA polymerase (New England Biolabs) or a KOD HiFi DNA polymerase (71085-3, Merck Millipore) are appropriate. Importantly, the PCR amplicons need to be clean-up before transfection. Sodium-acetate precipitation or PCR clean up kits could be used. At least 1015 μg are required for one transfection. 24 PCRs are usually required to obtain such a yield. • Parasites transfection and selection: 1015 μg of the selection cassette PCR amplicon is transfected along with 1020 μg of the CRISPR/Cas9 plasmid. Appropriate selection is then applied (see Section 20.5.2.2). Note that the CRISPR/Cas9 plasmid is only expressed transiently and will be lost. To improve selection, Cas9fluorescent plasmids can be used and transfected parasites can be FACS sorted 48 hours posttransfection. Clone drugresistant parasites by limiting dilution in 96 well plates and proceed to genotyping as previously described.

efficiency of selection by using a CRISPR/Cas9 plasmid containing a PYRr marker (Sugi et al., 2016). This technique can be used to evaluate the fitness of different mutant alleles, even if the function of the gene is quite important (i.e., “essential”), by transfecting several mutation type donors at the same time and following (by sequencing etc.) the kinetics of mutation enrichment (or depletion) over time (Sugi et al., 2016). It is possible to transfect sgRNA alone as an alternative approach. In wild-type T. gondii strains the double-stranded break in the targeted GOI is repaired by the NHEJ repair machinery leading to frequent indels resulting in premature stop codons (Fig. 20.3A). Two sgRNA could also be transfected to generate large gene deletion (Fig. 20.3B). Here 20% 30% of the parasites could harbor the expected mutation and could be isolated by limiting dilution at early passages. Cas9-fluorescent plasmids can be used to efficiently FACS sort/ clone the parasites 48 hours posttransfection. This allowed the isolation of previously difficult to obtain mutants such as MyoA (Frenal et al., 2014). This strategy is recommended for wild-type strains only. Indeed, in ΔKu80 strains, the absence of both the NHEJ repair machinery and an appropriate repair template is highly deleterious for the parasites and isolation of the targeted mutation is difficult.

This protocol is particularly suitable for ΔKu80 strains as double-stranded breaks induced by the CRISPR/Cas9 system are repaired through HR with the supplied repair template; however, this technique can be used with wild-type strains as well (but is predicted to have a lower efficiency). Pyrimethamine selection has been used to increase the

As detailed earlier the first approach involves several steps: (1) introduce an ectopic tet-regulatable copy of the target gene, (2) target the native locus by HR, and (3) knockdown of the expression of the ectopic copy using ATc treatment. The choice of selectable markers may differ from experiment to experiment [the tet-transactivator line (Meissner et al., 2002) is

20.5.7 Disruption of essential genes 20.5.7.1 Tetracycline inducible systems 20.5.7.1.1 Two-step strategy

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20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization

resistant to MPA], this example will use CAT, YFP, and DHFRTS. • Construct a plasmid for ectopic expression of the target gene (Meissner et al., 2002). If you omit the stop codon this should result in an N-terminal translational fusion to a cmyc epitope tag. • Transfect into the TATi transactivator line (Meissner et al., 2002), select stable transformants in the presence of 1 μM pyrimethamine, and clone by limiting dilution. • Test clones for transgene expression by IFA and Western blot using an anti-myc antibody (mAb 9E10, Roche). • Choose clones that express the transgene at a similar level as the native gene. Depending on the size of the target gene addition of the tag may result in a noticeable mobility shift on SDSPAGE. In this case an antibody against the target protein can be used to compare both proteins side by side. • It is critical to identify a tightly regulated clone before proceeding to the KO experiment. Careful characterization of clones will pay off with a clean interpretable phenotype. Test for regulation by culturing parasites in the presence or absence of 1 μg/mL of ATc (0.2 mg/mL stock in ethanol) followed by IFA and Western blot. Note that stable proteins might have to be diluted out by growth. Do your first screen after 2 days of ATc treatment and then titer the maximal or minimal treatment time for complete suppression using your tightest clone. Target the native locus as described earlier (using CAT/YFP positivenegative selection or CRISPR/Cas9), establish allelic replacement, and analyze regulation of the ectopic copy in confirmed KO clones by IFA and Western blot. To facilitate double HR, vectors should be linearized at both ends.

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20.5.7.1.2 Single-step approach

The CRISPR/Cas9 system offers alternative efficient strategies to generate tetracyclineinducible knockdowns as exemplified in Fig. 20.6C. Alternatively, an PCR amplicon of a cassette with a selectable marker and the tetregulatable promoter, generated with forward primers containing 30 bp homology upstream of the promoter region and reverse primer containing 30 bp homology starting at the ATG can be transfected along with an appropriate CRISPR/Cas9 plasmid (see, e.g., Lacombe et al., 2019). A complete protocol for CRISPR/Cas9mediated generation of tetracycline-inducible knockdown in T. gondii as described in Fig. 20.6C is available (Jacot et al., 2019). 20.5.7.2 Regulation of protein stability 20.5.7.2.1 Destabilization domain (ddFKBP)

The generation of transgenic parasites expressing DD-fusion can be selected either in the presence (knockdown of an essential gene) or in the absence of Shield-1 (expression of a toxic gene such as a dominant negative mutant). Conditional expression is performed with 1 μM Shield-1. 20.5.7.2.2 Auxin-based degron system

A detailed protocol for the generation of AID-based inducible knockouts using a CRISPR/Cas9 approach is available (Brown et al., 2018).

20.5.8 Insertional mutagenesis and tag rescue • Electroporate tachyzoites as using 50 μg of linearized (e.g., restricted with NotI) plasmid pDHFR*TScABP. Select for stable transformants in 1 μM pyrimethamine and apply the desired phenotypic screen. Clone mutants by limiting dilution, expand

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•

•

• • • • • • •

20. Genetic manipulation of Toxoplasma gondii

into T25 cultures and isolate genomic DNA as described earlier. Set up parallel 20 μL restriction digests using several restriction enzymes that cut once in your plasmid (e.g., EcoRI, HindIII, XhoI, XbaI for pDHFR*TScABP see Sullivan et al., 1999 for maps and a detailed discussion of enzyme choice). Use 2 μg genomic DNA for each digest and incubate overnight at 37 C. Purify DNA from digest using a Qiagen spin column following the manufacturer’s protocol and elute in 30 μL elution buffer. Mix 5 μL eluate with 2 μL 10 3 NEB ligase buffer, 13 μL H2O, and 1 μL T4 DNA ligase and incubate overnight at 16 C. Add 1 μL glycogen, 2 μL 3 M NaAc, pH 5.2, and 50 μL ethanol and precipitate DNA for 30 minutes at 220 C. Wash pellet with 1 mL 70% ethanol, air dry briefly, and resuspend pellet in 10 μL H2O. Electroporate 1 μL into 25 μL library efficient electrocompetent bacteria (we found DH12S to result in best recovery). Transfer into sterile microcentrifuge tube, add 200 μL LB medium and incubate for 1 hour at 37 C while shaking. Plate entire transformation onto an LB agar plate containing suitable antibiotic (in this case ampicillin). Tags can also be rescued by inverse PCR. See Sullivan et al. (1999) for primer design and a detailed protocol.

• The mutagenic potency can vary from batch to batch and has to be titrated by plaque assay. Prepare a stock solution (100 mg/mL in DMSO) and store multiple aliquots at 220 C. Perform triplicate plaque assays using 0, 25, 50, and 75 μL of mutagen. Optimal mutagenesis results in 70% parasite killing compared to untreated controls (the protocol below assumes 50 μL as the optimal dose). • Infect two confluent T25 HFF cultures with 1.2 mL of a freshly lysed culture 24 hours prior to the experiment. • Replace medium with 10 mL DMEM 0.1% FBS medium. • Incubate at 37 C for 30 minutes. • Add 50 L ENU to flask A and 50 μL sterile tissue culture grade DMSO to flask B. • Treat for 4 hours at 37 C. • Wash cultures three times with 10 mL cold sterile PBS and discard into a dedicated waste container. • Add 10 mL PBS, scrape cells with a cell scraper, liberate parasite by two passages through a 25-ga needle (see safety section), and filter through a 3 μm polycarbonate filter. • Transfer to 50 mL tube, add 40 mL PBS, and spin at 1500 3 g at 4 C for 20 minutes. • Resuspend in 5 mL PBS and count parasites. Proceed to cloning by limiting dilution. It is advisable to control the mutagenesis efficiency of each experiment by plaque assay.

20.5.9 Chemical mutagenesis

20.5.10 Complementation cloning using Toxoplasma gondii genomic libraries

• ENU is highly toxic and carcinogenic. Treat with extra care all materials that have come into contact with this chemical. Label tubes and flasks to warn members of your laboratory, and dispose contaminated solutions appropriately.

• Prepare 50 large and 10 small LB-agar petri dishes (10 μg/mL Kanamycin). • To titer the ToxoSuperCos library, prepare five 1.5 mL Eppendorf tubes with 135 μL LB (no antibiotics), one with 1 mL LB, and one empty tube.

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20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization

• Remove library from the 280 C freezer and keep on ice (work quickly to avoid thawing and immediately refreeze library). • Scrape a small amount of library (B20 μL) into the empty tube. • Add 1 μL of thawed scraped bacteria to 1 mL LB (1:103 dilution). • Keep the remainder of the thawed library at 4 C (stable for 12 days). • Prepare a dilution series (104108), plate 100 μL of each dilution on prewarmed small LB-Kan plates, grow overnight at 37 C, and count colonies to calculate the number of colony forming units (cfu)/mL. • To amplify the library DNA prewarm large LB-Kan plates at 37 C, prepare 10 mL of LB containing 50,000 cfu/mL, and plate 200 μL per plate. • Grow overnight 37 C (incubate longer if colonies are too small). • To harvest, add 2 mL of LB to the plate and scrape colonies using a cell scrape, transfer into a 250 mL centrifugation bottle (on ice) and wash with 1 mL of LB. Repeat for each plate and pool. • Pellet bacteria in a tabletop centrifuge, remove liquid and weigh the pellets (bacteria can be stored at 220 C at this step). • Purify cosmids using a commercial kit, for example, Qiagen large construct kit according to the manufacturer’s instructions, resuspend DNA pellet in 150 μL TE per column and store cosmid DNA at 4 C in the dark. • To complement T. gondii mutants perform 5 independent transfections as described earlier (8 3 107 parasites and 25 μg cosmid DNA per cuvette). Include at least one mock transfection to control for reversion. • Transfer independently into T175 HFF cultures, incubate overnight at permissive conditions then apply selective pressure. • For ts mutants plaques can be identified 1014 days after transfection.

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• Clone by limiting dilution, prepare genomic DNA, and rescue a sequence tag exploiting the Kan marker on the ToxoSuperCos backbone as described for insertional mutagenesis (use BglII, HindIII, and XhoI). • BLAST rescued sequences against ToxoDB. You should obtain hits to the same genomic region from independent complementation events. Check if your candidate region is represented among the sequenced and arrayed cosmids displayed on ToxoDB, acquire these cosmids and test for complementation.

20.5.11 Recombinering cosmids of Toxoplasma gondii genomic libraries • Find a cosmid that covers your gene (using www.toxodb.org), and identify the corresponding bacterial clone number (using http://toxomap.wustl.edu/cosmid.html). • Prepare the cosmid from an overnight 28 C30 C culture, confirm by digest and electroporate 100300 ng into E. coli strain EL250 (electroporate in 1 mm gap cuvette at 1.75 kV, 250 Ω, and 25 μF). • Induce the λ phage recombination machinery in a fresh 100 mL culture of EL250 containing cosmid (grown from 2 mL overnight culture at 28 C30 C to optical density OD 5 0.4) by immediately transferring it to 43 C and shaking 20 minutes at 100 rpm, following 20 minutes cooling in ice-water. • Use the cooled culture to make competent cells by three consecutive washes in ice-cold sterile ddH2O (in 50, 20, and 3 mL, centrifugations at 4000 rpm, 10 minutes at 4 C). Resuspend the competent pellet in 600 μL 10% sterile glycerol and aliquot 50 μL into ice-cold sterile microfuge tubes for storage at 280 C. • PCR amplifies a modification cassette (see Fig. 20.8 and Table 20.2 on how to design you desired manipulation) from 0.1 to 50 ng of plasmid template

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Promoter replacement

Gene replacement

Gene tagging

1. Cosmid Recombineering in EL250 PCR cassette

GENT

DHFR

PCR cassette

GENT

CAT

HA GENT

CAT

Parental cosmid KAN

KAN

KA N

Parental cosmid Kanamycin + gentamycin

GENT

DHFR

GENT

Modified cosmid

HA GENT

CAT gra

KA N

CAT

gra

KA N

KAN

Modified cosmid

2. Cosmid-based gene disruption in T. gondii

Modified cosmid

~20 kb flanks

GENT

GENT

DHFR

CAT Gra

Gra

GENT

CAT

HA GENT

CAT

HA GENT

CAT

Chloramphenicol or pyrimethamin

GENT

DHFR

FIGURE 20.8 Using cosmid recombineering to modify GOI. Schematic representation of the three available strategies for cosmid modification: promoter replacement, gene replacement and C-terminal gene tagging. The PCR cassette and recombination even into the cosmid are depicted on the top of each panel as step 1 with the resulting modified cosmid. Step 2 demonstrates the recombination into the genome using each modified cosmid (here shown linear) and the resulting modified locus. GOI, Gene of interest.

TABLE 20.2 Primers for PCR amplification of cosmid modification cassettes. Toxoplasma Type of selectable modification marker

F primer

R primer 0

Promoter replacement

DHFR

50 bp GOI at the 5 of the promoter 1 GAATGGTAACCGACAAACGCGTTC

GCTTTCGTCTGTCTTCAACCAGATCT 1 50 bp GOI just upstream of ATG

Gene replacement

BLE/CAT

50 bp GOI upstream of start codon 1 CCTCGACTACGGCTTCCATTGGCAAC

50 bp GOI downstream from stop codon 1 ATACGACTCACTATAGGGCGAATTGG

HA tagging

BLE/CAT

50 bp of GOI upstream of stop codon 50 bp GOI downstream from stop codon 1 AGGTACCCGTACGACGTCCCGGACTAC 1 ATACGACTCACTATAGGGCGAATTGG

GOI, Gene of interest.

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20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization

• Use 100300 ng gel-purified targeting cassette to electroporate (same as above) to one 50 μL aliquot, rescue in SOC media at 28 C30 C for one hour and plate on gentamycine 1 kanamycin to select for recombineering.

20.5.12 Safety concerns working with Toxoplasma gondii Several aspects of the parasite’s biology make work with T. gondii relatively safe. In immunocompetent persons the infection produces usually no or only modest symptoms. Depending on the region of the world 20% 70% of the population is already infected and resistant to reinfection. Lastly, the tachyzoites stage, which is most widely used in experimental work is not highly infective by aerosol or ingestion. However, T. gondii is a human pathogen with the ability to cause severe disease and should be handled with appropriate care (severe lab accidents have occurred in the past). We summarize a few ground rules in the following (this section does not represent a comprehensive laboratory safety manual). • Laboratory workers who belong to a specific risk group (active or potential severe immunosuppression, pregnancy) should not work with live parasites. • Safety procedures should be frequently reviewed with all members of the laboratory. • Handle parasites in designated biosafety cabinets. Label all work areas, flasks, tubes, and waste containers that might harbor infectious material accordingly. • Wear a lab coat, gloves, and goggles. Goggles are especially important for workers who do not wear glasses. An eye splash could potentially deliver a high inoculum of parasites.

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• The main route of infection with tachyzoites is direct inoculation by injury or through eye splash. Be extremely careful in all situations that involve sharps. Note that coverslips, microscope slides as well as plastic or glass tubes can break and produce sharp edges. Should you break something, sterilize using 70% ethanol before you attempt cleanup. Needle sticks are the most common source of laboratory infections. The safest approach is to minimize such situations is to avoid them. Consider if the use of sharps is really essential to your experiment. If you really have to needle pass infected cells to liberate parasites leave the plastic sheath on the needle and cut off its tip using sturdy scissors several mm before the tip of the actual needle. This can help to protect you from accidental sticks and provides extra safety at no additional cost or effort. • Be especially careful working with strains that encode resistance to drugs commonly used for the treatment of humans, including pyrimethamine, sulfadiazine, clindamycin, and azithromycin. • Sterilize all materials that were in contact with live parasites (autoclave all plastic tissue culture material, bleach all liquids accumulating in, e.g., vacuum bottles and frequently sterilize surfaces by spraying and wiping down with 70% ethanol). • Have a plan for a potential accident. While the goal is to prevent accidents, they might happen nonetheless. Establish local as well as national contacts to infectious disease specialists who could provide advice for diagnosis and treatment. [Reference laboratories include the Palo Alto Research Foundation (http://www.pamf.org/ serology) and the Laboratory of Parasitology and FAO/WHO International Centre for Research and Reference on Toxoplasmosis, Statens Seruminstitut, 2300 Copenhagen S, Denmark.]

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• Ensure good communication about lab safety and always disclose any contamination, accident or inoculation. Inform the head of your laboratory about any accident, even if you feel this was a minor incident.

Acknowledgments BS is supported by the National Institutes of Health and the Bill and Melinda Gates Foundation. DSF is supported by the Swiss National Foundation (FN310030_185325) and DJ by Carigest SA. SL is supported by NIH DP5 OD017892. LS is supported by a Royal Society of Edinburg Personal Research Fellowship. We thank Louis Weiss for providing protocols for the propagation of parasites in mice. We thank all current and former members of our laboratories and many investigators in the field for their protocols and discussion.

References Agop-Nersesian, C., Egarter, S., Langsley, G., Foth, B.J., Ferguson, D.J., Meissner, M., 2010. Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B. PLoS Pathog. 6, e1001029. Andenmatten, N., Egarter, S., Jackson, A.J., Jullien, N., Herman, J.P., Meissner, M., 2013. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods 10, 125127. Armstrong, C.M., Goldberg, D.E., 2007. An FKBP destabilization domain modulates protein levels in Plasmodium falciparum. Nat. Methods 4, 10071009. Arrizabalaga, G., Ruiz, F., Moreno, S., Boothroyd, J.C., 2004. Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation. J. Cell Biol. 165, 653662. Banaszynski, L.A., Chen, L.C., Maynard-Smith, L.A., Ooi, A.G., Wandless, T.J., 2006. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 9951004. Behnke, M.S., Wootton, J.C., Lehmann, M.M., Radke, J.B., Lucas, O., Nawas, J., et al., 2010. Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5, e12354. Biddau, M., Bouchut, A., Major, J., Saveria, T., Tottey, J., Oka, O., et al., 2018. Two essential thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii. PLoS Pathog. 14, e1006836.

Bisio, H., Lunghi, M., Brochet, M., Soldati-Favre, D., 2019. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. Nat. Microbiol. 4, 420428. Black, M.W., Boothroyd, J.C., 1998. Development of a stable episomal shuttle vector for Toxoplasma gondii. J. Biol. Chem. 273, 39723979. Black, M., Seeber, F., Soldati, D., Kim, K., Boothroyd, J.C., 1995. Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii. Mol. Biochem. Parasitol. 74, 5563. Black, M.W., Arrizabalaga, G., Boothroyd, J.C., 2000. Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress. Mol. Cell. Biol. 20, 93999408. Bohne, W., Wirsing, A., Gross, U., 1997. Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements. Mol. Biochem. Parasitol. 85, 8998. Brecht, S., Erdhart, H., Soete, M., Soldati, D., 1999. Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre. Gene 234, 239247. Breinich, M.S., Ferguson, D.J., Foth, B.J., van Dooren, G.G., Lebrun, M., Quon, D.V., et al., 2009. A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii. Curr. Biol. 19, 277286. Brooks, C.F., Johnsen, H., van Dooren, G.G., Muthalagi, M., Lin, S.S., Bohne, W., et al., 2010. The Toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival, Cell Host Microbe, 7. pp. 6273. Brown, K.M., Sibley, L.D., 2018. Essential cGMP signaling in Toxoplasma is initiated by a hybrid P-type ATPaseguanylate cyclase. Cell Host Microbe 24, 804816.e6. Brown, K.M., Long, S., Sibley, L.D., 2017. Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii. mBio 8. Available from: https:// doi.org/10.1128/mBio.00375-17. Brown, K.M., Long, S., Sibley, L.D., 2018. Conditional knockdown of proteins using auxin-inducible degron (AID) fusions in Toxoplasma gondii. Bio Protoc. 8. Available from: https://doi.org/10.21769/BioProtoc.2728. Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., et al., 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99, 78777882. Carey, K.L., Westwood, N.J., Mitchison, T.J., Ward, G.E., 2004. A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 101, 74337438. Chaturvedi, S., Qi, H., Coleman, D., Rodriguez, A., Hanson, P.I., Striepen, B., et al., 1999. Constitutive calcium-independent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab machinery. J. Biol. Chem. 274, 24242431.

Toxoplasma Gondii

References

Chen, T.R., 1977. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104, 255262. Chen, L.F., Han, X.L., Li, F.X., Yao, Y.Y., Fang, J.P., Liu, X. J., et al., 2018. Comparative studies of Toxoplasma gondii transcriptomes: insights into stage conversion based on gene expression profiling and alternative splicing. Parasit. Vectors 11, 402. Chiang, C.W., Carter, N., Sullivan JR, W.J., Donald, R.G., Roos, D.S., Naguib, F.N., et al., 1999. The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression. J. Biol. Chem. 274, 3525535261. Craver, M.P., Rooney, P.J., Knoll, L.J., 2010. Isolation of Toxoplasma gondii development mutants identifies a potential proteophosphogylcan that enhances cyst wall formation. Mol. Biochem. Parasitol. 169, 120123. Daher, W., Plattner, F., Carlier, M.F., Soldati-Favre, D., 2010. Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii. PLoS Pathog. 6, e1001132. Daher, W., Morlon-Guyot, J., Sheiner, L., Lentini, G., Berry, L., Tawk, L., et al., 2015. Lipid kinases are essential for apicoplast homeostasis in Toxoplasma gondii. Cell. Microbiol. 17, 559578. Delbac, F., Sanger, A., Neuhaus, E.M., Stratmann, R., Ajioka, J.W., Toursel, C., et al., 2001. Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division. J. Cell Biol. 155, 613623. Di Cristina, M., Carruthers, V.B., 2018. New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites. Parasitology 145, 11191126. Donald, R.G., Roos, D.S., 1993. Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drugresistance mutations in malaria. Proc. Natl. Acad. Sci. U.S.A. 90, 1170311707. Donald, R.G., Roos, D.S., 1994. Homologous recombination and gene replacement at the dihydrofolate reductasethymidylate synthase locus in Toxoplasma gondii. Mol. Biochem. Parasitol. 63, 243253. Donald, R.G., Roos, D.S., 1995. Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 92, 57495753. Donald, R., Carter, D., Ullman, B., Roos, D.S., 1996. Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. J. Biol. Chem. 271, 1401014019. Dvorin, J.D., Martyn, D.C., Patel, S.D., Grimley, J.S., Collins, C.R., Hopp, C.S., et al., 2010. A plant-like kinase

935

in Plasmodium falciparum regulates parasite egress from erythrocytes. Science 328, 910912. Egan, C.E., Dalton, J.E., Andrew, E.M., Smith, J.E., Gubbels, M.J., Striepen, B., et al., 2005. A requirement for the Vgamma1 1 subset of peripheral gammadelta T cells in the control of the systemic growth of Toxoplasma gondii and infection-induced pathology. J. Immunol. 175, 81918199. Farrell, A., Thirugnanam, S., Lorestani, A., Dvorin, J.D., Eidell, K.P., Ferguson, D.J., et al., 2011. A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis. Science 335, 218221. Fox, B.A., Belperron, A.A., Bzik, D.J., 1999. Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine. Mol. Biochem. Parasitol. 98, 93103. Fox, B.A., Belperron, A.A., Bzik, D.J., 2001. Negative selection of herpes simplex virus thymidine kinase in Toxoplasma gondii. Mol. Biochem. Parasitol. 116, 8588. Fox, B.A., Falla, A., Rommereim, L.M., Tomita, T., Gigley, J. P., Mercier, C., et al., 2011. Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryot. Cell 10, 11931206. Fox, B.A., Ristuccia, J.G., Gigley, J.P., Bzik, D.J., 2009. Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining. Eukaryot. Cell 8, 520529. Francia, M.E., Jordan, C.N., Patel, J.D., Sheiner, L., Demerly, J.L., Fellow, J.D., et al., 2012. Cell division in apicomplexan parasites is organized by a homolog of the striated rootlet fiber of algal flagella. PLoS Biol. 10, e1001444. Frankel, M.B., Mordue, D.G., Knoll, L.J., 2007. Discovery of parasite virulence genes reveals a unique regulator of chromosome condensation 1 ortholog critical for efficient nuclear trafficking. Proc. Natl. Acad. Sci. U.S.A. 104, 1018110186. Frenal, K., Marq, J.B., Jacot, D., Polonais, V., SoldatiFavre, D., 2014. Plasticity between MyoC- and MyoAglideosomes: an example of functional compensation in Toxoplasma gondii invasion. PLoS Pathog. 10, e1004504. Frenal, K., Jacot, D., Hammoudi, P.M., Graindorge, A., Maco, B., Soldati-Favre, D., 2017. Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat. Commun. 8, 15710. Gubbels, M.J., Striepen, B., 2004. Studying the cell biology of apicomplexan parasites using fluorescent proteins. Microsc. Microanal. 10, 568579.

Toxoplasma Gondii

936

20. Genetic manipulation of Toxoplasma gondii

Gubbels, M.J., Li, C., Striepen, B., 2003. High-throughput growth assay for Toxoplasma gondii using yellow fluorescent protein. Antimicrob. Agents Chemother. 47, 309316. Gubbels, M.J., Wieffer, M., Striepen, B., 2004. Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among Apicomplexa. Mol. Biochem. Parasitol. 137, 99110. Gubbels, M.J., Striepen, B., Shastri, N., Turkoz, M., Robey, E.A., 2005. Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii. Infect. Immun. 73, 703711. Gubbels, M.J., Vaishnava, S., Boot, N., Dubremetz, J.F., Striepen, B., 2006. A MORN-repeat protein is a dynamic component of the Toxoplasma gondii cell division apparatus. J. Cell Sci. 119, 22362245. Gubbels, M.J., Lehmann, M., Muthalagi, M., Jerome, M.E., Brooks, C.F., Szatanek, T., et al., 2008. Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii. PLoS Pathog. 4, e36. Hager, K.M., Striepen, B., Tilney, L.G., Roos, D.S., 1999. The nuclear envelope serves as an intermediary between the ER and golgi complex in the intracellular parasite Toxoplasma gondii. J. Cell. Sci. 112, 26312638. Hammoudi, P.M., Jacot, D., Mueller, C., Di Cristina, M., Dogga, S.K., Marq, J.B., et al., 2015. Fundamental roles of the golgi-associated Toxoplasma aspartyl protease, ASP5, at the host-parasite interface. PLoS Pathog. 11, e1005211. Hartley, J.L., Temple, G.F., Brasch, M.A., 2000. DNA cloning using in vitro site-specific recombination. Genome Res. 10, 17881795. Hassan, M.A., Melo, M.B., Haas, B., Jensen, K.D., Saeij, J.P., 2012. De novo reconstruction of the Toxoplasma gondii transcriptome improves on the current genome annotation and reveals alternatively spliced transcripts and putative long non-coding RNAs. BMC Genomics 13, 696. Heaslip, A.T., Dzierszinski, F., Stein, B., Hu, K., 2010. TgMORN1 is a key organizer for the basal complex of Toxoplasma gondii. PLoS Pathog. 6, e1000754. Heredero-Bermejo, I., Varberg, J.M., Charvat, R., Jacobs, K., Garbuz, T., Sullivan JR., W.J., et al., 2019. TgDrpC, an atypical dynamin-related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division. Mol. Microbiol. 111, 4664. Herm-Gotz, A., Weiss, S., Stratmann, R., Fujita-Becker, S., Ruff, C., Meyhofer, E., et al., 2002. Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor. EMBO J. 21, 21492158.

Herm-Gotz, A., Agop-Nersesian, C., Munter, S., Grimley, J. S., Wandless, T.J., Frischknecht, F., et al., 2007. Rapid control of protein level in the apicomplexan Toxoplasma gondii. Nat. Methods 4, 10031005. Hu, K., Roos, D.S., Murray, J.M., 2002. A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J. Cell Biol. 156, 10391050. Hu, K., Roos, D.S., Angel, S.O., Murray, J.M., 2004. Variability and heritability of cell division pathways in Toxoplasma gondii. J. Cell Sci. 117, 56975705. Hunt, A., Russell, M.R.G., Wagener, J., Kent, R., Carmeille, R., Peddie, C.J., et al., 2019. Differential requirements for cyclase-associated protein (CAP) in actin-dependent processes of Toxoplasma gondii. eLife 8, Available from: https://doi.org/10.7554/eLife.50598. Huynh, M.H., Carruthers, V.B., 2006. Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS Pathog. 2, e84. Huynh, M.H., Carruthers, V.B., 2009. Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot. Cell 8, 530539. Jacot, D., Tosetti, N., Pires, I., Stock, J., Graindorge, A., Hung, Y.F., et al., 2016. An apicomplexan actin-binding protein serves as a connector and lipid sensor to coordinate motility and invasion. Cell Host Microbe 20, 731743. Jacot D., Soldati-Favre D., 2019. CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knock-down in Toxoplasma gondii, in Toxoplasma gondii. Method Protocols 2071, 10643745. Jammallo, L., Eidell, K., Davis, P.H., Dufort, F.J., Cronin, C., Thirugnanam, S., et al., 2011. An insertional trap for conditional gene expression in Toxoplasma gondii: identification of TAF250 as an essential gene. Mol. Biochem. Parasitol. 175, 133143. Joiner, K.A., Roos, D.S., 2002. Secretory traffic in the eukaryotic parasite Toxoplasma gondii: less is more. J. Cell Biol. 157, 557563. Jullien, N., Sampieri, F., Enjalbert, A., Herman, J.P., 2003. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 31, e131. Karsten, V., Qi, H., Beckers, C.J., Joiner, K.A., 1997. Targeting the secretory pathway of Toxoplasma gondii. Methods 13, 103111. Karsten, V., Qi, H., Beckers, C.J., Reddy, A., Dubremetz, J.F., Webster, P., et al., 1998. The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms. J. Cell Biol. 141, 13231333. Kibe, M.K., Coppin, A., Dendouga, N., Oria, G., Meurice, E., Mortuaire, M., et al., 2005. Transcriptional regulation

Toxoplasma Gondii

References

of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii. Nucleic Acids Res. 33, 17221736. Kim, K., Boothroyd, J.C., 1995. Toxoplasma gondii: stable complementation of sag1 (p30) mutants using SAG1 transfection and fluorescence-activated cell sorting. Exp. Parasitol. 80, 4653. Kim, K., Soldati, D., Boothroyd, J.C., 1993. Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. Science 262, 911914. Kim, K., Eaton, M.S., Schubert, W., Wu, S., Tang, J., 2001. Optimized expression of green fluorescent protein in Toxoplasma gondii using thermostable green fluorescent protein mutants. Mol. Biochem. Parasitol. 113, 309313. Knoll, L.J., Boothroyd, J.C., 1998. Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase. Mol. Cell. Biol. 18, 807814. Knoll, L.J., Furie, G.L., Boothroyd, J.C., 2001. Adaptation of signature-tagged mutagenesis for Toxoplasma gondii: a negative screening strategy to isolate genes that are essential in restrictive growth conditions. Mol. Biochem. Parasitol. 116, 1116. Lacombe, A., Maclean, A., Ovciarikova, J., Tottey, J., Sheiner, L., 2019. In silico screen identifies a new Toxoplasma gondii mitochondrial ribosomal protein essential for mitochondrial translation. bioRxiv. Available from: https://doi.org/543520/543520. Lecordier, L., Mercier, C., Sibley, L.D., Cesbron-Delauw, M. F., 1999. Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell. Mol. Biol. Cell 10, 12771287. Limenitakis, J., Soldati-Favre, D., 2011. Functional genetics in Apicomplexa: potentials and limits. FEBS Lett. 585, 15791588. Long, S., Brown, K.M., Drewry, L.L., Anthony, B., Phan, I. Q.H., Sibley, L.D., 2017. Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog. 13, e1006379. Lunghi, M., Spano, F., Magini, A., Emiliani, C., Carruthers, V.B., Di Cristina, M., 2016. Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites. Curr. Genet. 62, 3138. Matrajt, M., Donald, R.G., Singh, U., Roos, D.S., 2002a. Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii. Mol. Microbiol. 44, 735747. Matrajt, M., Nishi, M., Fraunholz, M., Peter, O., Roos, D.S., 2002b. Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii. Mol. Biochem. Parasitol. 120, 285289.

937

Matrajt, M., Platt, C.D., Sagar, A.D., Lindsay, A., Moulton, C., Roos, D., 2004. Transcript initiation, polyadenylation, and functional promoter mapping for the dihydrofolate reductase-thymidylate synthase gene of Toxoplasma gondii. Mol. Biochem. Parasitol. 137, 229238. Mazumdar, J., Wilson, E.H., Masek, K., Hunter, C.A., Striepen, B., 2006. Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103, 1319213197. McFadden, D.C., Seeber, F., Boothroyd, J.C., 1997. Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro. Antimicrob. Agents Chemother. 41, 18491853. Meissner, M., Brecht, S., Bujard, H., Soldati, D., 2001. Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii. Nucleic Acids Res. 29, E115. Meissner, M., Schluter, D., Soldati, D., 2002. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837840. Mercier, C., Lefebvre-Van hende, S., Garber, G.E., Lecordier, L., Capron, A., Cesbron-Delauw, M.F., 1996. Common cis-acting elements critical for the expression of several genes of Toxoplasma gondii. Mol. Microbiol. 21, 421428. Mercier, C., Howe, D.K., Mordue, D., Lingnau, M., Sibley, L.D., 1998. Targeted disruption of the GRA2 locus in Toxoplasma gondii decreases acute virulence in mice. Infect. Immun. 66, 41764182. Messina, M., Niesman, I., Mercier, C., Sibley, L.D., 1995. Stable DNA transformation of Toxoplasma gondii using phleomycin selection. Gene 165, 213217. Metzger, D., Clifford, J., Chiba, H., Chambon, P., 1995. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc. Natl. Acad. Sci. U.S.A. 92, 69916995. Mital, J., Meissner, M., Soldati, D., Ward, G.E., 2005. Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol. Biol. Cell 16, 43414349. Nare, B., Allocco, J.J., Liberator, P.A., Donald, R.G., 2002. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine toxoplasmosis: gamma interferon is required for efficacy. Antimicrob. Agents Chemother. 46, 300307. Payne, T.M., Payne, A.J., Knoll, L.J., 2011. A Toxoplasma gondii mutant highlights the importance of translational regulation in the apicoplast during animal infection. Mol. Microbiol. 82, 12041216.

Toxoplasma Gondii

938

20. Genetic manipulation of Toxoplasma gondii

Pelletier, L., Stern, C.A., Pypaert, M., Sheff, D., Ngo, H.M., Roper, N., et al., 2002. Golgi biogenesis in Toxoplasma gondii. Nature 418, 548552. Pfefferkorn, E.R., 1988. Toxoplasma gondii viewed from a virological perspective. In: Englund, P.T., Sher, A. (Eds.), The Biology of Parasitism. Alan R. Liss, New York. Pfefferkorn, E.R., Guyre, P.M., 1984. Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon. Infect. Immun. 44, 211216. Pfefferkorn, E.R., Pfefferkorn, L.C., 1976. Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants. Exp. Parasitol. 39, 365376. Pfefferkorn, L.C., Pfefferkorn, E.R., 1980. Toxoplasma gondii: genetic recombination between drug resistant mutants. Exp. Parasitol. 50, 305316. Pieperhoff, M.S., Pall, G.S., Jimenez-Ruiz, E., Das, S., Melatti, C., Gow, M., et al., 2015. Conditional U1 gene silencing in Toxoplasma gondii. PLoS One 10, e0130356. Plattner, F., Yarovinsky, F., Romero, S., Didry, D., Carlier, M.F., Sher, A., et al., 2008. Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response. Cell Host Microbe 3, 7787. Radke, J.R., White, M.W., 1998. A cell cycle model for the tachyzoite of Toxoplasma gondii using the herpes simplex virus thymidine kinase. Mol. Biochem. Parasitol. 94, 237247. Radke, J.R., Guerini, M.N., White, M.W., 2000. Toxoplasma gondii: characterization of temperature-sensitive tachyzoite cell cycle mutants. Exp. Parasitol. 96, 168177. Radke, J.R., Striepen, B., Guerini, M.N., Jerome, M.E., Roos, D.S., White, M.W., 2001. Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii. Mol. Biochem. Parasitol. 115, 165175. Radke, J.R., Gubbels, M.J., Jerome, M.E., Radke, J.B., Striepen, B., White, M.W., 2004. Identification of a sporozoite-specific member of the Toxoplasma SAG superfamily via genetic complementation. Mol. Microbiol. 52, 93105. Roos, D.S., Donald, R.G., Morrissette, N.S., Moulton, A.L., 1994. Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol. 45, 2763. Roos, D.S., Sullivan, W.J., Striepen, B., Bohne, W., Donald, R.G., 1997. Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis. Methods 13, 112122. Rugarabamu, G., Marq, J.B., Guerin, A., Lebrun, M., Soldati-Favre, D., 2015. Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to

micronemal protein protease 1 activity during invasion. Mol. Microbiol. 97, 244262. Russo, I., Oksman, A., Vaupel, B., Goldberg, D.E., 2009. A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development. Proc. Natl. Acad. Sci. U.S.A. 106, 15541559. Sampels, V., Hartmann, A., Dietrich, I., Coppens, I., Sheiner, L., Striepen, B., et al., 2012. Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii. J. Biol. Chem. 287, 1628916299. Santos, J.M., Ferguson, D.J., Blackman, M.J., Soldati-Favre, D., 2011. Intramembrane cleavage of AMA1 triggers Toxoplasma to switch from an invasive to a replicative mode. Science 331, 473477. Seeber, F., 1997. Consensus sequence of translational initiation sites from Toxoplasma gondii genes. Parasitol. Res. 83, 309311. Seeber, F., Boothroyd, J.C., 1996. Escherichia coli betagalactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii. Gene 169, 3945. Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B. N., Palmer, A.E., Tsien, R.Y., 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 15671572. Sheiner, L., Demerly, J.L., Poulsen, N., Beatty, W.L., Lucas, O., Behnke, M.S., et al., 2011. A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor. PLoS Pathog. 7, e1002392. Sheiner, L., Fellows, J.D., Ovciarikova, J., Brooks, C.F., Agrawal, S., Holmes, Z.C., et al., 2015. Toxoplasma gondii Toc75 functions in import of stromal but not peripheral apicoplast proteins. Traffic 16, 12541269. Shen, B., Brown, K.M., Lee, T.D., Sibley, L.D., 2014. Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. mBio 5, e01114. Sibley, L.D., Messina, M., Niesman, I.R., 1994. Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy. Proc. Natl. Acad. Sci. U.S.A. 91, 55085512. Sidik, S.M., Hackett, C.G., Tran, F., Westwood, N.J., Lourido, S., 2014. Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9, e100450. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166, 14231435.e12.

Toxoplasma Gondii

References

Sidik, S.M., Huet, D., Lourido, S., 2018. CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii. Nat. Protoc. 13, 307323. Singh, U., Brewer, J.L., Boothroyd, J.C., 2002. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Mol. Microbiol. 44, 721733. Skariah, S., Bednarczyk, R.B., Mcintyre, M.K., Taylor, G.A., Mordue, D.G., 2012. Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates. J. Immunol. 188, 34043415. Skarnes, W.C., Rosen, B., West, A.P., Koutsourakis, M., Bushell, W., Iyer, V., et al., 2011. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337342. Soldati, D., Boothroyd, J.C., 1993. Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260, 349352. Soldati, D., Boothroyd, J.C., 1995. A selector of transcription initiation in the protozoan parasite Toxoplasma gondii. Mol. Cell Biol. 15, 8793. Soldati, D., Kim, K., Kampmeier, J., Dubremetz, J.-F., Boothroyd, J.C., 1995. Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection. Mol. Biochem. Parasitol. 74, 8797. Soldati, D., Dubremetz, J.F., Lebrun, M., 2001. Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int. J. Parasitol. 31, 12931302. Striepen, B., He, C.Y., Matrajt, M., Soldati, D., Roos, D.S., 1998. Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii. Mol. Biochem. Parasitol. 92, 325338. Striepen, B., Crawford, M.J., Shaw, M.K., Tilney, L.G., Seeber, F., Roos, D.S., 2000. The plastid of Toxoplasma gondii is divided by association with the centrosomes. J. Cell Biol. 151, 14231434. Striepen, B., Soldati, D., Garcia-Reguet, N., Dubremetz, J.F., Roos, D.S., 2001. Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii. Mol. Biochem. Parasitol. 113, 4554. Striepen, B., White, M.W., Li, C., Guerini, M.N., Malik, S.B., Logsdon JR., J.M., et al., 2002. Genetic complementation in apicomplexan parasites. Proc. Natl. Acad. Sci. U.S.A. 99, 63046309. Sullivan JR., W.J., Chiang, C.W., Wilson, C.M., Naguib, F. N., El kouni, M.H., Donald, R.G., et al., 1999. Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus. Mol. Biochem. Parasitol. 103, 114.

939

Sullivan JR., W.J., Dixon, S.E., Li, C., Striepen, B., Queener, S.F., 2005. IMP dehydrogenase from the protozoan parasite Toxoplasma gondii. Antimicrob. Agents Chemother. 49, 21722179. Sugi, T., Kato, K., Weiss, L.M., 2016. An improved method for introducing site-directed point mutation into the Toxoplasma gondii genome using CRISPR/Cas9. Parasitol. Int. 65, 558562. Suvorova, E.S., Croken, M., Kratzer, S., Ting, L.M., CONDE De felipe, M., Balu, B., et al., 2013. Discovery of a splicing regulator required for cell cycle progression. PLoS Genet. 9, e1003305. Tang, X., Liu, X., Tao, G., Qin, M., Yin, G., Suo, J., et al., 2016. “Self-cleaving” 2A peptide from porcine teschovirus-1 mediates cleavage of dual fluorescent proteins in transgenic Eimeria tenella. Vet. Res. 47, 68. Teale, W.D., Paponov, I.A., Palme, K., 2006. Auxin in action: signalling, transport and the control of plant growth and development. Nat. Rev. Mol. Cell Biol. 7, 847859. Thathy, V., Menard, R., 2002. Gene targeting in Plasmodium berghei. Methods. Mol. Med. 72, 317331. Tosetti, N., Pacheco, N.D., Soldati-Favre, D., Jacot, D., 2019. Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii. eLife 8. Available from: https://doi. org/10.7554/eLife.42669. Umejiego, N.N., Li, C., Riera, T., Hedstrom, L., Striepen, B., 2004. Cryptosporidium parvum IMP dehydrogenase: identification of functional, structural, and dynamic properties that can be exploited for drug design. J. Biol. Chem. 279, 4032040327. Upadhya, R., Kim, K., Hogue-Angeletti, R., Weiss, L.M., 2011. Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii. J. Microbiol. Methods 85, 103113. Uyetake, L., Ortega-Barria, E., Boothroyd, J.C., 2001. Isolation and characterization of a cold-sensitive attachment/invasion mutant of Toxoplasma gondii. Exp. Parasitol. 97, 5559. Vanchinathan, P., Brewer, J.L., Harb, O.S., Boothroyd, J.C., Singh, U., 2005. Disruption of a locus encoding a nucleolar zinc finger protein decreases tachyzoite-tobradyzoite differentiation in Toxoplasma gondii. Infect. Immun. 73, 66806688. Vandenhoff, M.J.B., Moorman, A.F.M., Lamers, W.H., 1992. Electroporation in intracellular buffer increases cellsurvival. Nucleic Acids Res. 20, 2902. Vinayak, S., Brooks, C.F., Naumov, A., Suvorova, E.S., White, M.W., Striepen, B., 2014. Genetic manipulation of the Toxoplasma gondii genome by fosmid recombineering. mBio 5, e02021. van Dooren, G.G., Tomova, C., Agrawal, S., Humbel, B.M., Striepen, B., 2008. Toxoplasma gondii Tic20 is essential

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for apicoplast protein import. Proc. Natl. Acad. Sci. U.S. A. 105, 1357413579. van Dooren, G.G., Reiff, S.B., Tomova, C., Meissner, M., Humbel, B.M., Striepen, B., 2009. A novel dynaminrelated protein has been recruited for apicoplast fission in Toxoplasma gondii. Curr. Biol. 19, 267276. van Poppel, N.F., Welagen, J., Duisters, R.F., Vermeulen, A. N., Schaap, D., 2006. Tight control of transcription in Toxoplasma gondii using an alternative tet repressor. Int. J. Parasitol. 36, 443452. Waller, R.F., Keeling, P.J., Donald, R.G., Striepen, B., Handman, E., Lang-Unnasch, N., et al., 1998. Nuclearencoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S. A. 95, 1235212357. Wang, Y., Wang, F., Wang, R., Zhao, P., Xia, Q., 2015. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci. Rep. 5, 16273. White, M.W., Jerome, M.E., Vaishnava, S., Guerini, M., Behnke, M., Striepen, B., 2005. Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant. Mol. Microbiol. 55, 10601071.

Wirtz, E., Leal, S., Ochatt, C., Cross, G.A., 1999. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89101. Xiao, Y., Karnati, S., Qian, G., Nenicu, A., Fan, W., Tchatalbachev, S., et al., 2012. Cre-mediated stress affects sirtuin expression levels, peroxisome biogenesis and metabolism, antioxidant and proinflammatory signaling pathways. PLoS One 7, e41097. Yang, S., Parmley, S.F., 1997. Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts. Gene 184, 112. Yeoh, L.M., Goodman, C.D., Hall, N.E., van Dooren, G.G., McFadden, G.I., Ralph, S.A., 2015. A serine-argininerich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii. Nucleic Acids Res. 43, 46614675. Yeoh, L.M., Lee, V.V., McFadden, G.I., Ralph, S.A., 2019. Alternative splicing in apicomplexan parasites. mBio 10. Available from: https://doi.org/10.1128/mBio.02866-18.

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21 Regulation of gene expression in Toxoplasma gondii Kami Kim1, Victoria Jeffers2 and William J. Sullivan, Jr.3 1

Department of Internal Medicine, University of South Florida, Tampa, FL, United States 2Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, United States 3Departments of Pharmacology & Toxicology, Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, IN, United States

21.1 Introduction Toxoplasma gondii is distinct from nearly all other members of the large coccidian family (phylum Apicomplexa) owing to the exceptional range of animals (virtually all warmblooded animals) that serve as host for its intermediate life cycle. Like other coccidians, Toxoplasma completes its definitive life cycle in a single animal host (Dubey et al., 1970). Both oocysts shed from the feline definitive host and tissue cysts produced in intermediate hosts can infect either definitive or intermediate hosts (Dubey, 1988) enabling Toxoplasma to increase its host range (Su et al., 2003). Development of sexual stages in the feline intestine results in oocysts that are shed into the environment (Long, 1982). Contamination of soil or water can lead to epidemics of human toxoplasmosis (Isaac-Renton et al., 1998; Choi et al., 1997; Bowie et al., 1997; Konishi and Takahashi,

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00021-9

1987; Stray-Pedersen and Lorentzen-Styr, 1980) and is an ongoing source of toxoplasmosis in South America (Lehmann et al., 2006; Blaizot et al., 2019). Moreover, oocysts are the primary source of Toxoplasma infections of livestock destined for slaughter and human consumption (Mateus-Pinilla et al., 1999; Andrews et al., 1997). Given the importance of Toxoplasma infections to human populations, understanding developmental mechanisms initiated by sporozoites or bradyzoites leading to tissue cyst formation is central to ultimately controlling transmission and chronic disease (see Chapter 18: Bradyzoite and sexual stage development). Studies of Toxoplasma primary infections in animals and of sporozoite- and bradyzoite-infected cultures in vitro (Dubey and Frenkel, 1976; Dubey, 1998; Jerome et al., 1998; Radke et al., 2003) indicate that development initiated by either the sporozoite or

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bradyzoite stage is similar and likely the consequence of a unified genetic program (Radke et al., 2003). Thus defining the changes in gene expression that accompany this development pathway is important to understand the underlying mechanisms responsible for toxoplasmosis caused by either route of infection. mRNA pools are dynamic indicating that regulation of mRNA steady state is a major mechanism employed to regulate developmental transitions in this parasite. Toxoplasma possesses a repertoire of epigenetic-based mechanisms to modulate transcription, as observed in other well-studied eukaryotes from yeast to multicellular animals. Finally, we discuss the emerging role of posttranscriptional regulation, which also appears to be active in this parasite.

21.2 Transcription in Toxoplasma Apicomplexan parasites exhibit complicated, multistage life cycles that involve a variety of hosts. Coincident with their complex life cycles are wholesale changes in gene expression associated with each developmental stage or host. Early efforts to accelerate gene discovery in Toxoplasma led to the sequence for .120,000 ESTs from RH- and ME49-strain tachyzoites, as well as ME49-strain bradyzoites and VEG strain oocysts (Ajioka et al., 1998; Manger et al., 1998; Li et al., 2003, 2004). A Toxoplasma SAGE project and a 10X-whole genome project for the Type II-Me49B7 followed by further genome sequence to provide 5X coverage of GT-1 (Type I) and VEG (Type III) and whole genome microarrays based on the Type II-Me49B7 reference strains were developed and made available to the Toxoplasma research community. The most recent releases of www.toxodb.org (see Chapter 23: ToxoDB: the functional genomic resource for Toxoplasma and related organisms) feature a revised, resequenced, and

reannotated ME49 reference genome, incorporating additional data including expression data derived from microarrays and next generation
derived RNA-seq, genome-wide chromatin immunoprecipitation (ChIP) studies, as well as proteomics studies (Lorenzi et al., 2016). This genome effort also incorporated genomes of 62 strains with the representation of the 16 major haplogroups (Lorenzi et al., 2016). In addition, gene IDs have been harmonized between the main reference strains ME49, GT-1, and VEG.

21.2.1 The parasite transcriptome and transcriptional regulation The first microarray transcriptome studies of the Apicomplexa in Plasmodium illustrated that more than 80% of transcripts had peak expression within a single timeframe in either the sexual stages or the intraerythrocytic cycle. The large changes in transcript levels in Plasmodium suggested mRNA expression is governed by “just-in-time” mechanisms; and the relatively low proportion of constitutive mRNAs in these parasites may reflect this concept (Llinas and DeRisi, 2004; Bozdech et al., 2003). Comparison of the changes in the Plasmodium transcriptome and proteome indicated that alterations in mRNA levels have a higher correlation to protein changes in this parasite than is observed in yeast or higher eukaryotes (Le Roch et al., 2004). Both microarrays and next-generation sequencing have been used to develop a comprehensive view of the T. gondii transcriptome and permit the characterization of candidate factors that regulate gene expression (Hassan et al., 2012; Minot et al., 2012; Rosowski and Saeij, 2012; Reid et al., 2012; Bahl et al., 2010; Behnke et al., 2010, 2014; Hehl et al., 2015). Initial transcriptome studies in T. gondii relied upon cDNA derived from EST projects that were subsequently used to construct a

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limited Toxoplasma cDNA microarray that focused on tachyzoite-bradyzoite transitions in cell culture models of bradyzoite differentiation (Cleary et al., 2002) and explored gene expression in mutants that was unable to differentiate (Singh et al., 2002; Matrajt et al., 2002). These studies supported a role for transcriptional mechanisms in determining developmental stage characteristics in Toxoplasma and evidence for coregulation of transcription in this parasite (Singh et al., 2002). In four mutants generated by chemical mutagenesis and selected against the ability to differentiate a common set of mRNAs was affected and unable to be induced, while other affected mRNA groups appeared to cluster with two or three mutants suggesting hierarchical gene expression may direct bradyzoite development (Singh et al., 2002). Subsequent SAGE and microarray projects supported the concept of coregulated sets of mRNA. A community effort led to the fabrication of a Affymetrix gene array that became the platform of choice for transcriptome analysis (Bahl et al., 2010) with numerous published expression datasets from different strains of parasites exposed to different experimental conditions, including bradyzoite inducing conditions (Behnke et al., 2008; Lescault et al., 2010; Buchholz et al., 2011) and sexual stages (Behnke et al., 2014). Most of these datasets are summarized at www.toxodb.org, and RNA-seq has now emerged as the technique of choice for transcriptome analysis. Most transcriptome studies have examined gene expression of tachyzoites and RNA-seq is now routinely used to compare wild-type and mutant strains or query the response to a specific treatment or condition (Croken et al., 2014). RNA-seq has also been very useful in comparison of merozoites and tachyzoites (Hehl et al., 2015). The studies comparing merozoites and tachyzoites showed that approximately 10% of annotated genes are differentially expressed using a very stringent

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( . 8 3 different) threshold (Hehl et al., 2015). RNA-seq of acute and chronic infection in mice (Pittman et al., 2014) was used for simultaneous profiling of both the mouse host and parasite with minimal manipulations. Dual transcriptome profiling studies have also been very useful for understanding how parasite effectors affect the host transcriptome (Naor et al., 2018). The most exciting technical advance has been single cell sequencing which has now been used to understand Plasmodium gametocyte differentiation program (Poran et al., 2017). Two groups have also applied single cell sequencing to T. gondii development (Xue et al., 2019; Waldman et al., 2019), and this technique will likely elucidate important differences in cell cycle development and developmental stages that could not previously be studied due to the limitations in biomass and the necessity to perform studies on heterogeneous samples. Nearly one-third of the most abundant Toxoplasma mRNA are Apicomplexa-specific genes that have simple genomic structures containing few, if any introns (Radke et al., 2005). Metabolic or structural genes typically contain introns. Many transcripts encoding proteins of the basal metabolic machinery and subcellular structures appear in Toxoplasma only when needed during parasite growth and development (Behnke et al., 2010), consistent with the “just-in-time” concept put forth from studies of Plasmodium (Llinas and DeRisi, 2004). Development-specific genes (sporozoite, tachyzoite, or bradyzoite), genes encoding proteins from biochemical pathways, and genes representing mRNA abundance classes are dispersed between all Toxoplasma chromosomes. Gene clusters are rare and most that have been characterized are clusters of virulence genes important for host interactions (AdomakoAnkomah et al., 2014; Blank and Boyle, 2018). mRNA expression patterns do not appear to be strongly influenced by physical proximity. For

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example, genes encoding enolase 1 and 2 are less than 1500 bp apart on chromosome VIII, yet expressed exclusively in bradyzoite or tachyzoite stages, respectively (Lyons et al., 2002). Thus local changes in chromatin structure or the recruitment of RNA polymerase to promoters has little influence on nearby genes. The basal transcriptional complex that controls the expression of protein-encoding genes (class II) in most eukaryotes is carried out by RNA polymerase II and its associated general transcription factors (GTFs). Comparisons of these transcription factors and the three nuclear polymerases (Ranish and Hahn, 1996) suggest that these mechanisms are conserved from the Archaea to mammals. In well-studied unicellular and multicellular eukaryotes, transcription involves a series of coregulatory complexes that work in concert to control the synthesis of RNA from a particular genomic region. Activating transcription factors (ATFs) bind to cis-acting promoter element(s) and recruit chromatin remodeling enzymes that relax the chromatin around the cis-element-containing region, as well as recruit the multisubunit Mediator complex that contacts the RNA polymerase II preinitiation complex (PIC) directly (Blazek et al., 2005). The accessibility of the cis-element to ATF binding is dependent upon the interaction with these remodeling enzymes, but can also be influenced by other factors such as the chromatin state at the regulatory sequence and the phase of the cell cycle (Fry and Peterson, 2002). In turn, the relaxed chromatin state allows for the formation of the PIC at the core promoter elements through activities contained within the Mediator that facilitate recruitment of RNA polymerase II and the GTFs. Current models of ATFs suggest that activation of RNA polymerase II by these factors occurs indirectly through their recruitment of ATP-dependent chromatin remodeling complexes (Blazek et al., 2005; Li et al., 2004; Featherstone, 2002). The analysis of protein-encoding genes in the Apicomplexa indicates that conventional

RNA polymerases with similarity to other crown eukaryotes are present. Homologs for all known required eukaryotic RNA polymerases have been found in the Toxoplasma genome: RNA polymerase I (transcribes ribosomal RNA), RNA polymerase II (transcribes protein-encoding transcripts), and RNA polymerase III (transcribes small RNA) (Li et al., 1989, 1991; Fox et al., 1993; Meissner and Soldati, 2005). The core elements of class II eukaryotic promoters include TATA box, Initiator, and downstream promoter elements that are recognized and bound by several GTFs: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (Blazek et al., 2005; Featherstone, 2002; Ruvalcaba-Salazar et al., 2005; Ranish and Hahn, 1996). The core of the GTF family includes the TATA Binding Protein, TFIID, and RNA polymerase II. Homologs for various subunits for the GTFs (TFIID, TFIIE, TFIIF, and TFIIH) and subunits of Mediator have been found in the Apicomplexa, and while GTFs are less conserved in the Apicomplexa, much of the basal transcriptional machinery and chromatin remodeling factors required for cooperative control of gene transcription in eukaryotes are present in these pathogens.

21.2.2 Gene-specific cis-elements Classical promoter-mapping strategies utilizing conventional protein reporters, including chloramphenicol acetyltransferase, β-galactosidase, green fluorescent protein, or firefly/renilla luciferase (luc), have been employed to map regulatory sequences in several promoters in Toxoplasma. Deletion studies to identify promoter cis-elements have been reported for various constitutive genes (GRAs and DHFR-TS), tachyzoite-specific genes (SAG1 and enolase 2), and bradyzoite specific genes (hsp30/BAG1, hsp70, LDH2, and enolase 1) and confirm that promoter elements are

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primarily located upstream from the translational start site (Nakaar et al., 1998; Kibe et al., 2005; Matrajt et al., 2004; Ma et al., 2004; Roos et al., 1997; Bohne et al., 1997; Mercier et al., 1996; Soldati and Boothroyd, 1995). Promoter elements were observed to be active in either DNA strand, but may have a limited working distance from the transcriptional start or lose their influence when located downstream of the coding region (Soldati and Boothroyd, 1995). The level of detail within these studies varies and minimal sequence elements were determined in only a few studies (Mercier et al., 1996; Matrajt et al., 2004); moreover, no published study has fully resolved the question of functional sufficiency for any putative cis-element. Nonetheless, it is evident that a 27 bp repeat sequence (6 3 repeat) in the SAG1 promoter is required for function and a sequence element (A/TGAGACG) found in the GRA promoters was demonstrated to be required for basal expression within the context of a 53 bp minimal promoter (Mercier et al., 1996). It is notable that the GAGACG is present in the central core of the SAG1 27 bp repeats is also found in regions implicated to contain regulatory cis-elements by deletion analysis of the NTPI/II and DHFR-TS promoters (Nakaar et al., 1998; Matrajt et al., 2004). Development-specific changes in mRNA levels are a dominant feature of the Apicomplexa transcriptome. Nearly onequarter of the transcripts detected in the Toxoplasma SAGE project was observed to be uniquely expressed during parasite development, and similar observations have emerged from functional genomics studies of the Plasmodium intraerythrocytic cycle (Le Roch et al., 2004; Bozdech and Ginsburg, 2005). Promoters controlling bradyzoite-specific genes, BAG1, hsp70, and LDH2, have been mapped using alkaline-stress induction at a similar resolution too (Ma et al., 2004; Yang and Parmley, 1997; Bohne et al., 1997). While these studies support the role of promoter

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elements in regulating stress
response in Toxoplasma, their resolution is too low to allow for the identification of common cis-elements. The reciprocal regulation of enolase 1 (bradyzoite-specific) and enolase 2 (tachyzoite-specific) is of particular interest given their close proximity in an ordered tandem array of enolase 2-1. Repression of enolase 1 expression in tachyzoites appears to require a distal region .600 bp from the enolase 1 ATG, and these elements are distinct from inductive elements that were mapped closer to the start of transcription (Kibe et al., 2005). Employing a dual luciferase model, bradyzoite-specific cis-elements within a Toxoplasma gene encoding a novel NTPase (Brady-NTPase; chromosome X, TGG_994683) were mapped (Behnke et al., 2008). A series of sequential and internal deletions followed by 6 bp substitution mutagenesis have identified a 15 bp cis-element that is responsible for induction of the Brady-NTPase promoter under a variety of drug and stress conditions that coinduce native bradyzoite gene expression. This element lies within the first 500 bp of the Brady-NTPase promoter, and 90% of the induction is lost when the element is mutated in the context of the full-length promoter fragment (1495 bp). Mutation of this element does not lead to increased expression in the tachyzoite stage indicating that it is a true inductive element. Approximately 2800 mRNAs have cyclical profiles during parasite division that cluster into two major transcriptional waves. Genes with maximum expression in the G1 subtranscriptome encode well-conserved metabolic and biosynthetic functions, while those mRNAs in the S/M subtranscriptome are enriched for genes encoding proteins involved in daughter budding and egress (Behnke et al., 2010). FIRE (finding important regulatory elements) analysis of the proximal promoter regions for all cyclical mRNAs was scanned for enrichment of possible DNA regulatory

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elements. Nine DNA motifs were identified by this analysis (Behnke et al., 2010) that was distributed in genes with peak transcription spanning the full tachyzoite cell cycle. DNA motifs that are overrepresented in the promoters flanking G1 genes are generally underrepresented in the promoters of S/M genes (and vice versa). One of the DNA motifs enriched in G1 promoters (50 -TGCATGC-30 ) is identical to the TgTRP2 cis-element required for transcription of ribosomal proteins (Van Poppel et al., 2006; Mullapudi et al., 2009) and is also identical to the 6 bp core DNA binding motif determined by PBM for AP2XI-3. The mRNA for this AP2 also peaks during the G1 period (Behnke et al., 2010). Single cell sequencing has been applied to tachyzoites (RH, ME49, Pru) and tachyzoites exposed to bradyzoite inducing conditions (ME49 and Pru) (Xue et al., 2019; Waldman et al., 2019) and has confirmed the existence of groups of gene expression corresponding to G1a, G1b, S, M, C states identified in earlier cell cycle gene analysis (Behnke et al., 2010). Bradyzoites were predominantly in the G1b state (Xue et al., 2019), supporting the concept of critical cell cycle checkpoints governing developmental transitions. These studies have also illustrated the heterogeneous states of populations of cells undergoing developmental transitions (Xue et al., 2019).

21.2.3 The evolution of APETALA2related proteins DNA-binding proteins in the Apicomplexa that are related to the APETALA-2 (AP2) class of plant transcription factors (ApiAP2 proteins) are thus far the most important set of proteins with critical roles in parasite gene expression (Balaji et al., 2005). AP2 domain
containing proteins were initially thought to be a plantspecific family of DNA binding proteins (Riechmann and Meyerowitz, 1998; Krizek,

2003). AP2 homologs were subsequently identified nonplant species such as cyanobacterium, ciliates, and viruses, indicating AP2 DNA-binding domains are widely conserved (Wuitschick et al., 2004; Magnani et al., 2004). Many of these proteins contain a second domain encoding a homing endonuclease function that confers the ability to operate as mobile genetic elements with the capacity to transpose, invade, and self-replicate by exploiting genome repair mechanisms (Chevalier and Stoddard, 2001; Koufopanou et al., 2002). Magnani et al. (2004) hypothesized that the plant AP2/ERF (ethylene response factor) family of transcription factors arose from the HNH-AP2 family of homing endonucleases present in bacteria or viruses and was incorporated into plant genomes via horizontal gene transfer (Magnani et al., 2004), or alternatively was acquired indirectly from an endosymbiotic event, likely with a cyanobacterium with an early plant progenitor. Over time the homing endonuclease function has been lost in plants as the AP2 DNA-binding domain has taken on specific regulatory roles in gene expression associated with plant development and stress response (Magnani et al., 2004; Altschul et al., 2010). Apicomplexan genomes, including Plasmodium spp., T. gondii, Cryptosporidium parvum, and Theileria spp., encode multiple ApiAP2 proteins that may have a similar endosymbiotic origin (Balaji et al., 2005; Altschul et al., 2010). Unlike plant AP2 factors, ApiAP2 proteins have undergone a lineage-specific expansion of a few progenitor genes leading to ApiAp2 factors carrying up to eight AP2 domains (Balaji et al., 2005; Altschul et al., 2010). Phylogenetic analysis of ApiAP2 proteins from Plasmodium (27 total), Theileria (19 total), and Cryptosporidium (21 total) suggests the common ancestor of these three protozoa contained nine conserved ApiAP2 proteins, with a higher number of orthologous pairs found in Plasmodium spp. and Theileria

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spp. genomes (Balaji et al., 2005). With the exception of the ApiAP2 domain, these proteins are otherwise not conserved and there are no other known protein domains (activation, localization, or protein
protein interaction) found in any of the Plasmodium spp. or Toxoplasma ApiAP2 proteins (Lindner et al., 2010; Altschul et al., 2010).

21.2.4 ApiAP2 structure determination and DNA binding The AP2 domain consists of approximately 60 amino acids and was first shown to confer DNA-binding specificity to the AP2/ethylene response element
binding family (EREBP) in plants (Jofuku et al., 1994; Ohme-Takagi and Shinshi, 1995; Balaji et al., 2005). AP2/EREBP proteins represent the second largest class of transcription factors in Arabidopsis thaliana, consisting of 145 proteins that are subdivided further into five subfamilies based on AP2 domain architecture (Sakuma et al., 2002). DREB (dehydration responsive element binding) and ERF (ERF is identical to EREBP) protein groups constitute the two largest subfamilies (56 DREB compared to 65 ERF) and contain one AP2 domain and a conserved WLG motif (Sakuma et al., 2002). While identical in domain architecture, these groups are defined by single amino acid changes within the DNA-binding domain that alter DNAbinding specificity (Sakuma et al., 2002). Proteins that include two AP2 domains (14 total proteins) are further divided into two subfamilies based on the presence or absence of a second nonAP2 DNA binding domain (Sakuma et al., 2002; Magnani et al., 2004). Alignment of ApiAP2 domains from Plasmodium, Cryptosporidium, and Theileria to the structure of the ERF1 from Arabidopsis indicates strong conservation of 12 residues that correspond to areas of hydrophobic interactions responsible for the backbone of the DNA

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binding domain rather than specifying DNA binding (Balaji et al., 2005; Fig. 21.1). Campbell et al. (2010) determined the DNA-binding specificity of 20 of the 27 Plasmodium falciparum ApiAP2s, revealing an unusual diversity of binding motifs that are either classically palindromic or nucleotide biased, and similar results are seen in T. gondii ApiAP2s (Kim, Sullivan, White, Llinas, unpublished). The structural basis of DNA-binding is evident in ApiAP2 factors that share sequence beyond these 12 core hydrophobic residues. For example, orthologs from P. falciparum (PF14_0633; PF3D7_1466400) and C. parvum (cgd2_3490) that share 68% similarity in the AP2 domain show nearly identical DNA-binding specificities, providing evidence that with respect to DNA recognition there is conservation of function across divergent apicomplexan species (De Silva et al., 2008). Whereas the majority of plant AP2 domains cluster tightly around DNA contact residues, ApiAP2 domains exhibit considerable flexibility within the domain, a feature that suggests a greater diversity in DNAbinding specificity (Balaji et al., 2005). The protein diversity in the ApiAP2 family of proteins is also large. ApiAP2 factors in P. falciparum and T. gondii range in size from 200 to greater than 4000 amino acids that are highly disordered outside the globular AP2 domain(s) (Campbell et al., 2010; see Fig. 21.1). Proteins of this secondary structure type are thought to undergo conformational changes upon interacting with other proteins (Xue et al., 2012), consistent with transcription factor functions. The high content of disordered proteins in parasitic eukaryotes (Xue et al., 2012) is thought to be a crucial adaptation to distinct environment niches. Structural determination of representatives of the Arabidopsis and Plasmodium AP2 families has provided valuable insight into the mechanism by which the AP2 domain binds target DNA sequences. The conserved secondary structure for the monomeric AP2 domain

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FIGURE 21.1 The AP2 family of Toxoplasma gondii. There are 67 members of the T. gondii ApiAP2 family as determined by a T. gondii community annotation effort (White, Sullivan, Kim, Croken and Wootton; see www.toxodb.org), named sequentially by chromosome assignment. Due to genome reannotation with improve gene models, the count of AP2 has been revised to 67 from 68. A schematic of the consensus DNA binding domain of the T. gondii family is shown in the upper left (Altschul et al., 2010). The inferred protein size and AP2 domain location within each TgAP2 are heterogeneous as can be seen in the stick figures representing TgAP2 whose mRNA vary during the cell cycle (right). The peak timing of mRNA of the cell cycle
regulated TgAP2 within the cell cycle is indicated, as determined by Affymetrix microarray analysis (Behnke et al., 2010). The mRNA expression of some additional Tg Api-AP2 members suggests that they are expressed in other developmental stages (Behnke et al., 2010; Xue et al., 2019; Waldman et al., 2019).

predicts three N-terminal antiparallel β-sheets and a C-terminal α-helix (Allen et al., 1998; Lindner et al., 2010). The NMR structure of Arabidopsis ERF1 GCC box binding domain (GBD is AP2 domain, target sequence 5 50 -A/ GCCGAC-30 ) in complex with DNA revealed a novel interaction. AtEFR1 binds DNA via interaction with the β-sheets at specific locations along the DNA backbone while being supported by the α-helix (Allen et al., 1998). This interaction, as depicted in the structure of the single AP2 domain
containing ERF1, is based on 11 highly conserved residues, seven of

which target specific interaction with the GCC box (50 -AGCCGCC-30 ). These residues are located within the β-sheets that bind to the sugar-phosphate backbone and comprise the framework for specific DNA interaction (Allen et al., 1998). The delineating feature of the DREB subfamily from the ERF subfamily of single domain AP2 proteins is a change in two conserved amino acids: V14 to A, E19 to D, respectively. This alters the target DNAbinding sequence (50 -TACCGACAT-30 ) illustrating that the diversity of recognition sequence is dictated by a limited number of

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evolutionarily conserved residues (Sakuma et al., 2002). Interestingly, the dual AP2 domain
containing AINTEGUMENTA contains the conserved arginine and tryptophan residues, but mutation of these residues has little to no effect on DNA binding, suggesting dual AP2 domain proteins exhibit a greater complexity in target DNA-binding sequences and each domain utilizes unique residues to facilitate binding [target sequence 5 50 -gCAC (A/G)N(A/T)TcCC(a/g)ANG(c/t)-30 ] (Krizek, 2003). These results from plants suggest dual AP2 domain proteins are more complex in their DNA binding, and this may also apply to ApiAP2 factors. In Plasmodium, initial characterization of the dual ApiAP2 protein PFF0200c indicated only one AP2 domain actively bound to a specific 10 mer sequence (De Silva et al., 2008). However, studies of the full-length protein in parasites clearly demonstrate that PfSIP2 (PFF0200c; PF3D7_0604100) requires both AP2 domains in order to bind the 16 bp bipartite SPE2 sequence motif (Voss et al., 2003; Flueck et al., 2010). This highlights the limitations associated with using any single approach to determine ApiAP2 function. The crystal structure of the ApiAP2 domain from P. falciparum (PF14_0633) provided further insight into AP2 function (Lindner et al., 2010). While ApiAP2 domains retain many of the canonical features previously described in the A. thaliana (Allen et al., 1998), key differences have been identified. In contrast to the A. thaliana structure, which acts as a monomer, PfAP2s are thought to dimerize via a domainswapping mechanism, with the α-helix of one promoter packed against a β-sheet of its partner (Lindner et al., 2010). This model suggests that DNA binding triggers stabilization of the homodimer or that an AP2 binds DNA as a monomer, elucidating a conformational change that then attracts the second monomer to bind (Lindner et al., 2010). In the case of Pf14_0633, dimerization is thought to be critical to combining distal regions of DNA to function in

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gene-specific transcription of sporozoite stage genes (Lindner et al., 2010). In addition, ApiAP2 may work in concert as proteomics studies of nuclear complexes have often identified more than one ApiAP2 in pull-downs of macromolecular complexes (Flueck et al., 2010; Lesage et al., 2018; Wang et al., 2014).

21.2.5 The function of ApiAP2 proteins Studies in Arabidopsis and other plant species have described major roles for AP2 proteins in a wide variety of developmental and stress responses. These transcription factors systematically regulate a diverse set of plant processes, including meristem, flower and seed development, and environmental responses to drought or attack from plant pathogens (Riechmann and Meyerowitz, 1998; Dietz et al., 2010). The Toxoplasma genome encodes 67 ApiAP2 domain
containing genes (Fig. 21.1; for product names, see www.ToxoDB.org and Altschul et al., 2010), more than twice the number found in P. falciparum (27 total) and other Apicomplexa (Balaji et al., 2005) (the gene models of the original 68 AP2 genes have been revised with improved gene prediction and resequencing of the genomes, and the final count is 67 ApiAP2s in Toxoplasma). These were originally named sequentially by chromosome location, but the names may need to be revised in light of new data from HiC analysis of the 3D organization of the nucleus that T. gondii has only 13 chromosomes (Bunnik et al., 2019). TgAP2s are expressed during parasite development (Behnke et al., 2010; Buchholz et al., 2011), and roughly a third (24 total) of TgAP2 genes is cell cycle regulated with mRNA expression profiles that span the tachyzoite division cycle (Behnke et al., 2010). Eleven TgAP2 mRNAs are induced during bradyzoite differentiation, suggesting a role in

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developmental gene expression. The remaining TgAP2 domain
containing proteins are either constitutively expressed (22 total) or are undetectable in tachyzoite and bradyzoites, indicating possible roles in the sporozoite or oocyst stages of development (six total) (Behnke et al., 2010). Of the ApiAP2 domain
containing genes, only about 50 have all structural features predicted to be necessary for DNA binding (Altschul et al., 2010). In most cases, there are no other obvious clues as to protein function, although studies to date for TgAP2 have shown nuclear localization. Yeast two hybrid studies in Plasmodium (LaCount et al., 2005), proteomics studies in Plasmodium (Zhang et al., 2011; Flueck et al., 2010) and Toxoplasma proteomics studies (Saksouk et al., 2005; Braun et al., 2010; Wang et al., 2014), support a role for ApiAP2 in gene regulation. In the rodent malaria Plasmodium berghei an ookinete-specific AP2 (AP2-O, PF11_0442 ortholog) is critical to mosquito mid-gut invasion. AP2-O directly interacts within the proximal promoter regions of 15 genes, including 10 that had previously been defined as ookinete specific or required for ookinete development within the mosquito mid-gut. AP2-O knockout parasites exhibited normal gametogenesis, however lacked the ability to infect mosquitos (Yuda et al., 2009). A second P. berghei AP2, AP2-Sporozoite (AP2-Sp), is a trans-acting factor whose cognate binding site is enriched in proximal promoter regions of known sporozoite specific genes, likely interacting with cis-elements to promote stage-specific gene expression (Yuda et al., 2010; Helm et al., 2010). The P. falciparum ortholog of this protein, PF14_0633, has a DNA-binding domain that shared a conserved DNA-binding motif (GCATGC) with both C. parvum (De Silva et al., 2008) as well as T. gondii orthologs. In contrast to Plasmodium the T. gondii gene appears to be essential, and to date, it has been refractory to disruption. While many of the

DNA binding motifs of ApiAP2 appear to be phylogenetically conserved, there is no conclusive evidence as to whether or not these factors have orthologous functions. Further evidence for transcription factor activity for AP2 proteins comes from a promoter-mapping study of a liver-stage exclusive promoter. Four repeats of the ApiAP2 PB000252.02.0 (PF11_0404 ortholog) DNAbinding motif were found in the minimal promoter. Interestingly, mutation of a single copy of the binding site within the liver stage promoter increased promoter luciferase gene expression, suggesting PbAP2 PB000252.02.0 has a role as a transcriptional repressor (Helm et al., 2010), which is a common mechanism used by plant AP2 factors to regulate the timing of developmental gene expression (Song et al., 2005; Schmid et al., 2003; Andriankaja et al., 2007). Finally, the Bilker and Soldati groups used the DNA binding domains of Api-AP2 to develop regulated promoters that work in both Plasmodium species as well as T. gondii, further bolstering the hypothesized role of these proteins in transcriptional regulation (Pino et al., 2012). Genetic studies suggest that Toxoplasma AP2 factor AP2IX-9, which is induced by alkaline stress, operates as a suppressor of bradyzoite differentiation through binding to specific bradyzoite gene promoters (Radke et al., 2013) Another AP2 factor AP2XI-4 is upregulated in bradyzoites and has been identified as an activator of bradyzoite differentiation (Walker et al., 2013). Thus it is expected that ApiAP2 factors in Toxoplasma, much like plant and Plasmodium spp. factors, will have an important role in regulating developmental gene expression in the intermediate and definitive life cycles. Analysis of ApiAP2 mRNA expression patterns in P. falciparum reveals an ordered timing of expression for 22 of 26 ApiAP2 proteins during the intraerythocytic development cycle (IDC) (Balaji et al., 2005; Campbell et al., 2010).

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The distribution of expression across the IDC suggests that ApiAP2 factors could be responsible for controlling the dynamic changes in gene expression during apicomplexan replication (Campbell et al., 2010). The enrichment of PfAP2 binding motifs in the promoters of cell cycle transcripts lends support to the idea that the trans-acting ApiAP2 regulator of groups of cell cycle genes will likely be coexpressed (Campbell et al., 2010). For example, the putative target genes for Pf13_0235, which includes ribosome function and heat shock genes, all have the same timing in the IDC. Also, enrichment of PfAP2 target sequences that are found in invasion and host cell entry genes may be regulated by Pf10_0075, and transcripts encoding DNA replication factors could be controlled by MAL8P1.153 (Campbell et al., 2010). In each case the binding motif of the coexpressed PfAP2 was enriched in the promoters of the inclusive mRNA cluster. The sequential profiles of 24 cell cycle
regulated TgAP2 factors provide attractive candidates for an interacting network operating during Toxoplasma replication to coordinate a similar cell cycle transcription cascade. Like early work on cell cycle Plasmodium ApiAP2s, the study of Toxoplasma cell cycle ApiAP2s has focused on determining the DNA-binding specificity for selected proteins and the DNA binding preferences of a substantial fraction of TgAP2 have now been identified using the protein binding array technology previously adapted to Plasmodium AP2 (Campbell et al., 2010; De Silva et al., 2008; White, Sullivan, Llinas, and Kim, unpublished). These motifs will be useful for genome-wide computational searches for putative regulatory targets within proximal promoters as determined by epigenomics ChIP-chip analysis (Gissot et al., 2007) and inferred transcription start sites (Yamagishi et al., 2010). While the cell cycle and developmental TgApiAP2s occupy largely unique groups, there are three TgAP2s (AP2VIIa-1, AP2VI-1, and

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AP2IX-4) that are periodically expressed in the tachyzoite cell cycle and are also elevated in late stage in vivo cysts (Behnke et al., 2010; Buchholz et al., 2011). These TgAP2 factors show peak mRNA levels in the S/M phase, which corresponds to a critical point in the tachyzoite cell cycle where parasites continue replication as a tachyzoite or differentiate into a bradyzoite (Radke et al., 2003). Although parasites pass through S phase to differentiate, the signals to differentiate are likely sensed in G1 phase (Kim, 2018). A handful of cell cycle
regulated AP2s have been studied in the context of the tachyzoite to bradyzoite transition, and it appears that they function as either activators or repressors, often working in opposition to determine the appropriate developmental pathway for the parasite; whether to continue to replicate, or to differentiate to the latency. AP2IV-4 is cell cycle regulated and peaks in the S/M stage. Although nonessential for tachyzoite replication, it is required for repression of bradyzoite specific genes during tachyzoite growth (Radke et al., 2018). Similarly, knock-out of AP2IX-4 did not affect tachyzoite growth rate but did lead to the upregulation of bradyzoitespecific genes, suggesting that it normally functions as a repressor during proliferation (Huang et al., 2017) However, the Type II AP2IX-4 mutant was also defective in forming bradyzoite cysts compared to the parental line, indicating that the role AP2IX-4 extends beyond simply repression of bradyzoite genes in tachyzoites (Huang et al., 2017). Two AP2 factors peak at the same point in the cell cycle (S/M) but exert opposing effects on gene expression (Hong et al., 2017). AP2IX-9 acts as a repressor to enhance bradyzoite formation, while AP2IV-3 functions as a gene activator to dampen the switch to latency and sustain parasite replication (Hong et al., 2017). Adding to the complexity of the role of AP2s in parasite transcriptional regulation, AP2s may also regulate each other by

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protein
protein interaction or dimerization. Gissot et al. identified AP2X-5 as a binding partner of AP2XI-5 (Lesage et al., 2018). Although AP2X-5 is constitutively expressed, AP2XI-5 is cell cycle regulated, peaking at the S/M stage. ChIP experiments suggest that AP2X-5 itself does not bind DNA, but knock out did lead to dysregulation of cell cycle genes that peak in S/M. Thus accumulation of AP2IX-5 at this point in the cell cycle may specifically recruit AP2IX-5 to these cell cycle
regulated gene loci (Lesage et al., 2018). TgAP2s have been identified as proteins that interact with chromatin remodelers HDAC3 (Saksouk et al., 2005) and GCN5b (Wang et al., 2014). Detailed analysis of the AP2 factors associated with GCN5b indicates that there are distinct complexes composed of different pairs of AP2 factors (Wang et al., 2014). Studies in T. gondii suggest that the activities of GCN5 and HDAC3 oppose each other (Sindikubwabo et al., 2017). A TgAP2 also was reported to be a component of the macromolecular complex that interacts with TgAgo an essential component of the RNAinduced silencing complex (RISC) that mediates the activity of many small RNAs in gene expression (Braun et al., 2010). TgAP2s are likely to regulate multiple critical processes in gene regulation and regulate developmental gene regulation in concert with the basal transcription machinery and chromatin remodeling factors (Fig. 21.2). Further work is needed to establish how these individual factors interact and respond to disparate environmental and host signals. In Plasmodium, AP-G has been identified as a master regulator of gametocyte differentiation (Poran et al., 2017; Sinha et al., 2014; Kafsack et al., 2014) whose expression is regulated by the same chromatin state that regulates expression of Plasmodium virulence genes, but none of the AP2 in T. gondii appear to act as a master regulator of bradyzoite formation. The different AP2 factors may act coordinately to regulate developmental

transitions, or perhaps their activity or their access to chromatin is regulated by an alternative factor such as the myb family transcription factor that is hypothesized to be a pioneer transcription factor as a master regulator of bradyzoite formation (Waldman et al., 2019). ApiAP2 proteins may have nontranscriptional roles. Genome-wide interaction studies of PfSIP2 (ChIP-chip), a dual domain PfAP2 protein, found that this factor localized to subtelomeric heterochromatin regions on all chromosomes (Flueck et al., 2010). After proteolytic processing, PfSIP-N exclusively localizes with the SPE2 DNA motif that is present in multiple copies in the promoter regions of upsB var genes associated with var gene silencing (Flueck et al., 2010; Voss et al., 2003). Overexpression of PfSIP2-N caused no changes in gene expression, lending support to a role in maintaining chromosome end biology (Flueck et al., 2010; Voss et al., 2003). This mechanism is supported by PfSIP2 orthologs that exist in other Plasmodium spp. that lack subtelomeric SPE2 motifs yet maintain the SPE2 motif in internal chromosome regions (Flueck et al., 2010). Taken together, PfSIP2 illustrates a novel function for ApiAP2 proteins in telomeric heterochromatin maintenance and gene silencing. A second PfAP2, Pf11_0091, binds to a motif within the var intron that is sufficient to localize DNA to a subnuclear compartment with silenced var genes in an actin-specific manner (Zhang et al., 2011). The var intron also has promoter activity (Calderwood et al., 2003), and as yet, the role of this ApiAP2 in transcriptional regulation has not been resolved.

21.2.6 Other factors that regulate gene expression Amongst the other factors that are developmentally regulated are the glycolytic enzymes, including ENO2 (tachyzoite) and ENO1 (bradyzoite). Intriguingly, these proteins are localized

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FIGURE 21.2 Model of developmental gene regulation by AP2 and chromatin factors. (A) Chromatin consists of heterochromatin and euchromatin. Heterochromatin is compact, with characteristic histone posttranslational modifications (e.g., histone H3K9 methylation) that prevent the accessibility of RNA polymerase and transcription factors. For simplicity, in this diagram chromatin remodelers are depicted as histone-modifying enzymes, but they include ATP-dependent remodelers, histone-modifying enzymes, and other specialized macromolecular complexes, which are important for maintaining a heterochromatin or euchromatin state. In the case of heterochromatin, HDAC3, which associates with a T. gondii corepressor complex (Saksouk et al., 2005), is one example of an enzyme that might be important for maintaining a closed chromatin structure by removing acetyl groups from histones. Euchromatin, an open chromatin state, is also maintained by macromolecular complexes, enabling access of sequence-specific transcription factors and the RNA polymerase complex. Activity of GCN5b, an acetyl transferase, is associated with active genes and GCN5b associates with AP2 factors and gene

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to the nucleus suggesting that they might also play a role in gene regulation in T. gondii. Several glycolytic enzymes have been identified as components of transcriptional complexes including LDH and GAPDH in the OCA-S complex (Zheng et al., 2003). As the expression of the ENO genes is also regulated at the mRNA level, the ENO1 promoter was used as bait to identify nuclear factors that interact with this promoter (Olguin-Lamas et al., 2011). Amongst the 35 nuclear proteins identified, most are hypothetical proteins, but those with inferred function included two Alba family DNA/RNA binding proteins, a potential histone chaperone (NF3), and other proteins predicted to interact with RNA and DNA. NF3 is nucleolar protein whose overexpression alters nucleolar morphology and inhibits parasite virulence. In the bradyzoite stage, NF3 is cytosolic. ChIP studies have confirmed that NF3 (Olguin-Lamas et al., 2011) is associated with chromatin.

21.3 Epigenetics in Toxoplasma

L

Epigenetic gene regulation refers to heritable changes in gene expression that are not genetically encoded in the DNA sequence of an organism. Among the mechanisms of epigenetic regulation are those affecting accessibility of factors to chromatin such as DNA methylation, histone modification, and nucleosome location. Noncoding RNA also affects a

myriad of nuclear and cytoplasmic processes that regulate epigenetic gene regulation. Current models of eukaryotic transcriptional activation implicate a significantly greater number of cofactors than was appreciated more than a decade ago. The simple binding of gene-specific ATFs to local sequences in nucleosomal DNA (Naar et al., 2001) is now recognized to be insufficient to recruit the RNA polymerase II-PIC. ATFs recruit chromatin remodelers to facilitate the assembly of the PIC on core promoter sequences (reviewed in Spector, 2003; Ehrenhofer-Murray, 2004; Li et al., 2004). These findings bring chromatin dynamics to the forefront of gene expression research, and the discovery that histone proteins can be chemically modified in ways that enhance or inactivate transcription, along with ATPases capable of repositioning nucleosomes, has prompted an intensive investigation into how these mechanisms act cooperatively to regulate gene expression. Although the order and assembly of transcriptional factors have not been demonstrated in apicomplexan parasites, the initial forays into understanding transcriptional regulation reveal that these parasites possess essential features of the basal transcription machinery as well as a significant collection of chromatin remodeling machinery. Research into chromatin remodeling mechanisms for the purpose of new drug target discovery is an important area of investigation, first illustrated by the HDAC (histone deacetylase) inhibitor, apicidin, which has broad-

transcription machinery (Wang et al., 2014). Studies in T. gondii suggest that the activities of GCN5 and HDAC3 oppose each other (Sindikubwabo et al., 2017). An intermediate chromatin state exists for poised genes, which have bivalent chromatin marks consistent with both heterochromatin and euchromatin. Such genes are not transcribed but are ready to be either repressed or activated. The prevalence of poised genes in T. gondii is not known, but some sexual-stage genes were dually marked with H3K9me3 and H3K14ac suggesting that a poised chromatin state may be characteristic of developmentally regulated genes (Sindikubwabo et al., 2017). (B) AP2 factors are considered the major transcription factors in T. gondii. Both repressive and activating AP2 have been characterized and implicated in regulation of the tachyzoitebradyzoite transition. AP2 factors that activate bradyzoite gene expression are opposed by AP2 that repress bradyzoite gene expression. Repressive AP2 could recruit repressive chromatin complexes to DNA after binding. Alternatively, the AP2 may compete for target motifs recognized by AP2 that activate bradyzoite gene expression. Source: From Kim, K., 2018. The epigenome, cell cycle, and development in Toxoplasma. Annu. Rev. Microbiol. 72, 479
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spectrum activity against a variety of apicomplexan parasites including human pathogens Toxoplasma and Plasmodium and veterinary pathogens from the Eimeria genera (DarkinRattray et al., 1996).

21.3.1 Chromatin and chromatin remodeling The fundamental building block of chromatin is the histone protein. Four canonical types of histones exist (H2A, H2B, H3, and H4) that form an octamer complexed with DNA (the nucleosome). Histone tails are subject to a diverse array of covalent modifications that have different consequences on gene transcription (Peterson and Laniel, 2004). Like many other eukaryotes, Toxoplasma H3 and H4 are exceptionally well conserved with each residue in the N-terminal tail reported to be susceptible to chemical modification being present (Sullivan, 2003; Nardelli et al., 2013). Histone variants, which may be substituted for canonical histones to modulate DNA-driven processes, are also conserved. Toxoplasma contains a homolog of variant H3.3 in addition to the canonical H3, the former being associated with genes undergoing transcription in other species (Sullivan, 2003). An ortholog of the centromeric H3 variant (CenH3) localizes to an apical subnuclear compartment and maps to centromeres by ChIP-chip (Brooks et al., 2011; see Fig. 21.4). Beyond the well-conserved H3 and H4 classes, the complement of histone proteins in Toxoplasma exhibits a number of unusual features (Dalmasso et al., 2011). Like yeast, Toxoplasma may not possess H1, the extra-nucleosomal “linker” histone involved in solenoid formation during chromatin condensation, although there is a small basic protein with homology to the H1 of kinetoplastids (Croken et al., 2012) present in the genome. Two distinct lineages of H2B are present, including the constitutively expressed TgH2B.Z, a parasite-specific H2B variant, and

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potential stage-regulated TgH2Ba and TgH2Bb (Dalmasso et al., 2006). The canonical H2A protein is TgH2A1 and both H2AZ and H2AX variants exist (Dalmasso et al., 2009). TgH2A1 and TgH2AX both possess a C-terminal SQ motif, consistent with predicted roles in the DNA damage response. Coimmunoprecipitation experiments are beginning to reveal the composition of Toxoplasma nucleosomes (Dalmasso et al., 2009). TgH2AZ dimerizes with TgH2B.Z, but not TgH2AX. TgH2AZ and TgH2B.Z localize with other acetylated histones to actively transcribed genes, whereas TgH2AX is present at repressed genes (Dalmasso et al., 2009). Interestingly, TgH2AX expression increases during bradyzoite conversion, consistent with the increase in repressed genes during the latent stage. These studies are consistent with the hypothesis that TgH2AZ and TgH2B.Z are involved in transcriptional activation, while TgH2AX and TgH2A1 may populate chromatin during stress (Dalmasso et al., 2009). Histone N-terminal tails are generally rich in positively charged amino acids and interact tightly with negatively charged DNA, facilitating condensation. The assembly of genomic DNA into histone nucleosomes and then into higher order chromatin structure is associated with transcriptional repression and “silenced” chromatin is thought to be the default mechanism guiding the formation of chromatin following DNA replication (Ehrenhofer-Murray, 2004). Thus active steps must be taken to alter the normal state of chromatin in order to achieve stable transcriptional activation. A myriad of chemical modifications to histones are now known and are proposed to operate in combinatorial fashion, constituting a “histone code” that reflects corresponding changes in the local activation (and inactivation) of specific genes (Strahl and Allis, 2000). The histone code of T. gondii has been characterized by mass spectrometry with T. gondii histones possessing novel modifications not previously described in protozoa

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(Nardelli et al., 2013; Silmon de Monerri et al., 2015; Fig. 21.3). Similar to Plasmodium (SalcedoAmaya et al., 2009) and in contrast to the metazoa, T. gondii nucleosomes consist primarily of euchromatic-acetylated chromatin consistent with chromatin that accessible to the transcriptional machinery (Nardelli et al., 2013). Numerous histone modifications, including

ubiquitination (Silmon de Monerri et al., 2015), are substoichiometric, suggesting that the combination of histone posttranslational modifications may reflect chromatin state. Consistent with the histone code hypothesis is the discovery of protein motifs capable of binding specific histone modifications. Examples include bromodomains that interact

FIGURE 21.3

The histone code of Toxoplasma gondii: a summary of PTM identified on T. gondii canonical histones and histone variants by mass spectrometry. PTM on canonical histones are shown in comparison with Plasmodium falciparum. Circles in different colors represent the modifications. PTM on canonical and histone variants are shown in comparison with P. falciparum. Identical amino acids are represented in gray. Circles above the sequence represent histone PTM in T. gondii (Nardelli et al., 2013). Histones depicted are histone 3 (H3); histone 4 (H4), histone H2A (H2A), histone (H2B) and variant histones H2Bv, H2A.Z, H2A.X and CenH3. The gray cylinders indicate the approximate location of the histone globular domain. Numbers above the sequences represent the amino acid position in T. gondii while 1 3 , 2 3 , and 3 3 above the red circles indicate mono-, di-, or trimethylation, respectively. PTM, Posttranslational modifications. Source: Courtesy Sheila Nardelli.

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with acetylated lysines, chromodomains that bind methylated lysines, and macro domains that recognize ADP-ribose moieties (Dhalluin et al., 1999; Bannister et al., 2001; Karras et al., 2005). The T. gondii genome encodes 12 predicted bromodomain-containing proteins, six of which display sequence homology to known bromodomain proteins in higher eukaryotes, such as the GCN5 homologs and TAF1, a member of the TFIID transcriptional initiation complex (reviewed in Jeffers et al., 2017b). The remaining six predicted proteins are conserved only within the Apicomplexans and their function remains to be determined. One of these parasite-specific bromodomain proteins is a homolog of the P. falciparum bromodomain protein PfBDP1, which forms a complex with the AP2 factor AP2-I to regulate expression of a subset of P. falciparum genes required for invasion (Josling et al., 2015; Santos et al., 2017). The function of TgBDP1 is still unknown, but it is likely that it also forms regulatory complexes with T. gondii AP2s to enhance expression of particular subsets of parasite genes. Treatment with the bromodomain domain inhibitor IBET151 completely inhibits tachyzoite replication, indicating that at least one of these bromodomain-containing proteins is essential for parasite viability (Jeffers et al., 2017a). The chromodomain protein TgChromo1 binds H3K9me3 and localizes to centromeres, as well as telomeres in T. gondii (Gissot et al., 2012) in RH strain. In a ChIP-chip study, only centromeres were demonstrated to be enriched in H3K9me2/3 (Brooks et al., 2011), but this may be artifact of the ChIP-chip technique, which masks repetitive regions of the genome such as telomeric regions because of potential for cross hybridization. Intriguingly, in Pru, a type II strain able to transition to bradyzoite forms, H3K9me3 was also reported to localize to “poised” regions of the genome that could be activated in response to signals (Sindikubwabo et al., 2017; see Fig. 21.2 for a model). Originally described in pluripotent

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stem cells (Bernstein et al., 2006), poised genes are those that have both gene activation and gene repression chromatin marks but are not transcribed, presumably because they are “poised” in cells awaiting activation signals. The studies with Pru were performed using ChIP-seq, and further work comparing strains using comparable techniques is needed. However, current data suggest that differences in differentiation competence of different T. gondii strains mirrors the complement of histone modifications seen on “poised” genes. In the following sections, we present a summary of histone modifications and discuss what is known about their occurrence in Toxoplasma.

21.3.2 Mapping the Toxoplasma epigenome Genome-wide approaches have been used to illuminate the chromatin modifications associated with gene activation as well as define functional regions of the T. gondii genome (Fig. 21.4). The major technique used has been ChIP(Chromatin Immunoprecipitation), which enriches the DNA
protein complexes of interest, followed by either ChIP-chip [hybridization of the enriched DNA to genome-wide arrays (Gissot et al., 2007)] or ChIP-seq high throughput sequencing (Nardelli et al., 2015). Because T. gondii histones and histone modifications seen in model organisms are conserved (see Fig. 21.3), commercial antibodies specific for histone modifications can be used for this technique. In other species, direct modification of DNA, primarily cytosine methylation, also affects the accessibility of macromolecular complexes to chromatin, but it appears that T. gondii DNA is not cytosine methylated (Gissot et al., 2008). More recent studies in P. falciparum suggest that modification of cytosine exists at very low levels, but the functional significance of these new modifications is not yet established (Ponts et al., 2013).

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FIGURE 21.4 Chromatin modifications that define the epigenome of Toxoplasma gondii. Genome-wide ChIP microarray hybridization studies (ChIP-chip) define the epigenome of T. gondii. Chromatin was harvested from intracellular tachyzoites and ChIPs were performed with antibodies specific for the indicated histone posttranslational modification. Enriched DNA was hybridized to a custom Nimblegen Toxoplasma genome tiled microarray and the results of hybridization are shown as log2 ratios of signal to input control DNA. cDNA was harvested in parallel and hybridized to the chip. H3K9ac, H3K4me3 are enriched at sites of active promoters, whereas H3K4me1 colocalizes with gene bodies of actively transcribed genes (cDNA track). Genes and exons are indicated with boxes above the baseline indicating genes predicted to be on the positive strand and boxes below the baseline indicating genes transcribed on the negative strand. The specialized centromeric histone CenH3 marks a gene-poor region of the chromosome and localizes to each chromosomal centromere with H3K9me2. ChIP, Chromatin immunoprecipitation.

21.3.2.1 Chromatin signatures in Toxoplasma biology ChIP with antibodies to either acetylated H3 or H4 (H3ac or H4ac) revealed that relative acetylation versus deacetylation is correlated with specific gene activation or repression in Toxoplasma, respectively (Saksouk et al., 2005; Gissot et al., 2007). This was shown in the context of stage conversion to latent bradyzoites, demonstrating that in parasites cultured as tachyzoites, H3 and H4 were acetylated at

tachyzoite-specific promoters such as SAG1 and SAG2A, while no acetylation is detected at bradyzoite-specific promoters such as LDH2 and BAG1 (Saksouk et al., 2005). Conversely, in a parasite population induced to enter the bradyzoite pathway, acetylation at tachyzoite promoters was diminished, while acetylation at bradyzoite promoters increased. As expected, intergenic regions upstream of constitutively expressed genes were found in the acetylated state in either population in vitro.

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Confirmation of the differential state of histone acetylation that is associated with specific remodeling enzymes was also observed in parasite transgenic lines expressing epitopetagged TgGCN5-A and TgHDAC3 proteins. Lysine acetyltransferase (KAT) TgGCN5-A was present at tachyzoite promoters in tachyzoites, but absent at bradyzoite promoters, whereas TgHDAC3 was associated with promoters that were downregulated in each respective developmental stage (Saksouk et al., 2005). Studies have also shown that the TgCARM1-mediated methylation of H3R17 is another signature of gene expression in Toxoplasma, with the presence of this protein at active genes in either the tachyzoite or bradyzoite stage (Saksouk et al., 2005). Interestingly, genes marked with methylated H3R17 also displayed enrichment of acetylated H3K18, a potentially synergistic signature for gene activation that relies on crosstalk between acetyland methyltransferase complexes. ChIP and mass spectrometry have shown that H3K4 can be mono-, di-, or trimethylated in Toxoplasma (Nardelli et al., 2013). Trimethylated H3K4 is enriched at tachyzoite promoters during the tachyzoite stage (Gissot et al., 2007; Fig. 21.4) and becomes enriched at bradyzoite promoters following differentiation, representing another mark of gene activation in this parasite (Saksouk et al., 2005). Genomewide ChIP studies have established that acetylation of H3K9, H4, and trimethylation of H3K4 occurs at promoters of actively expressed genes (Gissot et al., 2007). Trimethylation of H3K9 and H4K20 occurs at repressed genes in heterochromatic territories (Sautel et al., 2007), with the H3K9me2/3 marks enriched in centromeres (Brooks et al., 2011). Monomethylation of H3K4 and H4K31 is associated with gene bodies of actively transcribed genes (Sindikubwabo et al., 2017; Fig. 21.4). In contrast to Plasmodium, T. gondii does not encode major antigenic variant gene families whose silencing is associated with

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deposition of the heterochromatic histone marks H3K9me2/3 (Lopez-Rubio et al., 2007, 2009). The regions of the genome that encode these virulence genes are clustered in the nucleus of P. falciparum but clustering of virulence genes is not observed in T. gondii (Bunnik et al., 2019). In RH strain, a strain that does not develop to bradyzoites in culture, H3K9me3 was enriched only at centromeres and centromeres clustered in an apical region of the nucleus (Brooks et al., 2011). HiC analysis confirmed clustering of centromeres (Bunnik et al., 2019). In Pru, a developmentally competent strain, H3K9me3 was enriched at developmentally regulated genes (Sindikubwabo et al., 2017), suggesting there is strain variability in the histone modifications that may reflect biological competence for developmental transitions (see the previous discussion on poised genes). The histone modifications of some residues may be particularly important and act as a critical point of regulation of chromatin state. H3K9ac is a gene activation mark, whereas H3K9me3 may mark silenced genes. H4K31 can be modified by methylation, acetylation, succinylation, and ubiquitination (Sindikubwabo et al., 2017; Silmon de Monerri et al., 2015). H4K31 is on the lateral surface of H4, and H4K31ac localizes to promoters, whereas H4K31me localizes to gene bodies but inversely correlates with transcription (Sindikubwabo et al., 2017). As observed in other species, phosphorylation of TgH2AX has been linked to the parasite DNA damage response. H2AX is phosphorylated in response to double-stranded breaks at its C-terminal SQ(E/D)ϕ motif (ϕ denoting a hydrophobic residue) (Escargueil et al., 2008). Treatment of Toxoplasma with DNA-damaging agents methyl methanesulfonate or H2O2 led to increased phosphorylation of TgH2AX, as detected by immunoblotting with monoclonal antibody (Vonlaufen et al., 2010; Dalmasso et al., 2009). These studies indicate that phosphorylated TgH2AX can be used as a

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chromatin biomarker for DNA injury. Phosphorylation of H3S10 has also been reported in Toxoplasma, a mark that peaks during mitosis with monomethylation of H4K20 (Sautel et al., 2007). The function of H3S10 phosphorylation has been linked to chromosome condensation in fellow alveolate protozoan Tetrahymena (Wei et al., 1998).

21.3.3 Histone-modifying enzymes Chromatin remodelers generally fall into two distinct classes: those capable of covalently modifying histones or those that use ATP to reposition nucleosomes (SWI2/SNF2 family ATPases). Both types of chromatin remodeling machinery can be found in Toxoplasma and other protozoa as well (Dixon et al., 2010). Apicomplexan histones are subject to a wide variety of covalent modifications that include acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation. In the following subsections, we will discuss histone-modifying enzymes found in Toxoplasma and what we have learned to date about their role in parasite biology. 21.3.3.1 Histone acetylation In other eukaryotes, acetylation of lysine residues in the N-terminal histone tails is linked to gene activation. Conversely, the removal of acetyl groups is associated with transcriptional repression. A wide variety of HATs (histone acetyltransferases) and HDACs have been characterized among eukaryotes that control the acetylation status of nucleosomal histones and hence play an important role in the regulation of gene expression (Sterner and Berger, 2000; Thiagalingam et al., 2003). The Toxoplasma genome predicts at least seven HATs and seven HDACs present in the parasite. Proteins historically referred to as HATs and HDACs have been renamed KATs and KDACs (lysine acetyltransferases and

deacetylases, respectively) since many of them also act on nonhistone substrates (Allis et al., 2007). Given the discovery of widespread lysine acetylation in nonnuclear compartments of Toxoplasma, we propose this change in nomenclature be adopted for the parasite (Jeffers and Sullivan, 2012). Two MYST family KAT proteins (MOZ, Ybf2/Sas3, Sas2, Tip60) exist in Toxoplasma, each possessing a chromodomain and the atypical C2HC zinc finger domain upstream of the KAT domain (Smith et al., 2005; Vonlaufen et al., 2010). The predicted proteins, named TgMYST-A (TGME49_318330) and -B (TGME49_207080), have features consistent with the “MYST 1 CHD” subclass, homologous to yeast Esa1, human Tip60, and MOF (Utley and Cote, 2003). Previous studies demonstrate that this type of KAT has a preference for acetylating lysines in H4 and the observation that recombinant TgMYSTs also prefer H4 as substrate in assays using free core histones functionally validates this classification (Smith et al., 2005). Given this similarity, it was not surprising that genes encoding TgMYST-A and -B could not be disrupted by homologous recombination as the Esa1 homolog in yeast is also an essential gene (Smith et al., 1998). TgMYST-A is not amendable to stable overexpression unless the recombinant protein is mutated to nullify its HAT activity, suggesting a delicate balance of TgMYST-Amediated acetylation exists in Toxoplasma (Smith et al., 2005). Despite their histone acetylation abilities, both TgMYST KATs are predominantly cytoplasmic, suggesting they may act on nonhistone substrates (Jeffers and Sullivan, 2012). Overexpression of TgMYST-B is tolerated, but results in a significantly reduced proliferation rate unless enzymatic activity is ablated (Vonlaufen et al., 2010). Interestingly, the delayed replication may be connected to the dramatic resistance to DNA-damage observed for TgMYST-B overexpressing parasites.

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Consistent with heightened protection from DNA-damaging agents, parasites overexpressing TgMYST-B have increased levels of ataxia telangiectasia mutated (ATM) kinase and phosphorylated H2AX (Vonlaufen et al., 2010). Increased γH2AX leads to cell cycle arrest and a decrease in the number of cells in mitosis, likely explaining why parasites overexpressing TgMYST-B exhibit delayed replication (Vonlaufen et al., 2010; Rios-Doria et al., 2009). The connection between TgMYST-B and the ATM kinase
mediated DNA damage response was further supported when pharmacological inhibitors of ATM kinase or KATs rescued parasites overexpressing TgMYST-B from slowed replication (Vonlaufen et al., 2010). Two KAT proteins of the GCN5-class also exist in Toxoplasma designated TgGCN5-A (TGME49_254555) and TgGCN5-B (TGME49_243440), which is a highly unusual arrangement in a lower eukaryote. Aside from the close relative Neospora caninum, the presence of two GCN5 KATs in a single cell has not been documented for any other invertebrate. In contrast, mammalian species have two GCN5 KAT enzymes referred to as GCN5 and PCAF (p300/CBP associating factor). Deletion of mouse GCN5 is embryonic lethal, while the loss of PCAF has no discernible phenotype (Xu et al., 2000; Yamauchi et al., 2000). There is a striking parallel in Toxoplasma, in which TgGCN5-A is dispensable in tachyzoites yet TgGCN5-B appears to be essential (Bhatti et al., 2006; Sidik et al., 2016). The two TgGCN5s differ in other ways as well. GCN5 family members show a strong preference to acetylate H3, particularly lysine 14 (K14). Recombinant TgGCN5-A was found to have an exquisite selectivity to acetylate H3K18, whereas TgGCN5-B was more prototypical and capable of targeting H3K9, H3K14, and H3K18 in vitro (Bhatti et al., 2006; Saksouk et al., 2005). Another difference between the TgGCN5 KATs is their ability to bind with the ADA2 coactivator, for which two homologs

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have been identified in the Toxoplasma (TgADA2-A, DQ112184; TgADA2-B, DQ112185). By yeast two-hybrid assay, TgGCN5-B has been shown to interact with either TgADA2 homolog, while TgGCN5-A can only associate with TgADA2-B (Bhatti et al., 2006). It has been proposed that TgGCN5-B may be required for tachyzoite replication and TgGCN5-A may be required only for specific circumstances, such as the stress response. Studies of the TgGCN5-A knockout showed a significant recovery defect following exposure to alkaline pH stress, a condition commonly used to induce bradyzoite development in vitro. Microarray analyses revealed that parasites lacking TgGCN5-A fail to upregulate B75% of the genes normally induced during alkaline stress, including bradyzoite-specific induction markers BAG1 and LDH2 (Naguleswaran et al., 2010). While repeated attempts to knockout TgGCN5-B have failed, even in Δku80 parasites, an inducible dominant-negative strategy supports that TgGCN5-B is essential in tachyzoites (Wang et al., 2014). Furthermore, garcinol, which inhibits TgGCN5-B, prevents replication of tachyzoites as well as P. falciparum asexual replication, providing pharmacological evidence that GCN5 KATs are critical for parasite viability (Jeffers et al., 2016). A ChIP-chip analysis was performed to determine the genome-wide localization of TgGCN5-B in tachyzoites. TgGCN5-B was found at over 1000 genes; the highest confidence genes coinciding with TgGCN5-B localization were associated with gene expression and RNA processing, as well as metabolic genes, rather than virulence genes (Wang et al., 2014). The majority of the genes most significantly affected by garcinol were genes found to be under TgGCN5-B control as determined in the aforementioned ChIP-chip study (Jeffers et al., 2016). In each study the genes linked to TgGCN5-B do not belong to a single cellular

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pathway but associate with a wide variety of cellular functions. These findings suggest that multiple gene networks are modulated by TgGCN5-B. Recruitment of the TgGCN5-B complex to these gene networks appears to involve specific TgAP2 proteins, including AP2IX-7 and AP2X-8 (Wang et al., 2014). The TgGCN5-B complex may associate with different TgAP2s under different conditions, but this has yet to be determined experimentally. Apicomplexan GCN5 KATs contain an unusual N-terminal extension upstream of the well-conserved catalytic and bromodomains. Curiously, the length and amino acid composition vary greatly among the Apicomplexa, and even among the pair of GCN5s in Toxoplasma. Most GCN5s from early eukaryotes do not have appreciable sequence upstream of the KAT domain. In contrast, mammalian GCN5 and PCAF have N-terminal extensions, but they are very similar to each other. The function of the N-terminal extension may be to mediate protein
protein interactions (e.g., the binding of CBP) and/or substrate recognition, as GCN5 lacking the N-terminal extension can only acetylate free histones and not nucleosomal histones (Xu et al., 1998). The N-terminal extensions of TgGCN5-A and -B are required for nuclear localization, but dispensable for enzyme activity on free histones (Bhatti and Sullivan, 2005; Dixon et al., 2011). A six amino acid, basic-rich motif in the N-terminal extension of TgGCN5-A has been mapped as a necessary and sufficient nuclear localization signal (NLS) that interacts with the nuclear chaperone importin alpha (Bhatti and Sullivan, 2005). The NLS for TgGCN5-B is also rich in basic residues and found in the N-terminal extension, although its sequence is distinct from the NLS of TgGCN5-A (Dixon et al., 2011). Previous work has also noted that KDAC proteins exist in Plasmodium (Joshi et al., 1999; Freitas-Junior et al., 2005) and analysis of Toxoplasma genomic sequence indicates there are seven potential KDAC genes with one

experimentally characterized to date (TgHDAC3). Recombinant TgHDAC3 exhibits HDAC activity that is inhibited by butyrate, aroyl-pyrrole-hydroxy-amides, and trichostatin A and a native TgHDAC3-containing complex has been purified (TgCRC for CoRepressor Complex) (Saksouk et al., 2005). The TgCRC contains several protein components that are homologous to subunits found in the human N-CoR and SMRT complexes, as well as two large parasite-specific proteins of unknown function (Saksouk et al., 2005). Pull-down studies suggest HDAC3 interacts with both TgAgo1 (Braun et al., 2010) as well as TgAP2 (Saksouk et al., 2005), supporting a critical role of TgHDAC3 in gene regulation in T. gondii. With the exception of TgSIR2a, TgKDACs have been refractory to disruption, suggesting that they have essential functions in the biology of T. gondii. The genome-wide CRISPR/Cas9 screen also suggested that the five type I HDACs are essential for tachyzoite proliferation, but the type III HDACs SIR2 and SIR2b are not (Sidik et al., 2016). 21.3.3.2 Histone methylation The addition of methyl groups occurs on lysine and arginine residues of histones and can lead to gene activation or silencing (Zhang and Reinberg, 2001). There is an added layer of complexity in methyl-modifications as residues can be mono-, di-, or trimethylated. Toxoplasma possesses five protein arginine methyltransferase (PRMT) homologs, designated TgPRMT1-5. Recombinant TgPRMT1 (TGME49_219520) is capable of methylating H4R3, while TgPRMT4 (referred to as TgCARM1, coactivator associated arginine methyltransferase, TGME49_294270) methylates H3R17 (Saksouk et al., 2005), which parallels the substrate specificity of their human homologs. The importance of TgPRMT1 for histone methylation is not yet clear, as the phenotype of parasites lacking TgPRMT1 is a cell cycle phenotype that affects daughter cell counting (El Bissati et al., 2016). Proteomics analysis

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revealed that PRMT1 is the major R-methyltransferase in T. gondii but that most of its substrates are nonhistone proteins (Yakubu et al., 2017) (see Chapter 22: Proteomics and posttranslational protein modifications in Toxoplasma gondii). Human CARM1 has been associated with SWI2/SNF2 ATPases, including the Snf2Related CBP Activator Protein, or SRCAP (Xu et al., 2004; Monroy et al., 2003). Recombinant TgCARM1 incubated with parasite extract enriched for ATP-dependent nucleosome disruption activity indicated that TgCARM1 is likely to interact with a Toxoplasma SWI2/SNF2 member (Saksouk et al., 2005). An SRCAP SWI2/SNF2 homolog has been characterized in Toxoplasma (see the section below and Sullivan et al., 2003). This is the only Ino80 family member in T. gondi. It is predicted to exchange H2A and H2A.Z in nucleosomes and was essential in the CRISPR screen (Sidik et al., 2016). Together, these data suggest a conserved connection between CARM1 and SRCAP complexes. Lysine methyltransferases share a common feature known as the SET (Suv(39)-E(z)-TRX) domain (Dillon et al., 2005). The Toxoplasma genome reveals at least 19 candidates and a surprising amount of duplication and divergence within this protein family (Bougdour et al., 2010; Sivagurunathan et al., 2013). As has been observed with acetyl transferases, novel nonhistone substrates for methyltransferases have been described in many systems including T. gondii. Commercial methyl lysine antibodies often cross react with cytosolic or cytoskeletal structures in T. gondii (Sautel et al., 2007; Xiao et al., 2010), and one SET protein, AKMT or apical lysine methyltransferase, is implicated in methylation of apical cytoskeletal structures (Heaslip et al., 2011). In Plasmodium a PfSET10, a methylase with H3K4me specificity, has been implicated in maintaining the active var gene in a poised state during the cell cycle (Volz et al., 2012). It is likely that some of the TgSET will have analogous functions in

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lysine methylation of histones, particularly since histone lysine methylation is a common histone posttranslational modification (Nardelli et al., 2013; Fig. 21.3). Although bioinformatics studies have inferred specificity of these SET (Sivagurunathan et al., 2013), in most cases, these predictions have not been validated experimentally. ChIP-chip studies localize KMTox (formerly TgSET13) to genes related to antioxidant defenses, heat-shock proteins/chaperones, and genes involved in translation and carbohydrate metabolism. KMTox also protects against H2O2 exposure and was found to associate with 2cys peroxiredoxin-1 (TgPrx1) under oxidative conditions, bolstering the idea that KMTox contributes to the parasite’s antioxidant defense system (Sautel et al., 2009). Unlike its monomethylating human counterpart, biochemical and structural modeling analyses show that TgSET8 is capable of mono-, di-, and trimethylation of H4K20 (Sautel et al., 2007). Tachyzoites expressing a mutated version of TgSET8 (F1808Y) that abolishes the monomethylation of H4K20 are not able to progress through the cell cycle, suggesting that monomethyl H4K20 is required for parasite division (Bougdour et al., 2010). TgSET8 may also play important roles during the latent cyst stage, as suggested by high levels of monomethylated H4K20 in bradyzoites (Sautel et al., 2007). With regard to the removal of methyl groups, Toxoplasma appears to encode seven JmjC (Jumonji) domain
demethylating proteins. Only one has both the Jumonji N and C domains characteristic of JARID-like H3K4 and JMJD1-like H3K9 demethylases. The other members of this family belong to the JMJD6 family that demethylates H3R2 and H4R3 (Bougdour et al., 2010). Subsequent studies have questioned the exclusive role of the JmjC (Jumonji) domain proteins in protein demethylation—with new functions reported in RNA processing (Hong et al., 2010), so further studies will be needed to determine the function of

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these putative demethylases. This atypical expansion of demethylases in Toxoplasma may counter the extensive number of methyltransferases and the expansion of the methyltransferase and demethylase families is an aspect of T. gondii biology that differs from Plasmodium. Toxoplasma also appears to two encode homologs of lysine-specific demethyltransferases. The function of these proteins has not been characterized, although CRISPR screen suggested that both are important for tachyzoite fitness (Sidik et al., 2016). 21.3.3.3 Other histone covalent modifications Considerably less work has been done to dissect the roles of histone phosphorylation, ADP-ribosylation, and ubiquitylation in Toxoplasma. As mentioned, H3S10 phosphorylation has been reported, and Toxoplasma possesses two predicted proteins with strong similarity to histone kinase Snf1. Toxoplasma also appears to possess proteins containing PARP and PARG domains, required for the addition or removal (respectively) of ADPribose subunits (Dixon et al., 2010). There is also no shortage of ubiquitin-conjugating enzymes in this organism, including Ubc9, which is implicated in gene repression via the SUMOylation of H4 (Shiio and Eisenman, 2003). Histone ubiquitination has been confirmed by mass spectrometry (Nardelli et al., 2013; Silmon de Monerri et al., 2015). Ubiquitination of histones is substoichiometric (Silmon de Monerri et al., 2015) suggesting that these modifications are dynamic. A small ubiquitin-like modifier (SUMO)
conjugating system has been characterized in Toxoplasma (Braun et al., 2009), and SUMOylation has been reported on Plasmodium H2A and H2AZ. While SUMOylation of T. gondii histones is detectable by immunoblot (Nardelli et al., 2013), the exact modified residues have not yet been mapped. A number of novel histone modifications of unknown function have been

reported in the metazoa including propionylation and succinylation. These modifications are also present on T. gondii histones, and based upon conjectures in other organisms, these histone modifications may provide a mechanism by which changes in metabolism are sensed and can impact epigenetic gene regulation. In summary, previous reports coupled with bioinformatic analyses of the completed genome demonstrate that Toxoplasma is capable of mediating most known histone modifications. The extensive array of chromatin remodeling machinery suggests that histone modifications and epigenetics are likely to be instrumental during progression of the parasite life cycle. These observations underscore the antiquity of epigenetics in the evolution of the eukaryotic cell and indicate that much of this machinery has evolved along parasite-specific trajectories. 21.3.3.4 SWI2/SNF2 ATPases The second broad class of chromatin remodeling complexes in eukaryotes comprises the SWI2/SNF2 DNA-dependent ATPases that have roles in both transcriptional repression as well as activation (Mohrmann and Verrijzer, 2005). While the mechanism of action of these factors is incompletely understood, it is believed that the energy of ATP hydrolysis is used to reposition or relocate the nucleosome (Johnson et al., 2005). All members of the SWI2/SNF2 family contain a distinctive ATPase domain consisting of an N-terminal DEXDc portion and a C-terminal HELICc portion. Further classification based on sequence homology and additional structural features leads to four separate types: Snf2 members (contain a bromodomain), ISWI (contain a SANT domain), Mi-2 (contain a chromodomain), and Ino80/SRCAP/p400 (contain a lengthy insert between the DEXDc and HELICc domains). Previous reports have described SWI2/SNF2 factors in Apicomplexa, including an ISWI homolog in Plasmodium and

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an SRCAP homolog in Toxoplasma (TgSRCAP, AAL29689), Cryptosporidium, and Plasmodium (Ji and Arnot, 1997; Sullivan et al., 2003). Only TgSRCAP has been studied in any great detail. Like human SRCAP, TgSRCAP can function to enhance CREB-mediated transcription (CREB 5 cAMP response elementbinding protein) in the presence of the HAT CBP in vitro (Sullivan et al., 2003). Although cAMP-dependent gene expression is implicated in regulation of the tachyzoite-bradyzoite transition (Sugi et al., 2016), proteins with similarity to CBP or CREB are not present in Apicomplexa; therefore the role of TgSRCAP in Toxoplasma remains to be elucidated. SRCAP is important for exchange of H2A for H2A.Z in most species. To facilitate a better understanding of what TgSRCAP may do, a yeast two-hybrid screen was conducted using the lengthy “spacer” region separating the DEXDc and HELICc domains as “bait” (Nallani and Sullivan, 2005). The corresponding region in human SRCAP binds CBP (Johnston et al., 1999). Most of the strongest interacting proteins isolated and confirmed by in vitro coimmunoprecipitation are novel parasite-specific proteins having no homologs in other eukaryotes. A few of these are from genes predicted to encode domains suggestive of a role in DNA processes, including transcription. Of particular interest is the first protein described in Toxoplasma to contain Kelch repeats and a BTB/POZ domain (Nallani and Sullivan, 2005). POZ domains from several zinc finger proteins have been shown to mediate transcriptional repression and to interact with components of HDAC corepressor complexes. The gene was cloned (TGME49_298600) and termed TgLZTR since it is most similar to human Leucine-Zipper-like Transcriptional Regulator, a gene deleted in people with DiGeorge syndrome (Kurahashi et al., 1995). Future studies should elucidate the role of TgLZTR and whether it associates with TgSRCAP in vivo.

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In addition to TgSRCAP, the Toxoplasma genome contains at least 17 possible SWI2/ SNF2 homologs. Two bear high sequence similarity to Snf2 subclass members, with one harboring a bromodomain downstream of the ATPase domains. Another SWI2/SNF2 protein has a SANT domain, making it a likely ortholog of ISWI. A second SWI2/SNF2 has strong ISWI homology, but possesses an AT-hook domain instead of a SANT domain. There is also a predicted SWI2/SNF2 family member in Toxoplasma that contains a chromodomain, making it a probable Mi-2 ortholog. This protein was identified as part of the TgCRC (Saksouk et al., 2005). How this extensive family of SWI2/SNF2 ATPases contributes to gene expression in parasites remains an open area for future investigation.

21.3.4 Epigenetic mechanisms as drug targets Chromatin remodeling plays critical roles in various aspects of parasite physiology, prompting discussions about targeting this machinery for novel drug design. Histone acetylation has been validated as a drug target as early as 1996, when it was discovered that a fungal metabolite (now called apicidin) with potent antiprotozoal activity inhibited apicomplexan KDACs (Darkin-Rattray et al., 1996). Given the conservation of histone-modifying enzymes in human, to be viable drugs KDAC inhibitors must have selective toxicity arise. Selective toxicity may be achievable simply because the parasites replicate rapidly, so therapeutic levels of drug may have minimal effect on host cells. Second, parasite chromatin remodeling enzymes have significant divergent sequence outside of the catalytic domain that could be targeted selectively by small molecule inhibitors. Small divergence within catalytic domains can be sufficient to achieve selective toxicity. Apicomplexan HDAC3 is more susceptible to

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the KDAC inhibitor FR235222 due to the presence of a unique 2-amino acid insertion in the catalytic domain (Bougdour et al., 2009). In contrast to KDAC inhibitors, surprisingly few specific KAT inhibitors are available. Anacardic acid and curcumin inhibit Plasmodium replication and can inhibit PfGCN5 in vitro, but these compounds are believed to have many off-target effects (Cui et al., 2007, 2008). The natural product garcinol was shown to inhibit TgGCN5-B in vitro and disrupts the expression of genes known to be under the control of TgGCN5-B in vivo (Jeffers et al., 2016). Garincol inhibits replication of asexual stages of both Toxoplasma and Plasmodium, supporting the idea that GCN5 KATs are promising drug targets (Jeffers et al., 2016). As epigenetic mechanisms contribute to bradyzoite conversion, it may be possible to shortcircuit this important pathogenic process with small molecules. Tachyzoites treated with the KDAC inhibitor FR235222 convert into bradyzoites, and this compound may have activity against ex vivo Toxoplasma tissue cysts (Bougdour et al., 2009; Maubon et al., 2010). Genetic studies suggest that pharmacological interference of TgGCN5-A might be able to thwart bradyzoite cyst gene expression (Naguleswaran et al., 2010). Interference with parasite histone methylation may also disrupt control of differentiation, and inhibitors specific for Plasmodium methyltransferases are reported to alter expression of variant genes (Malmquist et al., 2012). While the reasons remain unclear, pretreating tachyzoites with a CARM1 (PRMT4) inhibitor leads to a higher frequency of bradyzoite development upon infecting host cells in vitro (Saksouk et al., 2005). An important consideration with respect to targeting histone modification enzymes is the recent set of studies demonstrating the abundance of nonhistone substrates for these enzymes (Smith et al., 2005). Several Toxoplasma KATs, namely, the MYST KATs,

are found predominantly outside of the parasite nucleus. Curiously, the Toxoplasma homolog of the elongator KAT Elp3 resides at the outer mitochondrion membrane as a tailanchored protein (Stilger and Sullivan, 2013). An acetylome has been published for Toxoplasma tachyzoites, revealing that lysine acetylation is widespread across proteins of diverse function and location within the parasite (Jeffers and Sullivan, 2012). The activity of KAT and KDAC inhibitors, therefore, may not be limited to dysregulation of gene expression but could exert their antiparasitic effect through interfering with acetylation of nonhistone substrates (Jeffers and Sullivan, 2012). While less extensively studied than lysine acetylation, methylation of T. gondii proteins of diverse function occurs on both lysines and arginines (Heaslip et al., 2011; Xiao et al., 2010; Yakubu et al., 2017) and inhibitors of these enzymes may also have specific antiparasitic activities. Inhibitors of Plasmodium lysine methyltransferases block growth of erythrocytic stages (Malmquist et al., 2012) and sublethal doses of these compounds promoted activation of quiescent Plasmodium cynomolgi hypnozoite forms (Dembele et al., 2014), implicating epigenetic mechanisms in hypnozoite formation. Finally, T. gondii histones and other proteins implicated in gene expression are extensively ubiquitinated (Silmon de Monerri et al., 2015) suggesting that ubiquitination may also be important modifications that could be targeted by novel inhibitors.

21.4 Posttranscriptional mechanisms in Toxoplasma 21.4.1 Translational control Examples of posttranscriptional mechanisms that regulate the level of specific proteins among protozoa have been described (Rochette et al., 2005; Larreta et al., 2004; Chow and

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Wirth, 2003; Garcia-Salcedo et al., 2002; Shapira et al., 2001). Transcription in the kinetoplastidae such Leishmania and Trypanosoma is polycistronic and posttranscriptional trans-splicing mechanisms are required to achieve mature mRNA (Campbell et al., 2003; Shapira et al., 2001). Thus in Leishmania and Trypanosoma species, mRNA levels are dictated mostly by posttranscriptional processing and the stability of the mRNA itself (Purdy et al., 2005a,b; Webb et al., 2005a,b; Cevallos et al., 2005; Haile et al., 2003). RNA-binding proteins with demonstrated roles in the regulation of translation and/or RNA stability have been found in Plasmodium, including Puf2 (Miao et al., 2010; Muller et al., 2011; Gomes-Santos et al., 2011), the DDX6-class RNA helicase, DOZI (development of zygote inhibited) (Mair et al., 2006), and Alba proteins (Chene et al., 2012; Goyal et al., 2012; Munoz et al., 2017). Other transcription-associated proteins with known roles in modulating mRNA decay and translation were also found in the genome (Coulson et al., 2004). The formation of stress-induced RNA granules has been reported for a number of parasite species, including Toxoplasma (Lirussi and Matrajt, 2011) and Plasmodium (Hanson and Mair, 2014; Bennink et al., 2018). Such RNA granules are proposed to be holding areas for translationally regulated mRNAs. As such, indications of posttranscriptional control have been described for protozoal genes with defined roles in differentiation (Vervenne et al., 1994; Dechering et al., 1997; Sullivan et al., 2004; Narasimhan et al., 2008; Miao et al., 2010), mitochondrial RNA processing (Rehkopf et al., 2000), and surface antigens (Lanzer et al., 1993; Levitt et al., 1993; Spano et al., 2002). In Toxoplasma, unbalanced ratios of mRNA and protein have been observed for SAG-related Toxoplasma surface proteins, designated SAG5A, SAG5B, and SAG5C (Spano et al., 2002), and for mRNAs encoding the proliferating cell nuclear antigens, TgPCNA1 and TgPCNA2 (Guerini et al., 2000). TgPCNA1

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mRNA was found to be sevenfold higher than that of TgPCNA2, yet TgPCNA 1 and 2 on Western blots were expressed at nearly equally levels in all strains examined (Guerini et al., 2000). In the context of stage-specific gene expression in Toxoplasma, mRNAs encoding bradyzoite-specific proteins G6-PI and MAG1 can also be detected during the tachyzoite stage, and mRNA encoding tachyzoite-specific proteins like LDH1 can also be detected in bradyzoites (Yang and Parmley, 1997; Dzierszinski et al., 2001; Weiss and Kim, 2000). These discrepancies in the levels of mRNA and protein indicate that posttranscriptional events occur in apicomplexan parasites. As in other eukaryotes, translational control through the phosphorylation of eukaryotic initiation factor-2 alpha subunit (eIF2α) is an important facet of gene regulation in Apicomplexa (Holmes et al., 2017). The eIF2 complex governs the rate-limiting step in the initiation of protein synthesis. In response to a wide variety of cellular stresses or developmental signals, eIF2α becomes phosphorylated, leading to a global cessation in translation except for a subset of mRNAs encoding transcription factors that reprogram the expressed genome to enable cell survival or differentiation mechanisms (Wek et al., 2006). The eIF2α translational control pathway was first characterized in Toxoplasma and linked to stress-induced bradyzoite development; moreover, TgIF2α remains in its phosphorylated stage during the latent cyst stage (Sullivan et al., 2004; Narasimhan et al., 2008). Inhibitors of TgIF2α dephosphorylation, such as salubrinal and guanabenz, trigger bradyzoite differentiation by inflating TgIF2 phosphorylation, supporting the idea that translational control is a major contributor to the development and maintenance of microbial latency (Joyce et al., 2011; Narasimhan et al., 2008; Konrad et al., 2013). Moreover, it appears that sustained TgIF2α phosphorylation is toxic to bradyzoite tissue cysts. In vitro generated

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tissue cysts appear to die in the presence of increasing concentrations of salubrinal or guanabenz (Benmerzouga et al., 2015; Konrad et al., 2013). Strikingly, guanabenz is also capable of significantly reducing brain cyst burden (a decrease of 70%
80%) in BALB/c mice with latent toxoplasmosis (Benmerzouga et al., 2015). The ability of guanabenz to significantly diminish cyst counts in vivo is particularly exciting as this drug, which readily crosses the blood
brain barrier, is already FDA-approved and could be rapidly repurposed as an antiparasitic. Studies in Plasmodium as well as kinetoplastid parasites and Entamoeba lend further support to the idea that TgIF2α phosphorylation is a crucial determinant of microbial latency (Zhang et al., 2010; Chow et al., 2011; Hendrick et al., 2016). Translational control through the phosphorylation of parasite eIF2α has significant roles beyond the modulation of latency and appears to be required even for normal progression through lytic cycles. Allelic replacement of the endogenous TgIF2α gene with a version incapable of being phosphorylated on the regulatory serine residue (Ser-71) exhibits fitness defects in vitro and in vivo that have been linked to viability following egress from its host cell (Joyce et al., 2010). A similar allelic replacement produces nonviable parasites in Plasmodium, demonstrating that PfIF2α phosphorylation is essential during the erythrocytic cycle (Zhang et al., 2012). The phosphorylation of eIF2α is also central to the development of drug-induced latency in apicomplexan parasites, which may provide a means to improve current therapeutic approaches. For example, pharmacological inhibition of eIF2α phosphorylation is able to prevent artemisinin-induced latency and therefore stop treatment failures in a mouse model of malarial infection (Zhang et al., 2017). Four eIF2α kinases have been identified in the Toxoplasma genome, designated TgIF2K-A through -D. TgIF2K-A localizes to the parasite

endoplasmic reticulum (ER) and interacts with GRP/BiP in a stress-dependent manner, making it a likely ortholog of PERK, an eIF2α kinase in higher eukaryotes that contributes to the unfolded protein response (UPR) that allows cells to adapt to ER stress (Narasimhan et al., 2008). Given the link between UPR and autophagy, it is likely that TgIF2K-A contributes to autophagy as part of the parasite ER stress response (Nguyen et al., 2017). TgIF2K-A also shows promise as a drug target, as does its Plasmodium homolog, PK4 (Zhang et al., 2012). Each of these apicomplexan PERK-like eIF2 kinases is inhibited by the PERK inhibitor GSK2606414, which also displays activity against both of these parasites (Augusto et al., 2018; Zhang et al., 2017). TgIF2K-B appears to be a cytoplasmic eIF2α kinase that is specific to Toxoplasma and its function has yet to be resolved. TgIF2K-C and -D are two GCN2-like kinases, which in other species are wellcharacterized responders to nutritional stress. Using knockout strategies, it has been determined that TgIF2K-D is the primary kinase phosphorylating TgIF2α upon egress from the host cell, and its loss phenocopies the nonphosphorylatable TgIF2α mutant (Konrad et al., 2011). In contrast, TgIF2K-C appears to manage the amino acid starvation response in intracellular tachyzoites (Konrad et al., 2014). A single GCN2-like kinase in Plasmodium called PfeIK1 has also been found to regulate the parasite’s response to amino acid starvation (Fennell et al., 2009). A key area of current investigation is linking translational control to transcriptional control. In other eukaryotes, following eIF2α phosphorylation, a select group of mRNAs is preferentially translated due to the presence of upstream open reading frames in the 50 -UTR (untranslated region) (Vattem and Wek, 2004). These messages tend to encode basic-leucine zipper transcription factors such as GCN4/ ATF4, which activate genes that facilitate the cellular adaptive response. Such transcription

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21.4 Posttranscriptional mechanisms in Toxoplasma

factors are not present in Apicomplexa, which probably uses a subset of AP2 factors to regulate transcription. Employing the ability to generate polyribosome profiles in Toxoplasma (Narasimhan et al., 2008), it has been shown that mRNAs encoding TgAP2 factors along with chromatin remodeling machinery are preferentially translated during stress in tachyzoites (Joyce et al., 2013). More recently, ribosomal profiling verified a dominant role for translational control in Toxoplasma and suggested that mRNA translation in the parasite is regulated by mRNA secondary structure and upstream open reading frames (Hassan et al., 2017).

21.4.2 Noncoding and small RNA One of the most exciting discoveries over the past two decades has been the role of small RNAs and longer RNAs in regulation of gene expression. T. gondii small RNAs have been cataloged (Wang et al., 2012; Braun et al., 2010) and RNA-seq studies have also identified long noncoding RNA (lncRNA) (Hassan et al., 2012). Many parasitic species use small RNAs to regulate gene expression. Unlike Plasmodium species, T. gondii encodes the essential components of the RISC, including a single Argonaute protein (Ago1), a Dicer protein (with RNAseIII catalytic domains), and an RNA-dependent RNA polymerase, suggesting that RNA silencing pathway is fully functional (Braun et al., 2010; Al Riyahi et al., 2006). The single T. gondii Argonaute protein is not predicted to have slicer activity, and thus the RISC complex is proposed to regulate gene expression by translation repression rather than RNA degradation (Braun et al., 2010). The mechanistic details of the RISC complex activity are not yet completely clear because methylation of TgAgo1 results in recruitment of a staphylococcal endonuclease (Musiyenko et al., 2012) that alters the activity of the RNA slicing complex as well as changing the specificity of the slicing from mismatches to perfect match RNA targets.

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Characterization of the small RNAome of T. gondii by RNA-seq led to the identification of 14 miRNA families that were unique without significant homology to known miRNA of plants and metazoan, but most were conserved in Neospora (Braun et al., 2010; Reid et al., 2012). A second study identified 17 conserved miRNA and 339 novel miRNA (Wang et al., 2012). Many miRNAs are recruited to the Ago complex to affect RNA turnover or translation. Some of the miRNA families of T. gondii were associated with polysomes, consistent with a role in translational repression. In addition, several of the miRNA families were differentially expressed in the Type I, II, and III lineages and differences were also seen in extracellular versus actively replicating parasites (Wang et al., 2012; Braun et al., 2010). The targets of these miRNA have been predicted, but not yet validated (Braun et al., 2010). A second group of small RNAs matched the repetitive elements REP1-3, mitochondrial-like sequences dispersed throughout the T. gondii genome (Braun et al., 2010). The dispersal of these elements has features suggestive of transposon dissemination and one proposed function of the RNAi pathway in T. gondii is to prevent expression of these elements (Braun et al., 2010). A third class of repeat associated small RNAs was identified that are proposed to maintain heterochromatin state at satellite DNA within the nucleus (Braun et al., 2010). At present the single Ago protein is proposed to mediate all the potential nuclear and cytoplasmic activities of the T. gondii RISC complex. Some of the miRNA families were associated with immunopurified TgAgo, as were proteins associated with RNA processing and the chromatin corepressor complex (Braun et al., 2010). Despite reports of successful RNAi in T. gondii (Al-Anouti et al., 2003; Al Riyahi et al., 2006), widespread RNAi has not been documented and efforts to by several groups to develop RNAi methodology for T. gondii gene

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21. Regulation of gene expression in Toxoplasma gondii

knock-down have failed (Matrajt, 2010), and reports of successful RNAi has been limited a few select genes (Al-Anouti et al., 2003, 2004; Ananvoranich et al., 2006). These observations suggest that the RNAi pathway has a specialized biological function in T. gondii. Consistent with this hypothesis, in the virulent RH strain, conditional disruption of the single argonaute gene AGO1 has a very modest phenotype (Musiyenko et al., 2012). Future evaluation and functional validation of the cataloged miRNA of T. gondii should establish the function and importance of these RNA species. There is also circumstantial evidence that lncRNA will have important roles in the biology of T. gondii. While the functions of lncRNA are only now emerging, P. falciparum has several lncRNA of interest, including a noncoding transcript associated with silenced var genes that is transcribed from a promoter located in the conserved var intron (Calderwood et al., 2003). In addition, there are lncRNA associated with telomeric regions of Plasmodium that have been hypothesized as important for chromosome stability (Calderwood et al., 2003; SierraMiranda et al., 2012; Broadbent et al., 2011). Long ncRNA are associated with developmental regulation in the metazoa (Braun et al., 2010). Several groups have discovered lncRNA that are upregulated during the process of bradyzoite differentiation (Matrajt, 2010). Some of these lncRNA are antisense to sense transcripts, raising the hypothesis that lncRNA may play a role in translational regulation or mRNA stability during the stress response. Finally, inspection of RNA-seq studies reveals many instances of antisense transcripts that could potentially regulate gene expression of specific genes that are important for the host
pathogen interaction (see www.toxodb.org for numerous RNA-seq datasets from various strains and developmental conditions). The antisense transcripts are often present in either 50 or 30 -UTR of genes. At present the significance of these antisense RNAs is not known,

but these could potentially act as an important regulator of cell cycle progression or developmental transitions.

21.4.3 Other posttranscriptional mechanisms Another completely unexplored area is the role of RNA processing, RNA trafficking and RNA stability in T. gondii gene regulation. While cell cycle transcription is likely to explain the cell cycle periodicity of mRNA, mRNA degradation must also have a prominent role in regulation of steady
state RNAs. As in other eukaryotes, T. gondii encodes numerous proteins with predicted RNA binding domains. One of these RRM1 has been demonstrated to have an essential and conserved role in RNA splicing (Suvorova et al., 2013). In other systems, alternative splicing and RNA stability are mechanisms by which gene expression is regulated posttranscriptionally, and these events can be influenced by the metabolic state of the cell. lncRNA have also been implicated in the regulation of splicing and RNA trafficking (Guttman and Rinn, 2012).

21.5 Conclusion and future directions Studies of global mRNA expression in Plasmodium and Toxoplasma indicate that transcriptional mechanisms play a major role in regulating the developmental program of these parasites. Coregulated genes are dispersed across parasite chromosomes and appear to be regulated by conserved conventional eukaryotic transcriptional machinery including chromatin remodelers. It is now clear that promoter structures in Apicomplexa contain cis-elements that are regulated by trans-acting factors. While the overall principles underlying regulation of gene expression in the Apicomplexa appear to

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References

be conserved, the Apicomplexa have evolved specific adaptations that reflect their unique biology. ApiAP2 DNA binding domains represent the largest family of putative transcription factors discovered in Apicomplexa parasites. These factors appear to operate as activators or suppressors of development and stressresponsive gene expression. AP2 proteins are not found in mammals. The plant-like AP2 DNA-binding domains in Apicomplexa are implicated as the major players in transcriptional regulation in these parasites and are likely to work in concert with other factors such as myb domain
containing proteins that may also play crucial roles (Waldman et al., 2019). AP2-related mechanisms may be a novel invention in the Apicomplexa and a target for therapeutic development. Epigenetic-based gene regulation provides an important contribution to Toxoplasma gene expression, with several links to stage-specific gene expression now well established for a variety of different types of histone-modifying enzymes (Dixon et al., 2010; see Fig. 21.2). In higher eukaryotes, chromatin remodeling machinery and DNA-binding transcription factors work in concert to modulate gene expression, and studies suggest that Toxoplasma is no exception (Wang et al., 2014). A great deal of work remains, however, in characterizing the large array of chromatin remodeling enzymes in Toxoplasma and understanding how these machineries are recruited to target promoters to work coordinately with TgAP2s or other possible DNA-binding factors. Equally important is the characterization of chromatin “reader” proteins that harbor motifs that can bind specific histone modifications. As initial results with pharmacological agents that interfere with chromatin modifiers have shown promise, epigenetic-based gene regulation remains a high priority for future investigation. It is also critical to characterize how cellular signals are interpreted by the parasite to result

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in a reprogramming of gene expression, particularly changes associated with stage conversion. These processes are likely to involve sensing of metabolic fluxes and small metabolites and integrating these changes to modulate gene expression. Considerable data now suggest that mechanisms of translational control and other means of posttranscriptional gene regulation will also be as important as conventional components of transcriptional control.

Acknowledgments The research in our laboratories is supported by the following grants: NIH AI116496, AI124723 (to W.J.S.), and AI148374 (K.K.).

References Adomako-Ankomah, Y., Wier, G.M., Borges, A.L., Wand, H.E., Boyle, J.P., 2014. Differential locus expansion distinguishes Toxoplasmatinae species and closely related strains of Toxoplasma gondii. mBio 5, e01003
e01013. Ajioka, J.W., Boothroyd, J.C., Brunk, B.P., Hehl, A., Hillier, L., Manger, I.D., et al., 1998. Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa. Genome Res. 8, 18
28. Al-Anouti, F., Quach, T., Ananvoranich, S., 2003. Doublestranded RNA can mediate the suppression of uracil phosphoribosyltransferase expression in Toxoplasma gondii. Biochem. Biophys. Res. Commun. 302, 316
323. Al-Anouti, F., Tomavo, S., Parmley, S., Ananvoranich, S., 2004. The expression of lactate dehydrogenase is important for the cell cycle of Toxoplasma gondii. J. Biol. Chem. 279, 52300
52311. Allen, M.D., Yamasaki, K., Ohme-Takagi, M., Tateno, M., Suzuki, M., 1998. A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. EMBO J. 17, 5484
5496. Allis, C.D., Berger, S.L., Cote, J., Dent, S., Jenuwien, T., Kouzarides, T., et al., 2007. New nomenclature for chromatin-modifying enzymes. Cell 131, 633
636. Al Riyahi, A., Al-Anouti, F., Al-Rayes, M., Ananvoranich, S., 2006. Single argonaute protein from Toxoplasma gondii is involved in the double-stranded RNA induced gene silencing. Int. J. Parasitol. 36, 1003
1014. Altschul, S.F., Wootton, J.C., Zaslavsky, E., Yu, Y.K., 2010. The construction and use of log-odds substitution scores for multiple sequence alignment. PLoS Comput. Biol. 6, e1000852.

Toxoplasma Gondii

972

21. Regulation of gene expression in Toxoplasma gondii

Ananvoranich, S., Al Rayes, M., Al Riyahi, A., Wang, X., 2006. RNA silencing of glycolysis pathway in Toxoplasma gondii. J. Eukaryot. Microbiol. 53 (Suppl. 1), S162
S163. Andrews, C.D., Dubey, J.P., Tenter, A.M., Webert, D.W., 1997. Toxoplasma gondii recombinant antigens H4 and H11: use in ELISAs for detection of toxoplasmosis in swine. Vet. Parasitol. 70, 1
11. Andriankaja, A., Boisson-Dernier, A., Frances, L., Sauviac, L., Jauneau, A., Barker, D.G., et al., 2007. AP2-ERF transcription factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif. Plant Cell 19, 2866
2885. Augusto, L., Martynowicz, J., Staschke, K.A., Wek, R.C., Sullivan Jr., W.J., 2018. Effects of PERK eIF2alpha kinase inhibitor against Toxoplasma gondii. Antimicrob. Agents Chemother. 62. Bahl, A., Davis, P.H., Behnke, M., Dzierszinski, F., Jagalur, M., Chen, F., et al., 2010. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii. BMC Genomics 11, 603. Balaji, S., Babu, M.M., Iyer, L.M., Aravind, L., 2005. Discovery of the principal specific transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains. Nucleic Acids Res. 33, 3994
4006. Bannister, A.J., Zegerman, P., Partridge, J.F., Miska, E.A., Thomas, J.O., Allshire, R.C., et al., 2001. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120
124. Behnke, M.S., Radke, J.B., Smith, A.T., Sullivan Jr., W.J., White, M.W., 2008. The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements. Mol. Microbiol. 68, 1502
1518. Behnke, M.S., Wootton, J.C., Lehmann, M.M., Radke, J.B., Lucas, O., Nawas, J., et al., 2010. Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5, e12354. Behnke, M.S., Zhang, T.P., Dubey, J.P., Sibley, L.D., 2014. Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development. BMC Genomics 15, 350. Benmerzouga, I., Checkley, L.A., Ferdig, M.T., Arrizabalaga, G., Wek, R.C., Sullivan Jr., W.J., 2015. Guanabenz repurposed as an antiparasitic with activity against acute and latent toxoplasmosis. Antimicrob. Agents Chemother. 59, 6939
6945. Bennink, S., Von bohl, A., Ngwa, C.J., Henschel, L., Kuehn, A., Pilch, N., et al., 2018. A seven-helix protein constitutes stress granules crucial for regulating translation during human-to-mosquito transmission of Plasmodium falciparum. PLoS Pathog. 14, e1007249.

Bernstein, B.E., Mikkelsen, T.S., Xie, X., Kamal, M., Huebert, D.J., Cuff, J., et al., 2006. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315
326. Bhatti, M.M., Sullivan Jr., W.J., 2005. Histone acetylase GCN5 enters the nucleus via importin-alpha in protozoan parasite Toxoplasma gondii. J. Biol. Chem. 280, 5902
5908. Bhatti, M.M., Livingston, M., Mullapudi, N., Sullivan Jr., W.J., 2006. Pair of unusual GCN5 histone acetyltransferases and ADA2 homologues in the protozoan parasite Toxoplasma gondii. Eukaryot. Cell 5, 62
76. Blaizot, R., Nabet, C., Blanchet, D., Martin, E., Mercier, A., Darde, M.L., et al., 2019. Pediatric Amazonian toxoplasmosis caused by atypical strains in French Guiana, 2002-2017. Pediatr. Infect. Dis. J. 38, e39
e42. Blank, M.L., Boyle, J.P., 2018. Effector variation at tandem gene arrays in tissue-dwelling coccidia: who needs antigenic variation anyway? Curr. Opin. Microbiol. 46, 86
92. Blazek, E., Mittler, G., Meisterernst, M., 2005. The mediator of RNA polymerase II. Chromosoma 113, 399
408. Bohne, W., Wirsing, A., Gross, U., 1997. Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements. Mol. Biochem. Parasitol. 85, 89
98. Bougdour, A., Maubon, D., Baldacci, P., Ortet, P., Bastien, O., Bouillon, A., et al., 2009. Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites. J. Exp. Med. 206, 953
966. Bougdour, A., Braun, L., Cannella, D., Hakimi, M.A., 2010. Chromatin modifications: implications in the regulation of gene expression in Toxoplasma gondii. Cell Microbiol. 12, 413
423. Bowie, W.R., King, A.S., Werker, D.H., Isaac-Renton, J.L., Bell, A., Eng, S.B., et al., 1997. Outbreak of toxoplasmosis associated with municipal drinking water. The BC Toxoplasma Investigation Team. Lancet 350, 173
177. Bozdech, Z., Ginsburg, H., 2005. Data mining of the transcriptome of Plasmodium falciparum: the pentose phosphate pathway and ancillary processes. Malar. J. 4, 17. Bozdech, Z., Llinas, M., Pulliam, B.L., Wong, E.D., Zhu, J., Derisi, J.L., 2003. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol. 1, E5. Braun, L., Cannella, D., Pinheiro, A.M., Kieffer, S., Belrhali, H., Garin, J., et al., 2009. The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii. Int. J. Parasitol. 39, 81
90. Braun, L., Cannella, D., Ortet, P., Barakat, M., Sautel, C.F., Kieffer, S., et al., 2010. A complex small RNA repertoire is generated by a plant/fungal-like machinery and effected by a metazoan-like Argonaute in the single-cell human parasite Toxoplasma gondii. PLoS Pathog. 6, e1000920.

Toxoplasma Gondii

References

Broadbent, K.M., Park, D., Wolf, A.R., Van tyne, D., Sims, J. S., Ribacke, U., et al., 2011. A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs. Genome Biol. 12, R56. Brooks, C.F., Francia, M.E., Gissot, M., Croken, M.M., Kim, K., Striepen, B., 2011. Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 108, 3767
3772. Buchholz, K.R., Fritz, H.M., Chen, X., Durbin-Johnson, B., Rocke, D.M., Ferguson, D.J., et al., 2011. Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii bradyzoites. Eukaryot. Cell 10, 1637
1647. Bunnik, E.M., Venkat, A., Shao, J., Mcgovern, K.E., Batugedara, G., Worth, D., et al., 2019. Comparative 3D genome organization in apicomplexan parasites. Proc. Natl. Acad. Sci. U.S.A. 116, 3183
3192. Calderwood, M.S., Gannoun-Zaki, L., Wellems, T.E., Deitsch, K.W., 2003. Plasmodium falciparum var genes are regulated by two regions with separate promoters, one upstream of the coding region and a second within the intron. J. Biol. Chem. 278, 34125
34132. Campbell, D.A., Thomas, S., Sturm, N.R., 2003. Transcription in kinetoplastid protozoa: why be normal? Microbes Infect. 5, 1231
1240. Campbell, T.L., De Silva, E.K., Olszewski, K.L., Elemento, O., Llinas, M., 2010. Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite. PLoS Pathog. 6, e1001165. Cevallos, A.M., Perez-Escobar, M., Espinosa, N., Herrera, J., Lopez-Villasenor, I., Hernandez, R., 2005. The stabilization of housekeeping transcripts in Trypanosoma cruzi epimastigotes evidences a global regulation of RNA decay during stationary phase. FEMS Microbiol. Lett. 246, 259
264. Chene, A., Vembar, S.S., Riviere, L., Lopez-Rubio, J.J., Claes, A., Siegel, T.N., et al., 2012. PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites. Nucleic Acids Res. 40, 3066
3077. Chevalier, B.S., Stoddard, B.L., 2001. Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res. 29, 3757
3774. Choi, W.Y., Nam, H.W., Kwak, N.H., Huh, W., Kim, Y.R., Kang, M.W., et al., 1997. Foodborne outbreaks of human toxoplasmosis. J. Infect. Dis. 175, 1280
1282. Chow, C.S., Wirth, D.F., 2003. Linker scanning mutagenesis of the Plasmodium gallinaceum sexual stage specific gene pgs28 reveals a novel downstream cis-control element. Mol. Biochem. Parasitol. 129, 199
208.

973

Chow, C., Cloutier, S., Dumas, C., Chou, M.N., Papadopoulou, B., 2011. Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation. Cell Microbiol. 13, 1059
1077. Cleary, M.D., Singh, U., Blader, I.J., Brewer, J.L., Boothroyd, J.C., 2002. Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryot. Cell 1, 329
340. Coulson, R.M., Hall, N., Ouzounis, C.A., 2004. Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum. Genome Res. 14, 1548
1554. Croken, M.M., Nardelli, S.C., Kim, K., 2012. Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives. Trends Parasitol. 28, 202
213. Croken, M.M., Ma, Y., Markillie, L.M., Taylor, R.C., Orr, G., Weiss, L.M., et al., 2014. Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage. PLoS One 9, e111297. Cui, L., Miao, J., Cui, L., 2007. Cytotoxic effect of curcumin on malaria parasite Plasmodium falciparum: inhibition of histone acetylation and generation of reactive oxygen species. Antimicrob. Agents Chemother. 51, 488
494. Cui, L., Miao, J., Furuya, T., Fan, Q., Li, X., Rathod, P.K., et al., 2008. Histone acetyltransferase inhibitor anacardic acid causes changes in global gene expression during in vitro Plasmodium falciparum development. Eukaryot. Cell 7, 1200
1210. Dalmasso, M.C., Echeverria, P.C., Zappia, M.P., Hellman, U., Dubremetz, J.F., Angel, S.O., 2006. Toxoplasma gondii has two lineages of histones 2b (H2B) with different expression profiles. Mol. Biochem. Parasitol. 148, 103
107. Dalmasso, M.C., Onyango, D.O., Naguleswaran, A., Sullivan Jr., W.J., Angel, S.O., 2009. Toxoplasma H2A variants reveal novel insights into nucleosome composition and functions for this histone family. J. Mol. Biol. 392, 33
47. Dalmasso, M.C., Sullivan Jr., W.J., Angel, S.O., 2011. Canonical and variant histones of protozoan parasites. Front. Biosci. (Landmark Ed) 16, 2086
2105. Darkin-Rattray, S.J., Gurnett, A.M., Myers, R.W., Dulski, P. M., Crumley, T.M., Allocco, J.J., et al., 1996. Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase. Proc. Natl. Acad. Sci. U.S.A. 93, 13143
13147. Dechering, K.J., Thompson, J., Dodemont, H.J., Eling, W., Konings, R.N., 1997. Developmentally regulated expression of pfs16, a marker for sexual differentiation of the human malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 89, 235
244.

Toxoplasma Gondii

974

21. Regulation of gene expression in Toxoplasma gondii

Dembele, L., Franetich, J.F., Lorthiois, A., Gego, A., Zeeman, A.M., Kocken, C.H., et al., 2014. Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nat. Med. 20, 307
312. De Silva, E.K., Gehrke, A.R., Olszewski, K., Leon, I., Chahal, J.S., Bulyk, M.L., et al., 2008. Specific DNAbinding by apicomplexan AP2 transcription factors. Proc. Natl. Acad. Sci. U.S.A. 105, 8393
8398. Dhalluin, C., Carlson, J.E., Zeng, L., He, C., Aggarwal, A. K., Zhou, M.M., 1999. Structure and ligand of a histone acetyltransferase bromodomain. Nature 399, 491
496. Dietz, K.J., Vogel, M.O., Viehhauser, A., 2010. AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling. Protoplasma 245, 3
14. Dillon, S.C., Zhang, X., Trievel, R.C., Cheng, X., 2005. The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol. 6, 227. Dixon, S.E., Stilger, K.L., Elias, E.V., Naguleswaran, A., Sullivan Jr., W.J., 2010. A decade of epigenetic research in Toxoplasma gondii. Mol. Biochem. Parasitol. 173, 1
9. Dixon, S.E., Bhatti, M.M., Uversky, V.N., Dunker, A.K., Sullivan Jr., W.J., 2011. Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B. Mol. Biochem. Parasitol. 175, 192
195. Dubey, J.P., 1988. Toxoplasmosis of Animals and Man. Dubey, J.P., 1998. Comparative infectivity of Toxoplasma gondii bradyzoites in rats and mice. J. Parasitol. 84, 1279
1282. Dubey, J.P., Frenkel, J.K., 1976. Feline toxoplasmosis from acutely infected mice and the development of Toxoplasma cysts. J. Protozool. 23, 537
546. Dubey, J.P., Miller, N.L., Frenkel, J.K., 1970. Toxoplasma gondii life cycle in cats. J. Am. Vet. Med. Assoc. 157, 1767
1770. Dzierszinski, F., Mortuaire, M., Dendouga, N., Popescu, O., Tomavo, S., 2001. Differential expression of two plantlike enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii. J. Mol. Biol. 309, 1017
1027. Ehrenhofer-Murray, A.E., 2004. Chromatin dynamics at DNA replication, transcription and repair. Eur. J. Biochem. 271, 2335
2349. El Bissati, K., Suvorova, E.S., Xiao, H., Lucas, O., Upadhya, R., Ma, Y., et al., 2016. Toxoplasma gondii arginine methyltransferase 1 (PRMT1) is necessary for centrosome dynamics during tachyzoite cell division. mBio 7, e02094-15. Escargueil, A.E., Soares, D.G., Salvador, M., Larsen, A.K., Henriques, J.A., 2008. What histone code for DNA repair? Mutat. Res. 658, 259
270.

Featherstone, M., 2002. Coactivators in transcription initiation: here are your orders. Curr. Opin. Genet. Dev. 12, 149
155. Fennell, C., Babbitt, S., Russo, I., Wilkes, J., RanfordCartwright, L., Goldberg, D.E., et al., 2009. PfeIK1, a eukaryotic initiation factor 2alpha kinase of the human malaria parasite Plasmodium falciparum, regulates stress
response to amino-acid starvation. Malar. J. 8, 99. Flueck, C., Bartfai, R., Niederwieser, I., Witmer, K., Alako, B.T., Moes, S., et al., 2010. A major role for the Plasmodium falciparum ApiAP2 protein PfSIP2 in chromosome end biology. PLoS Pathog. 6, e1000784. Fox, B.A., Li, W.B., Tanaka, M., Inselburg, J., Bzik, D.J., 1993. Molecular characterization of the largest subunit of Plasmodium falciparum RNA polymerase I. Mol. Biochem. Parasitol. 61, 37
48. Freitas-Junior, L.H., Hernandez-Rivas, R., Ralph, S.A., Montiel-Condado, D., Ruvalcaba-Salazar, O.K., RojasMeza, A.P., et al., 2005. Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 121, 25
36. Fry, C.J., Peterson, C.L., 2002. Transcription. Unlocking the gates to gene expression. Science 295, 1847
1848. Garcia-Salcedo, J.A., Nolan, D.P., Gijon, P., GomezRodriguez, J., Pays, E., 2002. A protein kinase specifically associated with proliferative forms of Trypanosoma brucei is functionally related to a yeast kinase involved in the co-ordination of cell shape and division. Mol. Microbiol. 45, 307
319. Gissot, M., Kelly, K.A., Ajioka, J.W., Greally, J.M., Kim, K., 2007. Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii. PLoS Pathog. 3, e77. Gissot, M., Choi, S.W., Thompson, R.F., Greally, J.M., Kim, K., 2008. Toxoplasma gondii and Cryptosporidium parvum lack detectable DNA cytosine methylation. Eukaryot. Cell 7, 537
540. Gissot, M., Walker, R., Delhaye, S., Huot, L., Hot, D., Tomavo, S., 2012. Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery. PLoS One 7, e32671. Gomes-Santos, C.S., Braks, J., Prudencio, M., Carret, C., Gomes, A.R., Pain, A., et al., 2011. Transition of Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio. PLoS Pathog. 7, e1002046. Goyal, M., Alam, A., Iqbal, M.S., Dey, S., Bindu, S., Pal, C., et al., 2012. Identification and molecular characterization of an Alba-family protein from human malaria parasite Plasmodium falciparum. Nucleic Acids Res. 40, 1174
1190.

Toxoplasma Gondii

References

Guerini, M.N., Que, X., Reed, S.L., White, M.W., 2000. Two genes encoding unique proliferating-cell-nuclear-antigens are expressed in Toxoplasma gondii. Mol. Biochem. Parasitol. 109, 121
131. Guttman, M., Rinn, J.L., 2012. Modular regulatory principles of large non-coding RNAs. Nature 482, 339
346. Haile, S., Estevez, A.M., Clayton, C., 2003. A role for the exosome in the in vivo degradation of unstable mRNAs. RNA 9, 1491
1501. Hanson, K.K., Mair, G.R., 2014. Stress granules and Plasmodium liver stage infection. Biol. Open 3, 103
107. Hassan, M.A., Melo, M.B., Haas, B., Jensen, K.D., Saeij, J.P., 2012. De novo reconstruction of the Toxoplasma gondii transcriptome improves on the current genome annotation and reveals alternatively spliced transcripts and putative long non-coding RNAs. BMC Genomics 13, 696. Hassan, M.A., Vasquez, J.J., Guo-Liang, C., Meissner, M., Nicolai siegel, T., 2017. Comparative ribosome profiling uncovers a dominant role for translational control in Toxoplasma gondii. BMC Genomics 18, 961. Heaslip, A.T., Nishi, M., Stein, B., Hu, K., 2011. The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase. PLoS Pathog. 7, e1002201. Hehl, A.B., Basso, W.U., Lippuner, C., Ramakrishnan, C., Okoniewski, M., Walker, R.A., et al., 2015. Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of nonoverlapping gene families to attach, invade, and replicate within feline enterocytes. BMC Genomics 16, 66. Helm, S., Lehmann, C., Nagel, A., Stanway, R.R., Horstmann, S., Llinas, M., et al., 2010. Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium. PLoS One 5, e13653. Hendrick, H.M., Welter, B.H., Hapstack, M.A., Sykes, S.E., Sullivan Jr., W.J., Temesvari, L.A., 2016. Phosphorylation of eukaryotic initiation factor-2alpha during stress and encystation in Entamoeba species. PLoS Pathog. 12, e1006085. Holmes, M.J., Augusto, L.D.S., Zhang, M., Wek, R.C., Sullivan Jr., W.J., 2017. Translational control in the latency of apicomplexan parasites. Trends Parasitol. 33, 947
960. Hong, D.P., Radke, J.B., White, M.W., 2017. Opposing transcriptional mechanisms regulate Toxoplasma development. mSphere 2. Available from: https://doi.org/ 10.1128/mSphere.00347-16. Hong, X., Zang, J., White, J., Wang, C., Pan, C.H., Zhao, R., et al., 2010. Interaction of JMJD6 with single-stranded RNA. Proc. Natl. Acad. Sci. U.S.A. 107, 14568
14572.

975

Huang, S., Holmes, M.J., Radke, J.B., Hong, D.P., Liu, T.K., White, M.W., et al., 2017. Toxoplasma gondii AP2IX-4 regulates gene expression during bradyzoite development. mSphere 2. Available from: https://doi.org/10.1128/ mSphere.00054-17. Isaac-Renton, J., Bowie, W.R., King, A., Irwin, G.S., Ong, C. S., Fung, C.P., et al., 1998. Detection of Toxoplasma gondii oocysts in drinking water. Appl. Environ. Microbiol. 64, 2278
2280. Jeffers, V., Sullivan Jr, W.J., 2012. Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii. Eukaryot. Cell 11, 735
742. Jeffers, V., Gao, H., Checkley, L.A., Liu, Y., Ferdig, M.T., Sullivan Jr., W.J., 2016. Garcinol inhibits GCN5mediated lysine acetyltransferase activity and prevents replication of the parasite Toxoplasma gondii. Antimicrob. Agents Chemother. 60, 2164
2170. Jeffers, V., Kamau, E.T., Srinivasan, A.R., Harper, J., Sankaran, P., Post, S.E., et al., 2017a. TgPRELID, a mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. mSphere 2. Available from: https://doi.org/10.1128/mSphere.00229-16. Jeffers, V., Yang, C., Huang, S., Sullivan Jr., W.J., 2017b. Bromodomains in protozoan parasites: evolution, function, and opportunities for drug development. Microbiol. Mol. Biol. Rev. 81. Available from: https:// doi.org/10.1128/MMBR.00047-16. Jerome, M.E., Radke, J.R., Bohne, W., Roos, D.S., White, M. W., 1998. Toxoplasma gondii bradyzoites form spontaneously during sporozoite- initiated development. Infect. Immun. 66, 4838
4844. Ji, D.D., Arnot, D.E., 1997. A Plasmodium falciparum homologue of the ATPase subunit of a multi-protein complex involved in chromatin remodelling for transcription. Mol. Biochem. Parasitol. 88, 151
162. Jofuku, K.D., den Boer, B.G., Van Montagu, M., Okamuro, J.K., 1994. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 6, 1211
1225. Johnston, H., Kneer, J., Chackalaparampil, I., Yaciuk, P., Chrivia, J., 1999. Identification of a novel SNF2/SWI2 protein family member, SRCAP, which interacts with CREB-binding protein. J. Biol. Chem. 274, 16370
16376. Johnson, C.N., Adkins, N.L., Georgel, P., 2005. Chromatin remodeling complexes: ATP-dependent machines in action. Biochem. Cell. Biol. 83, 405
417. Joshi, M.B., Lin, D.T., Chiang, P.H., Goldman, N.D., Fujioka, H., Aikawa, M., et al., 1999. Molecular cloning and nuclear localization of a histone deacetylase homologue in Plasmodium falciparum. Mol. Biochem. Parasitol. 99, 11
19.

Toxoplasma Gondii

976

21. Regulation of gene expression in Toxoplasma gondii

Josling, G.A., Petter, M., Oehring, S.C., Gupta, A.P., Dietz, O., Wilson, D.W., et al., 2015. A Plasmodium falciparum bromodomain protein regulates invasion gene expression. Cell Host Microbe 17, 741
751. Joyce, B.R., Queener, S.F., Wek, R.C., Sullivan Jr., W.J., 2010. Phosphorylation of eukaryotic initiation factor-2 {alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 107, 17200
17205. Joyce, B.R., Konrad, C., Wek, R.C., Sullivan Jr., W.J., 2011. Translation control is critical during acute and chronic stages of toxoplasmosis infection. Expert. Rev. Anti. Infect. Ther. 9, 1
3. Joyce, B.R., Tampaki, Z., Kim, K., Wek, R.C., Sullivan Jr., W.J., 2013. The unfolded protein response in the protozoan parasite Toxoplasma gondii features translational and transcriptional control. Eukaryot. Cell 12, 979
989. Kafsack, B.F., Rovira-Graells, N., Clark, T.G., Bancells, C., Crowley, V.M., Campino, S.G., et al., 2014. A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 507, 248
252. Karras, G.I., Kustatscher, G., Buhecha, H.R., Allen, M.D., Pugieux, C., Sait, F., et al., 2005. The macro domain is an ADP-ribose binding module. EMBO J. 24, 1911
1920. Kibe, M.K., Coppin, A., Dendouga, N., Oria, G., Meurice, E., Mortuaire, M., et al., 2005. Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii. Nucleic Acids Res. 33, 1722
1736. Kim, K., 2018. The epigenome, cell cycle, and development in Toxoplasma. Annu. Rev. Microbiol. 72, 479
499. Konishi, E., Takahashi, J., 1987. Some epidemiological aspects of Toxoplasma infections in a population of farmers in Japan. Int. J. Epidemiol. 16, 277
281. Konrad, C., Wek, R.C., Sullivan Jr., W.J., 2011. A GCN2-like eukaryotic initiation factor 2 kinase increases the viability of extracellular Toxoplasma gondii parasites. Eukaryot. Cell 10, 1403
1412. Konrad, C., Queener, S.F., Wek, R.C., Sullivan Jr., W.J., 2013. Inhibitors of eIF2alpha dephosphorylation slow replication and stabilize latency in Toxoplasma gondii. Antimicrob. Agents Chemother. 57, 1815
1822. Konrad, C., Wek, R.C., Sullivan Jr., W.J., 2014. GCN2-like eIF2alpha kinase manages the amino acid starvation response in Toxoplasma gondii. Int. J. Parasitol. 44, 139
146. Koufopanou, V., Goddard, M.R., Burt, A., 2002. Adaptation for horizontal transfer in a homing endonuclease. Mol. Biol. Evol. 19, 239
246. Krizek, B.A., 2003. AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain. Nucleic Acids Res. 31, 1859
1868.

Kurahashi, H., Akagi, K., Inazawa, J., Ohta, T., Niikawa, N., Kayatani, F., et al., 1995. Isolation and characterization of a novel gene deleted in DiGeorge syndrome. Hum. Mol. Genet. 4, 541
549. LaCount, D.J., Vignali, M., Chettier, R., Phansalkar, A., Bell, R., Hesselberth, J.R., et al., 2005. A protein interaction network of the malaria parasite Plasmodium falciparum. Nature 438, 103
107. Lanzer, M., Wertheimer, S.P., de Bruin, D., Ravetch, J.V., 1993. Plasmodium: control of gene expression in malaria parasites. Exp. Parasitol. 77, 121
128. Larreta, R., Soto, M., Quijada, L., Folgueira, C., Abanades, D.R., Alonso, C., et al., 2004. The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation. BMC Mol. Biol. 5, 3. Lehmann, T., Marcet, P.L., Graham, D.H., Dahl, E.R., Dubey, J.P., 2006. Globalization and the population structure of Toxoplasma gondii. Proc. Natl. Acad. Sci. U. S.A. 103, 11423
11428. Le Roch, K.G., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M., et al., 2004. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 14, 2308
2318. Lesage, K.M., Huot, L., Mouveaux, T., Courjol, F., Saliou, J. M., Gissot, M., 2018. Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii. Nucleic Acids Res. 46, 6057
6068. Lescault, P.J., Thompson, A.B., Patil, V., Lirussi, D., Burton, A., Margarit, J., et al., 2010. Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state. PLoS One 5, e14463. Levitt, A., Dimayuga, F.O., Ruvolo, V.R., 1993. Analysis of malarial transcripts using cDNA-directed polymerase chain reaction. J. Parasitol. 79, 653
662. Li, W.B., Bzik, D.J., Gu, H.M., Tanaka, M., Fox, B.A., Inselburg, J., 1989. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains. Nucleic Acids Res. 17, 9621
9636. Li, W.B., Bzik, D.J., Tanaka, M., Gu, H.M., Fox, B.A., Inselburg, J., 1991. Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III. Mol. Biochem. Parasitol. 46, 229
239. Li, L., Brunk, B.P., Kissinger, J.C., Pape, D., Tang, K., Cole, R.H., et al., 2003. Gene discovery in the apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Res. 13, 443
454.

Toxoplasma Gondii

References

Li, L., Crabtree, J., Fischer, S., Pinney, D., Stoeckert Jr., C.J., Sibley, L.D., et al., 2004. ApiEST-DB: analyzing clustered EST data of the apicomplexan parasites. Nucleic Acids Res. 32 (Database issue), D326
D328. Lindner, S.E., De Silva, E.K., Keck, J.L., Llinas, M., 2010. Structural determinants of DNA binding by a P. falciparum ApiAP2 transcriptional regulator. J. Mol. Biol. 395, 558
567. Lirussi, D., Matrajt, M., 2011. RNA granules present only in extracellular Toxoplasma gondii increase parasite viability. Int. J. Biol. Sci. 7, 960
967. Llinas, M., DeRisi, J.L., 2004. Pernicious plans revealed: Plasmodium falciparum genome wide expression analysis. Curr. Opin. Microbiol. 7, 382
387. Long, P.L., 1982. The Biology of the Coccida. University Park Press, Baltimore, MD. Lopez-Rubio, J.J., Gontijo, A.M., Nunes, M.C., Issar, N., Hernandez Rivas, R., Scherf, A., 2007. 50 Flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites. Mol. Microbiol. 66, 1296
1305. Lopez-Rubio, J.J., Mancio-Silva, L., Scherf, A., 2009. Genome-wide analysis of heterochromatin associates clonally variant gene regulation with perinuclear repressive centers in malaria parasites. Cell Host Microbe 5, 179
190. Lorenzi, H., Khan, A., Behnke, M.S., Namasivayam, S., Swapna, L.S., Hadjithomas, M., et al., 2016. Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes. Nat. Commun. 7, 10147. Lyons, R.E., Mcleod, R., Roberts, C.W., 2002. Toxoplasma gondii tachyzoite-bradyzoite interconversion. Trends Parasitol. 18, 198
201. Ma, Y.F., Zhang, Y., Kim, K., Weiss, L.M., 2004. Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus. Int. J. Parasitol. 34, 333
346. Magnani, E., Sjolander, K., Hake, S., 2004. From endonucleases to transcription factors: evolution of the AP2 DNA binding domain in plants. Plant Cell 16, 2265
2277. Mair, G.R., Braks, J.A., Garver, L.S., Wiegant, J.C., Hall, N., Dirks, R.W., et al., 2006. Regulation of sexual development of Plasmodium by translational repression. Science 313, 667
669. Malmquist, N.A., Moss, T.A., Mecheri, S., Scherf, A., Fuchter, M.J., 2012. Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms in Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S.A. 109, 16708
16713. Manger, I.D., Hehl, A., Parmley, S., Sibley, L.D., Marra, M., Hillier, L., et al., 1998. Expressed sequence tag analysis

977

of the bradyzoite stage of Toxoplasma gondii: identification of developmentally regulated genes. Infect. Immun. 66, 1632
1637. Mateus-Pinilla, N.E., Dubey, J.P., Choromanski, L., Weigel, R.M., 1999. A field trial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T. gondii exposure for swine. J. Parasitol. 85, 855
860. Matrajt, M., 2010. Non-coding RNA in apicomplexan parasites. Mol. Biochem. Parasitol. 174, 1
7. Matrajt, M., Nishi, M., Fraunholz, M.J., Peter, O., Roos, D. S., 2002. Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii. Mol. Biochem. Parasitol. 120, 285
289. Matrajt, M., Platt, C.D., Sagar, A.D., Lindsay, A., Moulton, C., Roos, D.S., 2004. Transcript initiation, polyadenylation, and functional promoter mapping for the dihydrofolate reductase-thymidylate synthase gene of Toxoplasma gondii. Mol. Biochem. Parasitol. 137, 229
238. Maubon, D., Bougdour, A., Wong, Y.S., Brenier-Pinchart, M.P., Curt, A., Hakimi, M.A., et al., 2010. Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form and study of new derivative compounds. Antimicrob. Agents Chemother. 54, 4843
4850. Meissner, M., Soldati, D., 2005. The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites. Microbes Infect. Available from: https://doi.org/ 10.1016/j.micinf.2005.04.019. Mercier, C., Lefebvre-Van Hende, S., Garber, G.E., Lecordier, L., Capron, A., Cesbron-Delauw, M.F., 1996. Common cis-acting elements critical for the expression of several genes of Toxoplasma gondii. Mol. Microbiol. 21, 421
428. Miao, J., Li, J., Fan, Q., Li, X., Li, X., Cui, L., 2010. The Puffamily RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum. J. Cell Sci. 123, 1039
1049. Minot, S., Melo, M.B., Li, F., Lu, D., Niedelman, W., Levine, S.S., et al., 2012. Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity. Proc. Natl. Acad. Sci. U.S.A. 109, 13458
13463. Mohrmann, L., Verrijzer, C.P., 2005. Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes. Biochim. Biophys. Acta 1681, 59
73. Monroy, M.A., Schott, N.M., Cox, L., Chen, J.D., Ruh, M., Chrivia, J.C., 2003. SNF2-related CBP activator protein (SRCAP) functions as a coactivator of steroid receptormediated transcription through synergistic interactions with CARM-1 and GRIP-1. Mol. Endocrinol. 17, 2519
2528.

Toxoplasma Gondii

978

21. Regulation of gene expression in Toxoplasma gondii

Mullapudi, N., Joseph, S.J., Kissinger, J.C., 2009. Identification and functional characterization of cis-regulatory elements in the apicomplexan parasite Toxoplasma gondii. Genome Biol. 10, R34. Muller, K., Matuschewski, K., Silvie, O., 2011. The Puffamily RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite. PLoS One 6, e19860. Munoz, E.E., Hart, K.J., Walker, M.P., Kennedy, M.F., Shipley, M.M., Lindner, S.E., 2017. ALBA4 modulates its stage-specific interactions and specific mRNA fates during Plasmodium yoelii growth and transmission. Mol. Microbiol. 106, 266
284. Musiyenko, A., Majumdar, T., Andrews, J., Adams, B., Barik, S., 2012. PRMT1 methylates the single Argonaute of Toxoplasma gondii and is important for the recruitment of Tudor nuclease for target RNA cleavage by antisense guide RNA. Cell Microbiol. Available from: https://doi.org/10.1111/j.1462-5822.2012.01763.x. Naar, A.M., Lemon, B.D., Tjian, R., 2001. Transcriptional coactivator complexes. Annu. Rev. Biochem. 70, 475
501. Naguleswaran, A., Elias, E.V., Mcclintick, J., Edenberg, H. J., Sullivan Jr., W.J., 2010. Toxoplasma gondii lysine acetyltransferase GCN5-A functions in the cellular response to alkaline stress and expression of cyst genes. PLoS Pathog. 6, e1001232. Nakaar, V., Bermudes, D., Peck, K.R., Joiner, K.A., 1998. Upstream elements required for expression of nucleoside triphosphate hydrolase genes of Toxoplasma gondii. Mol. Biochem. Parasitol. 92, 229
239. Nallani, K.C., Sullivan Jr., W.J., 2005. Identification of proteins interacting with Toxoplasma SRCAP by yeast twohybrid screening. Parasitol. Res. 95, 236
242. Naor, A., Panas, M.W., Marino, N., Coffey, M.J., Tonkin, C. J., Boothroyd, J.C., 2018. MYR1-dependent effectors are the major drivers of a host cell’s early response to Toxoplasma, including counteracting MYR1-independent effects. mBio 9. Available from: https://doi.org/ 10.1128/mBio.02401-17. Narasimhan, J., Joyce, B.R., Naguleswaran, A., Smith, A.T., Livingston, M.R., Dixon, S.E., et al., 2008. Translation regulation by eukaryotic initiation factor-2 kinases in the development of latent cysts in Toxoplasma gondii. J. Biol. Chem. 283, 16591
16601. Nardelli, S.C., Che, F.Y., Silmon de monerri, N.C., Xiao, H., Nieves, E., Madrid-Aliste, C., et al., 2013. The histone code of Toxoplasma gondii comprises conserved and unique posttranslational modifications. mBio 4, e0092213. Nardelli, S.C., Ting, L.M., Kim, K., 2015. Techniques to study epigenetic control and the epigenome in parasites. Methods Mol. Biol. 1201, 177
191.

Nguyen, H.M., Berry, L., Sullivan Jr., W.J., Besteiro, S., 2017. Autophagy participates in the unfolded protein response in Toxoplasma gondii. FEMS Microbiol. Lett. 364. Available from: https://doi.org/10.1093/femsle/ fnx153. Ohme-Takagi, M., Shinshi, H., 1995. Ethylene-inducible DNA binding proteins that interact with an ethyleneresponsive element. Plant Cell 7, 173
182. Olguin-Lamas, A., Madec, E., Hovasse, A., Werkmeister, E., Callebaut, I., Slomianny, C., et al., 2011. A novel Toxoplasma gondii nuclear factor TgNF3 is a dynamic chromatin-associated component, modulator of nucleolar architecture and parasite virulence. PLoS Pathog. 7, e1001328. Peterson, C.L., Laniel, M.A., 2004. Histones and histone modifications. Curr. Biol. 14, R546
R551. Pino, P., Sebastian, S., Kim, E.A., Bush, E., Brochet, M., Volkmann, K., et al., 2012. A tetracycline-repressible transactivator system to study essential genes in malaria parasites. Cell Host Microbe 12, 824
834. Pittman, K.J., Aliota, M.T., Knoll, L.J., 2014. Dual transcriptional profiling of mice and Toxoplasma gondii during acute and chronic infection. BMC Genomics 15, 806. Ponts, N., Fu, L., Harris, E.Y., Zhang, J., Chung, D.W., Cervantes, M.C., et al., 2013. Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum. Cell Host Microbe 14, 696
706. Poran, A., Notzel, C., Aly, O., Mencia-Trinchant, N., Harris, C.T., Guzman, M.L., et al., 2017. Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites. Nature 551, 95
99. Purdy, J.E., Donelson, J.E., Wilson, M.E., 2005a. Leishmania chagasi: the alpha-tubulin intercoding region results in constant levels of mRNA abundance despite protein synthesis inhibition and growth state. Exp. Parasitol. 110, 102
107. Purdy, J.E., Donelson, J.E., Wilson, M.E., 2005b. Regulation of genes encoding the major surface protease of Leishmania chagasi via mRNA stability. Mol. Biochem. Parasitol. 142, 88
97. Radke, J.R., Guerini, M.N., Jerome, M., White, M.W., 2003. A change in the premitotic period of the cell cycle is associated with bradyzoite differentiation in Toxoplasma gondii. Mol. Biochem. Parasitol. 131, 119
127. Radke, J.R., Behnke, M.S., Mackey, A.J., Radke, J.B., Roos, D.S., White, M.W., 2005. The transcriptome of Toxoplasma gondii. BMC. Biol. 3, 26. Radke, J.B., Lucas, O., De Silva, E.K., Ma, Y., Sullivan Jr., W.J., Weiss, L.M., et al., 2013. ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst. Proc. Natl. Acad. Sci. U.S.A. 110, 6871
6876.

Toxoplasma Gondii

References

Radke, J.B., Worth, D., Hong, D., Huang, S., Sullivan Jr., W. J., Wilson, E.H., et al., 2018. Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis. PLoS Pathog. 14, e1007035. Ranish, J.A., Hahn, S., 1996. Transcription: basal factors and activation. Curr. Opin. Genet. Dev. 6, 151
158. Rehkopf, D.H., Gillespie, D.E., Harrell, M.I., Feagin, J.E., 2000. Transcriptional mapping and RNA processing of the Plasmodium falciparum mitochondrial mRNAs. Mol. Biochem. Parasitol. 105, 91
103. Reid, A.J., Vermont, S.J., Cotton, J.A., Harris, D., HillCawthorne, G.A., Konen-Waisman, S., et al., 2012. Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy. PLoS Pathog. 8, e1002567. Riechmann, J.L., Meyerowitz, E.M., 1998. The AP2/EREBP family of plant transcription factors. Biol. Chem. 379, 633
646. Rios-Doria, J., Velkova, A., Dapic, V., Galan-Caridad, J.M., Dapic, V., Carvalho, M.A., et al., 2009. Ectopic expression of histone H2AX mutants reveals a role for its post-translational modifications. Cancer Biol. Ther. 8, 422
434. Rochette, A., Mcnicoll, F., Girard, J., Breton, M., Leblanc, E., Bergeron, M.G., et al., 2005. Characterization and developmental gene regulation of a large gene family encoding amastin surface proteins in Leishmania spp. Mol. Biochem. Parasitol. 140, 205
220. Roos, D.S., Sullivan, W.J., Striepen, B., Bohne, W., Donald, R.G., 1997. Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis. Methods 13, 112
122. Rosowski, E.E., Saeij, J.P., 2012. Toxoplasma gondii clonal strains all inhibit STAT1 transcriptional activity but polymorphic effectors differentially modulate IFNgamma induced gene expression and STAT1 phosphorylation. PLoS One 7, e51448. Ruvalcaba-Salazar, O.K., del Carmen Ramirez-Estudillo, M., Montiel-Condado, D., Recillas-Targa, F., Vargas, M., Hernandez-Rivas, R., 2005. Recombinant and native Plasmodium falciparum TATA-binding-protein binds to a specific TATA box element in promoter regions. Mol. Biochem. Parasitol. 140, 183
196. Saksouk, N., Bhatti, M.M., Kieffer, S., Smith, A.T., Musset, K., Garin, J., et al., 2005. Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii. Mol. Cell. Biol. 25, 10301
10314. Sakuma, Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K., Yamaguchi-Shinozaki, K., 2002. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-

979

inducible gene expression. Biochem. Biophys. Res. Commun. 290, 998
1009. Salcedo-Amaya, A.M., van Driel, M.A., Alako, B.T., Trelle, M.B., van den Elzen, A.M., Cohen, A.M., et al., 2009. Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S.A. 106, 9655
9660. Santos, J.M., Josling, G., Ross, P., Joshi, P., Orchard, L., Campbell, T., et al., 2017. Red blood cell invasion by the malaria parasite is coordinated by the PfAP2-I transcription factor. Cell Host Microbe 21, 731
741. e10. Sautel, C.F., Cannella, D., Bastien, O., Kieffer, S., Aldebert, D., Garin, J., et al., 2007. SET8-mediated methylations of histone H4 lysine 20 mark silent heterochromatic domains in apicomplexan genomes. Mol. Cell. Biol. 27, 5711
5724. Sautel, C.F., Ortet, P., Saksouk, N., Kieffer, S., Garin, J., Bastien, O., et al., 2009. The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences. Mol. Microbiol. 71, 212
226. Schmid, M., Uhlenhaut, N.H., Godard, F., Demar, M., Bressan, R., Weigel, D., et al., 2003. Dissection of floral induction pathways using global expression analysis. Development 130, 6001
6012. Shapira, M., Zilka, A., Garlapati, S., Dahan, E., Dahan, I., Yavesky, V., 2001. Post transcriptional control of gene expression in Leishmania. Med. Microbiol. Immunol. (Berl.) 190, 23
26. Shiio, Y., Eisenman, R.N., 2003. Histone sumoylation is associated with transcriptional repression. Proc. Natl. Acad. Sci. U.S.A. 100, 13225
13230. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166, 1423
1435.e12. Sierra-Miranda, M., Delgadillo, D.M., Mancio-Silva, L., Vargas, M., Villegas-Sepulveda, N., Martinez-Calvillo, S., et al., 2012. Two long non-coding RNAs generated from subtelomeric regions accumulate in a novel perinuclear compartment in Plasmodium falciparum. Mol. Biochem. Parasitol. 185, 36
47. Silmon de Monerri, N.C., Yakubu, R.R., Chen, A.L., Bradley, P.J., Nieves, E., Weiss, L.M., et al., 2015. The ubiquitin proteome of Toxoplasma gondii reveals roles for protein ubiquitination in cell-cycle transitions. Cell Host Microbe 18, 621
633. Sindikubwabo, F., Ding, S., Hussain, T., Ortet, P., Barakat, M., Baumgarten, S., et al., 2017. Modifications at K31 on the lateral surface of histone H4 contribute to genome structure and expression in apicomplexan parasites.

Toxoplasma Gondii

980

21. Regulation of gene expression in Toxoplasma gondii

eLife 6. Available from: https://doi.org/10.7554/ eLife.29391. Singh, U., Brewer, J.L., Boothroyd, J.C., 2002. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Mol. Microbiol. 44, 721
733. Sinha, A., Hughes, K.R., Modrzynska, K.K., Otto, T.D., Pfander, C., Dickens, N.J., et al., 2014. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature 507, 253
257. Sivagurunathan, S., Heaslip, A., Liu, J., Hu, K., 2013. Identification of functional modules of AKMT, a novel lysine methyltransferase regulating the motility of Toxoplasma gondii. Mol. Biochem. Parasitol. 189, 43
53. Smith, E.R., Eisen, A., Gu, W., Sattah, M., Pannuti, A., Zhou, J., et al., 1998. ESA1 is a histone acetyltransferase that is essential for growth in yeast. Proc. Natl. Acad. Sci. U.S.A. 95, 3561
3565. Smith, A.T., Tucker-Samaras, S.D., Fairlamb, A.H., Sullivan Jr., W.J., 2005. MYST family histone acetyltransferases in the protozoan parasite Toxoplasma gondii. Eukaryot. Cell 4, 2057
2065. Soldati, D., Boothroyd, J.C., 1995. A selector of transcription initiation in the protozoan parasite Toxoplasma gondii. Mol. Cell. Biol. 15, 87
93. Song, C.P., Agarwal, M., Ohta, M., Guo, Y., Halfter, U., Wang, P., et al., 2005. Role of an Arabidopsis AP2/ EREBP-type transcriptional repressor in abscisic acid and drought stress responses. Plant Cell 17, 2384
2396. Spano, F., Ricci, I., Di Cristina, M., Possenti, A., Tinti, M., Dendouga, N., et al., 2002. The SAG5 locus of Toxoplasma gondii encodes three novel proteins belonging to the SAG1 family of surface antigens. Int. J. Parasitol. 32, 121
131. Spector, D.L., 2003. The dynamics of chromosome organization and gene regulation. Annu. Rev. Biochem. 72, 573
608. Sterner, D.E., Berger, S.L., 2000. Acetylation of histones and transcription-related factors. Microbiol. Mol. Biol. Rev. 64, 435
459. Stilger, K.L., Sullivan Jr., W.J., 2013. Elongator protein 3 (Elp3) lysine acetyltransferase is a tail-anchored mitochondrial protein in Toxoplasma gondii. J. Biol. Chem. 288, 25318
25329. Strahl, B.D., Allis, C.D., 2000. The language of covalent histone modifications. Nature 403, 41
45. Stray-Pedersen, B., Lorentzen-Styr, A.M., 1980. Epidemiological aspects of Toxoplasma infections among women in Norway. Acta Obstet. Gynecol. Scand. 59, 323
326. Su, C., Evans, D., Cole, R.H., Kissinger, J.C., Ajioka, J.W., Sibley, L.D., 2003. Recent expansion of Toxoplasma through enhanced oral transmission. Science 299, 414
416.

Sugi, T., Ma, Y.F., Tomita, T., Murakoshi, F., Eaton, M.S., Yakubu, R., et al., 2016. Toxoplasma gondii cyclic AMPdependent protein kinase subunit 3 is involved in the switch from tachyzoite to bradyzoite development. mBio 7. Available from: https://doi.org/10.1128/mBio.00755-16. Sullivan Jr., W.J., 2003. Histone H3 and H3.3 variants in the protozoan pathogens Plasmodium falciparum and Toxoplasma gondii. DNA Seq. 14, 227
231. Sullivan Jr., W.J., Monroy, M.A., Bohne, W., Nallani, K.C., Chrivia, J., Yaciuk, P., et al., 2003. Molecular cloning and characterization of an SRCAP chromatin remodeling homologue in Toxoplasma gondii. Parasitol. Res. 90, 1
8. Sullivan Jr., W.J., Narasimhan, J., Bhatti, M.M., Wek, R.C., 2004. Parasite-specific eIF2 (eukaryotic initiation factor2) kinase required for stress-induced translation control. Biochem. J. 380, 523
531. Suvorova, E.S., Croken, M., Kratzer, S., Ting, L.M., Conde de Felipe, M., Balu, B., et al., 2013. Discovery of a splicing regulator required for cell cycle progression. PLoS Genet. 9, e1003305. Thiagalingam, S., Cheng, K.H., Lee, H.J., Mineva, N., Thiagalingam, A., Ponte, J.F., 2003. Histone deacetylases: unique players in shaping the epigenetic histone code. Ann. N.Y. Acad. Sci. 983, 84
100. Utley, R.T., Cote, J., 2003. The MYST family of histone acetyltransferases. Curr. Top. Microbiol. Immunol. 274, 203
236. Van Poppel, N.F., Welagen, J., Vermeulen, A.N., Schaap, D., 2006. The complete set of Toxoplasma gondii ribosomal protein genes contains two conserved promoter elements. Parasitology 133, 19
31. Vattem, K.M., Wek, R.C., 2004. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 101, 11269
11274. Vervenne, R.A., Dirks, R.W., Ramesar, J., Waters, A.P., Janse, C.J., 1994. Differential expression in blood stages of the gene coding for the 21-kilodalton surface protein of ookinetes of Plasmodium berghei as detected by RNA in situ hybridisation. Mol. Biochem. Parasitol. 68, 259
266. Volz, J.C., Bartfai, R., Petter, M., Langer, C., Josling, G.A., Tsuboi, T., et al., 2012. PfSET10, a Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state during parasite division. Cell Host Microbe 11, 7
18. Vonlaufen, N., Naguleswaran, A., Coppens, I., Sullivan Jr., W.J., 2010. MYST family lysine acetyltransferase facilitates ataxia telangiectasia mutated (ATM) kinasemediated DNA damage response in Toxoplasma gondii. J. Biol. Chem. 285, 11154
11161. Voss, T.S., Kaestli, M., Vogel, D., Bopp, S., Beck, H.P., 2003. Identification of nuclear proteins that interact differentially with Plasmodium falciparum var gene promoters. Mol. Microbiol. 48, 1593
1607.

Toxoplasma Gondii

References

Waldman, B.S., Schwarz, D., Wadsworth, M.H., Saeij, J.P., Shalek, A.K., Lourido, S., 2019. Identification of a master regulator of differentiation in Toxoplasma. bioRxiv ,https://www.biorxiv.org/content/10.1101/660753v1.. Walker, R., Gissot, M., Croken, M.M., Huot, L., Hot, D., Kim, K., et al., 2013. The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation. Mol. Microbiol. 87, 641
655. Wang, J., Liu, X., Jia, B., Lu, H., Peng, S., Piao, X., et al., 2012. A comparative study of small RNAs in Toxoplasma gondii of distinct genotypes. Parasites Vectors 5, 186. Wang, J., Dixon, S.E., Ting, L.M., Liu, T.K., Jeffers, V., Croken, M.M., et al., 2014. Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation. PLoS Pathog. 10, e1003830. Webb, H., Burns, R., Ellis, L., Kimblin, N., Carrington, M., 2005a. Developmentally regulated instability of the GPIPLC mRNA is dependent on a short-lived protein factor. Nucleic Acids Res. 33, 1503
1512. Webb, H., Burns, R., Kimblin, N., Ellis, L., Carrington, M., 2005b. A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes. RNA 11, 1108
1116. Wei, Y., Mizzen, C.A., Cook, R.G., Gorovsky, M.A., Allis, C.D., 1998. Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena. Proc. Natl. Acad. Sci. U.S.A. 95, 7480
7484. Weiss, L.M., Kim, K., 2000. The development and biology of bradyzoites of Toxoplasma gondii. Front. Biosci. 5, D391
D405. Wek, R.C., Jiang, H.Y., Anthony, T.G., 2006. Coping with stress: eIF2 kinases and translational control. Biochem. Soc. Trans. 34, 7
11. Wuitschick, J.D., Lindstrom, P.R., Meyer, A.E., Karrer, K. M., 2004. Homing endonucleases encoded by germ linelimited genes in Tetrahymena thermophila have APETELA2 DNA binding domains. Eukaryot. Cell 3, 685
694. Xiao, H., El Bissati, K., Verdier-Pinard, P., Burd, B., Zhang, H., Kim, K., et al., 2010. Post-translational modifications to Toxoplasma gondii alpha- and beta-tubulins include novel C-terminal methylation. J. Proteome Res. 9, 359
372. Xu, W., Edmondson, D.G., Roth, S.Y., 1998. Mammalian GCN5 and P/CAF acetyltransferases have homologous amino-terminal domains important for recognition of nucleosomal substrates. Mol. Cell. Biol. 18, 5659
5669. Xu, W., Edmondson, D.G., Evrard, Y.A., Wakamiya, M., Behringer, R.R., Roth, S.Y., 2000. Loss of Gcn5l2 leads to

981

increased apoptosis and mesodermal defects during mouse development. Nat. Genet. 26, 229
232. Xu, W., Cho, H., Kadam, S., Banayo, E.M., Anderson, S., Yates, J.R., et al., 2004. A methylation-mediator complex in hormone signaling. Genes Dev. 18, 144
156. Xue, B., Dunker, A.K., Uversky, V.N., 2012. Orderly order in protein intrinsic disorder distribution: disorder in 3500 proteomes from viruses and the three domains of life. J. Biomol. Struct. Dyn. 30, 137
149. Xue, Y., Theisen, T.C., Rastogi, S., Ferrel, A., Quake, S.R., Boothroyd, J.C., 2019. A single-parasite transcriptional landscape of asexual development in Toxoplasma gondii. bioRxiv 656165. ,https://doi.org/10.1101/656165.. Yakubu, R.R., Silmon de Monerri, N.C., Nieves, E., Kim, K., Weiss, L.M., 2017. Comparative monomethylarginine proteomics suggests that protein arginine methyltransferase 1 (PRMT1) is a significant contributor to arginine monomethylation in Toxoplasma gondii. Mol. Cell. Proteomics 16, 567
580. Yamagishi, J., Wakaguri, H., Ueno, A., Goo, Y.K., Tolba, M., Igarashi, M., et al., 2010. High-resolution characterization of Toxoplasma gondii transcriptome with a massive parallel sequencing method. DNA Res. 17, 233
243. Yamauchi, T., Yamauchi, J., Kuwata, T., Tamura, T., Yamashita, T., Bae, N., et al., 2000. Distinct but overlapping roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis. Proc. Natl. Acad. Sci. U.S.A. 97, 11303
11306. Yang, S., Parmley, S.F., 1997. Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts. Gene 184, 1
12. Yuda, M., Iwanaga, S., Shigenobu, S., Mair, G.R., Janse, C. J., Waters, A.P., et al., 2009. Identification of a transcription factor in the mosquito-invasive stage of malaria parasites. Mol. Microbiol. 71, 1402
1414. Yuda, M., Iwanaga, S., Shigenobu, S., Kato, T., Kaneko, I., 2010. Transcription factor AP2-Sp and its target genes in malarial sporozoites. Mol. Microbiol. 75, 854
863. Zhang, Y., Reinberg, D., 2001. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev. 15, 2343
2360. Zhang, M., Fennell, C., Ranford-Cartwright, L., Sakthivel, R., Gueirard, P., Meister, S., et al., 2010. The Plasmodium eukaryotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the mosquito salivary glands. J. Exp. Med. 207, 1465
1474. Zhang, Q., Huang, Y., Zhang, Y., Fang, X., Claes, A., Duchateau, M., et al., 2011. A critical role of perinuclear filamentous actin in spatial repositioning and mutually exclusive expression of virulence genes in malaria parasites. Cell Host Microbe 10, 451
463.

Toxoplasma Gondii

982

21. Regulation of gene expression in Toxoplasma gondii

Zhang, M., Mishra, S., Sakthivel, R., Rojas, M., Ranjan, R., Sullivan Jr., W.J., et al., 2012. PK4, a eukaryotic initiation factor 2alpha(eIF2alpha) kinase, is essential for the development of the erythrocytic cycle of Plasmodium. Proc. Natl. Acad. Sci. U.S.A. 109, 3956
3961. Zhang, M., Gallego-Delgado, J., Fernandez-Arias, C., Waters, N.C., Rodriguez, A., Tsuji, M., 2017. Inhibiting

the Plasmodium eIF2alpha kinase PK4 prevents artemisinin-induced latency. Cell Host Microbe 22, 766
776.e4. Zheng, L., Roeder, R.G., Luo, Y., 2003. S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 114, 255
266.

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C H A P T E R

22 Proteomics and posttranslational protein modifications in Toxoplasma gondii Louis M. Weiss1,2, Jonathan Wastling3, Victoria Jeffers4, William J. Sullivan, Jr5,6 and Kami Kim7,8 1

Department of Pathology Albert Einstein College of Medicine, Bronx, NY, United States Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, United States 3Faculty of Natural Sciences, University of Keele, Keele, Staffordshire, United Kingdom 4Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, United States 5Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, IN, United States 6Department of Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, IN, United States 7Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States 8Global Health Infectious Diseases Research Program, College of Public Health, University of South Florida, Tampa, FL, United States 2

22.1 Introduction to Toxoplasma gondii proteomics The identification and characterization of the function of proteins in Toxoplasma gondii has been a focus of many research groups since the development of techniques that enabled the study of individual genes and molecules. Among these active research, topics have been studies of the mechanism(s) of host cell invasion, the structure and composition of the apical organelles, stage conversion and the organization of the cytoskeleton. Proteomic

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00022-0

studies provide complementary data to those obtained by transcriptional “omics” approaches, for example, microarrays, serial analysis of gene expression, and RNA-Seq (Blader et al., 2001; Cleary et al., 2002; Matrajt et al., 2002; Sibley et al., 2002; Singh et al., 2002; Radke et al., 2005; Reid et al., 2012). As has been seen with other eukaryotes, observed gene transcription and observed protein expression do not necessary have a linear correlation (Nagaraj et al., 2011; Schwanhausser et al., 2011; Wastling et al., 2009; Krishna et al., 2015). Proteomics also provides information on

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the posttranslational modifications (PTMs), for example, phosphorylation and other modifications, which can affect a protein’s state of activation. Finally, the activity and function of a protein are often dependent on protein protein interactions, and proteomic studies can assist in identifying and understanding the nature of these interactions. Proteomics bridges the gap between genome and transcriptome data and the functional biological processes intrinsic to an organism. Developed in the late 1990s proteomics is now an accepted part of a “systems biology” approach to understanding biological problems (Wastling et al., 2012). Prior to the advent of genome-wide data and the evolution of the “post-genomic era”, a typical T. gondii experiment would have begun with the identification of a single gene or protein associated with a particular biology event. Detailed in-depth studies would then follow, including sequencing, cloning, and expression of the gene of interest, to provide localization and other functional data; mapping the gene by Southern blotting, its transcriptional expression by Northern blotting, and the expression of the protein for which it codes by immunoblotting. Many excellent studies founded on the above “hypothesis-driven” approach continue to provide evidence for our present understanding of T. gondii. A criticism of proteomics has been that it does not follow a classical “hypothesisdriven” research approach. On the other hand a key advantage of proteomics is that it does not prejudge the outcome of an analysis since it employs an unbiased survey of genes and proteins that are associated with specific biological events. Rather than hypothesis testing, proteomic experiments are an important contributor to hypothesis generation. Proteomic and other “omic” datasets can, therefore, be seen an integral component to an iterative process of hypothesis development and testing. Analysis of these datasets generates multiple hypotheses that can be tested directly in T.

gondii using classical experimentation. A general scheme for a proteomics experiment is shown in Fig. 22.1. Proteomics experiments entail experimental design, sample preparation (biological and protein), data acquisition, data processing and database searching, and interpretation and validation of results. There are many excellent reviews on proteomics in the literature (Aebersold and Mann, 2003; Bradshaw and Burlingame, 2005; Dhingra et al., 2005; Gorg et al., 2004; Johnson et al., 2004; Lane, 2005; Morrison et al., 2003; Phillips and Bogyo, 2005; Yates, 2004; Cox and Mann, 2011; Malmstrom et al., 2011). While the mass spectrometry (MS) data acquisition may represent the most challenging aspect to a biologist, biology is what drives good proteomics experiments. MS enables the mass measurement of ions derived from molecules and is capable of forming, separating, and detecting molecular ions based on their mass-to-charge ratio (m/z). Matrixassisted laser desorption/ionization MS (MALDI-MS) and electro-spray ionization tandem MS (ESI MS/MS) are the commonly used approaches for collection of proteomic data. The MS dataset consists of the precise mass (m/z ratios) of peptides obtained following the digestion of proteins with an enzyme, for example, trypsin. These masses give rise to a unique peptide mass fingerprint when using MALDI-MS, which can be matched to the theoretical predicted masses of peptides obtained by digestion with a particular enzyme. In ESI MS/MS the peptides can be further fragmented to obtain the amino acid sequence data (e.g., MS/MS) of a particular peptide, which can then be matched to the predicted peptide sequences in a genome. ESI MS/MS also provides data for determining peptide modifications, whereas deviation from the expected molecular mass with MALDI is indicative of some type of PTM, but would not identify the site of PTM. Well-defined modifications, for example, phosphorylation, sulfation, and

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FIGURE 22.1 A general scheme for a proteomics project. A typical workflow for an identification and quantification proteomics project is achieved by integrating two pipelines, the experiment pipeline and the bioinformatics pipeline. Experiment pipeline consists of four major steps: sample collection, protein extraction and purification, sample fractionation and digestion (in either protein or peptide space), and mass spectrometry analysis. The raw spectra data collected are then subjected to bioinformatics pipeline, where protein identifications are acquired based on the comparison between raw data and protein sequence database. Quantification packages are then used to extract and calculate relative or absolute quantifications of the analyte proteins. The biological meaning of the identification and quantification data is inferred by using tools that provide protein function and localization predictions and pathway analysis. Proteogenomic and database integration pipelines are also developed to facilitate data integration with online databases and improve genome.

myristylation, can often be inferred by comparing the partial sequence of the modified peptide obtained from MS/MS with the increase in mass of a peptide, as particular modifications increase the predicted size of protein by a characteristic mass. This ability to determine peptide modifications is critical for MS-based characterization of PTMs of proteins.

In addition to defining the proteins that are present, as well as their PTMs, proteomics can also provide quantitative data on the abundance of proteins in a sample. As it is difficult to get absolute quantification, most proteomic data provide relative quantitation. One can obtain quantitative data by two general approaches: label-based methods and label-free

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methods. One commonly used label-based method is Stable Isotope Labeling with Amino acids in Cell culture (SILAC) (Ong et al., 2002), which relies on the incorporation of a heavy amino acid (e.g., a 13C containing amino acid) to produce a labeled (heavy) protein sample. One can then analyze a sample containing proteins from cells that have been labeled and exposed to a particular treatment to those not exposed to that treatment in the same MS run. Relative abundance of particular peptides is inferred as the heavy and light peptides differ in m/z due to the 13C-containing amino acids and provide tags identifying the source of the particular peptide. The same approach can also be performed using chemical labeling techniques to define a sample, such as isotope-coded affinity tags, applied to cysteine residues (Gygi et al., 1999), or isobaric tags for relative and absolute quantification (iTRAQ), which uses a multiplexed set of isobaric reagents that yield amine-derivatized peptides for quantification (Ross et al., 2004). To allow multiple comparisons, the control condition is often tagged, which creates a reference sample that can be added to other samples for relative quantification of protein levels. With the increasing throughput and accuracy of modern mass spectrometers, label-free quantification methods that directly use raw spectral data from parallel MS runs to determine relative protein abundance are now more commonly used. This approach has been implemented by several commercial software packages such as Progenesis LC (liquid chromatography) MS (Nonlinear Dynamics) on all unique peptides and Protein Lynx Global Server (Waters) on the top 3 unique peptides. Open source packages such as MaxQuant (Cox and Mann, 2008), OpenMS (Sturm et al., 2008) and MSight (Palagi et al., 2005) are also available. A requirement of high-quality MS experiments is high-quality genome sequence and its associated protein prediction models, which

produces the virtual genome/proteome to which the observed experimental MS data is matched. T. gondii research has been revolutionized by the available genome sequences, transcriptome, and proteomic datasets that are publicly accessible at ToxoDB (http://www. toxodb.org) (Kissinger et al., 2003; Gajria et al., 2008). Genome sequence data are available for many of the known haplotypes of T. gondii (see Chapter 3: Molecular epidemiology and population structure of Toxoplasma gondii) as well as related Apicomplexa such as Neospora caninum, Hammondia hammondi, Sarcoystis neurona, and various Eimeria spp. In particular, ToxoDB has the complete genomes of ME49 (a Type II strain that serves as the reference genome), GT-1 (a Type I strain able to complete the entire life cycle both sexual and asexual), and VEG (a Type III strain). Alignments of the sequences of strains are available as genetic maps. Expression data and proteomic data are also available on this site as is the sequence data for chromosome Ia and Ib from RH strain (Gajria et al., 2008; Khan et al., 2006; Kissinger et al., 2003). Proteomic identification is entirely dependent on the accuracy of the associated gene models against which MS data is searched. Proteomic data represent experimentally defined translated and expressed genome sequences and can provide valuable information for genome annotation about intron exon boundaries, frameshifts, and N-terminal methionine excision, which are difficult to predict by in silico pipelines or transcriptomics studies (Silmon de Monerri and Weiss, 2015; Krishna et al., 2015; Krishna et al., 2011). Unmatched peptides contain information that may assist in correcting gene models and provide key input for training gene finding algorithms (Krishna et al., 2015; Silmon de Monerri and Weiss, 2015). A confounding issue in the results of proteomics experiments in any organism is the quality of the gene prediction databases at the time of the analysis. One cannot be certain that a

Toxoplasma Gondii

22.1 Introduction to Toxoplasma gondii proteomics

given protein will be present in the database, nor that a high-quality mass spectrum of a peptide(s) will identify the originating protein. For example, if intron exon junctions are predicted incorrectly, such that a protein is predicted to be two gene products instead of one, two identified peptides might be incorrectly mapped to two adjacent genes. Since current criteria for publishing MS data require at least two peptide hits per polypeptide, the data can be lost. Use of the highest resolution mass spectrometer available may permit the use of “one-hit wonders” for protein identification and facilitates de novo sequencing of peptides, which is not dependent upon databases. Many bioinformatic tools exist to evaluate the enormous datasets generated by proteomic studies; however, improved tools are still needed to integrate this data into gene annotation systems (Kremer et al., 2005; Souchelnytskyi, 2005; Hamady et al., 2005; Haley-Vicente and Edwards, 2003; Jones et al., 2004; Taylor et al., 2003; Krishna et al., 2015; Krishna et al., 2011). A comparison of proteomic data for various Apicomplexa including T. gondii with transcriptomic data (Wastling et al., 2009) revealed that proteomic data is often present for genetic loci lacking transcriptional evidence of expression. In a global study of T. gondii proteins, of the 2252 proteins identified, only 626 had EST data, 1131 had microarray data, and 72 had no demonstrated transcripts (Xia et al., 2008). Similar observations, demonstrating proteomic evidence without detectable mRNA transcripts, have been reported in mammalian cells, for example, HeLa cells (Cox and Mann, 2007). Differences in the expressed genes identified by proteomic and transcriptional datasets may be due to posttranslational control mechanisms as well as the “stock and go hypothesis” described in Plasmodium (Mair et al., 2006). Advances such as improved subfractionation of complex protein mixtures, protocols for separation and analysis of Apicomplexan subproteomes, and new bioinformatics resources

987

for computational analysis have been applied to the study of Apicomplexan proteins. Large scale proteomics approaches have been used to analyze various organisms such as Saccharomyces cerevisiae (Washburn et al., 2001), Mycoplasma mobile (Jaffe et al., 2004), Cryptosporidium parvum (Sanderson et al., 2008), T. gondii (Dybas et al., 2008; Xia et al., 2008), and Streptomyces luteogriseus (Wang et al., 2005b). Targeted studies of T. gondii rhoptry (Bradley et al., 2005), secretory (Zhou et al., 2005), and microneme (Zhou et al., 2004) proteins highlight the value of applying proteomics to explore important subproteomes. Proteomics has also been used in T. gondii to elucidate the role of PTMs, for example, glycosylation (Tomita et al., 2017; Wang et al., 2016; Luo et al., 2011; Fauquenoy et al., 2008), acetylation (Jeffers and Sullivan, 2012; Wang et al., 2014; Bouchut et al., 2015; Varberg et al., 2016), methylation (Yakubu et al., 2017), ubiquitination (Silmon de Monerri et al., 2015) SUMOylation (Braun et al., 2009), and phosphorylation (Al-Bajalan et al., 2017; Gaji et al., 2015; Jacot et al., 2014; Treeck et al., 2011). Proximity-dependent biotin identification coupled to MS (BioID MS) has provided an important tool for both defining protein complexes and for defining subproteomes of T. gondii, including inner membrane complex (IMC) proteins (Chen et al., 2015, 2017) and dense granule proteins (GRAs) (Nadipuram et al., 2016). BioID uses a bait protein of interest fused to a prokaryotic biotin ligase (BirA) to biotinylate prey protein in its proximity upon expression in the cell. The biotinylated complexes may then be purified by an avidin/ streptavidin based biotin affinity approach. The isolated biotinylated proteins can then be identified by MS to generate a list of potential interacting prey proteins (Roux et al., 2012). Since this is a proximity-based method where interactions are covalently fixed through biotinylation, it is ideal for detecting weak or transient interactions, and it has been found to be

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22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

more effective than immunoprecipitation followed by MS (IP-MS) in terms of identifying low-abundance proteins (Roux et al., 2012; Lambert et al., 2015). Tagging of proteins with BioID has been facilitated in T. gondii by using CRISPR approaches (Long et al., 2018). The proteome of any cell consists of a heterogeneous mixture of both soluble and hydrophobic proteins that are present in a large dynamic range. To overcome this challenge of complexity, it is useful to simplify the protein mixture prior to analysis. Further fractionation of the proteins or peptides is needed to reduce sample complexity prior to MS analysis. This separation may involve 2D-gel based analysis of proteins followed by MS analysis of digested spots or 1D-gel-based analysis of proteins with digestion of the bands followed by nano-LC and then MS analysis. It may involve a global shot gun approach (i.e., MudPIT) in which proteins are digested and then peptides separated by two serial liquid chromatographic procedures followed by MS analysis of the total protein profile. Each method has advantages, and these should be viewed as complementary strategies. MudPIT has been successfully used to obtain proteomes of a number of protozoan parasites, including Plasmodium falciparum (Florens et al., 2002; Lasonder et al., 2002) Trypanosoma cruzi (Paba et al., 2004), C. parvum (Sanderson et al., 2008) as well as the tachyzoites of T. gondii (Xia et al., 2008). Different instruments, such as MALDI-TOF/TOF and various electrospray ionization instruments (QqTOFs, ion traps), may produce overlapping, but not identical, protein lists. Data processing and database searches are the link between MS and biology. It is important to understand that the parameters that are set in the primary data processing (e.g., conversion to dta or mgf files) can affect search results, as can the settings used in the search engines. Furthermore, each search engine (e.g., Sequest, Mascot, X!Tandem, or Spectrum Mill) may provide overlapping, but different,

protein lists. Software programs can evaluate results obtained from different search protocols. Another tool used to evaluate the quality of the MS peptide hits is the False Discovery Rate (FDR). Data can be searched automatically against the gene prediction database and against a scrambled version of the database. The FDR is a measure of the percent of MS spectra that generate hits in the scrambled database and are thus suspect.

22.2 Toxoplasma gondii global proteomics The tachyzoite stage of T. gondii has been the main focus of the majority of global proteomic studies due to its ease of propagation in the laboratory allowing the generation of large amounts of biological material for analysis. Global proteomics experiments provide extensive lists of protein “hits”, without a biological reference except the tissue or organism. To identify all proteins in a cell or tissue, whatever the level of abundance (representation), requires extensive fractionation of proteins or peptides since the levels of expression may range across many orders of magnitude. Several rationales can justify a global proteomics effort. As discussed elsewhere in this chapter, gene prediction algorithms are still imperfect, particularly in organisms with many introns. It should be noted that it is possible for a particular protein to be completely missing from a gene prediction database. Therefore the MS data may need to be interpreted de novo and orthogonal validation carried out. Another reason for carrying out an exhaustive global proteomics experiment is to quantitate changes in protein expression between cells, tissues, or a developmental state, such as tachyzoites and bradyzoites. An early global proteomics effort (Cohen et al., 2002) defined a protocol for a reproducible 2DE map of RH tachyzoites, resolving over

Toxoplasma Gondii

22.2 Toxoplasma gondii global proteomics

1000 polypeptides. MS was used to analyze 71 of these proteins by MALDI-MS and MALDI postsource decay analysis (Cohen et al., 2002). Several protein spots were encoded by the same gene, indicating that PTM and/or alternative splicing events occurred. Around 30 tachyzoite proteins were identified, the more highly expressed being rhoptry, dense granule, and structural proteins. This early experiment demonstrated the viability of proteomic analyses for T. gondii, even in the absence of complete genome sequence. Xia et al. (2008) identified 2252 tachyzoite proteins, estimated to be about 30% of the predicted proteins in T. gondii in ToxoDB v4, using a multiplatform proteomics approach. This analysis identified 2477 intron spanning peptides providing evidence for correct splice site annotation. At least 15% of the identified peptides matched to alternative gene models. 2DE was used to identify 1217 spots using electrospray MS. A total of 616 nonredundant proteins were identified, of which 547 corresponded to a release4 gene annotation and 69 to alternative gene models or open reading frames. LC MS/MS was used to examine SDS-soluble RH tachyzoite proteins identifying a total of 2778 proteins that collapsed to a dataset of 1012 gene products (939 release4 and 73 alternative gene models) when redundancies were removed. MudPIT was used to analyze both Tris soluble and insoluble fractions from RH tachyzoites that resulted in a final dataset of 2409 proteins (2121 release4 and 288 alternative models). 1D-LC MS/MS techniques (Dybas et al., 2008) identified 2477 gene coding regions with 6438 possible alternative gene predictions from the tachyzoite stage of this parasite, representing about one-third of the expected genome. The commonly used gene prediction algorithms produced very disparate sets of protein sequences with overlaps ranging from 1.4% to 12% and observed false negative rates of 31% to 43%. A proteomics database (EPICdb) was

989

created that combined experimental (NCBI) and predicted T. gondii genes. The amino acid sequences of the hypothetical proteome of T. gondii were searched against the complete NCBI NR database, Apicomplexa proteins, and human proteins to identify unique and conserved proteins. 67% of the hypothetical T. gondii proteome had a homologous sequence in NR and 64% an Apicomplexan ortholog. Approximately 52% of T. gondii sequences were unique as compared to the human genome. 3838 (60%) of the experimentally identified proteins have been annotated as “hypothetical,” “putative,” or “predicted” in the NCBI NR database. In addition, 609 proteins identified by MS in T. gondii proteins were unique as compared to any known organisms. These findings provided proteomics evidence that improvements were needed in the T. gondii genome annotation available at that time (Madrid-Aliste et al., 2009). Environmentally resistant oocysts have also been examined by a global proteomics approach (Fritz et al., 2012; Zhou et al., 2016). Using a 1-DE LC MS/MS approach, 1021 T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 proteins were identified in the oocyst wall fraction of mature, sporulated oocysts (Fritz et al., 2012). Of these, 172 proteins had not been reported in other T. gondii proteomic studies, confirming the expression of stage specific proteins. This study identified a family of small, tyrosine-rich proteins present in the oocyst wall fractions, and late embryogenesis abundant domain containing proteins in the cytosolic fractions have been suggested to be involved in environmental resistance in oocysts. Zhou et al. (2016) used iTRAQ-based proteomic profiling to examine oocysts during sporulation and identified 2095 proteins of which 587 were differentially expressed. Analysis by STRING-10 and gene ontology (GO) terms suggested that metabolism related proteins were important in oocyst development and sporulation; in

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22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

particular, fatty acid metabolism appeared to be important in this process (Zhou et al., 2016). Wang et al. (2017) examined the global proteome of tachyzoites (T), bradyzoites (B), and sporulated oocysts (O) from T. gondii type II Prugniuad strain. A total of 6285 proteins were identified from these life cycle stages, of which differential expression was seen in 875 (O vs T), 656 (T vs C), and 538 (C vs O) proteins.

22.3 Toxoplasma gondii subproteomes Subproteome analysis relies on the preparation or enrichment of a smaller fraction of the total proteome, which decreases the complexity of the total proteome, thereby extending the dynamic range of the analytic strategy. This approach is particularly useful when the study is restricted to the proteins of a particular organelle, especially when a method for purifying the organelle has been established (Yates et al., 2005). An early example of this approach is the analysis of the excretory secretory products of T. gondii (Zhou et al., 2005). As for other proteomic data, validation is critical in order to have confidence that a protein is found in a particular organelle, especially as cytoplasmic proteins may interact with organelle proteins during purification. Careful sample preparation with confirmed enrichment of the structure of interest followed by validation of localization of proteins of interest by either tagging or immunolocalization are important quality-control steps in such studies. As an obligate intracellular parasite, T. gondii needs to successfully invade host cells and subsequently produce an intracellular environment in which it can acquire nutrients, yet avoid being killed by its host cell. The micronemes, rhoptries, and dense granules are specialized secretory organelles that are involved in cell invasion and the subsequent remodeling of host cells and were an early focus of proteomics.

Bradley et al. (2005) developed a procedure to purify rhoptry organelles. At the time of this proteomic study the known rhoptry proteins were ROP1, ROP2, ROP4, ROP8, and ROP9 (Beckers et al., 1994, 1997; Ossorio et al., 1992; Reichmann et al., 2002; Sadak et al., 1988). In addition, three other rhoptry proteins had been identified by others, including subtilisin-like protein TgSUB2, cathepsin B-like protein, Toxopain-1, and a sodium hydrogen exchanger TgNHE2 (Karasov et al., 2005; Miller et al., 2003; Que et al., 2002). To purify rhoptries, Bradley et al. modified a percoll gradient method developed by Dubremetz et al. (Leriche and Dubremetz, 1991) and subjected the fraction to sucrose gradient floatation (Bradley et al., 2005). Analysis of this fraction by immunoblots employing antibodies to known proteins from the rhoptries, dense granules, and mitochondria demonstrated enrichment of rhoptries while effectively removing the vast majority of dense granules and contaminating mitochondria. The majority of rhoptry proteins of interest (Bradley et al., 2005), as demonstrated using carbonate extraction and immunoblot, were found within the membrane fraction and not amenable to analysis by 2-DE. Therefore a 1D-LC MS/MS approach was used. The 1D gel was divided into 51 slices, subjected to in-gel trypsin digestion and MS/MS to obtain peptide fragmentation data suitable for proteomic database searching. 38 previously unidentified candidate rhoptry proteins were detected. A combination of approaches was used to verify the localization of the identified proteins, including epitope tagging and the production of antibodies against peptides and recombinant proteins. Twelve of 13 proteins for which antisera were produced localized to the rhoptries. Proteins identified in the rhoptry fraction were ROP1, ROP2, ROP4, ROP5, ROP8, ROP9, ROP10, ROP11, ROP12, ROP13, ROP14, ROP15, ROP16, RON1, RON2, RON3, RON4, Toxofilin, and Rab11 (Bradley et al., 2005).

Toxoplasma Gondii

22.3 Toxoplasma gondii subproteomes

A major discovery was the recognition of rhoptry neck proteins (RONs) that were later demonstrated to be critical in the formation of the moving junction during host cell invasion. El Hajj et al. (2006) used IP-MS analysis to identify and characterize the ROP2 family proteins consisting of ROP2, ROP4, ROP5, and ROP7. By BLAST analysis, they also identified ROP8 (ROP2L1), ROP11 (ROP2L2), ROP2L3, ROP2L4, ROP2L5, ROP2L6, and ROP16 as members of this family of proteins. The excreted secreted antigen (ESA) protein profile consists of microneme and GRAs (Charif et al., 1990). This repertoire of secreted proteins is surprisingly complex, with evidence for multiple redundant adhesive complexes whose components undergo extensive proteolysis during organelle maturation and host cell invasion (Zhou et al., 2004, 2005). Bioinformatic analysis of the T. gondii genome suggests that at least 800 genes encode proteins with putative secretory signal peptides (Zhou et al., 2005). ESA proteins were characterized, using 2D electrophoresis and MudPIT techniques (Zhou et al., 2004, 2005; Fauquenoy et al., 2008). Approximately 100 spots could be identified by 2D electrophoresis and most were successfully identified by protein microsequencing or MALDI-MS analysis (Zhou et al., 2004, 2005). Many proteins were present in multiple spots, consistent with the presence of PTMs and/or posttranslational processing of these proteins. MudPIT yielded additional novel adhesion proteins and hypothetical secretory proteins similar to proteins identified in Plasmodia (Fauquenoy et al., 2008). Many of the identified proteins were surface antigens and GRAs, demonstrating that the ESA preparation was not limited to micronemes. Zhou et al. (2005) characterized a subset of these novel ESA proteins by expressing them as fusion proteins with yellow fluorescent protein. This screen revealed shared and distinct localizations within the secretory compartments of T. gondii tachyzoites. Only 1 of the 38 rhoptry

991

proteins identified (Bradley et al., 2005) was found in the ESA fraction (Zhou et al., 2005), illustrating that these two subproteomes are distinct. Purified microneme proteins have also been analyzed using proteomic techniques (Carruthers, 2006; Carruthers and Tomley, 2008). Kawase et al. (2007) used A23187 to stimulate calcium-mediated excretion for proteins from T. gondii and identified 213 protein spots by 2DE. On comparison of spots identified from A23187 and DMSO treated parasites, a total of eight spots were increased. These proteins were identified as MIC2, MIC4, TgSPATR, AMA1, ROP9, MIC6, and MIC10. Hu et al. (2006) purified the conoid/apical complex of T. gondii and were able to identify 286 proteins using a MudPIT approach. These authors analyzed 10 12 replicate samples of conoid-enriched and conoid-depleted fractions resulting in the identification of 1157 proteins. Of these, about 30% were unique to each fraction and 35% were easily identified as contaminates from other subcellular organelles (e.g., ribosomal proteins, mitochondrial proteins, and microneme proteins). Validation of seven candidates was performed by either the production of antibodies to purified peptides or recombinant proteins or by molecular expression of tagged genes identified by proteomics. Of these, TgMORN1 localized to the posterior end of daughter cells and TgCAM1 and CAM2 to the conoid region. Che et al. analyzed a T. gondii membrane proteome, and identified 2241 T. gondii proteins with at least one predicted transmembrane segment. These proteins could be grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome (Che et al., 2010). To maximize the identification of membrane proteins, several different proteomic strategies were employed. In one approach, cell surface proteins were labeled with sulfo-NHS-SS-biotin and affinity purification was carried out by

Toxoplasma Gondii

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22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

using streptavidin beads. Proteins were then identified by 1-DE LC MS/MS. The other approach utilized three-layer “sandwich”-gel electrophoresis (TLSGE) (Liu et al., 2008). TLSGE concentrates relatively large volumes of protein samples on a small piece of protein gel, removing detergents and salts during electrophoresis. The concentrated protein fraction was then analyzed using MudPIT. 42% of the identified membrane proteins in this study had no known function, with many proteins being unique to T. gondii or to the Apicomplexa (Che et al., 2010). Multiple replicate samples of cytoskeletal preparations, cytosolic preparations, and membrane preparations were prepared from detergenttreated RH strain T. gondii lysed by French press and purified according to the method of Bradley et al. (2005) and analyzed by MS (Dybas et al., 2008). At least 1000 proteins were identified in this proteomic survey and are displayed at ToxoDB. Analysis by 2-DE demonstrated that in the T. gondii cytoskeletal fraction, protein isoforms and PTMs of proteins were common features. Complex modifications were present in α- and β-tubulins. The cytoskeleton of T. gondii, including T. gondii α- and β-tubulins, is extensively modified by PTM (Xiao et al., 2010). The modifications identified on α-tubulin included acetylation of Lys40, removal of the last Cterminal amino acid residue Tyr453 (detyrosinated tubulin), and truncation of the last five amino acid residues. Polyglutamylation was detected on both α- and β-tubulins. An antibody specific for mammalian α-tubulin lacking the last two C-terminal residues (Δ2-tubulin) labeled the apical region of this parasite. Detyrosinated tubulin was diffusely present in subpellicular microtubules and displayed an apparent accumulation at the basal end of tachyzoites. Methylation, a PTM not previously described on tubulin, was also identified (Xiao et al., 2010). Methylated tubulins were not detected in the human foreskin fibroblast host cells, suggesting that this may be a modification specific to the

Apicomplexa. Plessmann et al. (2004) also demonstrated that acetylation and glutamylation occur on the α-tubulin of T. gondii. Gomez de Leon et al. (2014) examined the proteome of the subpellicular cytoskeleton, isolated from detergent extracted RH tachyzoites (Patron et al., 2005). They identified 10 previously described IMC proteins, 7 new proteins with alveolin-like repeats, 10 conventional cytoskeletal proteins, 25 proteins of unknown function, and 37 proteins associated with other organelles (Gomez de Leon et al., 2014). Chen et al. (2015) utilized proximity-dependent BioID to characterize the composition of the T. gondii IMC utilizing ISP-3 as bait. Several previously described IMC proteins in both the alveoli and cytoskeletal network were identified (Chen et al., 2015). In addition, many new IMC proteins, apical cap proteins, and proteins that were unique to the sutures (ISC proteins) of the alveolar sacs were identified (Chen et al., 2015). Further extending this approach, ISC4 was labeled with BioID and used as bait for proteomic analysis (Chen et al., 2017), which resulted in the identification of five new proteins that localized the transverse sutures (called TSC proteins) as well as numerous other proteins found in the IMC. Several of the identified proteins were validated using immunolocalization and genetic techniques. BioID was also used to identify new GRA proteins within the PV using C-terminal BirA tagged GRA17 (a vacuolar pore component) as bait (Nadipuram et al., 2016). After labeling with biotin and purification by streptavidin chromatography and MS/MS analysis, a large number of known GRAs, for example, MAG1, MAF1, GRAs, cathepsin, and NTPaseII were identified. A total of 279 proteins were identified, with the highest ranking proteins being secreted proteins. BioID was then repeated using GRA13-BirA and GRA25-BirA. The proteins identified by GRA13-BirA, GRA25-BirA, and GRA17-BirA included most of the known GRA proteins (Nadipuram et al., 2016).

Toxoplasma Gondii

22.4 Toxoplasma gondii posttranslational modifications

They also identified a large number of hypothetical proteins, which were filtered for the presence of a predicted signal sequence, lack of a predicted endoplasmic reticulum retention signal (K/HDEL), and lack of other known identifiable domains. From this new GRA candidate list of 100 proteins, 13 of 15 selected for validation colocalized with GRA14 and were hence named GRA28 to GRA40 (Nadipuram et al., 2016). A similar approach using GRA1BirA and GRA1-APEX identified additional GRA proteins (Pan et al., 2018). Of 46 potential GRA proteins from their filtered datasets, 20 were known GRA proteins and 17 of the 20 were seen in both the GRA1-BioID and GRA-APEX proteomic data. Of the 26 other proteins, 5 were validated by immunolocalization to be new GRA proteins (TgGT1_200360, TGGT1_203600, TGGT1_309760, TGGT1_319340, and TGGT1_308970) (Pan et al., 2018). Tu et al. (2019a) used MS/MS to analyze tissue cyst wall fragments that were enriched from infected cells (disrupted by a ball-bearing homogenizer) by percoll gradient and IP with CST1 antibody magnetic particles. Known cyst wall proteins, including CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation, as well as GRA proteins not previously shown to associate with the cyst wall and uncharacterized hypothetical proteins. The hypothetical proteins were epitope tagged and immunolocalized, confirming that they were newly identified cyst wall/ matrix proteins (named CST2 to CST6) (Tu et al., 2019a). These proteins may be a subset of GRA proteins that specifically localize to the cyst wall during bradyzoite differentiation. In addition, BioID was used to identify additional new CST proteins by using CST1-BirA and other proteins known to localized to the cyst wall as bait (Tu et al., 2019b); these studies are defining the cyst wall protein interactome. The BioID approach identified CST2 to CST6 (Tu et al., 2019a) as well as several new proteins in the cyst wall and matrix (CST 7 to CST10).

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BioID employing TgHSP60-APEX and TgHSP60-BirA was used to identify T. gondii mitochondrial proteins (Seidi et al., 2018). A total of 421 potential mitochondrial proteins were identified (Seidi et al., 2018), including a divergent cytochrome c oxidase (TgAPiCox25) and other apicomplexan-specific components of COX. A total of 150 of the 421 proteins were hypothetical and an additional 140 had no previously defined role or experimentally determined localization in T. gondii. Twenty two of 37 of these uncharacterized proteins were localized to the mitochondrion by immunostaining.

22.4 Toxoplasma gondii posttranslational modifications PTMs are enzyme-catalyzed functional group modifications to proteins (Ohtsubo and Marth, 2006). The enzymes involved in producing PTMs (kinases, acetyl-transferases, ubiquitin ligases, methyl-transferases, etc.) are under tight dynamic transcriptional regulation and are often themselves regulated by PTMs, leading to a complex network of PTM interactions. PTMs increase the complexity of proteomes and play a role in almost all aspects of cell biology, allowing for fine-tuning of protein structure, function and localization. They can alter protein localization, affect protein protein interactions, activate or inactivate a protein’s function. There are an estimated 300 PTMs (Walsh et al., 2005), which are largely conserved across species. Among the most commonly characterized and studied PTMs are phosphorylation, glycosylation, acetylation, methylation, and ubiquitination (Witze et al., 2007). PTMs that have been found in global proteomic studies in T. gondii include phosphorylation, methylation, acetylation, palmitoylation, SUMOylation, and ubiquitination (see Table 22.1). Glycosylation is also a PTM seen in T. gondii (Luo et al., 2011). As PTMs can be transient and often substoichiometric (i.e., they occur on some but not all molecules of a protein),

Toxoplasma Gondii

TABLE 22.1

Global proteomic studies of posttranslational modifications (PTMs) in Toxoplasma gondii.

Parasite stage

PTM

PTM sites

Proteins

Percentage of proteome coverage

Intracellular tachyzoite

Lysine acetylation

411

274

Extracellular tachyzoite

Lysine acetylation

571

Intra- and extracellular tachyzoite

Arginine methylation

618

Intracellular tachyzoites

Detection/ purification method

Mass spectrometer

Reference

3.30

Antiacetylated lysine antibody

LTQ-Orbitrap Velos mass spectrometer

Jeffers and Sullivan (2012)

386

4.60

Antiacetylated lysine antibody (cell signaling technology)

LTQ-Orbitrap Velos mass spectrometer

Xue et al. (2013)

370

4.50

Methylation motif specific antibody immunoprecipitation Me-R4 100 and R*GG

LTQ-Orbitrap Elite mass spectrometer

Yakubu et al. (2017)

Cysteine palmitoylation, myristoylation, prenylation

401

4.80

ABE

Linear quadrupole ion trap mass spectrometer

Caballero et al. (2016)

Intracellular tachyzoites

Cysteine palmitoylation

282

3.40

17-ODYA bioorthogonal tagging

Quadrupole ion mobility time of flight mass spectrometer

Foe et al. (2015)

Purified and intracellular tachyzoites

Phosphorylation

T. gondii: 12,793

T. gondii: 2793

33.60

IMAC phosphopeptide enrichment

LTQ-Velos Orbitrap mass spectrometer

Treeck et al. (2011)

Purified T. gondii: 24,298

Purified T. gondii: 3506

42.10

Intra- and extracellular tachyzoites

Ubiquitination

800

454

5.40

Ubiquitin di-glycine LTQ-Orbitrap Elite mass Remnant Motif (K-ε-GG) spectrometer antibody

Silmon de Monerri et al. (2015)

Extracellular tachyzoites

SUMO

120

1.40

Whole cell extract of transgenic tachyzoites ectopically expressing HA Flag TgSUMO, antiFLAG M2 affinity gel

QTOF Ultima mass spectrometer

Braun et al. (2009)

Extracellular tachyzoites

Lysine succinylation

147

1.80

Antisuccinyl lysine antibody

Q Exactive Orbitrap mass spectrometer

Li et al. (2014)

425

ABE, Acyl-biotin exchange; IMAC, immobilized metal affinity chromatography.

22.4 Toxoplasma gondii posttranslational modifications

they can be difficult to detect and study, necessitating enrichment techniques for their detection (e.g., Doll and Burlingame, 2015; Mertins et al., 2013). Improvements in proteomic and MS methods, as well as sample preparation, have been exploited in a large number of proteome-wide surveys of PTMs in many different organisms (reviewed in Swaney and Villen, 2016; Mann and Jensen, 2003; Seo and Lee, 2004).

22.4.1 Phosphorylation Phosphorylation is a very common PTM that occurs when a phosphoryl group is transferred from ATP or GTP to an amino acid with the formation of a phosphoester bond (Ngounou Wetie et al., 2014; Olsen et al., 2006). While this PTM occurs in eukaryotes primarily on serine, threonine, or tyrosine residues, it also occurs on histidine, aspartate, and arginine residues (Elsholz et al., 2012; Laub and Goulian, 2007). About 2% 3% of eukaryotic genes code for protein kinases involved in this PTM (Thingholm et al., 2006; Manning et al., 2002) and more than 100 kinases have been identified in the Apicomplexa (Braconi Quintaje and Orchard, 2008; Jackson and Denu, 2001; Guan and Dixon, 1991; Doerig et al., 2015; Jacot and Soldati-Favre, 2012). Phosphorylation is reversed by phosphatases (Barford, 1996; Zhang et al., 2012), of which over 100 are encoded in the human genome (Venter et al., 2001), providing a reversible PTM modification that acts as an on/off switch for biological processes. This PTM plays a key role in many cellular processes including protein synthesis, protein degradation, inter/intracellular signaling, transcriptional and translational regulation, cell survival, apoptosis, metabolism, homeostasis, and differentiation (Johnson and Barford, 1993; Hunter, 2007; Thingholm et al., 2006). Phosphorylation is the most prevalent PTM in both T. gondii and P. falciparum, covering over 30% of the predicted proteomes (Treeck et al., 2011; Alam et al., 2015).

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22.4.2 Ubiquitination Ubiquitination involves the attachment of a 76-amino acid ubiquitin residue covalently by its C-terminal di-glycine (K-GG) to a lysine on the modified protein. This PTM has an important role in protein turnover, targeting proteins to the proteasome for degradation, but is also involved in cellular signaling, intracellular transport, protein protein interactions, and transcriptional regulation (Haglund and Dikic, 2005). Ubiquitination requires the action of ubiquitin-activating enzyme E1, ubiquitinconjugating enzyme E2, and ubiquitin-ligating enzyme E3. This PTM is reversible as deubiquitinating enzymes can remove the covalently attached ubiquitin (Nijman et al., 2005; Bhoj and Chen, 2009). Ubiquitination has been found on up to 5% of the predicted T. gondii proteome (Silmon de Monerri et al., 2015; Ponts et al., 2011). Proteins that have this PTM in T. gondii are highly enriched for GO terms related to structural function, ribosomal components, and dimerization (Silmon de Monerri et al., 2015). In addition, proteins involved in vesicular trafficking, ion transport, and translation are ubiquitinated. TgOTUD3A, an OTU (ovarian tumor) family deubiquitinase, is expressed in a cell cycle dependent manner and has activity against poly- but not monoubiquitinated targets with a preference for specific lysine linkages (K48 . K11 . K63) (Dhara and Sinai, 2016). These three polyubiquitin lysine linkage modifications (K48 . K11 . K63) were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle dependent manner (Dhara and Sinai, 2016).

22.4.3 Palmitoylation This PTM can also act as an on/off switch for protein function and localization, providing an important mechanism for regulating how proteins associate with membranes. Palmitoylation

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involves the addition of a 16-carbon palmitic acid to a cysteine residue via a labile acylthioester linkage (Corvi et al., 2012). G proteins, ion channels, receptors, cytoskeletal proteins, kinases, and signaling proteins have been found to be palmitoylated (Martin and Cravatt, 2009; Wan et al., 2007). Several T. gondii proteins display palmitoylation-dependent localization including IMC-subcompartment proteins (ISPs) (Fung et al., 2012) and TgHSP20 (De Napoli et al., 2013) that localize to the IMC. Proteins localizing to rhoptries and other organelles of the invasion machinery are also palmitoylated (Caballero et al., 2016). A metabolic-labeling approach confirmed palmitoylation of the T. gondii motor and glideosome-associated proteins (MLC1, MyoA, GAP45, GAP40, GAP50, GAP70, and GAP80) (Foe et al., 2015). Studies in Apicomplexa examining the palmitoylome have used acyl-biotin exchange (ABE) and/or metabolic labeling with palmitic acid analog followed by click chemistry (MLCC) to investigate this PTM (Jones et al., 2012; Foe et al., 2015; Caballero et al., 2016). The ABE approach identified 401 T. gondii palmitoylated proteins, representing 4.80% coverage of the entire proteome (Caballero et al., 2016), while the study using MLCC identified 282 proteins, representing 3.40% of the proteome (Foe et al., 2015).

22.4.4 Glycosylation Glycosylation is a very common PTM (Apweiler et al., 1999). Glycosyltransferases recognize specific protein motifs and transfer the first monosaccharide (or preformed oligosaccharide for N-glycosylation) onto a recognition site, following this initial sugar addition and other glycosyltransferases (and glycosidases for N-glycosylation) sequentially elongate the glycan PTM. This PTM plays a critical role in protein folding and stabilization, as well as cell cell adhesion and communication. There are two forms of glycosylation, N- and

O-linked glycosylation. Studies using lectin affinity chromatography have identified over a hundred glycosylated proteins in T. gondii (Luo et al., 2011; Wang et al., 2016). A proteomic analysis of the T. gondii glycoproteome confirmed the abundance of both O- and Nlinked glycoproteins with numerous modified proteins found in surface proteins, MIC, ROP, heat shock proteins, and hypothetical proteins (Luo et al., 2011). O-linked proteins were also purified using Vicia villosa lectin chromatography (Wang et al., 2016), demonstrating that glycosylation was present on proteins from the dense granules, micronemes, rhoptries, and IMC. O-GlcNAcylation is a dynamic PTM, known to occur in the cytosolic, nuclear, and mitochondrial compartments of eukaryotes (Butkinaree et al., 2010). O-GlcNAc is catalyzed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. OGT genes have been found in protists, for example, Giardia, Cryptosporidium, Toxoplasma, and Dictyostelium, confirming the presence of OGT enzymes these eukaryotes (Banerjee et al., 2009). O-GlcNacylation in T. gondii has been demonstrated by immunoblot showing numerous high molecular weight (above 130 kDa) OGlcNAc-modified proteins (Perez-Cervera et al., 2011). Using sWGA affinity chromatography, Aquino-Gil (Aquino-Gil et al., 2018) was able to identify a large number of proteins that were modified by O-Glc-NAc in T. gondii. In addition to proteins involved in stress response, cell shape organization, metabolism, and protein synthesis, they demonstrated the presence of O-GlcNAc on ROP proteins. These findings have not yet been reconciled with genetic and biochemical data indicating that the putative OGT is responsible for O-fucosylation rather O-Glc-NAcylation in T. gondii (Gas-Pascual et al., 2019). Although little is known about the functional consequences of glycosylation on T. gondii proteins, tunicamycin-treated parasites were found to have defects in invasion and

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motility, with TgGAP50 and TgMyoA implicated in these defects, suggesting a role for glycosylation in glideosome function (Luk et al., 2008; Fauquenoy et al., 2008). O-Glycosylation has been demonstrated on the mucin domain of CST1, a cyst wall protein, and this modification is involved in the stability of the tissue cyst wall (Tomita et al., 2013; Tomita et al., 2017). West et al. (Gas-Pascual et al., 2019) mapped probable connections between 59 glycogenes, their enzyme products, and the glycans to which they contribute, using a doubleCRISPR/Cas9 strategy and a MS-based glycomics workflow. Toxoplasma proteins appear to be decorated with three major species of N-glycans, each with an unexpected and unusual structure: Glc2Man6GlcNAc2, Glc1Man6GlcNAc2, and Man6GlcNAc2 (Gas-Pascual et al., 2019). Examining the results of disruption of 17 glycogenes, they identified novel Glc0-2-Man6GlcNAc2-type N-glycans, GalNAc2- and Glc-Fuctype O-glycans, and a nuclear O-Fuc type glycan in T. gondii (Gas-Pascual et al., 2019).

22.4.5 Methylation In eukaryotes, methylation occurs on lysine and arginine residues, and to a lesser extent on histidine, cysteine, aspartic acid, glutamic acid, serine, and threonine residues (Ishikawa and Melville, 1970; Paik et al., 2007), but in T. gondii, only lysine and arginine methylation have been explored. Protein methylation has a role in epigenetic and transcriptional regulation, RNA processing, metabolism, signal transduction, and DNA repair (Paik et al., 2007; Ishikawa and Melville, 1970; Bedford and Clarke, 2009). The role of methylation has not been fully explored in Apicomplexa, but T. gondii has an extensive repertoire of predicted lysine and arginine methyltransferases that is more extensive than for Plasmodium. The function of most of these proteins has not been explored. The SET domain (from Drosophila Su3-9 and “Enhancer of zeste” and Trithorax) was

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originally found in proteins that modify specific lysines on histones leading to gene activation (H3K4, H3K79, and H3K36) or gene repression (H3K9, H4K20, and H3K27). T. gondii histones are extensively modified (Nardelli et al., 2013) (see Chapter 21: Regulation of gene expression in Toxoplasma gondii). Initial phylogenetic analyses revealed that the 15 T. gondii SET proteins consist of 5 separate lineages of proteins, with those in the SMYD family being the most abundant (Sautel et al., 2009). While T. gondii encodes nearly 20 SET domain proteins, most T. gondii lysine methyltransferases (KMT) only SMYD family member KMTox (aka SET13 (Sautel et al., 2009), SET8 (Sautel et al., 2007), and apical lysine methyltransferase (AKMT) (Sivagurunathan et al., 2013) have been studied in detail. SET20 is responsible for mono-, di-, and trimethylation of H4K20 (Sautel et al., 2007). Both AKMT and KTox have in vitro KMT activity but their biological substrates have not been identified. As with other PTM, many modifications originally reported on histones have been found to be more ubiquitous and are implicated in other biological functions that are unrelated to gene expression. Only half of the original members of the SMYD family had a nuclear localization (Sautel et al., 2007, 2009). A more recent analysis of AKMT family members places the AKMT in a separate family unique to Apicomplexa that forms a distinct branch from the SMYD SET family (Sivagurunathan et al., 2013). AKMT regulates motility during invasion and egress (Heaslip et al., 2011; Pivovarova et al., 2018). Protein arginine methyltransferases (PRMTs) transfer methyl groups from S-adenosyl-methionine to arginine to form mono-, di-, and trimethyl arginine (Wang et al., 2005a). Arginine methylation is found on histones of many eukaryotes (Molina-Serrano et al., 2013), including T. gondii (Nardelli et al., 2013) (see Chapter 21: Regulation of gene expression in Toxoplasma gondii). This PTM also occurs on a large number of nonhistone proteins with

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diverse functions. In T. gondii, arginine methylation has been implicated in transcriptional regulation and splicing biology (Yakubu et al., 2017). Arginine methylation of transcription factors can inhibit their degradation by preventing phosphorylation events required for ubiquitinmediated destruction (Yamagata et al., 2008), pointing toward a high degree PTM crosstalk in the Apicomplexa. A T. gondii arginine monomethylome was obtained using IP with commercial monomethyl antibodies followed by MS/MS analysis (Yakubu et al., 2017). This study employed a sequential PTM enrichment workflow, wherein monomethyl arginine peptides from one dataset were purified from the flowthrough samples that had been depleted of ubiquitinated peptides in another experiment (Silmon de Monerri et al., 2015). A similar yield of monomethyl arginine peptides was obtained from both depleted and nondepleted samples indicating that there is little concordant proteome-wide modification of these two PTMs in T. gondii. Arginine monomethylated (MMA) proteins are involved in a wide range of functions and are enriched in DNA and RNA binding proteins, many of which are highly modified with up to seven MMA sites (Yakubu et al., 2017). The MMA proteome was found to cover almost 5% of the T. gondii proteome (Yakubu et al., 2017). Lysine (Kaur et al., 2016) and arginine methylome studies have been reported in P. falciparum, using antimonomethylarginine and antidimethylarginine cross-linked antibodies to detect mono-/di-/trimethyl arginine in the context of RGG, RGx, RxG, GxR, and WxxxR motifs (Zeeshan et al., 2017). In T. gondii, methylation has also been demonstrated on tubulin (Xiao et al., 2010), and PRMT1 has been implicated in centrosome dynamics (El Bissati et al., 2016).

22.4.6 Acetylation Acetylation is a dynamic and reversible modification that can affect protein stability, localization, activity, and protein protein

interactions (Xue et al., 2013). Histone acetyltransferases transfer the acetyl group from acetyl coenzyme A to the amino group of lysine, classically at the N-terminus of histones to form 3-N-acetyl lysine. This reaction can be reversed by histone deacetylases. Acetylation has been predominantly studied as a histone modification (see Chapter 21: Regulation of gene expression in Toxoplasma gondii), but it is now known to occur on transcription factors and other nuclear regulatory molecules, implicating it mainly in transcription and metabolic regulation (Jeffers and Sullivan, 2012; Wang et al., 2014). In P. falciparum (Cobbold et al., 2016) acetyllysine is found on ApiAP2 transcriptional regulators and alters AP2 DNAbinding capacity. In T. gondii, this PTM has been found on hundreds of nonhistone substrates and is predicted to play a role in mRNA translation, metabolism, DNA packaging, the cytoskeletal system and protein folding. A study using a pan-specific antiacetyl lysine antibody followed by MS analysis identified 411 acetylation marks on 274 proteins in tachyzoites (Jeffers and Sullivan, 2012). Acetylation is dependent on the acetylCoA/acetate pool, which implies that this PTM is subject to metabolic regulation and is a candidate regulator of parasite environmental sensing. Extracellular T. gondii tachyzoites are enriched in acetylated proteins involved in metabolism, translation, and chromatin biology (Xue et al., 2013). 96 proteins were found to be uniquely acetylated in intracellular T. gondii, 216 in extracellular parasites, and 177 proteins overlapping between the two conditions, indicating a change in acetylation between the two states (Jeffers and Sullivan, 2012; Xue et al., 2013). In addition to the subset of acetylation marks that were identified in intracellular, replicating tachyzoites (Jeffers and Sullivan, 2012), a further 339 novel acetylation marks were reported (Xue et al., 2013), totaling over 700 known acetylation marks in T. gondii to date. A sizeable proportion of acetylated proteins

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were specific to Apicomplexa, such as microneme and rhoptry proteins, ApiAP2 factors, and a large number of hypothetical proteins. A study in T. gondii of the most abundant nonhistone acetyl-lysine modification, K40 on alpha tubulin, determined that this PTM was deposited by the lysine acetyltransferase TgATAT (alpha tubulin acetyltransferase) during daughter cell formation (Varberg et al., 2016). Disruption of TgATAT by CRISPR/Cas9 targeting led to a loss of K40 acetylation and destabilization of microtubules, causing severe replication defects (Varberg et al., 2016).

22.4.7 Succinyllysine A study of the succinyllysine proteome of T. gondii identified 1.8% of the proteome as being lysine succinylated (Li et al., 2014). Interestingly, more than one quarter of succinyllysine proteins localize to the mitochondrion. Another quarter of succinyllysine proteins localize to the essential parasitespecific organelle, the apicoplast, which is important for fatty acid and isoprenoid synthesis (McFadden and Yeh, 2017).

22.4.8 SUMOylation SUMOylation is the covalent attachment of small ubiquitin-related modifier to a lysine residue. This PTM has been implicated in a large number of cellular functions in other eukaryotes, including transcription, DNA replication and repair, chromosome segregation, mitochondrial fission, ion transport, and signal transduction (Geiss-Friedlander and Melchior, 2007). About 1% of the T. gondii and P. falciparum predicted that proteomes have been shown to be SUMOylated (Braun et al., 2009). In P. falciparum, a SUMO ortholog was identified using polyclonal antibodies against synthetic peptides of the 100-amino amino acid PfSUMO and also by using parasites expressing FLAG-tagged PfSUMO followed by LC MS/

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MS for protein fractionation and sequence identification (Issar et al., 2008). A similar technique was used to map the SUMOylome in T. gondii (Braun et al., 2009). A total of 120 candidate proteins were identified by at least two peptides including proteins involved in chromatin and transcriptional machinery, ribosomal biogenesis, and translation, rhoptry proteins, and stress related proteins. SUMOylated proteins were also present in the cyst wall both in vitro and in vivo.

22.5 Studies on the function of posttranslational modifications in Toxoplasma. gondii biology 22.5.1 Posttranslational modifications in motility, invasion, and egress The glideosome, a macromolecular machine that facilitates gliding motility, comprises a motor complex and adhesive proteins anchored in the parasite membrane (reviewed in Keeley and Soldati, 2004). The motor complex (Meissner et al., 2002) consists of myosin heavy chain protein TgMyoA (Herm-Gotz et al., 2002; Nebl et al., 2011), two myosin light chains (TgMLC1 and TgELC1), and the gliding associated proteins (GAP) (Frenal et al., 2014). This structure is highly modified by palmitoylation, ubiquitin, phosphorylation, and methylation. Given the large number of PTM on glideosome proteins and invasion proteins, determining the role of PTMs in these aspects of parasite biology is challenging. Methylation is found on myosin heavy chain and myosin E, F, and J (Yakubu et al., 2017). Ubiquitin is found on TgGAP40, TgGAP45, TgGAP50, TgMLC1, and several myosins and has been speculated to be important for rearrangement of the cytoskeleton (Silmon de Monerri et al., 2015). These various glideosome PTMs likely engage in crosstalk to regulate motility. For example, TgGAP45 is ubiquitinated

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(Silmon de Monerri et al., 2015), myristoylated, palmitoylated (Frenal et al., 2010), and phosphorylated (Gilk et al., 2009). These PTMs probably help in targeting TgGAP45 to the mature glideosome (Gaskins et al., 2004; ReesChanner et al., 2006; Johnson et al., 2007). All glideosome proteins are phosphorylated in regions that are conserved among the Apicomplexa (Jacot and Soldati-Favre, 2012; Treeck et al., 2011; Gilk et al., 2009; Nebl et al., 2011; Tang et al., 2014; Green et al., 2008). Multiple phosphorylation sites on TgGAP45 control the final step of assembly of the glideosome machinery components (Gilk et al., 2009). Two sites on TgGAP45 (S163 and S167), when phosphorylated, prevent TgGAP45 association with TgGAP50, thus blocking the final assembly of the mature myosin A motor complex (Gilk et al., 2009). In contrast to TgGAP45 the impact of phosphorylation of TgMLC1 is unclear; systematic mutation of several phosphorylation sites on TgMLC1 did not impact glideosome function (Jacot et al., 2014). TgGAP45 tethers the IMC and plasma membrane and recruits MyoA and MLC1 (Frenal et al., 2010); however, deletion of a highly phosphorylated portion of TgGAP45 did not influence its ability to recruit motor components or alter the integrity of the IMC (Jacot et al., 2014). Overall, these findings suggest a role for this heavily phosphorylated region in protein protein interactions associated with parasite motility (Nebl et al., 2011). Palmitoylation is also seen on glideosome proteins, including MLC1, MyoA, GAP45, GAP40, GAP50, and GAP70/GAP80 (Foe et al., 2015; Caballero et al., 2016). Palmitoylated Cterminal cysteines are believed to be required for TgGAP45 to link the IMC to the plasma membrane, whereas N-terminal myristoylation and palmitoylation of TgGAP45 target the motor complex to the plasma membrane before anchoring it in the IMC (Frenal et al., 2010). Inhibition of palmitoylation in either T. gondii or P. falciparum reduces invasion and results in rhoptry mislocalization to the cytoplasm (Jones

et al., 2012; Caballero et al., 2016). This is consistent with the large number of invasionrelated proteins that are palmitoylated (Foe et al., 2015; Caballero et al., 2016). Blocking palmitoylation of AMA1 in T. gondii increases release of MIC2, a microneme protein, but not rhoptry or GRAs (Foe et al., 2015). Treatment of T. gondii and P. falciparum with the palmitoylation inhibitor 2-bromopalmitate significantly reduces invasion (Alonso et al., 2012; Jones et al., 2012). This treatment in T. gondii results in depalmitoylation of the cytoskeletal proteins IMC1, GAP45, HSP20, and Morn-1. Using a chemical genetics screen, Child et al. (2013) identified a T. gondii depalmitoylase (TgPPT1) that, when inhibited, led to an increase in motility and invasive capacity of tachyzoites. Together, these data suggest that parasite motility and invasion require tight regulation of palmitoylation.

22.5.2 Posttranslational modifications of the inner membrane complex The IMC is composed of flattened cisternae underneath the plasma membrane where it interacts with intermediate filament-like proteins and subpellicular microtubules (Morrissette et al., 1997; Chen et al., 2015, 2017). The IMC cisternae are linked by sutures, forming an interconnected network of vesicles, involved in host cell invasion and scaffolding during cytokinesis (Kono et al., 2013; Chen et al., 2017). Several studies show that palmitoylation is a critical PTM for protein localization to the IMC (Beck et al., 2010; Fung et al., 2012). ISP1, ISP2, and ISP3 are subject to modification, first by myristoylation to facilitate association with the IMC, followed by palmitoylation to anchor the proteins in membranes (Beck et al., 2010); however, ISP4 is solely dependent on palmitoylation for IMC targeting. ISPs undergo “kinetic trapping” and are weakly associated with the IMC membrane through cotranslational myristoylation and then are subsequently attached by palmitoylation

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(Beck et al., 2010). TgCDPK3 (Garrison et al., 2012; McCoy et al., 2012), GAP45 (Frenal et al., 2010), and HXGPRT (Chaudhary et al., 2005) also localize to the IMC upon palmitoylation. HSP20, shown to play a role in host cell invasion, localizes to the IMC following palmitoylation in T. gondii and P. falciparum (Montagna et al., 2012a,b; De Napoli et al., 2013) and its palmitoylation status determines IMC localization (Chaudhary et al., 2005; Rees-Channer et al., 2006; Garrison et al., 2012). Treatment of parasites with the palmitoylation inhibitor 2bromopalmitate caused TgHSP20 to redistribute from the IMC to cytosol (De Napoli et al., 2013). A nonpalmitoylatable version of HSP20 localized to the IMC in daughter cells and to the cytosol in mature cells, indicating that palmitoylation is required for retaining HSP20 in the IMC as T. gondii matures (De Napoli et al., 2013). In the T. gondii cytoskeleton, other IMC proteins, including most glideosome proteins and alpha- and beta-tubulin, are also ubiquitinated (Silmon de Monerri et al., 2015). Tubulin ubiquitination regulates its polymerization, inhibiting microtubule formation (Bheda et al., 2010). Several methylated IMC proteins were found to have a monomethyl-arginine PTM including IMC1, IMC4, SPM2, and possibly other hypothetical proteins (Yakubu et al., 2017). T. gondii phosphoproteomes identified three phosphorylated serines on TgISP2 and two phosphorylated serines on TgISP3 (Treeck et al., 2011). TgISPs can multimerize and adopt a pleckstrin homology domain that can bind membrane-associated inositol phosphates, suggesting that the phosphorylation of TgISP is involved in regulating cell division and protein recruitment (Tonkin et al., 2014).

22.5.3 Posttranslational modifications in transcriptional and posttranscriptional regulation ApiAP2 are also subject to PTMs, which likely alter their activity, protein protein

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interactions, protein stability, or DNA binding capacity (Seo and Lee, 2004). Acetylation of the putative ApiAP2 PF3D7_1007700 at its DNAbinding domain reduced the DNA-binding capability, suggesting that acetylation may play a role in regulating transcription (Cobbold et al., 2016). Almost all of the other 62 acetylation sites on the 16 identified ApiAP2s in P. falciparum are outside of the AP2 DNA-binding site and other known functional domains (Cobbold et al., 2016), suggesting that any alteration of DNA-binding activity by acetylation must occur due to allosteric interactions. In T. gondii, five ApiAP2 (AP2IX-5, AP2IX-7, AP2X-5, AP2XII-4, and AP2XII-8), are acetylated outside of the AP2 DNA-binding domain (Jeffers and Sullivan, 2012). Ubiquitination sites have been found in five ApiAP2 in T. gondii (Silmon de Monerri et al., 2015). The AP2X-7 (TGME49_214840) ubiquitin site K1368 is within its ApiAP2 domain, whereas for AP2X4, AP2X-9, AP2X-3, and AP2XII-8, the ubiquitin sites lie outside of the ApiAP2 domain (Silmon de Monerri et al., 2015). Arginine monomethylation was also found outside of the AP2 DNAbinding domain in 10 ApiAP2 proteins (Yakubu et al., 2017). The function and importance of most observed PTMs has not yet been experimentally tested. Differences in ribosomal protein acetylation may play a role in the global downregulation of translation in extracellular parasites (Xue et al., 2013). While in the extracellular environment, parasites are exposed to a number of stressors, such as a lack of nutrients, which can alter the pool of available acetyl-CoA. It is possible that P. falciparum and T. gondii use a conserved acetyl-CoA pathway to sense nutrient availability as a signal to influence downstream transcriptional activity, which is regulated through lysine acetylation. In P. falciparum, it has been found that SUMO/ubiquitin chains target modified proteins to the proteasome for degradation, which indicates a role for ubiquitination in the regulation of SUMO proteins (Ponts et al., 2011). Such

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atypical ubiquitin chains have also been found in other organisms (reviewed in Ikeda and Dikic, 2008). In P. falciparum, this proteasomedependent ubiquitination may contribute to the poor correlation between mRNA transcripts and protein levels (Le Roch et al., 2004). In T. gondii and P. falciparum, ubiquitination has been detected on subunits of RNA polymerase II (Ponts et al., 2011; Silmon de Monerri et al., 2015), which is associated with transcriptional arrest in yeast (Svejstrup, 2007; Daulny and Tansey, 2009). Overall, ubiquitin-like modifiers are implicated in transcriptional regulation in P. falciparum and T. gondii. Chromatin, especially histones, contains numerous PTMs that are known to affect the accessibility of DNA to transcription machinery (reviewed in Dahlin et al., 2015). Similar to other eukaryotes, Apicomplexan histones are highly modified with the following PTMs having been identified: phosphorylation (Nardelli et al., 2013), methylation (Saksouk et al., 2005), ubiquitination (Silmon de Monerri et al., 2015; Trelle et al., 2009), SUMOylation (Braun et al., 2009; Issar et al., 2008), acetylation (Miao et al., 2006; Trelle et al., 2009; Jeffers and Sullivan, 2012), succinylation (Xie et al., 2012), and crotonylation (Tan et al., 2011). Histone acetylation is seen in T. gondii and P. falciparum (Nardelli et al., 2013; Jeffers and Sullivan, 2012; Gissot et al., 2007; Trelle et al., 2009; Trenholme et al., 2014; Miao et al., 2006) and has been associated with transcriptional activation (Srivastava et al., 2014; Gissot et al., 2007), viability, and bradyzoite development (Dixon et al., 2010; Sullivan and Hakimi, 2006). T. gondii histone phosphorylation marks were found at S1 of histone 4 and T107 of histone 3 (Nardelli et al., 2013); however, no phosphorylation sites on H3 that have been linked to mitosis (PerezCadahia et al., 2009) were identified in T. gondii (Nardelli et al., 2013). Thirty five ubiquitination sites on eight histones, including H2AX, H2B, H2BA, and H3 have been identified in T. gondii (Silmon de Monerri et al., 2015).

RNA-binding proteins in T. gondii have been found to contain multiple PTMs, including phosphorylation and methylation (Yakubu et al., 2017; Lee, 2012), SUMOylation, and ubiquitination (Lovci et al., 2016). Strikingly, 22% of RNA-binding proteins are modified by monomethylation (MMA). Five proteins that contain an RNA-recognition motif (RRM) domain had MMA sites within the RRM domain (Yakubu et al., 2017). T. gondii splicing factor SF2 (TGME49_319530) had MMA within its RNA binding domain (Yakubu et al., 2017). In the human ortholog of this protein, MMA has a regulatory role in assembly of SF2 into interchromatin granule clusters (Larsen et al., 2016).

22.5.4 Posttranslational modifications as regulators of parasite differentiation The dynamic nature of PTMs in response to cellular environments suggests that they are candidates for regulators of parasite differentiation, and several studies have investigated global PTMs during differentiation in several Apicomplexa. These experiments can be difficult due to the amount of material required for global proteomic analysis. Differential protein expression has been surveyed by 2D-DIGE approaches in N. caninum (MaruganHernandez et al., 2010) and Besnoitia besnoiti (Fernandez-Garcia et al., 2013), demonstrating isoforms existence; however, little is known about the role of PTMs in these parasites. During P. falciparum asexual development, waves of transcription occur and nearly half of the proteins detected have multiple isoforms that are modified by PTMs (Foth et al., 2008, 2011). Several studies in P. falciparum have compared PTM levels between different stages within the asexual intraerythrocytic cycle. Peak levels of SUMOylation are found in trophozoites and schizonts due to increased oxidative stress (Reiter et al., 2013). SUMOylation is upregulated in response to cellular stress

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(Golebiowski et al., 2009; Lee et al., 2007) and is thought to play a role in promoting cell survival (Tempe et al., 2008). Studies of P. falciparum merozoites and schizont proteomes (Lasonder et al., 2012; Pease et al., 2013; Solyakov et al., 2011; Treeck et al., 2011; Collins et al., 2014) have identified differential phosphorylation between these stages (Lasonder et al., 2015). This includes exclusion of phosphotyrosine from merozoites and differential distribution of the core motif (K/R)xx(S/T) in merozoites and schizonts in vivo (Lasonder et al., 2015). Most studies of T. gondii PTM have focused upon tachyzoites. Although bradyzoites can be induced in vitro, obtaining large amounts of pure bradyzoites required for proteomic analysis is challenging and few laboratories have access to the sexual stages or oocysts. Several groups have compared PTMs in extracellular T. gondii tachyzoites to PTM in intracellular parasites. While extracellular parasites are searching for a host cell to invade, they encounter significant changes in nutrient availability, metabolic environment, and oxidative stress; changes in PTMs may protect the parasite in this harsh environment. Extracellular tachyzoites undergo transcriptional and translational arrest (Croken et al., 2014b) characterized by a unique transcriptional profile (Gaji et al., 2011; Lescault et al., 2010) and have been proposed to represent a unique tachyzoite state (Lescault et al., 2010). Extracellular tachyzoites have unique protein acetylation profiles (Xue et al., 2013) compared to intracellular parasites (Jeffers and Sullivan, 2012). The acetylome of extracellular parasites (Xue et al., 2013) contains 96 uniquely acetylated proteins, while intracellular parasites contain 216 uniquely acetylated proteins (Jeffers and Sullivan, 2012). The extracellular T. gondii tachyzoite acetylatome is enriched in proteins functioning in metabolism, translation, and chromatin biology (Xue et al., 2013). These studies indicate that significant changes to the proteome and their PTMs occur between intracellular and extracellular stages (Xue et al., 2013), similar

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to changes observed in mRNA expression (Gaji et al., 2011). Fewer ubiquitinated proteins were detected in extracellular tachyzoites compared to intracellular parasites (Silmon de Monerri et al., 2015), and proteins that are upregulated at the transcriptional level in G1 phase were enriched in the extracellular parasite ubiquitinome. Interestingly, for a number of proteins ubiquitinated at multiple sites in intracellular tachyzoites, several of those ubiquitination sites were not detected in extracellular stages, while other proteins (such as glycolytic proteins) were ubiquitinated only when parasites were extracellular (Silmon de Monerri et al., 2015). This suggests that ubiquitination has a role in regulating metabolism in extracellular tachyzoites. Extracellular tachyzoites contained 181 fewer monomethyl-arginine (MMA) sites compared to intracellular tachyzoites (Yakubu et al., 2017). This is consistent with reduced MMA during the G1 cell cycle arrest seen in extracellular parasites (Lescault et al., 2010) and with changes in arginine methylation seen in human cells during transcriptional arrest (Sylvestersen et al., 2014). It is possible that arginine methylation plays a role in transcriptional suppression due to stress. A mechanism by which T. gondii manages stress is through the phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (TgIF2α) at serine S71 (Joyce et al., 2010), which initiates translational control to conserve resources, while the cell reprograms its gene expression to cope with the cellular stress or developmental signal (Joyce et al., 2010). Phosphorylated eIF2α is also important for the formation and maintenance of latency stages in both T. gondii and P. falciparum (Holmes et al., 2017) (see also Chapter 21: Regulation of gene expression in Toxoplasma gondii).

22.5.5 Host parasite interactions T. gondii manipulates its host cell to provide a more hospitable environment for intracellular replication, for example, it is known to prevent

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apoptosis, reorganize cellular microtubules, associate cellular organelles with the parasitophorous membrane, and alter immune regulatory pathways. One of the mechanisms involved in this host cell reprogramming is the introduction of PTMs of cellular proteins. There are documented changes in the host proteome in parasitized cells, for example, differential phosphorylation (Nelson et al., 2008) due to secreted T. gondii proteins modifying host cell protein phosphorylation (Du et al., 2014; Alaganan et al., 2014; Braun et al., 2013). Many rhoptry (ROP) proteins are active serine threonine kinases, which phosphorylate host proteins. In T. gondii, the immune-related GTPases (IRGs) are recruited to the PV where they play an essential role in parasite destruction. ROP18 phosphorylates IRGs and prevents IRGmediated destruction (Fentress et al., 2010), and this PTM on IRBs is a major mechanism of T. gondii virulence in mice (Saeij et al., 2007; Taylor et al., 2006). Palmitoylation occurs on ROP5 (Caballero et al., 2016) and is likely a regulatory PTM involved in the ability of ROP5 to interact with ROP18 and other binding partners altering its affinity as a competitive inhibitor of IRG oligomerization (Fleckenstein et al., 2012; Niedelman et al., 2012). ROP5 is involved in invasion and virulence by increasing ROP18 kinase activity. ROP16 phosphorylates several host Signal Transducer and Activator of Transcription proteins (STAT1, STAT3, STAT5, and STAT6) (Rosowski and Saeij, 2012; Butcher et al., 2011; Yamamoto et al., 2009; Jensen et al., 2013; Ong et al., 2010), thereby downregulating the Th1 inflammatory response, dysregulating apoptosis (Carmen and Sinai, 2007), suppressing IFN-y-induced STAT1 activation (Kim et al., 2007), and inhibiting dendritic cell maturation (McKee et al., 2004). T. gondii GRA24 is exported into the host cell, where it forms a stable dimeric complex with p38α mitogenactivated protein kinase, leading to its translocation to the nucleus (Pellegrini et al., 2017) and prolonged parasite-specific autophosphorylation at p38α-Thr180 activates its kinase

activity (Braun et al., 2013). In addition to modifying host cell proteins, many of the proteins secreted into the host cell by either Plasmodium spp. or T. gondii are also highly phosphorylated (Jacot and Soldati-Favre, 2012; Treeck et al., 2011). In both Plasmodium spp. and T. gondii the exportome is known to contain some of the most highly phosphorylated proteins (Jacot and Soldati-Favre, 2012). The majority of these are hypothetical proteins with unknown functions. Studies have found differences at protein and transcript levels between infected and uninfected cells based on HFF gene expression using cDNA microarrays (Blader et al., 2001), expression profiles using human cDNA arrays (Saeij et al., 2007), expression levels of known parasite effectors using RNA sequencing data (Melo et al., 2013), and protein levels in host metabolic pathways using DIGE (Nelson et al., 2008) and 2D gel electrophoresis (Zhou et al., 2011). There is also evidence suggesting that T. gondii infection modulates the host cell acetylome. Changes in rat cortical astrocyte protein acetylation upon T. gondii infection were seen in 58 proteins, 34 of which showed at least a twofold increase in acetylation when assayed in the infected astrocytes (Bouchut et al., 2015). These proteins included lysine acetyltransferases (KATs), lysine deacytlases (HDACs), and transcription factors (Bouchut et al., 2015). In addition, there was a 600-fold increase in acetylation on histone H3.3, which suggests that T. gondii affects host cell epigenetic regulation as a means of controlling host cell gene expression (Bouchut et al., 2015).

22.6 Interactions of Toxoplasma gondii posttranslational modifications The combination of different PTMs on a protein is read by PTM-binding proteins and is important for protein protein interactions and for protein function. The studies mentioned in this chapter provide data supporting crosstalk

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22.6 Interactions of Toxoplasma gondii posttranslational modifications

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FIGURE 22.2

Interactions between Toxoplasma gondii proteins with PTM. T. gondii proteins with PTMs were compared to one another using a hypergeometric test of enrichment using methods described by Silmon de Monerri (Silmon de Monerri et al., 2015). Published PTM datasets were analyzed (Braun et al., 2009; Jeffers and Sullivan, 2012; Li et al., 2014; Treeck et al., 2011; Foe et al., 2015; Silmon de Monerri et al., 2015; Yakubu et al., 2017). The color key is in 2 log2 (Pvalue). PTMs, Posttranslational modifications. Source: Adapted with permission from Yakubu, R.R., Weiss, L.M., Silmon de Monerri, N.C., 2018. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies. Mol. Microbiol. 107, 1 23.

between the various PTMs seen on T. gondii proteins. Two studies have examined the interactions between different PTMs in T. gondii (Silmon de Monerri et al., 2015; Yakubu et al., 2018). Pairwise comparisons were performed between the PTM datasets using a statistical test of enrichment (Silmon de Monerri et al., 2015; Yakubu et al., 2018). The analysis of these comparisons is presented as a heatmap in Fig. 22.2. Significant overlap was seen between arginine monomethylated and phosphorylated proteins. While phosphorylation was significantly enriched in several of the other PTM

proteomes, proteins that are succinylated and phosphorylated did not significantly overlap. The ubiquitination and palmitoylation proteomes were also significantly enriched for the proteins in each dataset. However, while this analysis suggests that there is significant crosstalk between PTM proteomes in T. gondii, it has yet to be determined if PTMs harbor a large degree of redundancy or function in a tightly organized network of PTM cross talk. One caveat concerning these proteome-wide studies of PTMs is that they provide varying degrees of proteome coverage, and it is possible that the

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FIGURE 22.3 Toxoplasma gondii PTM display preferences for different metabolic pathways. T. gondii proteins with PTMs were compared were compared to sets of genes with different functions (classified by GO terms) using a hypergeometric test of enrichment using methodology described by Silmon de Monerri (Silmon de Monerri et al., 2015). The five most significantly enriched GO terms are shown in a clustered heatmap. Color key is in 2 log2 (P-value). Published PTM datasets were analyzed (Braun et al., 2009; Jeffers and Sullivan, 2012; Li et al., 2014; Treeck et al., 2011; Foe et al., 2015; Silmon de Monerri et al., 2015; Yakubu et al., 2017). GO, Gene ontology; PTMs, posttranslational modifications. Source: Adapted with permission from Yakubu, R.R., Weiss, L.M., Silmon de Monerri, N.C., 2018. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies. Mol. Microbiol. 107, 1 23.

data are skewed based on how representative these studies are with regard to modifications on the proteins that are captured. In other organisms, PTMs have been shown to accumulate on specific types of protein(s) in a particular pathway. To gain an understanding of the biological targets of PTM at

the pathway level, an enrichment analysis was performed using GO gene lists, which are gene annotations curated by function or pathway. Fig. 22.3 provides a heatmap of enrichment scores (P values derived from a statistical test of enrichment) of GO pathways for each set of proteins in a PTM proteome.

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22.6 Interactions of Toxoplasma gondii posttranslational modifications

This heatmap demonstrates the differential enrichment of PTM gene sets in the various GO pathways, which indicates various PTMs tend to target proteins in distinct biological pathways, for example, proteins in the ubiquitome, palmitoylome, and acetylome are significantly enriched in GO pathways related to protein biosynthesis (e.g., translation), and in contrast, the succinylome, phosphoproteome, arginine monomethylome, and SUMO proteome are not enriched in these GO pathways. As seen in other eukaryotes, the phosphoproteome is enriched in kinase and phosphotransferase functions and is enriched in nuclear proteins. A role for succinylation in metabolism and mitochondrial processes been described in other eukaryotes (Park et al., 2013; Rardin et al., 2013; Weinert et al., 2013; Hirschey and Zhao, 2015). In T. gondii, the succinylome was found to be enriched in GO pathways related to the tricarboxylic acid cycle, mitochondrial pathways, and metabolism. Overall, these data indicate that the various PTMs have unique functions in T. gondii biology and target specific pathways and compartments in this organism. PTMs typically occur in a spatiotemporal manner, and in other organisms, these have been identified as key cell cycle regulators. T. gondii has an 8-hour cell cycle, proceeding with G1 phase followed by S, M, and C phases; with a very short or absent G2 phase (Radke et al., 2001). Mitosis is closely linked to cytokinesis, due to endodyogeny (see Chapter 21: Regulation of gene expression in Toxoplasma gondii). There are distinct G1 and S/M regulated subtranscriptomes in this organism (Behnke et al., 2010). Similar to the classic eukaryotic cell cycle, checkpoints assess progress of the cell through various phases of the cell cycle in T. gondii. There are also START and M-phase checkpoints, as well as a mid-G1 checkpoint (Radke et al., 2001; Conde de Felipe et al., 2008). Ubiquitination is an essential regulator of checkpoint control during the cell

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cycle, as ubiquitination of checkpoint complexes enables progression through cell cycle checkpoints (Bassermann et al., 2014). Examination of enrichment of PTM-modified proteins in gene sets consisting of genes upregulated at different time points in G1 and S/M phase revealed a large number of ubiquitination targets in T. gondii that are regulated in a cell cycle dependent manner (Silmon de Monerri et al., 2015; Yakubu et al., 2018). Fig. 22.4 demonstrates the PTMs enriched in genes upregulated at 4.5 5.5 and 6.5 8 hours during G1 phase. The phosphoproteome and arginine monomethylome are uniquely enriched in the genes upregulated in mid-G1 (4.5 5.5 hours) and this, most likely, reflects a mid-G1 checkpoint in T. gondii (Radke et al., 2001; Conde de Felipe et al., 2008). This enrichment of upregulated mid-G1 proteins in the phosphoproteome and monomethylome probably indicates a need for increased signaling, gene regulation, and protein synthesis associated with the cell growth typically seen in G1. There is enrichment of all PTMs at 6.5 8 hours, at the end of G1 phase, and this probably precedes or coincides with a START (G1/S) checkpoint that is responsible for permitting parasites to reenter cell cycle (White et al., 2005). At this G1/S checkpoint the cell is preparing for replication and duplicates the Golgi and centrioles (Hartmann et al., 2006; Nishi et al., 2008; Pelletier et al., 2002) indicating a commitment to DNA replication. In genes upregulated between hours 3 and 4 of the S/M subtranscriptome ubiquitination, phosphorylation, and palmitoylation are significantly enriched. In the S/M phase, mitosis, chromosome replication, and budding occur. The S/M phase of the cell cycle is crucial for ensuring parasite integrity is maintained and mitosis occurs correctly. A checkpoint is present at this point in the late S phase, where chromosome replication slows or halts right before entering mitosis, associated with a DNA content of 1.8 (Radke et al., 2001;

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FIGURE 22.4 Toxoplasma gondii proteins with PTM are enriched in cell cycle regulated genes. T. gondii proteins with PTMs were compared against cell cycle gene sets by a hypergeometric test of enrichment. Gene sets were composed of genes that are transcriptionally upregulated in G1 or S/M subtranscriptomes at time points during the 8 h T. gondii cell cycle, as described by Croken (Croken et al., 2014a,b). (A) Two peaks of enrichment are seen in G1 phase (hours—4.5 5.5; hours—6.5 8) and (B) one peak of enrichment in S/M phase (hours—3 4). Adjusted P values (2(log2)-transformed) are plotted. Published PTM datasets were analyzed (Braun et al., 2009; Jeffers and Sullivan, 2012; Li et al., 2014; Treeck et al., 2011; Foe et al., 2015; Silmon de Monerri et al., 2015; Yakubu et al., 2017). PTMs, Posttranslational modifications. Source: Adapted with permission from Yakubu, R.R., Weiss, L.M., Silmon de Monerri, N.C., 2018. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies. Mol. Microbiol. 107, 1 23.

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22.7 Conclusion

Alvarez and Suvorova, 2017). The IMC is a critical structure in division during the S/M phase, and numerous IMC genes are upregulated in the mid-S/M phase and these upregulated IMCs display many PTMs. Over 35% of ubiquitinated proteins were demonstrated to be cell cycle regulated, with 63 proteins being both ubiquitinated and phosphorylated at the boundary of S/M phase, many of which are IMC and cytoskeletal proteins (Silmon de Monerri et al., 2015). One of the mechanisms of IMC regulation in cell division may be coordinated phosphorylation and ubiquitination. Several E3 ligases (ubiquitinating complexes) are upregulated at the mid-S/M time point (Silmon de Monerri et al., 2015). T. gondii encodes some eukaryotic ubiquitinating complexes, responsible for marking cell-cycle proteins and cyclins for destruction by the proteasome (Teixeira and Reed, 2013; Bassermann et al., 2014) such as APC components (Baker et al., 2007) and SCF proteins (Ponts et al., 2008). However, T. gondii ubiquitinated cyclin homologs, and CDK substrates were not identified in a study of ubiquitination (Silmon de Monerri et al., 2015). Overall, the analysis of these various PTM proteomes provides evidence that PTMs are highly coordinated throughout the T. gondii cell cycle and may play key roles in the regulation of cell cycle transitions (Silmon de Monerri et al., 2015; Yakubu et al., 2018).

22.7 Conclusion The availability of high-throughput technologies for sequencing, proteomics, transcriptomics, epigenomics, and metabolomics has provided a wealth of datasets for generating new hypothesis about parasite physiology. Proteomics data for T. gondii has also been helpful for improving genome annotation (Silmon de Monerri and Weiss, 2015).

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Improved methods to purify subproteomes, for example, the PVM and tissue cyst wall, as well as the use of BioID, are dramatically expanding our understanding of the structures formed by this organism and their relationship to its host cell. The application of selectively labeling techniques (i.e., iTRAQ and SILAC) combined with the ability to genetically manipulate T. gondii is yielding important insights into the global proteome under different conditions or life cycle stages, providing crucial data for systems biology applications. PTMs play important roles in virtually all aspects of Apicomplexan biology, including differentiation, invasion and egress, protein localization, and transcription. Although there are a number of proteome-wide studies with varying degrees of proteome coverage, we still do not know exactly how well we manage to capture all of the PTMs on the proteins that are surveyed. The vast majority of peptides in shotgun proteomics approaches are often unassigned, with modified peptides making up a large fraction of these spectra (Chick et al., 2015). Beyond the boundaries of the parasite’s vacuole, PTMs modify the host cell to create a hospitable environment for survival, replication, persistence, and dissemination. An exciting avenue of future research into PTMs will be the discovery of how the array of PTMs present in the parasite interacts to regulate its biology. This will involve the development of methods to perform improved highthroughput analysis of PTMs, as well as techniques to use proteomic technology to map protein interactions. All of this must be coupled with new bioinformatics platforms to handle the complex datasets and with standards for community wide deposition of data that will facilitate repeat analysis of data as new tools and insights are developed. Integration of the evolving genomic, proteomic, and metabolomic data promises fascinating insights into how T. gondii functions as a successful eukaryotic pathogen.

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Acknowledgments We are indebted to Rama R. Yakubu and Natalie C. Silmon de Monerri for their help on this chapter and efforts in proteomics in our laboratories. The work was supported by NIH NIAID R01AI134753 (LMW), R21 AI123495 (LMW), R01 AI087625 (KK), R01 AI116496 (WJS), and R01 AI124723 (WJS).

References Aebersold, R., Mann, M., 2003. Mass spectrometry-based proteomics. Nature 422, 198 207. Al-Bajalan, M.M.M., Xia, D., Armstrong, S., Randle, N., Wastling, J.M., 2017. Toxoplasma gondii and Neospora caninum induce different host cell responses at proteome-wide phosphorylation events; a step forward for uncovering the biological differences between these closely related parasites. Parasitol. Res. 116, 2707 2719. Alaganan, A., Fentress, S.J., Tang, K., Wang, Q., Sibley, L. D., 2014. Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse. Proc. Natl. Acad. Sci. U.S.A. 111, 1126 1131. Alam, M.M., Solyakov, L., Bottrill, A.R., Flueck, C., Siddiqui, F.A., Singh, S., et al., 2015. Phosphoproteomics reveals malaria parasite protein kinase G as a signalling hub regulating egress and invasion. Nat. Commun. 6, 7285. Alonso, A.M., Coceres, V.M., De Napoli, M.G., Nieto Guil, A.F., Angel, S.O., Corvi, M.M., 2012. Protein palmitoylation inhibition by 2-bromopalmitate alters gliding, host cell invasion and parasite morphology in Toxoplasma gondii. Mol. Biochem. Parasitol. 184, 39 43. Alvarez, C.A., Suvorova, E.S., 2017. Checkpoints of apicomplexan cell division identified in Toxoplasma gondii. PLoS Pathog. 13, e1006483. Apweiler, R., Hermjakob, H., Sharon, N., 1999. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta 1473, 4 8. Aquino-Gil, M.O., Kupferschmid, M., Shams-Eldin, H., Schmidt, J., Yamakawa, N., Mortuaire, M., et al., 2018. Apart from rhoptries, identification of Toxoplasma gondii’s O-GlcNAcylated proteins reinforces the universality of the O-GlcNAcome. Front. Endocrinol. (Lausanne) 9, 450. Baker, D.J., Dawlaty, M.M., Galardy, P., Van Deursen, J.M., 2007. Mitotic regulation of the anaphase-promoting complex. Cell. Mol. Life Sci. 64, 589 600.

Banerjee, S., Robbins, P.W., Samuelson, J., 2009. Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum. Glycobiology 19, 331 336. Barford, D., 1996. Molecular mechanisms of the protein serine/threonine phosphatases. Trends Biochem. Sci. 21, 407 412. Bassermann, F., Eichner, R., Pagano, M., 2014. The ubiquitin proteasome system—implications for cell cycle control and the targeted treatment of cancer. Biochim. Biophys. Acta 1843, 150 162. Beck, J.R., Rodriguez-Fernandez, I.A., De Leon, J.C., Huynh, M.H., Carruthers, V.B., Morrissette, N.S., et al., 2010. A novel family of Toxoplasma IMC proteins displays a hierarchical organization and functions in coordinating parasite division. PLoS Pathog. 6, e1001094. Beckers, C.J., Dubremetz, J.F., Mercereau-Puijalon, O., Joiner, K.A., 1994. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J. Cell. Biol. 127, 947 961. Beckers, C.J., Wakefield, T., Joiner, K.A., 1997. The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein. Mol. Biochem. Parasitol. 89, 209 223. Bedford, M.T., Clarke, S.G., 2009. Protein arginine methylation in mammals: who, what, and why. Mol. Cell 33, 1 13. Behnke, M.S., Wootton, J.C., Lehmann, M.M., Radke, J.B., Lucas, O., Nawas, J., et al., 2010. Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5, e12354. Bheda, A., Gullapalli, A., Caplow, M., Pagano, J.S., Shackelford, J., 2010. Ubiquitin editing enzyme UCH L1 and microtubule dynamics: implication in mitosis. Cell Cycle 9, 980 994. Bhoj, V.G., Chen, Z.J., 2009. Ubiquitylation in innate and adaptive immunity. Nature 458, 430 437. Blader, I.J., Manger, I.D., Boothroyd, J.C., 2001. Microarray analysis reveals previously unknown changes in Toxoplasma gondii-infected human cells. J. Biol. Chem. 276, 24223 24231. Bouchut, A., Chawla, A.R., Jeffers, V., Hudmon, A., Sullivan Jr., W.J., 2015. Proteome-wide lysine acetylation in cortical astrocytes and alterations that occur during infection with brain parasite Toxoplasma gondii. PLoS One 10, e0117966. Braconi Quintaje, S., Orchard, S., 2008. The annotation of both human and mouse kinomes in UniProtKB/SwissProt: one small step in manual annotation, one giant

Toxoplasma Gondii

References

leap for full comprehension of genomes. Mol. Cell. Proteomics 7, 1409 1419. Bradley, P.J., Ward, C., Cheng, S.J., Alexander, D.L., Coller, S., Coombs, G.H., et al., 2005. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii. J. Biol. Chem. 280, 34245 34258. Bradshaw, R.A., Burlingame, A.L., 2005. From proteins to proteomics. IUBMB Life 57, 267 272. Braun, L., Cannella, D., Pinheiro, A.M., Kieffer, S., Belrhali, H., Garin, J., et al., 2009. The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii. Int. J. Parasitol. 39, 81 90. Braun, L., Brenier-Pinchart, M.P., Yogavel, M., CurtVaresano, A., Curt-Bertini, R.L., Hussain, T., et al., 2013. A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation. J. Exp. Med. 210, 2071 2086. Butcher, B.A., Fox, B.A., Rommereim, L.M., Kim, S.G., Maurer, K.J., Yarovinsky, F., et al., 2011. Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1dependent growth control. PLoS Pathog. 7, e1002236. Butkinaree, C., Park, K., Hart, G.W., 2010. O-linked beta-Nacetylglucosamine (O-GlcNAc): extensive crosstalk with phosphorylation to regulate signaling and transcription in response to nutrients and stress. Biochim. Biophys. Acta 1800, 96 106. Caballero, M.C., Alonso, A.M., Deng, B., Attias, M., De Souza, W., Corvi, M.M., 2016. Identification of new palmitoylated proteins in Toxoplasma gondii. Biochim. Biophys. Acta 1864, 400 408. Carmen, J.C., Sinai, A.P., 2007. Suicide prevention: disruption of apoptotic pathways by protozoan parasites. Mol. Microbiol. 64, 904 916. Carruthers, V.B., 2006. Proteolysis and Toxoplasma invasion. Int. J. Parasitol. 36, 595 600. Carruthers, V.B., Tomley, F.M., 2008. Microneme proteins in apicomplexans. Subcell. Biochem. 47, 33 45. Charif, H., Darcy, F., Torpier, G., Cesbron-Delauw, M.F., Capron, A., 1990. Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites. Exp. Parasitol. 71, 114 124. Chaudhary, K., Donald, R.G., Nishi, M., Carter, D., Ullman, B., Roos, D.S., 2005. Differential localization of alternatively spliced hypoxanthine-xanthine-guanine phosphoribosyltransferase isoforms in Toxoplasma gondii. J. Biol. Chem. 280, 22053 22059. Che, F.Y., Madrid-Aliste, C., Burd, B., Zhang, H., Nieves, E., Kim, K., et al., 2010. Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii. Mol. Cell. Proteomics 10, M110.000745.

1011

Chen, A.L., Kim, E.W., Toh, J.Y., Vashisht, A.A., Rashoff, A.Q., Van, C., et al., 2015. Novel components of the Toxoplasma inner membrane complex revealed by BioID. mBio 6, e02357-14. Chen, A.L., Moon, A.S., Bell, H.N., Huang, A.S., Vashisht, A.A., Toh, J.Y., et al., 2017. Novel insights into the composition and function of the Toxoplasma IMC sutures. Cell. Microbiol. 19. Available from: https://doi.org/ 10.1111/cmi.12678. Chick, J.M., Kolippakkam, D., Nusinow, D.P., Zhai, B., Rad, R., Huttlin, E.L., et al., 2015. A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides. Nat. Biotechnol. 33, 743 749. Child, M.A., Hall, C.I., Beck, J.R., Ofori, L.O., Albrow, V.E., Garland, M., et al., 2013. Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion. Nat. Chem. Biol. 9, 651 656. Cleary, M.D., Singh, U., Blader, I.J., Brewer, J.L., Boothroyd, J.C., 2002. Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryot. Cell 1, 329 340. Cobbold, S.A., Santos, J.M., Ochoa, A., Perlman, D.H., Llinas, M., 2016. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite. Sci. Rep. 6, 19722. Cohen, A.M., Rumpel, K., Coombs, G.H., Wastling, J.M., 2002. Characterisation of global protein expression by twodimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii. Int. J. Parasitol. 32, 39 51. Collins, M.O., Wright, J.C., Jones, M., Rayner, J.C., Choudhary, J.S., 2014. Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation. J. Proteomics 103, 1 14. Conde De Felipe, M.M., Lehmann, M.M., Jerome, M.E., White, M.W., 2008. Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization. Mol. Biochem. Parasitol. 157, 22 31. Corvi, M.M., Alonso, A.M., Caballero, M.C., 2012. Protein palmitoylation and pathogenesis in apicomplexan parasites. J. Biomed. Biotechnol. 2012, 483969. Cox, J., Mann, M., 2007. Is proteomics the new genomics? Cell 130, 395 398. Cox, J., Mann, M., 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367 1372. Cox, J., Mann, M., 2011. Quantitative, high-resolution proteomics for data-driven systems biology. Annu. Rev. Biochem. 80, 273 299.

Toxoplasma Gondii

1012

22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

Croken, M.M., Ma, Y., Markillie, L.M., Taylor, R.C., Orr, G., Weiss, L.M., et al., 2014a. Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage. PLoS One 9, e111297. Croken, M.M., Qiu, W., White, M.W., Kim, K., 2014b. Gene set enrichment analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program. BMC Genomics 15, 515. Dahlin, J.L., Chen, X., Walters, M.A., Zhang, Z., 2015. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: lessons learned from Rtt109 histone acetyltransferases. Crit. Rev. Biochem. Mol. Biol. 50, 31 53. Daulny, A., Tansey, W.P., 2009. Damage control: DNA repair, transcription, and the ubiquitin-proteasome system. DNA Repair (Amst.) 8, 444 448. De Napoli, M.G., De Miguel, N., Lebrun, M., Moreno, S.N., Angel, S.O., Corvi, M.M., 2013. N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization. Biochim. Biophys. Acta 1833, 1329 1337. Dhara, A., Sinai, A.P., 2016. A cell cycle-regulated Toxoplasma deubiquitinase, TgOTUD3A, targets polyubiquitins with specific lysine linkages. mSphere 1, e00085-16. Available from: https://doi.org/10.1128/mSphere.00085-16. Dhingra, V., Gupta, M., Andacht, T., Fu, Z.F., 2005. New frontiers in proteomics research: a perspective. Int. J. Pharm. 299, 1 18. Dixon, S.E., Stilger, K.L., Elias, E.V., Naguleswaran, A., Sullivan, W.J., 2010. A decade of epigenetic research in Toxoplasma gondii. Mol. Biochem. Parasitol. 173, 1 9. Doerig, C., Rayner, J.C., Scherf, A., Tobin, A.B., 2015. Posttranslational protein modifications in malaria parasites. Nat. Rev. Microbiol. 13, 160 172. Doll, S., Burlingame, A.L., 2015. Mass spectrometry-based detection and assignment of protein posttranslational modifications. ACS Chem. Biol. 10, 63 71. Du, J., An, R., Chen, L., Shen, Y., Chen, Y., Cheng, L., et al., 2014. Toxoplasma gondii virulence factor ROP18 inhibits the host NF-kappaB pathway by promoting p65 degradation. J. Biol. Chem. 289, 12578 12592. Dybas, J.M., Madrid-Aliste, C.J., Che, F.Y., Nieves, E., Rykunov, D., Angeletti, R.H., et al., 2008. Computational analysis and experimental validation of gene predictions in Toxoplasma gondii. PLoS One 3, e3899. El Bissati, K., Suvorova, E.S., Xiao, H., Lucas, O., Upadhya, R., Ma, Y., et al., 2016. Toxoplasma gondii arginine methyltransferase 1 (PRMT1) is necessary for centrosome dynamics during tachyzoite cell division. mBio 7, e02094-15. El Hajj, H., Demey, E., Poncet, J., Lebrun, M., Wu, B., Galeotti, N., et al., 2006. The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic

characterization and molecular modeling. Proteomics 6, 5773 5784. Elsholz, A.K., Turgay, K., Michalik, S., Hessling, B., Gronau, K., Oertel, D., et al., 2012. Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 109, 7451 7456. Fauquenoy, S., Morelle, W., Hovasse, A., Bednarczyk, A., Slomianny, C., Schaeffer, C., et al., 2008. Proteomics and glycomics analyses of N-glycosylated structures involved in Toxoplasma gondii—host cell interactions. Mol. Cell. Proteomics 7, 891 910. Fentress, S.J., Behnke, M.S., Dunay, I.R., Mashayekhi, M., Rommereim, L.M., Fox, B.A., et al., 2010. Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence. Cell Host Microbe 8, 484 495. Fernandez-Garcia, A., Alvarez-Garcia, G., MaruganHernandez, V., Garcia-Lunar, P., Aguado-Martinez, A., Risco-Castillo, V., et al., 2013. Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis. Parasitology 140, 999 1008. Fleckenstein, M.C., Reese, M.L., Konen-Waisman, S., Boothroyd, J.C., Howard, J.C., Steinfeldt, T., 2012. A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins. PLoS Biol. 10, e1001358. Florens, L., Washburn, M.P., Raine, J.D., Anthony, R.M., Grainger, M., Haynes, J.D., et al., 2002. A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520 526. Foe, I.T., Child, M.A., Majmudar, J.D., Krishnamurthy, S., Van Der Linden, W.A., Ward, G.E., et al., 2015. Global analysis of palmitoylated proteins in Toxoplasma gondii. Cell Host Microbe 18, 501 511. Foth, B.J., Zhang, N., Mok, S., Preiser, P.R., Bozdech, Z., 2008. Quantitative protein expression profiling reveals extensive post-transcriptional regulation and posttranslational modifications in schizont-stage malaria parasites. Genome Biol. 9, R177. Foth, B.J., Zhang, N., Chaal, B.K., Sze, S.K., Preiser, P.R., Bozdech, Z., 2011. Quantitative time-course profiling of parasite and host cell proteins in the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 10, M110.006411. Frenal, K., Polonais, V., Marq, J.B., Stratmann, R., Limenitakis, J., Soldati-Favre, D., 2010. Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe 8, 343 357. Frenal, K., Marq, J.B., Jacot, D., Polonais, V., Soldati-Favre, D., 2014. Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion. PLoS Pathog. 10, e1004504.

Toxoplasma Gondii

References

Fritz, H.M., Bowyer, P.W., Bogyo, M., Conrad, P.A., Boothroyd, J.C., 2012. Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance. PLoS One 7, e29955. Fung, C., Beck, J.R., Robertson, S.D., Gubbels, M.J., Bradley, P.J., 2012. Toxoplasma ISP4 is a central IMC subcompartment protein whose localization depends on palmitoylation but not myristoylation. Mol. Biochem. Parasitol. 184, 99 108. Gaji, R.Y., Behnke, M.S., Lehmann, M.M., White, M.W., Carruthers, V.B., 2011. Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression. Mol. Microbiol. 79, 192 204. Gaji, R.Y., Johnson, D.E., Treeck, M., Wang, M., Hudmon, A., Arrizabalaga, G., 2015. Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid initiation of motility during Toxoplasma gondii egress. PLoS Pathog. 11, e1005268. Gajria, B., Bahl, A., Brestelli, J., Dommer, J., Fischer, S., Gao, X., et al., 2008. ToxoDB: an integrated Toxoplasma gondii database resource. Nucleic Acids Res. 36, D553 D556. Garrison, E., Treeck, M., Ehret, E., Butz, H., Garbuz, T., Oswald, B.P., et al., 2012. A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma. PLoS Pathog. 8, e1003049. Gas-Pascual, E., Ichikawa, H.T., Sheikh, M.O., Serji, M.I., Deng, B., Mandalasi, M., et al., 2019. CRISPER/Cas9 and glycomics tools for Toxoplasma glycobiology. J. Biol. Chem. 294, 1104 1125. Available from: https:// doi.org/10.1101/401828. Gaskins, E., Gilk, S., Devore, N., Mann, T., Ward, G., Beckers, C., 2004. Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J. Cell. Biol. 165, 383 393. Geiss-Friedlander, R., Melchior, F., 2007. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 8, 947 956. Gilk, S.D., Gaskins, E., Ward, G.E., Beckers, C.J., 2009. GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex. Eukaryot. Cell 8, 190 196. Gissot, M., Kelly, K.A., Ajioka, J.W., Greally, J.M., Kim, K., 2007. Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii. PLoS Pathog. 3, e77. Golebiowski, F., Matic, I., Tatham, M.H., Cole, C., Yin, Y., Nakamura, A., et al., 2009. System-wide changes to SUMO modifications in response to heat shock. Sci. Signal. 2, ra24. Gomez de Leon, C.T., Diaz Martin, R.D., Mendoza Hernandez, G., Gonzalez Pozos, S., Ambrosio, J.R., Mondragon Flores, R., 2014. Proteomic characterization

1013

of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites. J. Proteomics 111, 86 99. Gorg, A., Weiss, W., Dunn, M.J., 2004. Current twodimensional electrophoresis technology for proteomics. Proteomics 4, 3665 3685. Green, J.L., Rees-Channer, R.R., Howell, S.A., Martin, S.R., Knuepfer, E., Taylor, H.M., et al., 2008. The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. J. Biol. Chem. 283, 30980 30989. Guan, K.L., Dixon, J.E., 1991. Evidence for protein-tyrosinephosphatase catalysis proceeding via a cysteinephosphate intermediate. J. Biol. Chem. 266, 17026 17030. Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., Aebersold, R., 1999. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994 999. Haglund, K., Dikic, I., 2005. Ubiquitylation and cell signaling. EMBO J. 24, 3353 3359. Haley-Vicente, D., Edwards, D.J., 2003. Proteomic informatics: in silico methods lead to data management challenges. Curr. Opin. Drug. Discov. Dev. 6, 322 332. Hamady, M., Cheung, T.H., Resing, K., Cios, K.J., Knight, R., 2005. Key challenges in proteomics and proteoinformatics. Progress in proteins. IEEE Eng. Med. Biol. Mag. 24, 34 40. Hartmann, J., Hu, K., He, C.Y., Pelletier, L., Roos, D.S., Warren, G., 2006. Golgi and centrosome cycles in Toxoplasma gondii. Mol. Biochem. Parasitol. 145, 125 127. Heaslip, A.T., Nishi, M., Stein, B., Hu, K., 2011. The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase. PLoS Pathog. 7, e1002201. Herm-Gotz, A., Weiss, S., Stratmann, R., Fujita-Becker, S., Ruff, C., Meyhofer, E., et al., 2002. Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor. EMBO J. 21, 2149 2158. Hirschey, M.D., Zhao, Y., 2015. Metabolic regulation by lysine malonylation, succinylation, and glutarylation. Mol. Cell. Proteomics 14, 2308 2315. Holmes, M.J., Augusto, L.D.S., Zhang, M., Wek, R.C., Sullivan Jr., W.J., 2017. Translational control in the latency of apicomplexan parasites. Trends Parasitol. 33, 947 960. Hu, K., Johnson, J., Florens, L., Fraunholz, M., Suravajjala, S., Dilullo, C., et al., 2006. Cytoskeletal components of an invasion machine—the apical complex of Toxoplasma gondii. PLoS Pathog. 2, e13. Hunter, T., 2007. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol. Cell 28, 730 738. Ikeda, F., Dikic, I., 2008. Atypical ubiquitin chains: new molecular signals. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Rep. 9, 536 542.

Toxoplasma Gondii

1014

22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

Ishikawa, Y., Melville, D.B., 1970. The enzymatic alpha-Nmethylation of histidine. J. Biol. Chem. 245, 5967 5973. Issar, N., Roux, E., Mattei, D., Scherf, A., 2008. Identification of a novel post-translational modification in Plasmodium falciparum: protein sumoylation in different cellular compartments. Cell. Microbiol. 10, 1999 2011. Jackson, M.D., Denu, J.M., 2001. Molecular reactions of protein phosphatases—insights from structure and chemistry. Chem. Rev. 101, 2313 2340. Jacot, D., Soldati-Favre, D., 2012. Does protein phosphorylation govern host cell entry and egress by the Apicomplexa? Int. J. Med. Microbiol. 302, 195 202. Jacot, D., Frenal, K., Marq, J.B., Sharma, P., Soldati-Favre, D., 2014. Assessment of phosphorylation in Toxoplasma glideosome assembly and function. Cell. Microbiol. 16, 1518 1532. Jaffe, J.D., Berg, H.C., Church, G.M., 2004. Proteogenomic mapping as a complementary method to perform genome annotation. Proteomics 4, 59 77. Jeffers, V., Sullivan, W.J., 2012. Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii. Eukaryot. Cell 11, 735 742. Jensen, K.D., Hu, K., Whitmarsh, R.J., Hassan, M.A., Julien, L., Lu, D., et al., 2013. Toxoplasma gondii rhoptry 16 kinase promotes host resistance to oral infection and intestinal inflammation only in the context of the dense granule protein GRA15. Infect. Immun. 81, 2156 2167. Johnson, L.N., Barford, D., 1993. The effects of phosphorylation on the structure and function of proteins. Annu. Rev. Biophys. Biomol. Struct. 22, 199 232. Johnson, J.R., Florens, L., Carucci, D.J., Yates III., J.R., 2004. Proteomics in malaria. J. Proteome Res. 3, 296 306. Johnson, T.M., Rajfur, Z., Jacobson, K., Beckers, C.J., 2007. Immobilization of the type XIV myosin complex in Toxoplasma gondii. Mol. Biol. Cell. 18, 3039 3046. Jones, A., Hunt, E., Wastling, J.M., Pizarro, A., Stoeckert Jr., C.J., 2004. An object model and database for functional genomics. Bioinformatics 20, 1583 1590. Jones, M.L., Collins, M.O., Goulding, D., Choudhary, J.S., Rayner, J.C., 2012. Analysis of protein palmitoylation reveals a pervasive role in Plasmodium development and pathogenesis. Cell Host Microbe 12, 246 258. Joyce, B.R., Queener, S.F., Wek, R.C., Sullivan, W.J., 2010. Phosphorylation of eukaryotic initiation factor-2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 107, 17200 17205. Karasov, A.O., Boothroyd, J.C., Arrizabalaga, G., 2005. Identification and disruption of a rhoptry-localized homologue of sodium hydrogen exchangers in Toxoplasma gondii. Int. J. Parasitol. 35, 285 291.

Kaur, I., Zeeshan, M., Saini, E., Kaushik, A., Mohmmed, A., Gupta, D., et al., 2016. Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages. Sci. Rep. 6, 35432. Kawase, O., Nishikawa, Y., Bannai, H., Zhang, H., Zhang, G., Jin, S., et al., 2007. Proteomic analysis of calciumdependent secretion in Toxoplasma gondii. Proteomics 7, 3718 3725. Keeley, A., Soldati, D., 2004. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa. Trends Cell Biol. 14, 528 532. Khan, A., Bohme, U., Kelly, K.A., Adlem, E., Brooks, K., Simmonds, M., et al., 2006. Common inheritance of chromosome Ia associated with clonal expansion of Toxoplasma gondii. Genome Res. 16, 1119 1125. Kim, S.K., Fouts, A.E., Boothroyd, J.C., 2007. Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: insights from a genome-wide transcriptional profiling. J. Immunol. 178, 5154 5165. Kissinger, J.C., Gajria, B., Li, L., Paulsen, I.T., Roos, D.S., 2003. ToxoDB: accessing the Toxoplasma gondii genome. Nucleic Acids Res. 31, 234 236. Kono, M., Prusty, D., Parkinson, J., Gilberger, T.W., 2013. The apicomplexan inner membrane complex. Front. Biosci. (Landmark Ed) 18, 982 992. Kremer, A., Schneider, R., Terstappen, G.C., 2005. A bioinformatics perspective on proteomics: data storage, analysis, and integration. Biosci. Rep. 25, 95 106. Krishna, R., Wastling, J.M., Jones, A.R. 2011. Automated integration of Mass Spectrometry based proteomics evidence for improvement of gene annotations. In: Eighth BSPR-EBI Meeting. Vistable to the Hidden Proteome Wellcome Trust Conference Center, Cambridge, UK. Krishna, R., Xia, D., Sanderson, S., Shanmugasundram, A., Vermont, S., Bernal, A., et al., 2015. A large-scale proteogenomics study of apicomplexan pathogensToxoplasma gondii and Neospora caninum. Proteomics 15, 2618 2628. Lambert, J.P., Tucholska, M., Go, C., Knight, J.D., Gingras, A.C., 2015. Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes. J. Proteomics 118, 81 94. Lane, C.S., 2005. Mass spectrometry-based proteomics in the life sciences. Cell. Mol. Life Sci. 62, 848 869. Larsen, S.C., Sylvestersen, K.B., Mund, A., Lyon, D., Mullari, M., Madsen, M.V., et al., 2016. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells. Sci. Signal. 9, 1 15. Lasonder, E., Ishihama, Y., Andersen, J.S., Vermunt, A.M., Pain, A., Sauerwein, R.W., et al., 2002. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 419, 537 542.

Toxoplasma Gondii

References

Lasonder, E., Green, J.L., Camarda, G., Talabani, H., Holder, A.A., Langsley, G., et al., 2012. The Plasmodium falciparum schizont phosphoproteome reveals extensive phosphatidylinositol and cAMP-protein kinase A signaling. J. Proteome Res. 11, 5323 5337. Lasonder, E., Green, J.L., Grainger, M., Langsley, G., Holder, A.A., 2015. Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites. Proteomics 15, 2716 2729. Laub, M.T., Goulian, M., 2007. Specificity in twocomponent signal transduction pathways. Annu. Rev. Genet. 41, 121 145. Le Roch, K.G., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M., et al., 2004. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 14, 2308 2318. Lee, E.K., 2012. Post-translational modifications of RNAbinding proteins and their roles in RNA granules. Curr. Protein Pept. Sci. 13, 331 336. Lee, Y.J., Miyake, S., Wakita, H., Mcmullen, D.C., AZUMa, Y., Auh, S., et al., 2007. Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells. J. Cereb. Blood Flow. Metab. 27, 950 962. Leriche, M.A., Dubremetz, J.F., 1991. Characterization of the protein contents of rhoptries and dense granules of Toxoplasma gondii tachyzoites by subcellular fractionation and monoclonal antibodies. Mol. Biochem. Parasitol. 45, 249 259. Lescault, P.J., Thompson, A.B., Patil, V., Lirussi, D., Burton, A., Margarit, J., et al., 2010. Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state. PLoS One 5, e14463. Li, X., Hu, X., Wan, Y., Xie, G., Li, X., Chen, D., et al., 2014. Systematic identification of the lysine succinylation in the protozoan parasite Toxoplasma gondii. J. Proteome Res. 13, 6087 6095. Liu, T., Martin, A.M., Sinai, A.P., Lynn, B.C., 2008. Threelayer sandwich gel electrophoresis: a method of salt removal and protein concentration in proteome analysis. J. Proteome Res. 7, 4256 4265. Long, S., Brown, K.M., Sibley, L.D., 2018. CRISPR-mediated tagging with BirA allows proximity labeling in Toxoplasma gondii. Bio Protoc 8, e2768. Available from: https://doi.org/10.21769/BioProtoc.2768. Lovci, M.T., Bengtson, M.H., Massirer, K.B., 2016. Posttranslational modifications and RNA-binding proteins. Adv. Exp. Med. Biol. 907, 297 317.

1015

Luk, F.C., Johnson, T.M., Beckers, C.J., 2008. N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii. Mol. Biochem. Parasitol. 157, 169 178. Luo, Q., Upadhya, R., Zhang, H., Madrid-Aliste, C., Nieves, E., Kim, K., et al., 2011. Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry. Microbes Infect. 13, 1199 1210. Madrid-Aliste, C.J., Dybas, J.M., Angeletti, R.H., Weiss, L. M., Kim, K., Simon, I., et al., 2009. EPIC-DB: a proteomics database for studying Apicomplexan organisms. BMC Genomics 10, 38. Mair, G.R., Braks, J.A., Garver, L.S., Wiegant, J.C., Hall, N., Dirks, R.W., et al., 2006. Regulation of sexual development of Plasmodium by translational repression. Science 313, 667 669. Malmstrom, L., Malmstrom, J., Aebersold, R., 2011. Quantitative proteomics of microbes: Principles and applications to virulence. Proteomics 11, 2947 2956. Mann, M., Jensen, O.N., 2003. Proteomic analysis of posttranslational modifications. Nat. Biotechnol. 21, 255 261. Manning, G., Whyte, D.B., Martinez, R., Hunter, T., Sudarsanam, S., 2002. The protein kinase complement of the human genome. Science 298, 1912 1934. Martin, B.R., Cravatt, B.F., 2009. Large-scale profiling of protein palmitoylation in mammalian cells. Nat. Methods 6, 135 138. Marugan-Hernandez, V., Alvarez-Garcia, G., Risco-Castillo, V., Regidor-Cerrillo, J., Ortega-Mora, L.M., 2010. Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE. Proteomics 10, 1740 1750. Matrajt, M., Donald, R.G., Singh, U., Roos, D.S., 2002. Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii. Mol. Microbiol. 44, 735 747. McCoy, J.M., Whitehead, L., Van Dooren, G.G., Tonkin, C. J., 2012. TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells. PLoS Pathog. 8, e1003066. McFadden, G.I., Yeh, E., 2017. The apicoplast: now you see it, now you don’t. Int. J. Parasitol. 47, 137 144. McKee, A.S., Dzierszinski, F., Boes, M., Roos, D.S., Pearce, E.J., 2004. Functional inactivation of immature dendritic cells by the intracellular parasite Toxoplasma gondii. J. Immunol. 173, 2632 2640. Meissner, M., Schluter, D., Soldati, D., 2002. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837 840. Melo, M.B., Nguyen, Q.P., Cordeiro, C., Hassan, M.A., Yang, N., Mckell, R., et al., 2013. Transcriptional

Toxoplasma Gondii

1016

22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways. PLoS Pathog. 9, e1003779. Mertins, P., Qiao, J.W., Patel, J., Udeshi, N.D., Clauser, K. R., Mani, D.R., et al., 2013. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat. Methods 10, 634 637. Miao, J., Fan, Q., Cui, L., Li, J., Li, J., Cui, L., 2006. The malaria parasite Plasmodium falciparum histones: organization, expression, and acetylation. Gene 369, 53 65. Miller, S.A., Thathy, V., Ajioka, J.W., Blackman, M.J., Kim, K., 2003. TgSUB2 is a Toxoplasma gondii rhoptry organelle processing proteinase. Mol. Microbiol. 49, 883 894. Molina-Serrano, D., Schiza, V., Kirmizis, A., 2013. Crosstalk among epigenetic modifications: lessons from histone arginine methylation. Biochem. Soc. Trans. 41, 751 759. Montagna, G.N., Buscaglia, C.A., Munter, S., Goosmann, C., Frischknecht, F., Brinkmann, V., et al., 2012a. Critical role for heat shock protein 20 (HSP20) in migration of malarial sporozoites. J. Biol. Chem. 287, 2410 2422. Montagna, G.N., Matuschewski, K., Buscaglia, C.A., 2012b. Small heat shock proteins in cellular adhesion and migration: evidence from Plasmodium genetics. Cell Adhes. Migr. 6, 78 84. Morrison, R.S., Kinoshita, Y., Johnson, M.D., Conrads, T.P., 2003. Proteomics in the postgenomic age. Adv. Protein Chem. 65, 1 23. Morrissette, N.S., Murray, J.M., Roos, D.S., 1997. Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J. Cell. Sci. 110 (Pt 1), 35 42. Nadipuram, S.M., Kim, E.W., Vashisht, A.A., Lin, A.H., Bell, H.N., Coppens, I., et al., 2016. In vivo biotinylation of the Toxoplasma parasitophorous vacuole reveals novel dense granule proteins important for parasite growth and pathogenesis. mBio 7, e00808-16. Available from: https://doi.org/10.1128/mBio.00808-16. Nagaraj, N., Wisniewski, J.R., Geiger, T., Cox, J., Kircher, M., Kelso, J., et al., 2011. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548. Nardelli, S.C., Che, F.Y., Silmon de Monerri, N.C., Xiao, H., Nieves, E., Madrid-Aliste, C., et al., 2013. The histone code of Toxoplasma gondii comprises conserved and unique posttranslational modifications. mBio 4, e0092213. Nebl, T., Prieto, J.H., Kapp, E., Smith, B.J., Williams, M.J., Yates, J.R., et al., 2011. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex. PLoS Pathog. 7, e1002222.

Nelson, M.M., Jones, A.R., Carmen, J.C., Sinai, A.P., Burchmore, R., Wastling, J.M., 2008. Modulation of the host cell proteome by the intracellular apicomplexan parasite Toxoplasma gondii. Infect. Immun. 76, 828 844. Ngounou Wetie, A.G., Woods, A.G., Darie, C.C., 2014. Mass spectrometric analysis of post-translational modifications (PTMs) and protein-protein interactions (PPIs). Adv. Exp. Med. Biol. 806, 205 235. Niedelman, W., Gold, D.A., Rosowski, E.E., Sprokholt, J.K., Lim, D., Farid Arenas, A., et al., 2012. The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferongamma response. PLoS Pathog. 8, e1002784. Nijman, S.M., Luna-Vargas, M.P., Velds, A., Brummelkamp, T.R., Dirac, A.M., Sixma, T.K., et al., 2005. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773 786. Nishi, M., Hu, K., Murray, J.M., Roos, D.S., 2008. Organellar dynamics during the cell cycle of Toxoplasma gondii. J. Cell. Sci. 121, 1559 1568. Ohtsubo, K., Marth, J.D., 2006. Glycosylation in cellular mechanisms of health and disease. Cell 126, 855 867. Olsen, J.V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., et al., 2006. Global, in vivo, and sitespecific phosphorylation dynamics in signaling networks. Cell 127, 635 648. Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., et al., 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376 386. Ong, Y.C., Reese, M.L., Boothroyd, J.C., 2010. Toxoplasma rhoptry protein 16 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6. J. Biol. Chem. 285, 28731 28740. Ossorio, P.N., Schwartzman, J.D., Boothroyd, J.C., 1992. A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry. Mol. Biochem. Parasitol. 50, 1 15. Paba, J., Ricart, C.A., Fontes, W., Santana, J.M., Teixeira, A. R., Marchese, J., et al., 2004. Proteomic analysis of Trypanosoma cruzi developmental stages using isotopecoded affinity tag reagents. J. Proteome Res. 3, 517 524. Paik, W.K., Paik, D.C., Kim, S., 2007. Historical review: the field of protein methylation. Trends Biochem. Sci. 32, 146 152. Palagi, P.M., Walther, D., Quadroni, M., Catherinet, S., Burgess, J., Zimmermann-Ivol, C.G., et al., 2005. MSight: an image analysis software for liquid chromatographymass spectrometry. Proteomics 5, 2381 2384. Pan, M., Li, M., Li, L., Song, Y., Hou, L., Zhao, J., et al., 2018. Identification of novel dense-granule proteins in Toxoplasma gondii by two proximity-based biotinylation

Toxoplasma Gondii

References

approaches. J. Proteome Res. 18, 319 330. Available from: https://doi.org/10.1021/acs.jproteome.8b00626. Park, J., Chen, Y., Tishkoff, D.X., Peng, C., Tan, M., Dai, L., et al., 2013. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways. Mol. Cell 50, 919 930. Patron, S.A., Mondragon, M., Gonzalez, S., Ambrosio, J.R., Guerrero, B.A., Mondragon, R., 2005. Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites. Int. J. Parasitol. 35, 883 894. Pease, B.N., Huttlin, E.L., Jedrychowski, M.P., Talevich, E., Harmon, J., Dillman, T., et al., 2013. Global analysis of protein expression and phosphorylation of three stages of Plasmodium falciparum intraerythrocytic development. J. Proteome Res. 12, 4028 4045. Pellegrini, E., Palencia, A., Braun, L., Kapp, U., Bougdour, A., Belrhali, H., et al., 2017. Structural basis for the subversion of MAP kinase signaling by an intrinsically disordered parasite secreted agonist. Structure 25, 16 26. Pelletier, L., Stern, C.A., Pypaert, M., Sheff, D., Ngo, H.M., Roper, N., et al., 2002. Golgi biogenesis in Toxoplasma gondii. Nature 418, 548 552. Perez-Cadahia, B., Drobic, B., Davie, J.R., 2009. H3 phosphorylation: dual role in mitosis and interphase. Biochem. Cell. Biol. 87, 695 709. Perez-Cervera, Y., Harichaux, G., Schmidt, J., DebierreGrockiego, F., Dehennaut, V., Bieker, U., et al., 2011. Direct evidence of O-GlcNAcylation in the apicomplexan Toxoplasma gondii: a biochemical and bioinformatic study. Amino Acids 40, 847 856. Phillips, C.I., Bogyo, M., 2005. Proteomics meets microbiology: technical advances in the global mapping of protein expression and function. Cell. Microbiol. 7, 1061 1076. Pivovarova, Y., Liu, J., Lesigang, J., Koldyka, O., Rauschmeier, R., Hu, K., et al., 2018. Structure of a novel dimeric SET domain methyltransferase that regulates cell motility. J. Mol. Biol. 430, 4209 4229. Plessmann, U., Reiter-Owona, I., Lechtreck, K.F., 2004. Posttranslational modifications of alpha-tubulin of Toxoplasma gondii. Parasitol. Res. 94, 386 389. Ponts, N., Saraf, A., Chung, D.W., Harris, A., Prudhomme, J., Washburn, M.P., et al., 2011. Unraveling the ubiquitome of the human malaria parasite. J. Biol. Chem. 286, 40320 40330. Ponts, N., Yang, J., Chung, D.W., Prudhomme, J., Girke, T., Horrocks, P., et al., 2008. Deciphering the ubiquitinmediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence. PLoS One 3, e2386. Que, X., Ngo, H., Lawton, J., Gray, M., Liu, Q., Engel, J., et al., 2002. The cathepsin B of Toxoplasma gondii,

1017

toxopain-1, is critical for parasite invasion and rhoptry protein processing. J. Biol. Chem. 277, 25791 25797. Radke, J.R., Striepen, B., Guerini, M.N., Jerome, M.E., Roos, D.S., White, M.W., 2001. Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii. Mol. Biochem. Parasitol. 115, 165 175. Radke, J.R., Behnke, M.S., Mackey, A.J., Radke, J.B., Roos, D.S., White, M.W., 2005. The transcriptome of Toxoplasma gondii. BMC. Biol. 3, 26. Rardin, M.J., He, W., Nishida, Y., Newman, J.C., Carrico, C., Danielson, S.R., et al., 2013. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks. Cell. Metab. 18, 920 933. Rees-Channer, R.R., Martin, S.R., Green, J.L., Bowyer, P.W., Grainger, M., Molloy, J.E., et al., 2006. Dual acylation of the 45 kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites. Mol. Biochem. Parasitol. 149, 113 116. Reichmann, G., Dlugonska, H., Fischer, H.G., 2002. Characterization of TgROP9 (p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified by T cell clone. Mol. Biochem. Parasitol. 119, 43 54. Reid, A.J., Vermont, S.J., Cotton, J.A., Harris, D., HillCawthorne, G.A., Konen-Waisman, S., et al., 2012. Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy. PLoS Pathog. 8, e1002567. Reiter, K., Mukhopadhyay, D., Zhang, H., Boucher, L.E., Kumar, N., Bosch, J., et al., 2013. Identification of biochemically distinct properties of the small ubiquitinrelated modifier (SUMO) conjugation pathway in Plasmodium falciparum. J. Biol. Chem. 288, 27724 27736. Rosowski, E.E., Saeij, J.P., 2012. Toxoplasma gondii clonal strains all inhibit STAT1 transcriptional activity but polymorphic effectors differentially modulate IFNgamma induced gene expression and STAT1 phosphorylation. PLoS One 7, e51448. Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., et al., 2004. Multiplexed protein quantitation in Saccharomyces cerevisiae using aminereactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154 1169. Roux, K.J., Kim, D.I., Raida, M., Burke, B., 2012. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell. Biol. 196, 801 810. Sadak, A., Taghy, Z., Fortier, B., Dubremetz, J.F., 1988. Characterization of a family of rhoptry proteins of Toxoplasma gondii. Mol. Biochem. Parasitol. 29, 203 211. Saeij, J.P., Coller, S., Boyle, J.P., Jerome, M.E., White, M.W., Boothroyd, J.C., 2007. Toxoplasma co-opts host gene

Toxoplasma Gondii

1018

22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

expression by injection of a polymorphic kinase homologue. Nature 445, 324 327. Saksouk, N., Bhatti, M.M., Kieffer, S., Aaron, T., Musset, K., Garin, J., et al., 2005. Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii. Mol. Cell. Biol. 25, 10301 10314. Sanderson, S.J., Xia, D., Prieto, H., Yates, J., Heiges, M., Kissinger, J.C., et al., 2008. Determining the protein repertoire of Cryptosporidium parvum sporozoites. Proteomics 8, 1398 1414. Sautel, C.F., Cannella, D., Bastien, O., Kieffer, S., Aldebert, D., Garin, J., et al., 2007. SET8-mediated methylations of histone H4 lysine 20 mark silent heterochromatic domains in apicomplexan genomes. Mol. Cell. Biol. 27, 5711 5724. Sautel, C.F., Ortet, P., Saksouk, N., Kieffer, S., Garin, J., Bastien, O., et al., 2009. The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences. Mol. Microbiol. 71, 212 226. Schwanhausser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., et al., 2011. Global quantification of mammalian gene expression control. Nature 473, 337 342. Seidi, A., Muellner-Wong, L.S., Rajendran, E., Tjhin, E.T., Dagley, L.F., Aw, V.Y., et al., 2018. Elucidating the mitochondrial proteome of Toxoplasma gondii reveals the presence of a divergent cytochrome c oxidase. eLife 7, e38131. Available from: https://doi.org/10.7554/eLife.38131. Seo, J., Lee, K.J., 2004. Post-translational modifications and their biological functions: proteomic analysis and systematic approaches. J. Biochem. Mol. Biol. 37, 35 44. Sibley, L.D., Mordue, D.G., Su, C., Robben, P.M., Howe, D. K., 2002. Genetic approaches to studying virulence and pathogenesis in Toxoplasma gondii. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 357, 81 88. Silmon de Monerri, N.C., Weiss, L.M., 2015. Integration of RNA-seq and proteomics data with genomics for improved genome annotation in Apicomplexan parasites. Proteomics 15, 2557 2559. Silmon de Monerri, N.C., Yakubu, R.R., Chen, A.L., Bradley, P.J., Nieves, E., Weiss, L.M., et al., 2015. The ubiquitin proteome of Toxoplasma gondii reveals roles for protein ubiquitination in cell-cycle transitions. Cell Host Microbe 18, 621 633. Singh, U., Brewer, J.L., Boothroyd, J.C., 2002. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Mol. Microbiol. 44, 721 733. Sivagurunathan, S., Heaslip, A., Liu, J., Hu, K., 2013. Identification of functional modules of AKMT, a novel

lysine methyltransferase regulating the motility of Toxoplasma gondii. Mol. Biochem. Parasitol. 189, 43 53. Solyakov, L., Halbert, J., Alam, M.M., Semblat, J.P., DorinSemblat, D., Reininger, L., et al., 2011. Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum. Nat. Commun. 2, 565. Souchelnytskyi, S., 2005. Bridging proteomics and systems biology: what are the roads to be traveled? Proteomics 5, 4123 4137. Srivastava, S., Bhowmick, K., Chatterjee, S., Basha, J., Kundu, T.K., Dhar, S.K., 2014. Histone H3K9 acetylation level modulates gene expression and may affect parasite growth in human malaria parasite Plasmodium falciparum. FEBS J. 281, 5265 5278. Sturm, M., Bertsch, A., Gropl, C., Hildebrandt, A., Hussong, R., Lange, E., et al., 2008. OpenMS—an opensource software framework for mass spectrometry. BMC Bioinformatics 9, 163. Sullivan Jr., W.J., Hakimi, M.A., 2006. Histone mediated gene activation in Toxoplasma gondii. Mol. Biochem. Parasitol. 148, 109 116. Svejstrup, J.Q., 2007. Contending with transcriptional arrest during RNAPII transcript elongation. Trends Biochem. Sci. 32, 165 171. Swaney, D.L., Villen, J., 2016. Proteomic analysis of protein posttranslational modifications by mass spectrometry. Cold Spring Harb. Protoc. 2016, pdb.top077743. Sylvestersen, K.B., Horn, H., Jungmichel, S., Jensen, L.J., Nielsen, M.L., 2014. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest. Mol. Cell. Proteomics: MCP 13, 2072 2088. Tan, M., Luo, H., Lee, S., Jin, F., Yang, J.S., Montellier, E., et al., 2011. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 146, 1016 1028. Tang, Q., Andenmatten, N., Hortua Triana, M.A., Deng, B., Meissner, M., Moreno, S.N., et al., 2014. Calciumdependent phosphorylation alters class XIV a myosin function in the protozoan parasite Toxoplasma gondii. Mol. Biol. Cell. 25, 2579 2591. Taylor, C.F., Paton, N.W., Garwood, K.L., Kirby, P.D., Stead, D.A., Yin, Z., et al., 2003. A systematic approach to modeling, capturing, and disseminating proteomics experimental data. Nat. Biotechnol. 21, 247 254. Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., et al., 2006. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314, 1776 1780. Teixeira, L.K., Reed, S.I., 2013. Ubiquitin ligases and cell cycle control. Annu. Rev. Biochem. 82, 387 414. Tempe, D., Piechaczyk, M., Bossis, G., 2008. SUMO under stress. Biochem. Soc. Trans. 36, 874 878.

Toxoplasma Gondii

References

Thingholm, T.E., Jorgensen, T.J., Jensen, O.N., Larsen, M.R., 2006. Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat. Protoc. 1, 1929 1935. Tomita, T., Bzik, D.J., Ma, Y.F., Fox, B.A., Markillie, L.M., Taylor, R.C., et al., 2013. The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence. PLoS Pathog. 9, e1003823. Tomita, T., Sugi, T., Yakubu, R., Tu, V., Ma, Y., Weiss, L.M., 2017. Making home sweet and sturdy: Toxoplasma gondii ppGalNAc-Ts glycosylate in hierarchical order and confer cyst wall rigidity. mBio 8, e02048-16. Available from: https://doi.org/10.1128/mBio.02048-16. Tonkin, M.L., Beck, J.R., Bradley, P.J., Boulanger, M.J., 2014. The inner membrane complex sub-compartment proteins critical for replication of the apicomplexan parasite Toxoplasma gondii adopt a pleckstrin homology fold. J. Biol. Chem. 289, 13962 13973. Treeck, M., Sanders, J.L., Elias, J.E., Boothroyd, J.C., 2011. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries. Cell Host Microbe 10, 410 419. Trelle, M.B., Salcedo-Amaya, A.M., Cohen, A.M., Stunnenberg, H.G., Jensen, O.N., 2009. Global histone analysis by mass spectrometry reveals a high content of acetylated lysine residues in the malaria parasite Plasmodium falciparum. J. Proteome Res. 8, 3439 3450. Trenholme, K., Marek, L., Duffy, S., Pradel, G., Fisher, G., Hansen, F.K., et al., 2014. Lysine acetylation in sexual stage malaria parasites is a target for antimalarial small molecules. Antimicrob. Agents Chemother. 58, 3666 3678. Tu, V., Mayoral, J., Sugi, T., Tomita, T., Han, B., Ma, Y.F., et al., 2019a. Enrichment and proteomic characterization of the cyst wall from in vitro Toxoplasma gondii cysts. mBio 10, e00469-19. Available from: https://doi.org/ 10.1128/mBio.00469-19. Tu, V., Tomita, T., Sugi, T., Mayoral, J., Han, B., Yakubu, R., et al., 2019. The Toxoplasma gondii cyst wall interactome. mBio, in press. Varberg, J.M., Padgett, L.R., Arrizabalaga, G., Sullivan Jr., W.J., 2016. TgATAT-mediated alpha-tubulin acetylation is required for division of the protozoan parasite Toxoplasma gondii. mSphere 1, e00088-15. Available from: https://doi.org/10.1128/mSphere.00088-15. Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R. J., Sutton, G.G., et al., 2001. The sequence of the human genome. Science 291, 1304 1351. Walsh, C.T., Garneau-Tsodikova, S., Gatto Jr., G.J., 2005. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. Engl. 44, 7342 7372.

1019

Wan, J., Roth, A.F., Bailey, A.O., Davis, N.G., 2007. Palmitoylated proteins: purification and identification. Nat. Protoc. 2, 1573 1584. Wang, J., Dixon, S.E., Ting, L.M., Liu, T.K., Jeffers, V., Croken, M.M., et al., 2014. Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation. PLoS Pathog. 10. Wang, C., Leffler, S., Thompson, D.H., Hrycyna, C.A., 2005a. A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases. Biochem. Biophys. Res. Commun. 331, 351 356. Wang, R., Prince, J.T., Marcotte, E.M., 2005b. Mass spectrometry of the M. smegmatis proteome: protein expression levels correlate with function, operons, and codon bias. Genome Res. 15, 1118 1126. Wang, K., Peng, E.D., Huang, A.S., Xia, D., Vermont, S.J., Lentini, G., et al., 2016. Identification of novel O-linked glycosylated Toxoplasma proteins by Vicia villosa lectin chromatography. PLoS One 11, e0150561. Wang, Z.X., Zhou, C.X., Elsheikha, H.M., He, S., Zhou, D. H., Zhu, X.Q., 2017. Proteomic Differences between developmental stages of Toxoplasma gondii revealed by iTRAQ-based quantitative proteomics. Front. Microbiol. 8, 985. Washburn, M.P., Wolters, D., Yates III., J.R., 2001. Largescale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242 247. Wastling, J.M., Xia, D., Sohal, A., Chaussepied, M., Pain, A., Langsley, G., 2009. Proteomes and transcriptomes of the Apicomplexa—where’s the message? Int. J. Parasitol. 39, 135 143. Wastling, J.M., Armstrong, S.D., Krishna, R., Xia, D., 2012. Parasites, proteomes and systems: has Descartes’ clock run out of time? Parasitology 139, 1103 1118. Weinert, B.T., Scholz, C., Wagner, S.A., Iesmantavicius, V., Su, D., Daniel, J.A., et al., 2013. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell Rep 4, 842 851. Witze, E.S., Old, W.M., Resing, K.A., Ahn, N.G., 2007. Mapping protein post-translational modifications with mass spectrometry. Nat. Methods 4, 798 806. Xia, D., Sanderson, S.J., Jones, A.R., Prieto, J.H., Yates, J.R., Bromley, E., et al., 2008. The proteome of Toxoplasma gondii: integration with the genome provides novel insights into gene expression and annotation. Genome Biol. 9, R116. Xiao, H., El Bissati, K., Verdier-Pinard, P., Burd, B., Zhang, H., Kim, K., et al., 2010. Post-translational modifications to Toxoplasma gondii alpha- and beta-tubulins include novel C-terminal methylation. J. Proteome Res. 9, 359 372.

Toxoplasma Gondii

1020

22. Proteomics and posttranslational protein modifications in Toxoplasma gondii

Xie, Z., Dai, J., Dai, L., Tan, M., Cheng, Z., Wu, Y., et al., 2012. Lysine succinylation and lysine malonylation in histones. Mol. Cell. Proteomics 11, 100 107. Xue, B., Jeffers, V., Sullivan, W.J., Uversky, V.N., 2013. Protein intrinsic disorder in the acetylome of intracellular and extracellular Toxoplasma gondii. Mol. Biosyst. 9, 645 657. Yakubu, R.R., Silmon de Monerri, N.C., Nieves, E., Kim, K., Weiss, L.M., 2017. Comparative monomethylarginine proteomics suggests that protein arginine methyltransferase 1 (PRMT1) is a significant contributor to arginine monomethylation in Toxoplasma gondii. Mol. Cell. Proteomics . Available from: https://doi.org/10.1074/ mcp.M117.066951. Yakubu, R.R., Weiss, L.M., Silmon de Monerri, N.C., 2018. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies. Mol. Microbiol. 107, 1 23. Yamagata, K., Daitoku, H., Takahashi, Y., Namiki, K., Hisatake, K., Kako, K., et al., 2008. Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol. Cell 32, 221 231. Yamamoto, M., Standley, D.M., Takashima, S., Saiga, H., Okuyama, M., Kayama, H., et al., 2009. A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3. J. Exp. Med. 206, 2747 2760. Yates III., J.R., 2004. Mass spectral analysis in proteomics. Annu. Rev. Biophys. Biomol. Struct. 33, 297 316.

Yates III., J.R., Gilchrist, A., Howell, K.E., Bergeron, J.J., 2005. Proteomics of organelles and large cellular structures. Nat. Rev. Mol. Cell Biol. 6, 702 714. Zeeshan, M., Kaur, I., Joy, J., Saini, E., Paul, G., Kaushik, A., et al., 2017. Proteomic identification and analysis of arginine-methylated proteins of Plasmodium falciparum at asexual blood stages. J. Proteome Res. 16, 368 383. Available from: https://doi.org/10.1021/acs. jproteome.5b01052. Zhang, Q.C., Petrey, D., Deng, L., Qiang, L., Shi, Y., Thu, C. A., et al., 2012. Structure-based prediction of proteinprotein interactions on a genome-wide scale. Nature 490, 556 560. Zhou, X.W., Blackman, M.J., Howell, S.A., Carruthers, V.B., 2004. Proteomic analysis of cleavage events reveals a dynamic two-step mechanism for proteolysis of a key parasite adhesive complex. Mol. Cell. Proteomics 3, 565 576. Zhou, X.W., Kafsack, B.F., Cole, R.N., Beckett, P., Shen, R. F., Carruthers, V.B., 2005. The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins. J. Biol. Chem. 280, 34233 34244. Zhou, D.H., Yuan, Z.G., Zhao, F.R., Li, H.L., Zhou, Y., Lin, R.Q., et al., 2011. Modulation of mouse macrophage proteome induced by Toxoplasma gondii tachyzoites in vivo. Parasitol. Res. 109, 1637 1646. Zhou, C.X., Zhu, X.Q., Elsheikha, H.M., He, S., Li, Q., Zhou, D.H., et al., 2016. Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts during sporulation. J. Proteomics 148, 12 19.

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23 ToxoDB: the functional genomic resource for Toxoplasma and related organisms* Omar S. Harb1, Jessica C. Kissinger2 and David S. Roos1 1

Department of Biology, University of Pennsylvania, Philadelphia, PA, United States 2Center for Tropical & Emerging Global Diseases, Department of Genetics & Institute of Bioinformatics, University of Georgia, Athens, GA, United States

23.1 Introduction ToxoDB (http://ToxoDB.org) continues to expand in functionality and data content. In addition to including genomes and functional data from Toxoplasma gondii strains, ToxoDB also contains genomes and data from additional Sarcocystidae, such as Neospora caninum and Sarcocystis neurona, and Eimeriidae, such as Eimeria tenella and Cyclospora cayetanensis. From its inception, one of the major goals of ToxoDB has been to provide the global Toxoplasma research community with a cutting edge and useful resource that supports daily laboratory research activities. Importantly, paramount to its success is accessibility to online data interrogation tools without the need for sophisticated computational skills beyond basic knowledge of Internet browser utility. This is an update of the chapter that appeared in the second edition of this book series.

The idea of ToxoDB began to evolve in 1998, when it became necessary for a number of research groups to computationally query expressed sequence tag (EST) data using basic local alignment search tool (BLAST) (Ajioka et al., 1998; Altschul et al., 1990; Kissinger et al., 2003). It soon became clear that making large-scale data available to the entire community has tremendous benefits in bolstering basic research and moving the field forward. Moreover, as high-throughput techniques became more readily available coupled with vast expansions in computational power, it became critical to not only make data available but also easily searchable. The establishment of the National Institute of Allergy and Infectious Diseases-funded Bioinformatics Resource Centers (https://www.niaid.nih. gov/research/bioinformatics-resource-centers) (Greene et al., 2007) provided a structured mechanism to deliver a ToxoDB from a

* On behalf of the EuPathDB group.

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23. ToxoDB: the functional genomic resource for Toxoplasma and related organisms

laboratory research project into a production operation as part of the Eukaryotic Pathogen Database resources (EuPathDB: http://eupathdb. org) (Aurrecoechea et al., 2017). The mission of EuPathDB focuses on primarily providing a service to the communities it serves. EuPathDB is charged with ensuring that genomic (and other large-scale) datasets pertaining to supported pathogens are conveniently accessible to the worldwide community of biomedical researchers. ToxoDB is designed to make bioinformatics easily accessible to the bench scientist and offers following three key features that keep it relevant and useful to the community: 1. Integration of diverse large-scale “omics” datasets in a timely manner. This necessitates continuous evolution and readiness to develop and incorporate new types of data. In addition, the bimonthly release schedule of all EuPathDB databases means that there are six annual opportunities for new data to be integrated based on community prioritization. 2. A search system and advanced browsing capabilities enable scientists to integrate and ask their own questions about large-scale datasets. The goal is not to simply replicate the final conclusions of the publication but to complement any publication with the ability to make new discoveries and develop new hypotheses. The underlying data can be queried using a user-friendly graphic web interface. 3. Primary data analysis with the ability to integrate results in ToxoDB enables scientists to analyze their own data using the Galaxy platform then integrate results into ToxoDB where they can interrogate them in the context of all other data in the database. An important aspect of data integration is that EuPathDB staff work closely with data

providers who frequently make data available prior to publication. EuPathDB adheres to a strict standard operating procedure vis-a-vis data release that ensures proper quality assurance of data, and that the data provider has the final say in when their data is publicly released. The full data release policy may be accessed here: http://toxodb.org/EuPathDB_ datasubm_SOP.pdf. The EuPathDB group is organized into teams each of which carries out essential functions needed for the final release of a production database (e.g., the production ToxoDB). Teams include database administrators, system administrators, software engineers, user interface developers, data loaders, data developers, and community outreach specialists. The organization of the teams and methods of effective communication have evolved over the years to produce a robust mechanism for continuous data integration, software and website innovations, and community support. Importantly, the EuPathDB group does not include any students or postdoctoral fellows ensuring that EuPathDB continues to focus on community needs rather than personal research projects (Roos, 2011).

23.2 Data content As with any rapidly evolving online resource, it is critical that the reader consults the ToxoDB website to view the most recent updates. One place to get the most up-to-date information about what is new in each release is the news section: https://toxodb.org/news. ToxoDB also includes a searchable dataset page that allows a user to quickly find the datasets they are looking for by searching using key words: https://toxodb.org/toxo/ app/search/dataset/AllDatasets/result.

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23.4 Functional data in ToxoDB

Finally, a data summary view of all integrated data is available here: https://toxodb.org/ toxo/processQuestion.do?questionFullName 5 OrganismQuestions.GenomeDataTypes.

23.3 Genome in ToxoDB While ToxoDB focuses on integrating Toxoplasma genomes, it also includes genomes and annotation from related organisms whose integration provides additional avenues for discovery based on evolutionary relatedness. Table 23.1 includes a list of all the integrated genomes as of release 43 of ToxoDB. The reader is also encouraged to explore genome sequences of evolutionarily related organisms through the EuPathDB portal site (http://eupathdb.org) including the free-living nonparasitic Chromera velia and Vitrella brassicaformis (Woo et al., 2015). Gene models have been greatly improved due to the availability of RNA-sequence (RNA-seq) data to help guide gene model predictions. Curation of genes has also been improved by a community effort to provide expert manual annotation of genes and through the user comment system. Updated information regarding genomes in ToxoDB is available from the home page by clicking on the Data Summary link on the left side of the home page (Fig. 23.1).

23.4 Functional data in ToxoDB In addition to the genomes and annotation described earlier, ToxoDB contains a variety of functional data types generated by research groups across the globe. Increasingly, data provided to ToxoDB is done so prepublication to enable ample time for integration in the database and to ensure data release occurs concomitantly

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with publication. Detailed information about each dataset available in ToxoDB, and links to associated queries may be accessed here: https://toxodb.org/toxo/app/search/dataset/ AllDatasets/result. Functional datasets currently available in ToxoDB include the following: 1. Proteomics data made up of peptide sequences from mass spectrometry experiments that are mapped to translated genes or open reading frames. Genes with peptide matches from the available proteomics experiments may be identified, and peptide data are displayed on gene pages and in the genome browser (GBrowse). Experiments are available from multiple strains, life cycle stages, subcellular fractions as well as phosphopeptide data. In addition to peptide level data, quantitative proteomics data are also available. 2. ESTs include sequence data from cloned cDNA libraries retrieved from the GenBank Expressed Sequence Tag database (dbEST) and mapped to the genomes. Genes with EST evidence may be identified based on all or specific EST libraries (Boguski et al., 1993). In addition, EST alignments are available as a data track in the GBrowse. EST libraries are available for T. gondii, S. neurona, N. caninum, and Eimeria spp. 3. Microarray (glass slides or high-density arrays) data in ToxoDB are reanalyzed using a standard analysis method to ensure consistency in data representation. Microarray data for individual genes are displayed on gene pages under the expression section as graphs and tables. In addition, mapped probes for each microarray platform can be viewed in the GBrowse. Depending on the original microarray experiment, ToxoDB offers searches based on fold induction, percentile

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23. ToxoDB: the functional genomic resource for Toxoplasma and related organisms

TABLE 23.1 Genomes in ToxoDB, updated with release 43 (April 2019). Organism

Gene Annotation count

Genome size (Mbps)

Number of contigs

Available organellar genome Citation

Toxoplasma gondii ME49

Yes

8920

65.67

2265

Apicoplast

Lorenzi et al. (2016)

T. gondii VEG

Yes

8563

64.52

1323

Apicoplast

Lorenzi et al. (2016)

T. gondii GT1

Yes

8637

63.95

2063

Apicoplast

Lorenzi et al. (2016)

T. gondii GAB2-2007GAL-DOM2

Yes

9296

63.55

2511

Apicoplast

Lorenzi et al. (2016)

T. gondii TgCatPRC2

Yes

10,300

64.19

3064

Apicoplast

Lorenzi et al. (2016)

T. gondii ARI

Yes

10,148

64.69

2745

Apicoplast

Lorenzi et al. (2016)

T. gondii FOU

Yes

10,297

64.53

2871

Apicoplast

Lorenzi et al. (2016)

T. gondii VAND

Yes

9426

64.27

2141

Apicoplast

Lorenzi et al. (2016)

T. gondii RUB

Yes

10,213

64.96

2431

Apicoplast

Lorenzi et al. (2016)

T. gondii MAS

Yes

10,176

63.32

2183

Apicoplast

Lorenzi et al. (2016)

T. gondii p89

Yes

9874

64.16

2153

Apicoplast

Lorenzi et al. (2016)

T. gondii RH

partial

63

4.03

3

Apicoplast

Lorenzi et al. (2016)

T. gondii TgCATBr9

No

0

61.82

6287

Apicoplast

Lorenzi et al. (2016)

T. gondii TgCATBr5

No

0

61.64

6995

Lorenzi et al. (2016)

T. gondii CAST

No

0

63.05

5717

Lorenzi et al. (2016)

T. gondii COUG

No

0

63.70

8562

Lorenzi et al. (2016)

T. gondii CtCo5

No

0

62.62

6177

Lorenzi et al. (2016)

T. gondii TgCkUg2

No

0

41.93

36,913

Bontell et al. (2009)

Neospora caninum Liverpool

Yes

7276

59.10

66

Reid et al. (2012)

Hammondia hammondi H.H.34

Yes

8178

67.70

14,861

Cyclospora cayetanensis CHN_HEN01

Yes

7901

44.03

2297

Liu et al. (2016)

Cystoisospora suis Wien I

Yes

11,785

83.64

14,630

Palmieri et al. (2017)

Sarcocystis neurona SN3

Yes

7089

124.41

873

S. neurona SO SN1

Yes

7177

130.22

3066

Blazejewski et al. (2015)

Eimeria tenella Houghton

Yes

8634

51.86

4664

Reid et al. (2014)

Eimeria acervulina Houghton

Yes

7045

45.83

3415

Pain, unpublished

Apicoplast

Apicoplast

Lorenzi et al. (2016)

Blazejewski et al. (2015)

(Continued)

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23.4 Functional data in ToxoDB

TABLE 23.1 (Continued) Organism

Gene Annotation count

Genome size (Mbps)

Number of contigs

Available organellar genome Citation

Eimeria brunetti Houghton

Yes

8898

66.89

8575

Eimeria falciformis Bayer Haberkorn 1970

Yes

6102

43.67

753

Eimeria maxima Weybridge

Yes

6258

45.98

3564

Pain, unpublished

Eimeria mitis Houghton

Yes

10,265

72.24

15,978

Pain, unpublished

Eimeria necatrix Houghton

Yes

8872

55.01

3707

Pain, unpublished

Eimeria praecox Houghton

Yes

7906

60.08

21,348

Pain, unpublished

Pain, unpublished Apicoplast

Heitlinger et al. (2014)

FIGURE 23.1 The ToxoDB home page. (A) The banner section contains several useful links including Login and Registration options, and quick ID and Text Search tools. The banner is present throughout ToxoDB web pages. (B) The left-hand section includes expandable menus with news regarding releases and useful links to community-related items and educational material, and a link to the Data Summary table for all EuPathDB sites. (C) The central section contains links to all searches and tools in three categories—searches that return genes, searches that return other data types (Popset Isolate Sequences, RFLP Genotype Isolates, Genomic Sequences, Genomic Segments, SNPs, ESTs, open reading frames, Metabolic Pathways, and Compounds), and Tools [Sequence Retrieval, BLAST, Companion (Steinbiss et al., 2016), EuPaGDT (Peng and Tarleton, 2015)]. EST, Expressed sequence tag; SNPs, single-nucleotide polymorphisms.

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4.

5.

6.

7.

8.

23. ToxoDB: the functional genomic resource for Toxoplasma and related organisms

expression, or similarity of expression. These searches return genes whose expression pattern satisfies the chosen search parameters. Chromatin immunoprecipitation microarray (ChIP-chip) and chromatin immunoprecipitation sequence (ChIP-seq) data for a variety of DNA-binding proteins are available and may be displayed in the GBrowse. In addition, genes may be identified based on their proximity to called binding peaks. RNA-seq reads are mapped to the genome, and expression graphs and tables are available on gene pages. Genes may be identified based on their RNA-seq expression profile. In addition, tracks representing depth of coverage and splice junction sites based on intron spanning may be displayed in the GBrowse. Single-nucleotide polymorphisms (SNPs) data are available on gene pages or in the GBrowse. Genes may be identified based on their SNP characteristics (e.g., synonymous, nonsynonymous, or noncoding). In addition, several searches return SNPs based on their IDs, gene IDs, and genomic location. Isolate genotype and metadata (e.g., geographic location and host species) is integrated from GenBank, and several available searches return isolates based on a variety of criteria, including isolate ID, taxon/strain, host, isolation source, locus sequence, geographic location, text searches, and BLAST similarity. Isolate data based on restriction fragment length polymorphism data are also available. Metabolic pathway data are represented as genes (via enzyme commission numbers) mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) or MetaCyc pathways (Caspi et al., 2016; Kanehisa et al., 2017). All pathways are displayed using Cytoscape (Lopes et al., 2010).

9. Phenotype data from clustered regularly interspaced short palindromic repeats screens enable users to identify genes based on their relative fitness (Sidik et al., 2018). In addition to the functional data described earlier, several data types are generated by inhouse analyses. These include transmembrane domain (TM) and signal peptide (SP) predictions, molecular weight and isoelectric point calculations, open reading frame predictions, synteny mapping, splice junction predictions based on RNA-seq data, Orthology, InterPro domains, and gene ontology (GO).

23.5 The ToxoDB home page The ToxoDB home page contains three main sections (Fig. 23.1) as follows: 1. The top banner (Fig. 23.1A) section is a constant presence throughout ToxoDB web pages. It contains a clickable ToxoDB logo (links back to the home page), database release version number and date, and Gene ID and text search boxes. In addition, the banner contains many useful help links and links to Register, Login, “Contact Us” form, and social media pages (EuPathDB Facebook, Twitter, and YouTube channel): a. Facebook: https://www.facebook.com/ eupathdb/ b. Twitter: https://twitter.com/EuPathDB c. YouTube: https://www.youtube.com/ EuPathDB/ The banner also contains a gray tool bar that provides access to all searches, tools, primary data analysis tools, the “My Datasets” section, help, and information via expandable menus. 2. The left-hand section (Fig. 23.1B) includes expandable menus including a Data Summary section that links to a table of all data available in EuPathDB databases, a News and Tweets section with information

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23.6 The search strategy system

regarding data release news, and other important community news including a live stream of EuPathDB tweets and retweets, a Community Resources section with links to related sites and community files, an Education and Tutorials section that includes links to online video tutorials and links to workshop exercises, and an about ToxoDB section that includes general information regarding EuPathDB publications, scientific working group, and advisory team, funding, and website usage statistics. 3. The central section (Fig. 23.1C) includes links to data searches and tools. This includes three main categories: (1) the first column includes access to all searches that return genes. (2) The middle column includes access to searches that return other data types such as isolates, SNPs, DNA motifs, ESTs, open

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reading frames, metabolic pathways, and compounds. (3) The third column includes links to frequently accessed tools such as BLAST, the sequence retrieval tool, and the genome bowser.

23.6 The search strategy system 23.6.1 Running your first search The first step in running a search in ToxoDB is to decide what type of data you are interested in searching. As described earlier, there are several types of data. For example, if you are interested in searching for genes, you would choose one of the searches available under the heading “Search for Genes” on the home page (Fig. 23.2A). Each of the search

FIGURE 23.2 Running a first search. (A) Searches may be selected by clicking on the arrow symbol next to search categories (arrow pointing up) then selecting a one of the available searches. (B) A screen shot of the search window that is displayed following clicking on the “Transmembrane Domain Count” query. Parameters such as organism and the number of transmembrane domains may be modified. Clicking on the “Get Answer” button runs this query (red box).

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categories is expandable by clicking on the arrow to the left of the category name to reveal additional subcategories (Fig. 23.2A, red arrow pointing up). In addition, a “search for searches” feature is available to allow you to quickly find what you are looking for without having to expand all categories (Fig. 23.2A, red box). Selecting one of the searches loads a search page with search options. In the example in Fig. 23.2, a search for genes based on the “Transmembrane Domain Count” found under the “Protein targeting and localization” is selected (Fig. 23.2A) to reveal search parameters that may be configured by selected or deselecting the check boxes and changing the maximum and minimum TM counts (Fig. 23.2B). Once the parameters are selected, a search may be run by clicking on the “Get Answer” button (Fig. 23.2B, red box). In the example shown in Fig. 23.2, the search would return all genes from all organisms in ToxoDB that contain at least one TM domain.

23.6.2 Understanding and configuring the results page Once a search is run, results are displayed on a results page that is divided into three sections (Fig. 23.3, top to bottom). In the top section (Fig. 23.3A) is the search strategy graphical system. Results are displayed graphically in rectangles that contain the number of results and the name of the query. The strategy is interactive and expandable (described below). In the example as shown in Fig. 23.3, the strategy is composed of a single step representing the search of all genes from all organisms in ToxoDB containing at least one TM domain (returning 40,020 genes). The middle section (Fig. 23.3B) contains an organism filter table that allows a user to filter the results of a query based on species and/or strain. This offers quick toggling between species without the need to rerun the query. Results of filtering

are displayed dynamically in the bottom section of the results page. The bottom section (Fig. 23.3C) displays the actual results of a query in a dynamic and customizable table. The table allows you to add more columns including expression graphs (click on the “Select Columns” button, red box in Fig. 23.3C), remove columns by clicking on the “x” icon to the right of the column name, move columns (drag and drop), sort items in a column (click on the up or down arrows to the left of the columns names), view as many or as few items per page (click on advanced paging), add items to your basket (click on the basket icon on the left of each row, requires login—green basket indicates an item is in your basket), and visit hyperlinked items such as gene pages by clicking on the gene ID (first column). In addition, the contents of individual columns may be displayed graphically (histogram or word cloud) by clicking on the graph icon (in the column heading, red circle in Fig. 23.3C). All results may be downloaded in multiple formats by clicking on the “Download” link (underlined in Fig. 23.3C). Download formats include excel formatted tables with any of the available results columns, general feature format version 3 (GFF3), or customized fast adaptive shrinkage threshold algorithm sequence. Results of a search may also be displayed graphically mapped to chromosomes or scaffolds/contigs or analyzed using enrichment tools such as GO and metabolic pathway enrichment (arrow heads and described next).

23.6.3 Building a multistep search strategy Moving beyond an initial search is achieved by clicking on the “Add Step” button and growing a search strategy (Fig. 23.3A). Once the “Add Step” button is selected, a popup window offers the user access to all searches in ToxoDB (Fig. 23.4A). Any search may be

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FIGURE 23.3 Screen shot of the results page. (A) The search strategy is a graphical display of the results and search workflow. Each search is a step in the strategy, in this example running a search for genes with transmembrane domains returns over 40,000 genes from all organisms in ToxoDB (note that this number will change as the number of organisms loaded in the database changes). (B) The distribution of genes among species and strains in ToxoDB is displayed in a filter table. Clicking any of the organism specific results will filter the results in the search strategy and in the results table. (C) The results are displayed in a dynamic results table that allows sorting, rearranging, analyzing, and adding columns. Inserts of red boxes show the results of a word cloud analysis on the product description column (click on the graph icon in red circle) and the list of available columns to load when the “Add Columns” option is selected (red box). Individual genes may be added to the basket (green basket icons to the left of gene IDs). Data may be downloaded in multiple formats (Download link underlined) in addition to tailored sequence data based on user defined coordinates (e.g., for the list of genes retrieve only the 500 nucleotides upstream of the translation start site). Arrow heads point to additional analysis tabs: “Genome View” displays all genes in the results list graphically on chromosomes, scaffolds, or contigs, and “Analyze Results” provides access to enrichment analysis tools such as GO and metabolic pathway enrichment (see Fig. 23.10). GO, Gene ontology.

selected, configured, and combined with the results from the previous step in a strategy. In the example shown in Fig. 23.4, all genes in ToxoDB with SPs are identified and combined with the TM step using a union operation (this would define all genes with an SP, a TM

domain, or both). A search strategy may be extended with as many steps as needed. For example, to identify genes from Fig. 23.4D that are upregulated in all enteroepithelial stages compared to tachyzoites and tissue cysts, a transcript expression search may be added to the

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FIGURE 23.4 Expanding a search strategy. (A) To combine results form a search with results from another available search click on the red “Add Step” button. (B) This displays a popup window containing all available searches in ToxoDB. (C) Selecting a new search to run, displays a popup window with the search specific parameters and options for how to combine the results of the new search with those in the previous one (intersect, union, minus, and genomic colocation). (D) Clicking on the “Add Step” button runs the new search and combines them graphically (in this example a union operation was applied). Results of the union are shown below the organism filter table as described in Fig. 23.3.

strategy. Fig. 23.5 shows the results obtained when adding a search for all genes upregulated by 10-fold in early and late enteroepithelial stage parasites compared to tachyzoites and tissue cysts based on an RNA-seq experiment (Ramakrishnan et al., 2019) (Fig. 23.5A). Note that in this example, the direction of regulation chosen was “upregulated,” the reference samples were tachyzoites and tissue cysts, and the comparator samples were the cat stages EES1 5 (very early to very late enteroepithelial stages). In addition, a 10-fold difference between the maximum expression value from the reference samples and the minimum expression value

from the comparator samples was selected (Fig. 23.5A). Results of this search were combined with the previous results using the intersect operation (Fig. 23.5B). The results indicate that 181 genes meet these criteria. Note that these genes are only for T. gondii ME49, since all functional data are by default mapped to the reference ME49 strain in ToxoDB. To define orthologous genes in ToxoDB, a step may be added, and the results transformed to their orthologs (Fig. 23.5D). This yields a total of 3871 genes across all organisms in ToxoDB including possible paralogs within ME49 (Fig. 23.5E). Any step or operation (union, intersect, minus) may

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FIGURE 23.5 Creating and revising multistep strategies. Following the same logic as in Fig. 23.4, additional steps may be added. (A) Fold change search parameters for a dataset that includes RNA-seq data from Feline enterocyte, tachyzoite, and bradyzoite stages (Ramakrishnan et al., 2019) are shown. This search allows a user to select samples from an experiment and return results based on their differential expression. (B) New searches are combined using Boolean operations, results from this search are combined with the previous results using an intersect operation. (C) The strategy grows by an additional step with results updated graphically and tabularly. (D) Most datasets in ToxoDB are mapped to the ME49 reference genome, thus the returned gene list includes only ME49 genes. However, to find orthologs of the ME49 genes in all species and strains in ToxoDB, the results may be transformed into orthologs by clicking on the add step box and selecting the “Transform by Orthology” option then selecting the Organisms you wish to transform to. (E) The strategy is now comprised of four steps showing all genes in ToxoDB that contain Transmembrane Domains and/or signal peptides and are upregulated feline enterocytic stages. All strategies can be renamed, duplicated, saved, shared, and deleted (red box). (F) Mousing over strategy panel reveals edit links on all strategy steps. Clicking on the edit link of a step reveals a popup window with additional options allowing a step in a strategy to be revised, deleted, and expanded into substrategies. (G) In this example the first step is revised to include genes with at least 12 Transmembrane Domains. (H) Revising the first step results in updating all the subsequent steps in the strategy. RNA-seq, RNA-sequence.

be revised at any given time with results of a strategy automatically reflecting the change (Fig. 23.5H). In the example as shown in Fig. 23.5E, the first step of the strategy can be revised by clicking on the edit button that appears upon mouse over. Once the edit button is clicked, a popup window appears, which

includes search details and a number of step option including revise (Fig. 23.5F). Clicking on revise opens up the search prefilled with the search parameters. To include proteins with at least 12 TM domain, modify the minimum TM number to 12 (Fig. 23.5G). Strategies themselves can also be renamed, duplicated, saved, shared,

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23. ToxoDB: the functional genomic resource for Toxoplasma and related organisms

or deleted (red box in Fig. 23.5E). Clicking on the share link generates a unique URL that can be shared with others. For example, the strategy in Fig. 23.5E can be accessed by using this link: https://toxodb.org/toxo/im.do? s 5 09ab66e606006ccc.

23.6.4 Defining genes based on their phylogenetic profile Orthology in ToxoDB is defined based on protein groupings produced by the OrthoMCL database (Chen, 2006; Fischer et al., 2011; Li et al., 2003). This database includes, in addition to all organisms in EuPathDB resources, 150 organisms representing important branches of the tree of life. Taking advantage of OrthoMCL groups, one can define orthologs within ToxoDB as described earlier in Fig. 23.5E or

define a phylogenetic profile of genes present in ToxoDB. For example, to determine which genes in Fig. 23.5 do not have orthologs in mammals, an “Orthology Phylogenetic Profile” step may be added to the strategy (Fig. 23.6A and B). The parameters for this search (Fig. 23.6C) allow defining orthology groups based on the presence (clicking once on the gray circles next to entire phyla or organisms; check mark) or absence (clicking twice on the gray circles next to entire phyla or organisms; red “x”) of the desired phyla. Unselected gray circles in Fig. 23.6C indicate no preference for ortholog presence or absence. In the example shown in Fig. 23.6C, genes in ToxoDB with no orthologs in any mammals in OrthoMCL were defined. Adding this step to the strategy results in a total of 26,309 genes that are conserved in ToxoDB but absent in mammals (Fig. 23.6D).

FIGURE 23.6 Illustration of the phylogenetic profile query. (A) Defining genes based on their phylogenetic profile is achieved by adding a step (or starting a new search). (B) The “Orthology Phylogenetic Profile” query is located under the “Orthology and Synteny” category the popup window. (C) Organisms may be included (check mark) or excluded (red “x”) to define the type of genes by clicking on the circles. In this example, only genes without orthologs in mammals are selected. (D) Results of this query are added to the growing strategy revealing 2630 genes in ToxoDB that meet all the criteria of this search strategy. Search strategies may be saved and shared with others using unique URLs (generated by clicking on the share link—red arrow in A): https://toxodb.org/toxo/im.do?s 5 09ab66e606006ccc.

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23.7 Genomic colocation

23.7 Genomic colocation Genomic colocation is a tool in ToxoDB that allows finding results based on defining a relationship between -mappable features (e.g., genes, SNPs, and DNA motifs). For instance,

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this tool can be used to define all genes in Toxoplasma that contain a polymorphism in the 10 nucleotides upstream of the translation start site. To do this, two searches need to be combined with the genomic colocation tool as illustrated in Fig. 23.7. In this example the first

FIGURE 23.7 The genomic colocation tool allows combining any feature that can be mapped to a genomic sequence (i.e., genes, DNA motifs, and SNPs). (A) SNP searches are found under the “SNPs” category in the “Search for Other Data Types” section on the home page. (B) The SNPs within a group of isolates was selected and allows the identification of all SNPs with selected isolates that meet the search parameters. In this example, all isolates were selected and SNPs with at least 80% read frequency were returned. (C) This search returned 2196888 SNPs mapped onto T. gondii ME49. (D) To return genes based on their relative location to the returned SNPs, a second search by clicking on the “Add Step” box then selecting the genes by organism search found under the “Taxonomy” category. (E) T. gondii ME49 is selected from the organism list, the red arrow points to a filter that allows you to quickly find the organism of choice, in this case by simply typing me49. Since the first search returned SNPs and the second search will return genes, notice that the only option to combine the searches is via a genomic colocation search. (F) Clicking on the continue button reveals a popup window containing a dynamic logic statement, which allows the selection of the relationship between the SNPs and the genes by virtue of their genomic location. In this example the query is asked to return all genes that contain a SNP in the 10 nucleotides upstream of their translation starts. (G) Once the parameters of the colocation query are submitted, results are displayed as part of the search strategy. An icon representing colocation is displayed. Any strategy may be shared using a unique URL that can be generated by clicking on the share link: https://toxodb.org/toxo/im.do?s 5 e7b0a983c16c14b3. SNPs, singlenucleotide polymorphisms.

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search identifies all SNPs among all available Toxoplasma strains using the SNP query for differences within a group of isolates (Fig. 23.7A). This search allows the selection of the desired strains (in this case all were selected) (Fig. 23.7B). In addition, the percent isolates with a SNP call was set to 80%. This parameter applies to the selected set of aligned isolate sequences. At any given nucleotide position, some isolates in your group may not have data supporting a base call because the Read Frequency Threshold was not met or fewer than our minimum of five reads aligned. “Percent isolates with a base call” defines the fraction of the selected isolates that must have a base call before a SNP is returned for that nucleotide position based on the remaining isolates that do have data. See the description below for more information. Fig. 23.7C shows the result of this search which returned 2196888 SNPs distributed across all Toxoplasma chromosomes (SNPs are all represented on the reference strain ME49). To define genes that are within 10 nucleotides of SNPs, a step is added to find all genes in T. gondii ME49 (Fig. 23.7D and E). A quick way to find the organism of interest in any of the searches in ToxoDB is by using the filter box at the top of the search (arrow in Fig. 23.7E). Note that the only option to combine this search with the SNP search is the genomic colocation option (bottom of Fig. 23.7E). Choosing this option and selecting continue opens a popup window with customizable colocation parameters that include a dynamic logic statement that is updated based on the chosen parameters (Fig. 23.7F). The logic statement can be modified as follows:

2. Next the region relative to the genes is customized using the gray highlighted region in the lower right-hand side of Fig. 23.7F. In this case the upstream option is chosen, and the number of nucleotides is changed to 10. Note that both the graphic and the logic statement are dynamically updated. 3. Next the type of relationship between the Genes and the SNPs is selected from the drop-down menu (box 2, Fig. 23.7F). The available options include “overlaps,” “is contained in,” or “contains.” For this query the latter was chosen since the interest is to define an upstream 10 nucleotide region that contains SNPs. 4. The parameters for the SNP results may now be modified; these include the strand (box 3, Fig. 23.7F) and the region (lower left side of Fig. 23.7F)—similar to Step 2, above. In this case the parameters will not be modified since the SNP may be on either strand, and the exact region of the SNP (the SNP itself) is desired to be within the upstream 10 nucleotides.

1. Select the type of results of interest. In this case the objective is to return genes, which can be selected from the first drop-down menu in the logic statement (box 1, Fig. 23.7F).

ToxoDB includes a GBrowse developed by the Generic Model Organism Database project (Stein, 2002). GBrowse allows users to display genomic DNA from genomes in ToxoDB and dynamically decorate regions of interest

After modifying the parameters the logic statement reads “Return each gene from Step 2” whose upstream region (defined as 10 nucleotides) contains the exact region of a SNP in Step 1 on either strand. Clicking on “Get Answer” returns all genes that meet the colocation criteria shown in Fig. 23.7E (results include in addition to gene IDs, the number, and location of matches).

23.8 The genome browser

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23.8 The genome browser

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FIGURE 23.8 GBrowse allows graphical display of data on the genome. (A) The top section of GBrowse includes tabs that link to a track selection page, snapshots page, custom tracks page, and a configuration section. (B) The section below the table includes, a “Landmark or Region” window for enter specific coordinates to visit or gene IDs, a scrolling a zooming button and links to several useful tools (drop-down menu on the right-hand side). Tools include a PCR primer design tool, restriction site display tool, and several data download options. (C) The Overview and Region section graphically shows the entire reference sequence (i.e., an entire chromosome) and an intermediate size sequence region, respectively. (D) The Details section includes a representation of the coordinates being looked at and any data track that has been loaded. (E) A screen shot of the “Select Tracks” section showing the various expandable/collapsible sections (“ 1 ”/“ 2 ,” red arrow). In this image the protein expression section is expanded revealing a number of experimental data that may be loaded by selected the check boxes. Tracks start to load in the browser section as soon as they are selected. GBrowse, Genome browser.

(which may be as big as an entire chromosome) with additional data tracks. Tracks include a plethora of data such as gene models, proteomics data, transcriptional data (e.g., RNA-seq reads, microarray probes, ChIP-chip, and ChIP-seq profiles), synteny between strains and species in ToxoDB, and splice site junctions (critical for supporting annotated gene models or defining alternative models). GBrowse has multiple entry points from within ToxoDB web pages, including from the home page (tools section in Fig. 23.1) and from

various sections on gene pages. When accessed from the home page, or gene pages, the annotated genes track is turned on by default (Fig. 23.8). The top section of the GBrowse window includes tabs that allow you to navigate to the “Browser” (this is the section that displays data tracks), the “Select Tracks” section allows choosing desired data tracks to display in the “Browser” section, the “Snapshots” section allows saving specific GBrowse views for later access, the “Custom Tracks” section allows the loading of user specific tracks

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(this includes RNA-seq read files), and the “Preferences” section allows setting general preferences for your GBrowse experience (Fig. 23.8A). Below the tab is the search section (Fig. 23.8B) that enables specifying the exact coordinates of sequence to be viewed (gene IDs may also be entered in the Landmark or Region box) and access to several configuration tools that include a restriction site configuration (choose which restriction sites to view in the browser), design polymerase chain reaction (PCR) primers (interactive graphical tool for designing primers based on a region of interest), and various track download options. In addition, this section includes the scrolling tool that enables zooming and left/right scrolling. The overview and region sections (Fig. 23.8C) provide a graphical display of the genomic location of the region in view (the overview represents the largest contiguous genomic

sequence to which the region of interest belongs to, in this case T. gondii chromosome VI). The Details section (Fig. 23.8D) includes the specific region being viewed and any tracks that have been loaded. To load tracks in GBrowse, click on the “Select Tracks” tab (Fig. 23.8A) to reveal available tracks that are distributed into subsections (Fig. 23.8E). Each subsection may be expanded or collapsed by toggling the 1 / 2 buttons (arrow in Fig. 23.8E). Desired data tracks can be selected with a checkbox and multiple tracks may be loaded simultaneously. At any point during track selection, one may view the laded tracks by clicking on the “Browser” tab (Fig. 23.8A). The choice of tracks to turn on depends on the question being asked. Examples of GBrowse track combinations are shown in Fig. 23.9 with links (in URL and QR code formats).

FIGURE

23.9 Example GBrowse track combinations. Three track combinations have been generated and bookmarked. To load these GBrowse views in your browser, you may use the provided URLs or simply scan the QR code using a smart phone.

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23.9 Data analysis and integration into ToxoDB

23.9 Data analysis and integration into ToxoDB There are multiple ways a user can analyze their own data. This section describes three main tools.

23.9.1 Gene list analysis Tools available under this section allow a user to analyze their gene lists and determine if they are enriched base on GO terms, metabolic pathways, or text. Gene lists may be

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derived from results of searches within ToxoDB or from a user uploaded gene list. To upload a gene list, click on the “Annotation, curation and identifiers” category (Fig. 23.1C), then paste or upload your list of gene IDs. The results of this search will be returned as a strategy with the IDs from your lists that are also in the database. To analyze a gene list, click on the “Analyze Results” tab below the organism filter table (Fig. 23.3C, red arrow head). This action will reveal the available enrichment tools, which include GO, metabolic pathway, and text enrichment (Fig. 23.10A). Clicking on

FIGURE 23.10 Any gene results may be analyzed by clicking on the “Analyze Results” tab. (A) There are three analysis options: GO, metabolic pathway, and word enrichment. (B) Clicking on the GO enrichment option reveals parameters that can be defined by the user including the desired ontology (cellular component, molecular function, and biological process) and P-value cutoff. (C) Clicking on the submit button runs the enrichment analysis and returns results in a table which includes multiple columns including the GO IDs, P-value, and corrected P-values. GO, Gene ontology.

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23. ToxoDB: the functional genomic resource for Toxoplasma and related organisms

any of the tools will reveal a set of configuration parameters. GO enrichment parameters can be modified to select the structured GO vocabulary to search (i.e., cellular component, molecular function, or biological process), the evidence source (i.e., curated or computed), and the P-value cutoff to use. In addition, a user may limit analysis to using the GO slim subset (Fig. 23.10B). Results of the enrichment analysis appear below the parameters and include columns for the enriched GO IDs and terms, the P-values and the adjusted Bonferroni P-values.

23.9.2 Analyze my experiment Users can access this feature from the gray menu bar in the banner section of ToxoDB web pages (Fig. 23.1A). Clicking on this link provides users with access to the EuPathDB Galaxy platform where they can securely and privately upload and analyze their own data. Galaxy tools provide access to the hundreds of bioinformatics tools to run directly or as part of user-developed custom workflows and are extensively described elsewhere (Afgan et al., 2018). The EuPathDB Galaxy platform includes all EuPathDB genomes preloaded and ready for use. Readers interested in exploring this tool further are encouraged to explore the galaxy exercises available through the EuPathDB workshop pages: https://workshop.eupathdb.org/. The following are links to RNA-seq and variant calling exercises from a recent EuPathDB workshop: • RNA-seq part I: http://tinyurl.com/ y25gbwey • RNA-seq part II: http://tinyurl.com/ y69ggcqs • Variant calling part I: http://tinyurl.com/ y37n63bs

• Variant calling part II: http://tinyurl.com/ yy2cfqj6

23.9.3 Galaxy result integration (my datasets) The ability to analyze primary data such as RNA or DNA sequencing data is critical to many researchers, and the availability of the galaxy platform makes such analysis more accessible to the general biomedical scientist. However, once data is analyzed, another hurdle is the ability to query the analyzed data and compare to other available data. To this end, EuPathDB has developed a mechanism for private integration of analysis results into EuPathDB resources. This allows a researcher to use the search strategy system to start querying and running additional analyses on their data within the familiar ToxoDB platform. Fig. 23.11 illustrates the export of analyzed RNA-seq data from cyst and tachyzoite stages to the ToxoDB “My Datasets” section (Fig. 23.11A). Once data have been exported, a private dataset page is generated that allows a user to interact with the data including editing descriptive attributes, sharing with other users, and deploying searches and visualizations (Fig. 23.11B). Coverage plots of RNA-seq data can be visualized in the GBrowse by clicking on the “Send to GBrowse” button (red box in Fig. 23.11B and C). Importantly, RNA-seq data can be queried through a custom generate search page (Fig. 23.11D) with results appearing as part of a search strategy (Fig. 23.11E). Users can combine their results with any of the available searches in ToxoDB, run analyses such as GO enrichment on their search results or visualize the results in tabular format where custom-generated graphs become available (Fig. 23.11E). In addition, a custom section on gene pages provides graphical representation of the experiment (Fig. 23.11F).

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23.10 Future directions

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FIGURE 23.11 Screen shots showing the user dataset integration tool which allows export of certain data types from the EuPathDB galaxy to ToxoDB. (A) To export RNA-seq results, select the “RNA-seq to EuPathDB” export tool from the menu of options on the left-hand side (red box). Two types for RNA-seq data may be exported: coverage graphs in BigWig format and tabular gene fragments per kilobase of transcript per million mapped reads expression values. Once a dataset is exported, a private dataset page is generated that includes dataset information and links to available visualizations and searches using the user’s data. (B) Coverage plots of the mapped RNA-seq reads can be viewed in GBrowse by clicking on the send to GBrowse link. (C) A screen shot showing coverage plots from a user analyzed dataset. (D) A dynamically generated query that can be used to interrogate the user’s data. (E) Query results are returned in the familiar strategy panel with a gene results table. A word cloud can be rabidly deployed based on terms in the gene product descriptions (arrow and insert). A dynamically generated graph representing expression values from the experiment are available both in the results table and in the “User Dataset” section of the gene page (shown in F). RNA-seq, RNA-sequence.

23.10 Future directions It is expected that the volume and complexity of datasets will continue expanding over the coming years. An expansion of ToxoDB to include compound record pages and metabolic pathways that are fully integrated into the search strategy system is well underway and should be forthcoming in the near future. This would lay the ground work to incorporate metabolomic data and enable queries for compounds and reactions. In addition, the availability of host response data to parasite infection in HostDB (http://hostdb.org)

provides an opportunity to generate integrated searches that leverage both the host and the parasite data to identify clusters of related or connected genes. Finally, we anticipate developing many more tools for primary user data analysis such as full integration of differential expression data and variant data.

Acknowledgments EuPathDB is funded with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. HHSN272200900038C.

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EuPathDB wishes to acknowledge all data providers and user comments submitters. The collaborative nature of the Toxoplasma research community and their willingness to support data integration and dissemination is admirable. All integrated data are cited in ToxoDB, and direct links to publications are provided. The bioinformatic resource center has been refunded and now includes support for Vectors and integration of Vectorbase.org, protozoan parasites and integration of all EuPathDB resources including FungiDB. The resource name has been updated to the Eukaryotic Pathogen, Vector and Host Informatics Resources (VEuPathDB) supported under NIH contract 75N93019C00077.

References Afgan, E., Baker, D., Batut, B., van den Beek, M., Bouvier, ˇ D., Cech, M., et al., 2018. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537 W544. Available from: https://doi.org/10.1093/nar/gky379. Ajioka, J.W., Boothroyd, J.C., Brunk, B.P., Hehl, A., Hillier, L., Manger, I.D., et al., 1998. Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the apicomplexa. Genome Res. 8, 18 28. Available from: https://doi.org/10.1101/gr.8.1.18. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403 410. Available from: https://doi.org/10.1016/ S0022-2836(05)80360-2. Aurrecoechea, C., Barreto, A., Basenko, E.Y., Brestelli, J., Brunk, B.P., Cade, S., et al., 2017. EuPathDB: the eukaryotic pathogen genomics database resource. Nucleic Acids Res. 45, D581 D591. Available from: https://doi. org/10.1093/nar/gkw1105. Blazejewski, T., Nursimulu, N., MBio, V.P., 2015. Systemsbased analysis of the Sarcocystis neurona genome identifies pathways that contribute to a heteroxenous life cycle. Am. Soc. Microbiol. 6, 1. Available from: https:// doi.org/10.1128/mBio.02445-14. Boguski, M.S., Lowe, T., Tolstoshev, C.M., 1993. dbEST— database for “expressed sequence tags”. Nat. Genet. 4, 332 333. Bontell, I.L., Hall, N., Ashelford, K.E., Dubey, J.P., Boyle, J. P., Lindh, J., et al., 2009. Whole genome sequencing of a natural recombinant Toxoplasma gondii strain reveals chromosome sorting and local allelic variants. Genome Biol. 10 (5), R53. Available from: https://doi.org/ 10.1186/gb-2009-10-5-r53. Caspi, R., Billington, R., Ferrer, L., Foerster, H., Fulcher, C. A., Keseler, I.M., et al., 2016. The MetaCyc database of metabolic pathways and enzymes and the BioCyc

collection of pathway/genome databases. Nucleic Acids Res. 44, D471 D480. Available from: https://doi.org/ 10.1093/nar/gkv1164. Chen, F., 2006. OrthoMCL-DB: querying a comprehensive multi-species collection of ortholog groups. Nucleic Acids Res 34, D363 D368. Available from: https://doi. org/10.1093/nar/gkj123. Fischer, S., Brunk, B.P., Chen, F., Gao, X., Harb, O.S., Iodice, J.B., et al., 2011. Using OrthoMCL to assign proteins to OrthoMCL-DB groups or to cluster proteomes into new ortholog groups. Curr Protoc Bioinformatics. Available from: https://doi.org/10.1002/0471250953. bi0612s35 Chapter 6: Unit 6.12.1 19. Greene, J.M., Collins, F., Lefkowitz, E.J., Roos, D., Scheuermann, R.H., Sobral, B., et al., 2007. National institute of allergy and infectious diseases bioinformatics resource centers: new assets for pathogen informatics. Infect Immun 75:3212 3219. Available from: https://doi:10.1128/IAI.00105-07. Heitlinger, E., Spork, S., Lucius, R., Dieterich, C., 2014. The genome of Eimeria falciformis - reduction and specialization in a single host apicomplexan parasite. BMC Genomics 15, 696. Available from: https://doi.org/ 10.1186/1471-2164-15-696. Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y., Morishima, K., 2017. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 45, D353 D361. Available from: https://doi.org/ 10.1093/nar/gkw1092. Kissinger, J.C., Gajria, B., Li, L., Paulsen, I.T., Roos, D.S., 2003. ToxoDB: accessing the Toxoplasma gondii genome. Nucleic Acids Res. 31, 234 236. Available from: https://doi.org/10.1093/nar/gkg072. Li, L., Stoeckert, C.J., Roos, D.S., 2003. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13, 2178 2189. Available from: https:// doi.org/10.1101/gr.1224503. Liu, S., Wang, L., Zheng, H., Xu, Z., Roellig, D.M., Li, N., et al., 2016. Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. BMC Genomics 17, 316. Available from: https://doi.org/ 10.1186/s12864-016-2632-3. Lopes, C.T., Franz, M., Kazi, F., Donaldson, S.L., Morris, Q., Bader, G.D., 2010. Cytoscape Web: an interactive webbased network browser. Bioinformatics 26, 2347 2348. Available from: https://doi.org/10.1093/bioinformatics/btq430. Lorenzi, H., Khan, A., Behnke, M.S., Namasivayam, S., Swapna, L.S., Hadjithomas, M., et al., 2016. Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii

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References

genomes. Nat. Commun. 7, 10147. Available from: https://doi.org/10.1038/ncomms10147. Palmieri, N., Shrestha, A., Ruttkowski, B., Beck, T., Vogl, C., Tomley, F., et al., 2017. The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates. Int. J. Parasitol. 47, 189 202. Available from: https://doi.org/10.1016/j. ijpara.2016.11.007. Peng, D., Tarleton, R., 2015. EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens. Microb. Genom. 1, e000033. Available from: https://doi.org/10.1099/mgen.0.000033. Ramakrishnan, C., Maier, S., Walker, R.A., Rehrauer, H., Joekel, D.E., Winiger, R.R., et al., 2019. An experimental genetically attenuated live vaccine to prevent transmission of Toxoplasma gondii by cats. Sci. Rep. 9, 1474. Available from: https://doi.org/10.1038/s41598-01837671-8. Reid, A.J., Vermont, S.J., Cotton, J.A., Harris, D., 2012. Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy. PLoS Pathog. 8, e1002567. Available from: https://doi.org/ 10.1371/journal.ppat.1002567.

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Reid, A.J., Blake, D.P., Ansari, H.R., Billington, K., Browne, H.P., Bryant, J., et al., 2014. Genomic analysis of the causative agents of coccidiosis in domestic chickens. Genome Res. 24, 1676 1685. Available from: https:// doi.org/10.1101/gr.168955.113. Roos, D., 2011. David Roos. Interview by H. Craig Mak. Nat. Biotechnol. 29, 141 142. Available from: https:// doi.org/10.1038/nbt.1774. Sidik, S.M., Huet, D., Lourido, S., 2018. CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii. Nat. Protoc. 13, 307 323. Available from: https://doi.org/ 10.1038/nprot.2017.131. Stein, L.D., 2002. The generic genome browser: a building block for a model organism system database. Genome Res. 12, 1599 1610. Available from: https://doi.org/ 10.1101/gr.403602. Steinbiss, S., Silva-Franco, F., Brunk, B., Foth, B., HertzFowler, C., Berriman, M., et al., 2016. Companion: a web server for annotation and analysis of parasite genomes. Nucleic Acids Res. 44, W29 34. Available from: https://doi.org/10.1093/nar/gkw292. Woo, Y.H., Ansari, H., Otto, T.D., Klinger, C.M., Kolisko, M., 2015. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. eLife 4, e06974.

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C H A P T E R

24 Cerebral toxoplasmosis Anita A. Koshy1,2,3, Tajie H. Harris4 and Melissa B. Lodoen5,6 1

Department of Neurology, University of Arizona, Tucson, AZ, United States 2Department of Immunobiology, University of Arizona, Tucson, AZ, United States 3BIO5 Institute, University of Arizona, Tucson, AZ, United States 4Department of Neuroscience, Center for Brain Immunology and Glia, University of Virginia, Charlottesville, VA, United States 5Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, United States 6Institute for Immunology, University of California, Irvine, CA, United States

24.1 Introduction Toxoplasma gondii is one of the world’s most successful parasites in part because of its ability to infect and persist in most warm-blooded animals. A unique characteristic of T. gondii is its ability to persist in the central nervous system (CNS)—composed of the brain, retina, and spinal cord—of a variety of hosts, including humans and rodents. In humans, this tropism for and persistence in the CNS underlies much of the symptomatic disease seen in the immunodeficient (e.g., AIDS patients and fetuses) and, even, in the immunocompetent (e.g., patients with chorioretinitis). As clinical disease in humans is extensively reviewed in Chapter 4, Human Toxoplasma infection, this chapter will focus on our mechanistic understanding of cerebral toxoplasmosis or T. gondii infection of the brain. For simplicity, we will use CNS and brain interchangeably throughout

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00024-4

the chapter though most of the referenced studies in humans and mice focus on the brain. Here we will address how the parasite enters the CNS, what CNS cells are infected, and how cerebral infection is controlled. At the end of the chapter, we will review the potential for latent T. gondii infection to affect rodent and human cognition and behavior.

24.2 Models for understanding cerebral toxoplasmosis As it is currently not possible to perform mechanistic studies of symptomatic or asymptomatic cerebral toxoplasmosis in humans, in vitro studies on relevant human and murine cells and rodent models of toxoplasmosis in vivo are the mainstays of such studies. Like humans, mice are natural intermediate hosts in which the CNS is the major organ of

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© 2020 Elsevier Ltd. All rights reserved.

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encystment (Alvarado-Esquivel et al., 2015; Remington and Cavanaugh, 1965). This advantage should not be underappreciated, as for many pathogens, mice are not natural hosts or the disease is completely different in mice, leading investigators to use immunocompromised mice or human mouse hybrid systems (Crawford et al., 2015; Ku et al., 2005; Zerboni et al., 2005). Of course, mice and humans differ in many ways, including that inbred mice preclude the genetic diversity naturally found in humans, an issue that is only partially addressed by using outbred CD1 mice. Despite these disadvantages, and because no in vitro system (including organoids) can adequately mimic the parasite brain immune system interaction, most of our in vivo understanding of CNS toxoplasmosis comes from studies in inbred mice. An in-depth review of toxoplasmosis animal models, including cerebral toxoplasmosis is presented in Chapter 7, Toxoplasma animal models and therapeutics.

24.3 Mouse and parasite genotype affect central nervous system outcomes More than 30 years ago, investigators recognized that different inbred mouse strains showed clear phenotypic differences to peroral infection with Me49, a commonly used type II strain. Me49 was relatively avirulent in Swiss Webster, SWR/J, and Balb/c mice (0% mortality) while the same dose of Me49 showed a high rate of lethality (80%) in C57BL/6J and C57BL/6-beige mice (McLeod et al., 1984). The resistant mice had lower CNS cyst burdens and limited evidence of encephalitis—inflammation in the brain parenchyma—while the sensitive mice showed higher cyst burdens and marked encephalitis. Over the ensuing decade, elegant studies determined that these phenotypic differences segregated by the H-2 locus genotype with the H-2d (Ld) haplotype linked to limited CNS disease and the H-2b

and H-2k (Lb and Lk) loci linked to aggressive CNS disease (Brown and McLeod, 1990). As the H-2 locus determines which MHC I alleles are expressed, these findings suggested that the determinant of CNS disease might lie in the antigen restriction of these different MHC alleles. This possibility was confirmed by two groups who showed that peptides from T. gondii proteins ROP7 and GRA6 were exclusively presented by Ld MHC (Blanchard et al., 2008; Frickel et al., 2008). While these studies linked the H-2 MHC locus to cerebral toxoplasmosis outcomes, another study using congenic BALB and B10 mouse strains with different H-2 loci (Lb, Lk, Lq) showed that the same MHC locus produced disparate outcomes depending on the background strain (BALB vs B10) (Deckert-Schlu¨ter et al., 1994b), suggesting an effect of non-MHC loci on disease outcomes. Similarly, many years ago it was recognized that infection with different T. gondii strains leads to different outcomes in mice (Reikvam and Lorentzen-Styr, 1976; Sibley and Boothroyd, 1992). The genes that account for the strain-specific differences in acute virulence in mice have been identified and are extensively covered in Chapter 14 “Toxoplasma secretory proteins and their roles in parasite cell cycle and infection”; Chapter 17 “Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages”; and Chapter 25 “Innate immunity to Toxoplasma gondii.” Our understanding of T. gondii strain-specific effects on cerebral toxoplasmosis is very limited because most studies have been performed with a single T. gondii strain, usually from the type II strain family. In studies that have used more than one strain, strain-specific outcomes have been observed (Suzuki et al., 1989a,b; Suzuki and Joh 1994; Brooks et al., 2015; Cabral et al., 2017; Tuladhar et al., 2019), though the work to understand the cellular, molecular, and

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24.5 Parasite entry into the central nervous system

genetic underpinnings of these differences is just beginning. Finally, in addition to differences between strain types, even two strains from the same strain type/clade can produce different outcomes (Blanchard et al., 2015). Collectively, these discrepancies mean that variations in experimental paradigms (e.g., mouse or parasite strain used) may result in conflicting results.

24.4 Overview of the central nervous system The CNS is a highly organized and complex organ. The complexity of the brain includes its variability in cell types as well as how those cell types vary by brain region. The chief cell classes in the brain are neurons and glia. Neurons are the major “signaling” cells of the brain that underlie the processing network that allows integration of complex information (from internal or external stimuli) to initiate simple and complex behavioral responses. While neurons are categorized by size, morphology, neurotransmitter production and release, and even expression patterns, a more generalizable principle is that most neuron cell bodies reside in the gray matter of the CNS, with the long axonal processes coming together to form the white matter of the CNS. Glial cells are composed of astrocytes, oligodendrocytes, and microglia. Oligodendrocytes are cells that wrap neuron axons with myelin. A single oligodendrocyte can ensheath many neurons. Astrocytes, once considered simple support cells for neurons, are recognized to play many critical roles in the brain, including being an essential part of the brain immune response. Like neurons, astrocytes have different subtypes (Miller and Raff, 1984; Zamanian et al., 2012), though the complexity and biological significance of these subtypes is not well defined. During

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neuroinflammatory insults astrocytes upregulate many genes that lead to a variety of cellular changes, including the increased expression of glial fibrillary acid protein (GFAP), a structural protein that is often used to identify reactive astrocytes. In a normal, uninjured brain, many astrocytes do not express GFAP (Sofroniew and Vinters, 2010). Microglia were originally thought to come from the ectoderm that gives rise to other CNS cell types but now have definitively been shown to arise from mesoderm and specifically from early hematopoietic precursors (Ginhoux et al., 2013). Thus, microglia are the tissue-resident macrophages of the CNS, akin to Langerhans cells in the skin and Kupffer cells in the liver but are unique in that they appear to be a self-renewing population. Unlike neurons, at baseline, in the uninflamed brain, glia are homogenously distributed across the CNS. These differences in distribution often lead to confusion about the number of glia to neurons. Careful counts in uninflamed brain suggest that this ratio varies across the brain, with white matter having a very high ratio of glia to neurons and the cerebral cortex having an almost 1:1 ratio across a range of mammals, including humans (Herculano-Houzel, 2014).

24.5 Parasite entry into the central nervous system 24.5.1 Toxoplasma gondii dissemination to the central nervous system As an ingested pathogen, the initial site of T. gondii infection in many individuals is the intestine, from which parasites rapidly spread to the lymphatics and to distal organs via the bloodstream. Once T. gondii is ingested as tissue cysts or oocyst in contaminated food or water, T. gondii bradyzoites or sporozoites, respectively, are released into the gut. These

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forms then differentiate into the rapidly dividing tachyzoite form, allowing the parasite to replicate within the small intestine (Gregg et al., 2013). T. gondii tachyzoites can then migrate across the intestinal epithelium, principally between the interepithelial cell junctions, without disrupting the integrity of the barrier (Barragan et al., 2005). Infected neutrophils have also been detected transporting T. gondii within the small intestine (Coombes et al., 2013). It remains unclear whether T. gondii dissemination from the intestine is accomplished by extracellular parasites powering their own motility or within infected, motile cells or even from epithelial cell infection with egress across the basolateral membrane, but eventually the parasites gain access to the circulation, thereby facilitating their dissemination to a variety of other organs, including the CNS. T. gondii tachyzoites can be found in the peripheral blood of humans during acute infection, reactivation of retinal disease, and even, rarely, during chronic disease (Silveira et al., 2011). In mice, within days after oral infection, extracellular and intracellular tachyzoites within infected monocytes are detected in the peripheral blood (Courret et al., 2006), indicating rapid systemic spread of T. gondii. Parasites are also found in circulating cells expressing markers for T cells, neutrophils, and dendritic cells (DCs). In C57BL/6 mice intraperitonally infected with T. gondii, free tachyzoites in the blood peak at 6-day postinfection (dpi) and then decline, likely due to rising levels of circulating T. gondii-specific antibody, as these free tachyzoites persist at high levels in the blood of mice that are deficient in B cells, T cells, and IgM secretion (Konradt et al., 2016). In these studies, the mean circulation half-life of free tachyzoites in the blood was calculated to be approximately 3 minutes (Konradt et al., 2016), suggesting that although free parasites are present in the peripheral blood, their time spent in this compartment is relatively transient.

24.5.2 Unique features of the blood brain barrier During acute infection and during reactivation of chronic infection in immunocompromised mice, foci of T. gondii are found surrounding microvessels (Dellacasa-Lindberg et al., 2007; Konradt et al., 2016). This proximity to microvessels indicates that T. gondii likely enters the brain by the vasculature, requiring parasites to breach the blood brain barrier (BBB), a formidable biological barrier that is impenetrable to many pathogens in the circulation. The blood vessels of this unique barrier comprise endothelial cells, which are surrounded by pericytes and astrocyte endfeet (Serlin et al., 2015). Collectively, these cellular structures form the neurovascular unit, a highly restrictive barrier that protects the brain by limiting the passage of immune cells, solutes, and toxins from the bloodstream into the brain during homeostasis. A key feature of the brain capillary endothelial cells is that they express tight junction proteins, such as occludin and claudin-5, thereby interconnecting the endothelial cells and contributing to the low permeability of this barrier (Potente and Ma¨kinen, 2017; Zhao et al., 2015). The exchange of nutrients and waste is accomplished by dedicated transporters in the barrier endothelial cells, as BBB endothelial cells support notably low levels of vesicle trafficking and transcytosis, thereby restricting the flow of molecules across the barrier (Ayloo and Gu, 2019). Despite the importance of maintaining a high degree of barrier integrity for healthy brain function, the BBB is notably dynamic and selectively permeable, especially during states of systemic inflammation. T. gondii infection can activate the endothelium, resulting in upregulation of adhesion molecules (e.g., ICAM-1, VCAM-1) (Lachenmaier et al., 2011; Silva et al., 2010) and promoting permeability of the BBB (Pober and Sessa, 2007). These features of activated endothelium allow immune cells and

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potentially pathogens to adhere to and transmigrate across the BBB, as discussed next.

24.5.3 Breaching the blood brain barrier The mechanism by which T. gondii enters the human brain remains incompletely understood. Most research in this area has utilized mouse models of infection or in vitro transmigration assays and focused on two potential mechanisms of CNS entry: the extracellular route of infection, whereby free tachyzoites transmigrate across the BBB or invade the vascular endothelium, or the intracellular route, in which infected immune cells shuttle the parasite across the BBB and into the brain parenchyma. In order for extracellular parasites in the circulation to transmigrate into tissues, parasites must be able to attach to blood vessel endothelial cells in rapidly flowing blood. Work in microfluidic chambers suggests that T. gondii tachyzoites are capable of adhering to human vascular endothelium under physiologic shear stress conditions and that parasite adhesion is most effective at low shear stress, similar to that encountered in postcapillary venules (Harker et al., 2014). Akin to attachment and invasion in static conditions (see Chapter 14 "Toxoplasma secretory proteins and their roles in parasite cell cycle and infection"), MIC2 adhesin was found to mediate initial T. gondii adhesion to vascular endothelium under fluidic shear stress. Compared to static conditions, shear stress enhances both T. gondii gliding and invasion of the endothelial cells, suggesting parasitic biomechanical sensing of the external environment (Harker et al., 2014). Interestingly, T. gondii infection of endothelial cells may serve as a critical step in invasion into the brain and other organs. A recent study showed that during in vivo infection via oral or intraperitoneal administration of T. gondii,

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parasites were found in endothelial cells within the lung and brain and that parasite replication and lysis of the endothelial cells appeared to lead to T. gondii entry to the brain parenchyma (Konradt et al., 2016). Consistent with these findings, tachyzoites have been shown to invade and replicate preferentially in human retinal vascular endothelial cells compared to dermal endothelial cells (Zamora et al., 2008). Studies comparing the replication rate of T. gondii in a variety of human brain cell types have demonstrated that T. gondii proliferates more efficiently in endothelial cells than in neurons or microglia (Mammari et al., 2014). Recent work indicates a role for epidermal growth factor receptor (EGFR) signaling in parasite replication in brain and retinal endothelial cells, as inhibiting EGFR signaling in these cells enhances parasite clearance via an autophagy pathway and reduces brain and retinal invasion by T. gondii in vivo (Corcino et al., 2019). Collectively, these studies suggest that infection of the microvascular endothelial cells serves as an important portal for entry to the CNS. In addition to the invasion of endothelial cells, T. gondii may hijack the migratory potential of motile immune cells to cross the BBB via a “Trojan horse” mechanism. Indeed, it has been demonstrated that T. gondii infection of a variety of peripheral immune cells, including monocytes, macrophages, DCs, neutrophils, and NK cells, increases the motility of these cells (Harker et al., 2015), potentially as a mechanism of enhancing parasite spread in the infected host. In studies on the dissemination of T. gondii following oral infection of mice, CD11b1 monocytes were found harboring T. gondii in the blood, and adoptive transfer experiments of fluorescently labeled monocytes into mice prior to infection resulted in the detection of these labeled, infected cells in brain homogenates generated from these mice (Courret et al., 2006). Studies performed on an in vitro model of the rodent BBB, in which primary glia and brain

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endothelial cells were seeded into a transwell system, revealed that T. gondii-infected CD11b1/CD11c2 peripheral blood mononuclear cells predominantly crossed this transwell barrier (Lachenmaier et al., 2011). Studies of infected primary human monocytes and DCs have also demonstrated a marked effect of T. gondii infection on adhesion and migration. Human peripheral blood monocytes infected with T. gondii can adhere to human vascular endothelium under shear stress conditions and transmigrate across the endothelial barrier (Harker et al., 2013). Crawling and transmigration of infected monocytes in flow conditions are dependent on Mac-1 on the monocyte and ICAM-1 on the endothelium (Ueno et al., 2014). The hypermigratory phenotype of T. gondii-infected monocytes is associated with reduced integrin clustering, impaired phosphorylation of the focal adhesion kinases and PYK2, and reduced recruitment of talin, paxillin, and vinculin to nascent focal adhesions (Cook et al., 2018). The effects of T. gondii infection on the cytoskeletal dynamics of infected monocytes are also observed in infected DCs (Bierly et al., 2008; Lambert et al., 2009). T. gondii strain types I, II, and III all induce hypermotility in infected DCs (Lambert et al., 2009). The hypermotility phenotype involves rapid actin cytoskeleton remodeling, integrin redistribution, and the loss of adhesive podosomes (Weidner et al., 2013). The T. gondii 14-3-3 adaptor protein is a key effector protein that induces hypermotility of macrophages, DCs, and microglia, by localizing to the parasitophorous vacuolar membrane and recruiting host 14-3-3 proteins to this site (Weidner et al., 2016). Interestingly, DCs were found to secrete the neurotransmitter GABA in response to T. gondii infection, and GABAergic signaling through the GABAA receptor potentiates DC hypermotility and the trafficking of infected DCs to the CNS, revealing an activating function in cell migration for

this classically inhibitory neurotransmitter (Fuks et al., 2012). The activation of GABAA receptors was found to induce a transient Ca21 flux via the voltage-dependent Ca21 channel Cav1.3, which was critical for GABAergicinduced DC hypermotility (Kanatani et al., 2017). More recently, this mechanism was also observed in T. gondii-infected microglia (Bhandage et al., 2019).

24.6 Brain regions and host cells infected in the brain 24.6.1 Human toxoplasmosis Our understanding of cerebral toxoplasmosis in humans primarily comes from AIDS patients with active toxoplasmic encephalitis (TE). At the onset of the AIDS epidemic, when TE was relatively common, several studies used imaging and autopsies to identify TE lesions (Arendt et al., 1999; Lang et al., 1989; Porter and Sande, 1992; Post et al., 1983; Strittmatter et al., 1992). While the anatomic regions in which TE lesions were found varied by study, most TE lesions were found in gray matter with relatively few lesions found in white matter. Based upon the studies with higher patient numbers (studies with data on $ 100 patients) (Arendt et al., 1999; Porter and Sande, 1992), the cerebral cortex is involved more often than the basal ganglia or thalamus (two deep gray matter areas) that are involved more often than the cerebellum that is involved more often than the brainstem or white matter. The spinal cord is rarely involved. Host cell T. gondii interactions have not been well studied in either immunocompromised or immunocompetent humans. A single in vitro study on human neurons and astrocytes suggests that T. gondii invades and encysts in both cell types but proliferates more readily in astrocytes compared to neurons (Halonen et al.,

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1996). In a small number of autopsies on severely immunocompromised patients, parasites/cysts were identified within astrocytes, neurons, oligodendrocytes, vascular endothelium, and pericytes (Bertoli et al., 1995; Ghatak and Sawyer, 1978; Ghatak and Zimmerman, 1973; Hirano, 2005). In immunocompetent patients, a recent report states that T. gondii was found in both astrocytes and neurons in routine autopsies, but it is unclear from the methodology of this study how cellular location was determined (Alvarado-Esquivel et al., 2015).

24.6.2 Rodent cerebral toxoplasmosis Our understanding of T. gondii’s distribution in the CNS of rodents comes primarily from studies that have used cyst location to determine which regions of the brain are commonly infected (Afonso et al., 2012; Berenreiterova´ et al., 2011; Dellacasa-Lindberg et al., 2007; Di Cristina et al., 2008; Dubey et al., 2016; Evans et al., 2014; Gonzalez et al., 2007; Haroon et al., 2012; Vyas et al., 2007). Huge variability exists in the identified regions that may be accounted for by differences in rodent model (rat vs mouse; immunocompetent vs immunocompromised); time postinfection analyzed; T. gondii strain utilized; parasite detection techniques (immunohiostochemistry, bioluminescent imaging); and depth of examination (i.e., single section/mouse vs multiple). The Berenreiterova´ et al. study should be highlighted because of the depth of examination (8 μm sections across the whole brain, B1600 sections/mouse, n 5 5 mice) and the amount of raw data presented. This study notes a high level of cysts in several areas of the cortex, basal ganglia, hippocampus, and amygdala and low levels of cysts in the cerebellum, the pons, caudate putamen, and white matter tracts. The findings of high cyst levels within the cortex and low levels within the brainstem/white matter tracts are consistent with human data and work from another group

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that localized neurons injected with T. gondii proteins rather than cysts (Mendez et al., 2018). Like human CNS cells, T. gondii readily invades and encysts in rodent astrocytes, neurons, and microglia in vitro (Fischer et al., 1997; Lu¨der et al., 1999), though neurons may be particularly efficient at causing parasites to encyst (Lu¨der et al., 1999). In vivo, in postnatally acquired T. gondii or congenital toxoplasmosis, T. gondii cysts are primarily found in neurons, though cysts are occasionally located in astrocytes or unidentified cell types (Cabral et al., 2016; Ferguson and Hutchison, 1987a,b; Melzer et al., 2010; Sims et al., 1988, 1989). This preferential location of cysts in neurons despite an ability to invade and encyst in other CNS cells types was consistent with in vitro data showing that IFN-γ-stimulated astrocytes and microglia can limit or clear intracellular parasites (Degrandi et al., 2013; Halonen et al., 2001; Lu¨der et al., 1999; Martens et al., 2005), but IFN-γ-stimulated neurons cannot (Schlu¨ter et al., 2001a). Recent work using an in vivo Cre reporter system that allows the identification of CNS cells injected with parasite proteins, even if the cells were never invaded or cleared the intracellular parasite, suggests another option. This work found that parasites primarily interact with and infect neurons (Fig. 24.1), whereas only a small percentage of astrocytes and oligodendrocytes are injected with parasite protein, suggesting that T. gondii encysts in neurons because parasites principally interact with neurons (Cabral et al., 2016). Why T. gondii predominantly infects or injects neurons remains unclear, though this interaction may be driven by physical properties such as the neuron size or differences in the distribution of cell types in CNS areas with high or low vascularization. If physical factors drive the CNS cell-T. gondii interaction, such a mechanism could explain a recent study showing that in ocular toxoplasmosis cysts are predominantly in Muller cells, specialized large glial cells that lie within the

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GFP1 neuron with Toxoplasma gondii cyst in a distal neuronal process. A Cre reporter mouse that only expresses a GFP after Cremediated recombination was infected with a mCherry-expressing T. gondii strain that triggers Cre-mediated recombination via the injection of a T. gondii::Cre fusion protein. At 3 weeks postinfection, the brain was harvested, sectioned into B200 μm thick sections, processed to render the tissue optically clear, and then imaged at 40 3 on a confocal microscope. A maximal projection image of a cyst within a neuronal process is shown. Green 5 GFP expression within neurons, Red 5 mCherry-expressing parasites. Scale bars, 20 μm. GFP, Green fluorescent protein. Source: Image with permission from Cabral, C.M., Tuladhar, S., Dietrich, H.K., Nguyen, E., MacDonald, W.R., Trivedi, T., et al., 2016. Neurons are the primary target cell for the brain-tropic intracellular parasite Toxoplasma gondii. PLoS Pathog. 12, e1005447.

FIGURE 24.1

cellular layer of the retina, rather than retinal neurons (Song et al., 2018).

while limiting immune pathology. As very little is known about the role of oligodendrocytes in cerebral toxoplasmosis, oligodendrocytes are not discussed.

24.7 Control of cerebral toxoplasmosis 24.7.1.1 Neurons Once T. gondii has entered the brain, as in other organs, there must be a coordinated immune response that enables killing and clearance of parasites but also does not create such a massive inflammatory response that the host dies. This balance between controlling the parasite and avoiding immune pathology is particularly important in the CNS, an organ with limited regenerative capacity and that resides within a fixed space.

24.7.1 Parenchymal central nervous system cells Though infiltrating immune cells are essential to the control of T. gondii once it enters the brain, neurons, astrocytes, and microglia are also important in coordinating parasite control

For many years, neurons have been considered bystanders in most neuroinflammatory responses, though this view is changing as accumulating evidence in virology suggests that neurons do respond to inflammatory cytokines (e.g., IFN-γ) (Binder and Griffin, 2001; Rose et al., 2007) and can clear infecting viruses (Cavanaugh et al., 2015; Cho et al., 2013; Griffin and Metcalf, 2011). These findings suggest that neurons could have a role in direct killing of T. gondii as well as indirectly helping control CNS toxoplasmosis through secretion of cytokines and chemokines. While several studies have shown that T. gondii can infect human neurons (Halonen et al., 1996; Passeri et al., 2016; Tanaka et al., 2016), no studies have addressed if human neurons can kill or restrict the replication of intracellular parasites. Only a single in vitro

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study has addressed these issues in murine neurons (Schlu¨ter et al., 2001a). In this study, IFNγ-stimulated murine primary neuron cultures were infected with a T. gondii strain (RH) that is resistant to IRG proteins, the major IFN-γ-inducible mechanism by which murine cells clear T. gondii (Fleckenstein et al., 2012; Steinfeldt et al., 2010), making the negative results inconclusive. Given the recent finding that T. gondii interacts with far more neurons than anticipated and that most of these neurons are uninfected (Koshy et al., 2012), work to address (or readdress) the ability of human and murine neurons to clear or growth restrict intracellular parasites should be pursued. Primary pure murine neuron cultures have been shown to respond to IFN-γ with the increases in STAT1, pSTAT1, CXCL10, SOCS-1, and IL-6 (Rose et al., 2007; Schlu¨ter et al., 2001a), though with delayed kinetics and some differences in downstream IFN-γ-dependent genes compared to mouse embryonic fibroblasts (Rose et al., 2007). In response to T. gondii infection, neurons also show changes in immune response genes [e.g., IL-6, MIP-1B (Schlu¨ter et al., 2001a)], a limited number of which appear to be dependent upon TLR-2 signaling (Umeda et al., 2017). Only one study has used conditional knockouts to look at the role of neuronal immune signaling in controlling cerebral toxoplasmosis in vivo (Ha¨ndel et al., 2012). In this study, infecting mice that lacked gp130, the IL-6 family receptor, only in neurons led to a decrease in long-term survival rates and increases in CNS parasite burdens, infiltrating immune cells, and neuron death, suggesting that neuron IL-6 family member signaling plays an important role in generating an effective neuroinflammatory response while avoiding immune pathology. In addition to direct control of intracellular parasites or cytokine/chemokine secretion and signaling, neurons may also participate in the immune response by restimulating CD81 T effector cells through MCH antigen T-cell

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receptor interactions. Such an interaction has been controversial for decades, but work in viral infections has shown that infected neurons interact with CD81 T cells in vivo (McDole et al., 2010) and can restimulate activated CD81 T cells in vitro (Chevalier et al., 2011). One study using adoptive transfer of CD81 T cells from wild-type or perforindeficient mice to chronically infected, immunocompromised mice found a decrease in cyst burden in the mice that received wildtype, but not perforin-deficient, CD81 T cells (Suzuki et al., 2010). While this study indirectly suggested that CD81 T cells can interact with infected neurons, another study failed to identify T cell neuron interactions when examining ex vivo infected brain using 2-photon microscopy (Schaeffer et al., 2009). A recent study has addressed this issue more directly by showing that neurons infected with ovalbumin (OVA)-expressing parasites can restimulate CD81 T cells specific for OVA. In addition, to show that neuron MHC T cell interactions were relevant in vivo, the study utilized C57Bl/6 transgenic mice in which all cells express a floxed copy of the Ld locus, the MHC I locus that determines the mouse strain susceptibility to cerebral toxoplasmosis (see Section 24.3) (Brown and McLeod, 1990). When the Ld gene was selectively removed from neurons, using a neuron-specific Cre driver, the resulting mice had a higher cyst burden, suggesting that neurons can activate effector T cells via appropriate presentation of antigen in MHC I molecules (Salvioni et al., 2019). Collectively, these data suggest that neurons play a central role in the control of cerebral toxoplasmosis. 24.7.1.2 Astrocytes Unlike neurons, astrocytes are more globally recognized to play an essential role in most neuroinflammatory processes, including infection (Sofroniew and Vinters, 2010). Thus, more work has examined the role of astrocytes in

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cerebral toxoplasmosis. Both human primary astrocytes and human astrocytoma (tumor) cell lines have been used in studies of the T. gondiiastrocyte interface (Da¨ubener et al., 1996, 1993; Pelloux et al., 1996), but here we focus on data on from primary astrocytes as tumor cell lines, by definition, have aberrant cellular signaling and responses. Human astrocytes have been shown to growth restrict intracellular parasites potentially through the production of nitric oxide and/or tryptophan-depletion via upregulation of indoleamine 2,3-dioxygenase, the enzyme that catalyzes the first step of tryptophan metabolism (Oberdo¨rfer et al., 2003; Peterson et al., 1995). Of note, the Peterson 1995 study used a T. gondii strain resistant to IRGs (RH) but still noted growth restriction in human astrocytes in the setting of IFN-γ stimulation. This finding is consistent with recent work showing that human non-CNS cells control parasites by non-IRG mechanisms that may not show the same strain-specific susceptibility seen in murine cells (Niedelman et al., 2013, 2012; Qin et al., 2017). Conversely, murine cells heavily rely on IRGs for intracellular clearance of parasites, which negates the findings of an early study that used RH to infect murine astrocytes and found no clearance of intracellular parasites even with IFN-γ stimulation (Peterson et al., 1993). Multiple subsequent studies using IRG-susceptible parasite strains have clearly established that murine astrocytes are capable of clearing parasites via the IRGs and other IFN-γ-induced GTPases (Degrandi et al., 2013; Halonen et al., 2001; Martens et al., 2005). In addition to upregulating and deploying the IRGs or NO against intracellular parasites, astrocytes increase the expression of many other genes in response to T. gondii infection and stimulation by cytokines such as IFN-α, IFN-γ, and TNF-α (Brenier-Pinchart et al., 2004; Wilson et al., 2005; Hidano et al., 2016). Upregulated chemokines/cytokines include MCP1/CCL2, CCL5, and CXCl10 that are

chemoattractants for immune cells such as T cells and monocytes (Brenier-Pinchart et al., 2004; Cekanaviciute et al., 2014; Hidano et al., 2016) as well as neuroprotective proteins such as prostaglandin E2, which may serve to protect neurons from the toxic inflammatory environment (Rozenfeld et al., 2003). This broad response suggests that astrocytes play an essential role in coordinating the CNS immune response. Further support for the essentiality of astrocytic responses in cerebral toxoplasmosis comes from studies in conditional and full knockout mice. In wild-type mice, reactive GFAP1 astrocytes surround foci of T. gondii infection and inflammation (Cekanaviciute et al., 2014; Stenzel et al., 2004). In mice that lack GFAP, T. gondii infection produces high CNS parasite burdens both early and late in CNS infection; diffuse immune cell infiltration; and areas of necrosis with free tachyzoites at 45 dpi (Stenzel et al., 2004), a time point at which free tachyzoites are no longer found in immunocompetent mice (Ferguson et al., 1991). Similar but more severe findings were observed in two studies in which astrocytes were selectively targeted to be unable to respond to either IL-6 family members (astrocytic gp130 conditional knockout mice) (Dro¨gemu¨ller et al., 2008) or initiate astrocytic STAT1 signaling, the major mediator of IFN-γ cellular responses (astrocytic STAT1 conditional knockout mice) (Hidano et al., 2016). In both conditional knockout mice strains, T. gondii infection led to increased mortality during chronic infection (301 dpi) and increased CNS parasite burdens compared to the control mice (mice with the appropriate floxed gene but no Cre recombinase expression). A similar but milder phenotype of increased parasite burden with increased CD4 T-cell infiltration was seen in a study using mice in which astrocytes constitutively expressed CCL21, a T-cell chemoattractant (Ploix et al., 2011). A final study used mice in which a dominant negative TGFβ

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receptor was expressed in a subset of astrocytes. This partial abrogation of astrocytic TGFβ signaling had no effect on early or late CNS parasite burden but did increase the number of T cells, activated macrophages/microglia, and reactive astrocytes in the brain in early CNS infection. This increased neuroinflammatory response correlated with increased neuronal stress and possible death at early and late time points (Cekanaviciute et al., 2014). Collectively, these studies show that astrocytes are critical for producing an appropriate and effective CNS immune response to T. gondii in vivo. 24.7.1.3 Microglia Microglia are the sole brain-resident immune cell population and thus are purported to be the first line of defense against CNS infection. The prevailing view for a large part of the 20th century was that microglia were neuroectodermal cells similar to neurons, astrocytes, and oligodendrocytes (Ginhoux et al., 2013; Rezaie and Male, 2002). However, after a series of elegant experiments using mice deficient in key regulators of myeloid cell development and survival, microglia have been confirmed to be myeloid cells, derived from the mesoderm, that migrate into the CNS very early in development (Beers et al., 2006; Dai et al., 2002; Ginhoux et al., 2010; Kierdorf et al., 2013; McKercher et al., 1996; Wang et al., 2012; Wegiel et al., 1998). Microglia are found in all regions of the CNS and constitute between 10% and 20% of total brain cells. In vivo imaging has shown that microglial processes are highly dynamic and constantly survey the brain parenchyma (Nimmerjahn et al., 2005; Davalos et al., 2005). In addition, microglia rapidly respond to parenchymal injury suggesting that microglia monitor the CNS for pathological changes (Davalos et al., 2005; Nimmerjahn et al., 2005). Initial studies assessing the role of microglia during infection with T. gondii identified

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“microglial nodules” in the parenchyma using histological methods (Ferguson et al., 1991; Frenkel, 1949, p. 19; Graham et al., 1984; Tognetti et al., 1982). Despite being called microglial nodules a variety of cells, including mononuclear cells, lymphocytes, and astrocytes, may also be part of these nodules that are associated with extracellular parasites (Frenkel, 1949; Stenzel et al., 2004). Within the nodules myeloid cells are hypertrophic and amoeboid, which is suggestive of microglia activation. Thus microglia/macrophages are found in association with parasites and may have an active role in clearing parasites from the infected brain (Conley and Jenkins, 1981; Frenkel, 1949; John et al., 2011; van Horssen et al., 2012). Assessing the role of microglia in the control of cerebral toxoplasmosis has been extremely challenging because peripheral monocytes infiltrate into the CNS during cerebral toxoplasmosis and then convert to macrophages, which like microglia express CD11b, CD45, F4/80, and Iba-1 (a surface molecule commonly used for identifying microglia and macrophages by immunohistochemistry). In addition, in vitro cultures of “microglia” are usually generated from neonatal mice, which do not resemble microglia isolated from the adult brain (Butovsky et al., 2014). The cultures are also purified in a way that brain perivascular macrophages might also be present and incorrectly identified as microglia. Thus, as newer techniques offer mechanisms for exclusively identifying or targeting microglia using Cre/loxP strategies in vivo and culturing microglia from adult mice in vitro (Ajami et al., 2007; Bohlen et al., 2019; Elmore et al., 2014; Yona et al., 2013), we will gain a clearer understanding of the contribution of microglia to the control of cerebral toxoplasmosis. With these caveats in place, below we summarize what is currently known about the role of microglia in cerebral toxoplasmosis, which primarily comes from studies of rodent microglia, most commonly mouse.

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Microglial appear to help control cerebral toxoplasmosis through direct or cell-intrinsic mechanisms (e.g., restricting growth of intracellular parasites) as well as indirect or cellextrinsic mechanisms (e.g., cytokine secretion). In the setting of priming with IFN-γ and the TLR agonist, lipopolysaccharide (LPS), murine neonatal microglia exhibit a reduced infection rate as well as restricting intracellular growth of T. gondii via a nitric oxide (NO) dependent mechanism (Chao et al., 1993a,b). Consistent with this finding, iNOS knockout mice have an increased parasite burden, develop necrotic CNS lesions, and succumb during the later stages of acute infection (Scharton-Kersten et al., 1997). In addition, administration of a small molecule inhibitor of NO synthesis during chronic infection results in the reactivation of T. gondii, leading to an increase in CNS parasite burden and death (Schlu¨ter et al., 1999). However, whether NO production from microglia is critical for control of T. gondii in vivo remains unknown. Beyond NO, other IFNγ-induced antimicrobial pathways, including GTPases (GBPs and IRGs) (Hunter and Sibley, 2012), may be induced in murine microglia to control T. gondii. Interestingly, rat neonatal microglia also restrict intracellular parasite growth but do not require IFN-γ priming (Lu¨der et al., 1999). Finally, one study in embryonic human microglia suggests that IFN-γ decreases the infection rate and that TNF-α and IL-6 contribute to parasite control (Chao et al., 1994). In addition to the direct action on parasites, microglia may also produce cytokines and chemokines that affect the neuroinflammatory response. In the setting of non-T. gondii infections (mycobacteria, Japanese encephalitis virus, and Staphylococcus aureus), activation of microglial NLRP3 is known to induce microglial secretion of IL-1β without triggering pyroptosis (Halle et al., 2008; Hanamsagar et al., 2011; Kaushik et al., 2012; Lee et al., 2013; Shi et al., 2012). In the T. gondii-infected brain, cells

consistent with microglia express IL-1β, indicating possible inflammasome involvement (Biswas et al., 2015; Schlu¨ter et al., 1997). Currently, there are no studies that assess the role of microglial inflammasome activation in vivo during cerebral toxoplasmosis. During murine toxoplasmosis, IL-6 production by microglia in response to T. gondii has been demonstrated in vitro as well as ex vivo (Biswas et al., 2015; Fischer et al., 1993). In IFN-γ/LPS-primed, T. gondii-infected primary human fetal microglia, IL-6 neutralization abrogated the antimicrobial activity of these cells (Chao et al., 1994), while IL-6 neutralization had no effect on the antimicrobial response of primed, T. gondii-infected murine neonatal microglia (Chao et al., 1993b). In addition to microglia, astrocytes and infiltrating monocyte-derived macrophages also express IL-6 (Biswas et al., 2015; Fischer et al., 1997). Thus, the protective effect of IL-6 during TE may represent the combined contribution of several cellular sources of this cytokine. Microglia have been shown to produce IL12 in vivo during infection with T. gondii (Biswas et al., 2015; Schlu¨ter et al., 2001b). IL-12 is a critical cytokine in the induction of IFN-γ by T and NK cells and thus may be important for the persistent production of IFNγ by T cells in the brain. While the original study using IL-12-depleting antibodies beginning on day 28 postinfection did not affect survival through day 40 postinfection (Gazzinelli et al., 1994), subsequent work in IL-12 knockout mice treated with recombinant IL-12 only for the first 14 days of infection found that mice survived until approximately 60 days postinfection but had poor IFN-γ responses and increased parasite levels in the brain (Yap et al., 2000). These results suggest that IL-12 is required for the establishment of a strong IFNγ response during the acute phase of infection and must also be maintained throughout chronic infection, which may be a role for microglia.

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During infection, microglia have been implicated in the production of several chemokines that lead to monocyte and T-cell recruitment to the CNS, which is critical for local control of T. gondii. Inflammatory monocytes, NK cells and a subset of T cells express CCR2, the receptor for CCL2 (Saederup et al., 2010). CCR2- and CCL2-knockout mice fail to control T. gondii replication in the brain and succumb to infection during the acute phase (Benevides et al., 2008; Robben et al., 2005). Mice lacking CCR2 recruit fewer monocytes to the brain, which are necessary for host resistance during chronic infection (Benevides et al., 2008; Biswas et al., 2015; Robben et al., 2005). Similarly, antibody depletion of CCR21 cells during the chronic phase leads to an increased CNS parasite burden and decreased survival of infected mice (Biswas et al., 2015). As demonstrated in the intestine, the resident macrophage population was insufficient to control T. gondii in the absence of CCR2-expressing infiltrating monocytes (Dunay et al., 2008). Therefore, by producing CCL2 microglia may call in inflammatory monocytes required to control chronic T. gondii infection of the brain. In addition to producing CCL2, microglia also produce CCL5/RANTES, which is recognized by CCR1 and CCR5, chemokine receptors on monocytes, T cells, and NK cells (Glass et al., 2005; Schaller et al., 2008; Shi and Pamer, 2011; Strack et al., 2002b). Mice deficient in either CCR1 or CCR5 succumb during acute infection secondary to an inability to control parasites in the periphery, which also results in a higher CNS parasite burden (Khan et al., 2001, 2006; Luangsay et al., 2003). The specific role of microglial-derived CCL5 in recruiting peripheral immune cells to the CNS during infection has not yet been addressed. During toxoplasmosis astrocytes secrete CXCL10 and microglia/macrophages secrete CXCL9 and CXCL10 (Strack et al., 2002a,b). Both CXCL9 and CXCL10 signal through CXCR3, which is expressed on activated and

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memory T cells as well as NK cells (Mu¨ller et al., 2010). CXCL10 is necessary for the recruitment of CD41 and CD81 T cells to inflamed tissues during acute infection with T. gondii and for the recruitment and/or retention of antigen-specific CD81 T cells in the CNS during chronic infection (Cohen et al., 2013; Harris et al., 2012; Khan et al., 2000). In addition to recruiting T cells, CXCL10 increases the speed of T-cell migration in the brain, reducing the time it takes for these cells to find infected cells (Harris et al., 2012). CXCL9 also contributes to the recruitment of CD41 and CD81 T cells to the T. gondii-infected brain and reduces parasite load (Ochiai et al., 2015). In summary, microglia have been proposed to play many roles in controlling CNS infection with T. gondii, including directly killing parasites via NO and potentially modulate the CNS immune response by releasing cytokines and chemokines. However, as noted before, much of this knowledge has been gleaned from work unable to differentiate microglia from macrophages. Thus the exact functions of microglia during infection of the brain with T. gondii remain unclear, though newer in vivo and in vitro techniques promise to shed light on this question.

24.7.2 Systemic immune cells 24.7.2.1 Immune cell infiltration into the central nervous system Effective control of T. gondii infection in the CNS involves the trafficking and entry of peripheral immune cells to the brain. The adhesion and migration of immune cells into tissues require an orchestrated series of receptor ligand interactions on the endothelial cell surface and typically occur in postcapillary venules, where blood flow is relatively slow (Ley et al., 2007). T cell recruitment to the CNS is critical for controlling TE during chronic infection (Gazzinelli et al., 1992). The upregulation of

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ICAM-1 and VCAM-1 on cerebral blood vessels likely plays an important role in the adhesion and entry of CD41 and CD81 T cells to the brain (Deckert-Schlu¨ter et al., 1994a). Circulating IFNγ and signaling through the IFN-γ receptor contribute to the upregulation of ICAM-1 and VCAM-1 on cerebral blood vessels in mouse models of TE (Deckert-Schlu¨ter et al., 1999; Wang et al., 2007). Both CD41 and CD81 T cell numbers in the brain are reduced and parasite loads are elevated in chronically infected reactivated mice in which the VLA-4 integrin on T cells, which binds to VCAM-1, is blocked (Wang et al., 2007; Sa et al., 2014; Wilson et al., 2009). Notably, T. gondii infection of transgenic mice in which VCAM-1 is deleted on endothelial cells (VCAMfl/fl MxCre mice) leads to increased mortality during chronic infection, but leukocyte entry to the CNS is not compromised, indicating that other adhesion molecules can compensate for the loss of VCAM-1 on brain endothelial cells (Deckert et al., 2003). In addition to T cell-mediated immune control in the CNS, infiltrating peripheral blood monocytes also contribute to host defenses during chronic T. gondii infection. In mice, monocyte populations are defined, in part, on the basis of their expression of Ly6C and the chemokine receptors CCR2 and CX3CR1 (Geissmann et al., 2003). During T. gondii infection, both inflammatory (Ly6ChiCCR21) and patrolling (Ly6CloCX3CR1int) populations of monocytes are recruited to the CNS within 2 weeks of infection, and whole-brain imaging analysis reveals preferential accumulation of these cells in the olfactory tubercle, suggesting regional vulnerability to neuroinflammation during T. gondii infection (Schneider et al., 2019). These monocyte populations are also detected in the brain during chronic infection, and the inflammatory subset is critical for parasite control, as depletion with anti-CCR2 mAb results in increased parasite burden and reduced survival of the mice (Biswas et al., 2015). The recruitment of inflammatory

monocytes to the CNS is dependent on PSGL-1 (P-selectin glycoprotein ligand-1), which can interact with selectins on the endothelium, as PSGL-1 blockade similarly results in reduced inflammatory monocytes in the brain during chronic infection (Biswas et al., 2015). Neutrophils may also facilitate monocyte trafficking to the CNS during T. gondii infection, as the depletion of these cells is associated with slightly reduced monocyte entry to the brain, lower levels of IFN-γ, and elevated parasite loads in the CNS (Biswas et al., 2017). 24.7.2.2 Innate immune cells 24.7.2.2.1 Monocyte-derived macrophages and dendritic cells

During chronic T. gondii infection numerous myeloid cells infiltrate the CNS as monocytes and differentiate into monocyte-derived macrophages and DCs (Biswas et al., 2015; John et al., 2009; Nance et al., 2012; Schaeffer et al., 2009). The blood-derived cells have been defined as CD11b-expressing cells that express high levels of CD45, differentiating these cells from microglia that express an intermediate level of CD45. Given the similarities of microglia and macrophages, these cell types may have similar mechanisms that contribute to the control chronic T. gondii infection, including the production of IL-1β, IL-6, IL-12, NO, and chemokines as discussed in the section on microglia. Studies have also described a macrophage population in the brain that expresses CD206 and arginase-1, consistent with alternatively activated macrophages (AAMacs). Chitinase activity from AAMacs was shown to be required for optimal control of T. gondii in the brain, defining a protective role for this macrophage subset (Nance et al., 2012). As noted above, dissecting out the role of macrophage versus microglia is challenging at this time but new techniques may allow future experiments that specifically target each population individually.

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A small portion of brain-infiltrating myeloid cells express CD11c and high levels of MHC II, consistent with the phenotype of DCs (John et al., 2009), which are regarded as the most efficient antigen present cells (APCs) for naive T-cell activation. Two studies identified interactions between antigen-specific T cells and CD11c-expressing cells in the CNS (John et al., 2009; Schaeffer et al., 2009), suggesting that DCs may be the main APCs in cerebral toxoplasmosis, despite brain-derived CD11c-expressing cells serving as poor APCs to T cells ex vivo (John et al., 2009). Consistent with this possibility, recent work in an experimental autoimmune encephalomyelitis model found that microglia are poor APCs and that conventional DCs are the major APC in this model of neuroinflammation (Mundt et al., 2019). For cerebral toxoplasmosis, more work is required to fully determine the importance of antigen presentation in the brain and which cell types play a critical role. 24.7.2.2.2 Neutrophils and other granulocytes

Several studies have documented the infiltration of neutrophils into the brain during chronic T. gondii infection (Biswas et al., 2017; O’Brien et al., 2019). Typically, neutrophil infiltration is associated with an inability to control parasite replication or inflammation in the brain. For example, neutrophil accumulation has been observed following IFN-γ depletion that leads to uncontrolled parasite replication or in IL-27-deficient mice, which have unrestrained inflammatory responses to the parasite (Stumhofer et al., 2010). As noted above, recent studies have also demonstrated that neutrophil depletion affects monocyte recruitment during chronic T. gondii infection (Biswas et al., 2017). Currently whether neutrophil recruitment to the CNS helps or is detrimental in cerebral toxoplasmosis is unknown. The roles of other granulocytes in the CNS also remain largely unexplored. Mast cells reside in the meninges and may shape the inflammatory

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response to T. gondii, while basophils and eosinophils have not been reported to infiltrate the infected brain. 24.7.2.3 Adaptive immune cells 24.7.2.3.1 T cells

T cells are required to control T. gondii in the brain (Dupont et al., 2012; Gazzinelli et al., 1992). In humans, the necessity for T cells was made evident in the 1980s by the remarkable number of AIDS patients with CD4 levels ,100 who developed TE (Luft and Remington, 1992). A similar phenotype is observed when chronically infected mice are depleted of CD41 and CD81 T cells. Following T-cell depletion, mice rapidly succumb to infection and are unable to control parasite reactivation within the brain (Gazzinelli et al., 1992). Similarly, the depletion of IFN-γ during chronic infection also results in mortality due to uncontrolled parasite replication in the brain (Suzuki et al., 1989a,b; Gazzinelli et al., 1992). Together, these findings implicate T-cell production of IFN-γ as the critical immune mechanism necessary to control T. gondii. Beyond cytokine production, perforin-mediated cytolysis of infected cells is an additional mechanism of protection by CD81 T cells (Denkers et al., 1997; Suzuki et al., 2010). Perforin knockout mice succumb to infection in the chronic phase and have significantly higher parasite burden in the brain than wild-type mice (Denkers et al., 1997). These results suggest that both IFN-γ production and perforin-mediated cytolysis contribute to parasite control during chronic infection. To address the antigen specificity of T cells that infiltrate into the CNS during chronic infection, parasites expressing the model antigen OVA were used to infect mice. OVA-specific CD81 T cells were only able to infiltrate the brains of mice infected with transgenic OVAexpressing parasites, not the parental strain. These experiments suggest that the CD81 T-cell repertoire may be largely parasite specific

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(Wilson et al., 2009). The specificity of CD81 T cells in the brain during infection has also been explored using tetramer reagents, which detect clear populations specific for T. gondii (Frickel et al., 2008; Wilson et al., 2010). The mechanisms that promote the recruitment and retention of antigen-specific cells in the CNS remain unclear and underscore the complexity of developing a T-cell response in the brain. Several multiphoton imaging studies have revealed factors that dictate the behavior of parasite-specific CD81 T cells in the brain during T. gondii infection. Two studies explored the interactions of OVA-specific CD81 T cells (OT-I) with CD11c-expressing cells in the CNS. The OT-I T cells make contacts with potential APCs in the CNS, including in regions of parasite reactivation, where sustained interactions were observed, suggesting that CD81 T cells may receive cues from APCs in distinct regions of the brain (John et al., 2011; Schaeffer et al., 2009). Imaging studies also revealed that once CD81 T cells reach the brain, the migratory behavior of the cells is akin to a Le´vy walk, where the T cells make numerous small movements and more rarely move great distances. Surprisingly, the migration pattern was not mediated by chemokine signals, but rather, chemokine signals enhanced the speed of T cells migrating in the brain, which was predicted to increase the capacity of T cells to encounter rare infected cells in the brain (Harris et al., 2012). Together these studies reveal that the protective CD81 T-cell response to T. gondii is dependent on numerous factors that activate T cells to express IFN-γ and perforin, provide signals that promote the retention of antigen-specific cells, and chemokines that promote the motility of T cells through the tissue. The role of CD41 T cells in the infected brain has been less thoroughly explored. Early studies that used antibodies to deplete CD41 T cells alone did not observe an increase in mortality in chronically infected mice, which

suggested that CD41 T cells may not play a major role in controlling T. gondii in the brain (Gazzinelli et al., 1992). Mice lacking CD41 T cells vary in their resistance to infection with T. gondii, which may be dependent on parasite strain. CD4-deficient mice infected with transgenic Prugniaud (Pru) parasites survive infection nearly as well as wild-type mice (Schaeffer et al., 2009). On the other hand, CD4 knockout mice infected with Me49 parasites survive for only about 40 days postinfection (Johnson and Sayles, 2002). In addition to producing IFN-γ during the chronic phase of infection, CD41 T cells may play a larger role in providing help for B cell. In studies examining CD4-deficient mice infected with Me49, knockout mice had an increased CNS parasite burden, relatively normal IFN-γ responses, and low parasitespecific IgG titers in comparison to wild-type mice, suggesting that CD41 T cells are necessary for generating optimal antibody responses during T. gondii infection. Indeed, transfer of immune serum to chronically infected CD4 knockout mice extended survival (Johnson and Sayles, 2002). A role for CD41 T cells in supporting a long-term CD81 T-cell response to T. gondii was revealed in vaccination studies using attenuated strains of T. gondii. In one study, CD4 knockout animals had decreased CD81 T-cell responses at 180 days postimmunization, as shown by cytolysis assays and IFN-γ production. Moreover, transfer of CD81 T cells from immunized CD4 knockout mice to naı¨ve mice did not protect them from a lethal T. gondii challenge (Casciotti et al., 2002). In subsequent studies, mice depleted of CD41 T cells prior to immunization failed to generate a strong antigen-specific CD81 T-cell response (Jordan et al., 2009). Together, these studies demonstrate how protective immunity to T. gondii may depend on CD41 T cells. 24.7.2.3.2 Regulatory T cells

Regulatory T cells (Tregs) have been shown to play an integral role in balancing inflammatory

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immune responses to CNS infection (CervantesBarraga´n et al., 2012). To begin to define the role of Tregs during T. gondii infection, mice were treated with IL-2 complexes during acute infection, which increased Treg numbers during early infection. The increase in Treg number correlated with a protection from lethal immunopathology but increased parasite cyst burden in the brain during chronic infection (Oldenhove et al., 2009). This study highlights that Tregs are involved in maintaining a balance between protective immunity and immunopathology. During chronic T. gondii infection, Tregs are recruited to the inflamed brain and are largely Th1-polarized, expressing T-bet, CXCR3, and IFN-γ, as well as the suppressive cytokine, IL-10 (O’Brien et al., 2017). A major question regarding the role of Tregs during infection is whether or not the Tregs are specific for the pathogen. A T. gondiispecific MHC II tetramer can readily identify effector CD41 T cells, but Tregs in the CNS were not recognized by the same reagent, suggesting that Tregs in the brain may not be specific for T. gondii. Upon further characterization of the Treg population by TCRβ sequencing, minimal sequence overlap was observed between effector CD41 T cells and Tregs in the inflamed CNS, suggesting that these two populations arise from distinct lineages and may recognize distinct antigens. In addition to potential differences in antigen specificity, differences in the localization of effector CD41 T cells and Tregs within the CNS were also observed. CD41 effector T cells were readily found in the brain parenchyma, meninges, and perivascular spaces, whereas Tregs were restricted to the meninges and perivascular spaces. The meninges and perivascular spaces during chronic infection were also enriched for MHC II and CD11c-expressing APCs. Multiphoton microscopy of Tregs and CD11cexpressing cells revealed that these cells form long-lived interactions that are dependent on the adhesion molecule LFA-1, suggesting that Tregs may suppress APC responses in specific locations within the CNS. Taken together, Th1

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polarized Tregs are recruited to the brain during chronic T. gondii infection where they largely localize to regions of immune cell entry to the brain (O’Brien et al., 2017). Future studies are required to determine the specific roles of Tregs once they reach the brain using depletion studies and cell-type-specific knockout mice. Tregs produce high levels of the immunosuppressive cytokine IL-10. To explore the role of IL-10 exclusively during the chronic phase of infection, recent studies utilized antibodies that block the IL-10 receptor (IL-10R). Consistent with previous reports in IL-10deficient mice (Wilson et al., 2005), IL-10R blockade leads to severe immunopathology associated with increases in the inflammatory response, including increased APC activation, expansion of CD41 T cells, and neutrophil recruitment to the brain. In addition to IL-10, ICOS (inducible T cell costimulator) also limits neuroinflammation during chronic infection. ICOS-ligand (ICOSL) blockade during chronic infection leads to the expansion of effector T cells specifically in the brain without affecting IL-10 production or APC activation. ICOSL blockade also leads to changes in effector T cells in the brain, including increased expression of IL-2-associated signaling molecules CD25, phosphorylated STAT5 (pSTAT5), Ki67, and Bcl-2 in effector T cells in the brain (O’Brien et al., 2019). There are likely numerous factors that regulate chronic inflammation during T. gondii infection in addition to Tregs, IL-10, IL-27, and ICOS. T cells during chronic infection also express PD-1, a molecule associated with T-cell exhaustion (Wilson et al., 2009; Bhadra et al., 2011). Blockade of PD-1 can restore functionality to T cells in severe cases of chronic T. gondii infection (Bhadra et al., 2011). 24.7.2.3.3 B cells

Though most research focuses on the T-cell response to T. gondii in the CNS, B cells are likely also important during chronic T. gondii

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infection. The μMT mouse strain lacks B cells and, akin to CD4 knockout mice, succumbs to infection during the chronic stages with a high CNS parasite burden. The main role of B cells is likely the production of antibody, as μMT mice survive T. gondii infection if given serum from immune mice (Kang et al., 2000). More recent studies have shown that IL-21 is critical for driving this antibody response, promoting germinal center B cells, T follicular helper cells and parasite-specific class-switched antibody (Stumhofer et al., 2013). Together, these studies highlight the potential importance of B cells during chronic T. gondii infection, though it is also possible that the lack of anti-T. gondii antibodies leads to persistent parasitemia (Konradt et al., 2016) and increased seeding of the CNS with parasites. Thus many questions regarding the role and function of B cells during CNS infection remain, including whether B cells traffic to the CNS to produce antibody locally.

24.8 Physiologic effects of Toxoplasma gondii on the central nervous system Given T. gondii’s ability to infect and persist in the CNS, as well as the neuroinflammatory response the infection provokes, considerable work has been done to understand how T. gondii infection may alter host behavior and CNS physiology.

24.8.1 Effects on animal behavior Most studies on animal behavior have been done on rodents (mice and rats). As reviewed by Worth et al., many studies have identified a wide-range of behavioral changes in infected rodents (e.g., loss of aversion to cat urine; decreased motor coordination; and memory and learning deficits), but other studies have shown no impairments in the same tasks and behaviors (Worth et al., 2014). The inconsistencies in the

findings may arise from many factors, such as rodent or parasite strain, mode or timing of infection, or behavior assessed, but the lack of consistency makes it difficult to draw definitive conclusions about the extent to which T. gondii infection changes rodent behavior (Worth et al., 2014). Despite this inconsistency, several hypotheses for the cause of these behavioral changes have been suggested. These hypotheses [as reviewed in Tedford and McConkey (2017)] include (1) the location of persistent cysts; (2) changes in neurotransmitter levels, especially dopamine; (3) parasites producing excess dopamine; (4) neuroinflammation affecting specific pathways; and (5) infection-associated hormonal changes. While these hypotheses are not mutually exclusive and some data underlie each one, contradictory data for most hypotheses also exist. In terms of cysts or cyst location causing behavioral changes, Ingram et al. observed that Balb/c mice infected with a strain of T. gondii that only produces an acute infection, not a persistent CNS infection, still showed the loss of aversion to cat urine (Ingram et al., 2013). Similarly, two studies identified increases in dopamine or its metabolites in the brains of T. gondii-infected mice (Ihara et al., 2016; Stibbs, 1985), but another study failed to confirm these findings (Wang et al., 2015). Finally, while T. gondii does have two tyrosine hydroxylases (AAH1, AAH2) that that are able to convert tyrosine to L-dopa, the precursor molecule to dopamine (Gaskell et al., 2009), a study using a strain of T. gondii in which AAH2 was knocked out [AAH1 appears to be essential (Wang et al., 2015)] found that Balb/c mice infected with either the parental or knockout strains showed behavioral abnormalities and neurochemical changes classically linked to dopamine pathways and secretion (McFarland et al., 2018). Thus, at this time, no definitive mechanisms have been identified to explain the T. gondii-associated behavior changes identified in some studies.

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24.8.2 Effects on rodent neurophysiology and structure Several studies have identified other neurophysiologic and structural changes in T. gondii-infected mice. A study using MRI to evaluate the brains of mice infected with T. gondii for 8 months found an increase in the size of the ventricles (the fluid-filled spaces in the middle of the brain), as well as mild asymmetries of the brain parenchyma. Though these findings are most easily explained by a loss of parenchymal cells, histology on the mice showed no loss of neurons, axonal injury, or “extensive” demyelination (Hermes et al., 2008). However, in another study that used an MRI technique more sensitive to disruptions in white matter tracts, mice infected with T. gondii for 4 5 months and that had abnormalities in tactile sensation showed white matter tract disruptions in the somatosensory cortex (SSC), cortex highly involved in processing tactile sensation. Follow-up studies of the SSC showed a decrease in dendritic arborizations, spine number, and essential synaptic proteins, suggesting synaptic disruption of the SSC (Parlog et al., 2015). Finally, two recent studies suggested that T. gondii infection increases CNS excitability through two distinct but nonexclusive mechanisms, offering possible mechanistic insights into why patients with TE have seizures (Luft and Remington, 1992). David et al. showed that Me49 infected C57BL/6 mice had decreased astrocytic uptake of extracellular glutamate, the major excitatory neurotransmitter of the brain, and corresponding increases in brain electrical activity (David et al., 2016). Brooks et al. found that Me49 (type II) but not CEP (type III)-infected mice showed a mislocalization of glutamate decarboxylase (GAD) in hippocampal neurons at 1 mpi. As GAD is the key enzyme in converting glutamate to GABA—the major inhibitory neurotransmitter in the brain—this

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finding suggested that the Me49-infected mice would have increased brain excitability and thus an increased propensity for seizures, which indeed was documented with skull electroencephalograms (Brooks et al., 2015).

24.8.3 Effects on human behavior T. gondii seropositivity has been associated with a number of human diseases, disorders, and behaviors, including an increased risk of car accidents (Flegr et al., 2002), Alzheimer’s disease (Kusbeci et al., 2011), depression (Alvarado-Esquivel et al., 2016), and schizophrenia (Sutterland et al., 2015). One of the major issues with these studies is that, by necessity, they rely upon a correlation between T. gondii seropositivity and the given disease or behavior. Correlations do not prove causation, and it is equally plausible that the personality traits or disease process studied increases the propensity for acquiring a T. gondii infection rather than the other way around (Worth et al., 2014). In addition, many of the studies have relatively small numbers of people (Kusbeci et al., 2011), do not include baseline data on known risk factors for a given disease/disorder, and fail to control or consider these factors (Flegr et al., 2002). In addition, several studies have shown no correlation between T. gondii seropositivity and some of the same diseases and disorders (Gale et al., 2016; Mahami-Oskouei et al., 2016; Perry et al., 2016; Torniainen-Holm et al., 2019). Finally, for many of these diseases, the incidence of the disease (e.g., schizophrenia) does not vary between countries with high and low T. gondii seropositivity (Mayeux and Stern, 2012; Pappas et al., 2009; van Os and Kapur, 2009). Thus, currently, the ability of “latent” T. gondii infection to potentiate or cause changes in human neurocognition/ behavior remains unclear.

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24.9 Conclusion T. gondii’s tropism for and persistence in the CNS underlies T. gondii’s ability to pass between intermediate hosts and to cause devastating neurologic disease in those with underdeveloped immune responses or who acquire a severe immunodeficiency. The findings described above reveal our current understanding of the complex interactions between the immune system, the parasite, and the brain, which govern T. gondii’s entry and persistence in the CNS. Yet for many areas, our knowledge falls short. Future work will undoubtedly address many of these outstanding questions such as determining regional susceptibility to inflammation, the immune capabilities of neurons, segregating the roles of microglia versus infiltrating peripheral monocytes/macrophages, and defining antigen presentation in the brain.

Acknowledgement This work was funded in part by the National Institutes of Health (R01NS091067 (THH), R56NS106028 (THH), R01AI120846 (MBL)), and the American Cancer Society (RSG-14-202-01-MPC (MBL)).

References Afonso, C., Paixa˜o, V.B., Costa, R.M., 2012. Chronic Toxoplasma infection modifies the structure and the risk of host behavior. PLoS One 7, e32489. Available from: https://doi.org/10.1371/journal.pone.0032489. Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W., Rossi, F.M. V., 2007. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat. Neurosci. 10, 1538 1543. Available from: https://doi. org/10.1038/nn2014. Alvarado-Esquivel, C., Sa´nchez-Anguiano, L.F., MendozaLarios, A., Herna´ndez-Tinoco, J., Pe´rez-Ochoa, J.F., Antuna-Salcido, E.I., et al., 2015. Prevalence of Toxoplasma gondii infection in brain and heart by immunohistochemistry in a hospital-based autopsy series in Durango, Mexico. Eur. J. Microbiol. Immunol. (Bp.) 5, 143 149. Available from: https://doi.org/10.1556/ 1886.2015.00014.

Alvarado-Esquivel, C., Sanchez-Anguiano, L.F., Hernandez-Tinoco, J., Berumen-Segovia, L.O., TorresPrieto, Y.E., Estrada-Martinez, S., et al., 2016. Toxoplasma gondii infection and mixed anxiety and depressive disorder: a case-control seroprevalence study in Durango, Mexico. J. Clin. Med. Res. 8, 519 523. Available from: https://doi.org/10.14740/ jocmr2576w. Arendt, G., von Giesen, H.J., Hefter, H., Neuen-Jacob, E., Roick, H., Jablonowski, H., 1999. Long-term course and outcome in AIDS patients with cerebral toxoplasmosis. Acta Neurol. Scand 100, 178 184. Ayloo, S., Gu, C., 2019. Transcytosis at the blood-brain barrier. Curr. Opin. Neurobiol. 57, 32 38. Available from: https://doi.org/10.1016/j.conb.2018.12.014. Barragan, A., Brossier, F., Sibley, L.D., 2005. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell. Microbiol 7, 561 568. Available from: https://doi.org/10.1111/j.14625822.2005.00486.x. Beers, D.R., Henkel, J.S., Xiao, Q., Zhao, W., Wang, J., Yen, A.A., et al., 2006. Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U.S.A. 103, 16021 16026. Available from: https://doi.org/10.1073/ pnas.0607423103. Benevides, L., Milanezi, C.M., Yamauchi, L.M., Benjamim, C.F., Silva, J.S., Silva, N.M., 2008. CCR2 receptor is essential to activate microbicidal mechanisms to control Toxoplasma gondii infection in the central nervous system. Am. J. Pathol. 173, 741 751. Available from: https://doi.org/10.2353/ajpath.2008.080129. Berenreiterova´, M., Flegr, J., Kubˇena, A.A., Nˇemec, P., 2011. The distribution of Toxoplasma gondii cysts in the brain of a mouse with latent toxoplasmosis: implications for the behavioral manipulation hypothesis. PLoS One 6, e28925. Available from: https://doi.org/10.1371/journal.pone.0028925. Bertoli, F., Espino, M., Arosemena, J.R., Fishback, J.L., Frenkel, J.K., 1995. A spectrum in the pathology of toxoplasmosis in patients with acquired immunodeficiency syndrome. Arch. Pathol. Lab. Med. 119, 214 224. Bhadra, R., Gigley, J.P., Weiss, L.M., Khan, I.A., 2011. Control of Toxoplasma reactivation by rescue of dysfunctional CD8 1 T-cell response via PD-1-PDL-1 blockade. Proc. Natl. Acad. Sci. U.S.A. 108, 9196 9201. Available from: https://doi.org/10.1073/pnas.1015298108. Bhandage, A.K., Kanatani, S., Barragan, A., 2019. Toxoplasma-Induced Hypermigration of Primary Cortical Microglia Implicates GABAergic Signaling. Front Cell Infect Microbiol 9, 73. Available from: https://doi.org/10.3389/fcimb.2019.00073.

Toxoplasma Gondii

References

Bierly, A.L., Shufesky, W.J., Sukhumavasi, W., Morelli, A. E., Denkers, E.Y., 2008. Dendritic cells expressing plasmacytoid marker PDCA-1 are Trojan horses during Toxoplasma gondii infection. J. Immunol. 181, 8485 8491. Binder, G.K., Griffin, D.E., 2001. Interferon-gammamediated site-specific clearance of alphavirus from CNS neurons. Science 293, 303 306. Available from: https:// doi.org/10.1126/science.1059742. Biswas, A., Bruder, D., Wolf, S.A., Jeron, A., Mack, M., Heimesaat, M.M., et al., 2015. Ly6C(high) monocytes control cerebral toxoplasmosis. J. Immunol. 194, 3223 3235. Available from: https://doi.org/10.4049/ jimmunol.1402037. Biswas, A., French, T., Du¨sedau, H.P., Mueller, N., RiekBurchardt, M., Dudeck, A., et al., 2017. Behavior of neutrophil granulocytes during Toxoplasma gondii infection in the central nervous system. Front. Cell. Infect. Microbiol. 7, 259. Available from: https://doi.org/ 10.3389/fcimb.2017.00259. Blanchard, N., Gonzalez, F., Schaeffer, M., Joncker, N.T., Cheng, T., Shastri, A.J., et al., 2008. Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum. Nat. Immunol. 9, 937 944. Available from: https://doi.org/10.1038/ni.1629. Blanchard, N., Dunay, I.R., Schlu¨ter, D., 2015. Persistence of Toxoplasma gondii in the central nervous system: a fine-tuned balance between the parasite, the brain and the immune system. Parasite Immunol. 37, 150 158. Available from: https://doi.org/10.1111/pim.12173. Bohlen, C.J., Bennett, F.C., Bennett, M.L., 2019. Isolation and culture of microglia. Curr. Protoc. Immunol. 125, e70. Available from: https://doi.org/10.1002/cpim.70. Brenier-Pinchart, M.-P., Blanc-Gonnet, E., Marche, P.N., Berger, F., Durand, F., Ambroise-Thomas, P., et al., 2004. Infection of human astrocytes and glioblastoma cells with Toxoplasma gondii: monocyte chemotactic protein-1 secretion and chemokine expression in vitro. Acta Neuropathol. 107, 245 249. Available from: https://doi.org/10.1007/s00401-003-0804-0. Brooks, J.M., Carrillo, G.L., Su, J., Lindsay, D.S., Fox, M.A., Blader, I.J., 2015. Toxoplasma gondii infections alter GABAergic synapses and signaling in the central nervous system. MBio 6, e01428-15. Available from: https://doi.org/10.1128/mBio.01428-15. Brown, C.R., McLeod, R., 1990. Class I MHC genes and CD8 1 T cells determine cyst number in Toxoplasma gondii infection. J. Immunol. 145, 3438 3441. Butovsky, O., Jedrychowski, M.P., Moore, C.S., Cialic, R., Lanser, A.J., Gabriely, G., et al., 2014. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat. Neurosci. 17, 131 143. Available from: https://doi.org/10.1038/nn.3599.

1063

Cabral, C.M., Tuladhar, S., Dietrich, H.K., Nguyen, E., MacDonald, W.R., Trivedi, T., et al., 2016. Neurons are the primary target cell for the brain-tropic intracellular parasite Toxoplasma gondii. PLoS Pathog. 12, e1005447. Available from: https://doi.org/10.1371/journal. ppat.1005447. Cabral, C.M., McGovern, K.E., MacDonald, W.R., Franco, J., Koshy, A.A., 2017. Dissecting amyloid beta deposition using distinct strains of the neurotropic parasite Toxoplasma gondii as a novel tool. ASN Neuro 9, 1759091417724915. Available from: https://doi.org/ 10.1177/1759091417724915. Casciotti, L., Ely, K.H., Williams, M.E., Khan, I.A., 2002. CD8(1)-T-cell immunity against Toxoplasma gondii can be induced but not maintained in mice lacking conventional CD4(1) T cells. Infect. Immun 70, 434 443. Cavanaugh, S.E., Holmgren, A.M., Rall, G.F., 2015. Homeostatic interferon expression in neurons is sufficient for early control of viral infection. J. Neuroimmunol. 279, 11 19. Available from: https:// doi.org/10.1016/j.jneuroim.2014.12.012. Cekanaviciute, E., Dietrich, H.K., Axtell, R.C., Williams, A. M., Egusquiza, R., Wai, K.M., et al., 2014. Astrocytic TGF-β signaling limits inflammation and reduces neuronal damage during central nervous system Toxoplasma infection. J. Immunol. 193, 139 149. Available from: https://doi.org/10.4049/jimmunol.1303284. Cervantes-Barraga´n, L., Firner, S., Bechmann, I., Waisman, A., Lahl, K., Sparwasser, T., et al., 2012. Regulatory T cells selectively preserve immune privilege of selfantigens during viral central nervous system infection. J. Immunol. 188, 3678 3685. Available from: https:// doi.org/10.4049/jimmunol.1102422. Chao, C.C., Anderson, W.R., Hu, S., Gekker, G., Martella, A., Peterson, P.K., 1993a. Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism. Clin. Immunol. Immunopathol. 67, 178 183. Chao, C.C., Hu, S., Gekker, G., Novick, W.J., Remington, J. S., Peterson, P.K., 1993b. Effects of cytokines on multiplication of Toxoplasma gondii in microglial cells. J. Immunol. 150, 3404 3410. Chao, C.C., Gekker, G., Hu, S., Peterson, P.K., 1994. Human microglial cell defense against Toxoplasma gondii. The role of cytokines. J. Immunol. 152, 1246 1252. Chevalier, G., Suberbielle, E., Monnet, C., Duplan, V., Martin-Blondel, G., Farrugia, F., et al., 2011. Neurons are MHC class I-dependent targets for CD8 T cells upon neurotropic viral infection. PLoS Pathog. 7, e1002393. Available from: https://doi.org/10.1371/journal. ppat.1002393. Cho, H., Proll, S.C., Szretter, K.J., Katze, M.G., Gale Jr, M., Diamond, M.S., 2013. Differential innate immune

Toxoplasma Gondii

1064

24. Cerebral toxoplasmosis

response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded RNA viruses. Nat. Med. 19, 458 464. Available from: https://doi.org/10.1038/nm.3108. Cohen, S.B., Maurer, K.J., Egan, C.E., Oghumu, S., Satoskar, A.R., Denkers, E.Y., 2013. CXCR3-dependent CD41 T cells are required to activate inflammatory monocytes for defense against intestinal infection. PLoS Pathog. 9, e1003706. Available from: https://doi.org/10.1371/ journal.ppat.1003706. Conley, F.K., Jenkins, K.A., 1981. Immunohistological study of the anatomic relationship of toxoplasma antigens to the inflammatory response in the brains of mice chronically infected with Toxoplasma gondii. Infect. Immun. 31, 1184 1192. Cook, J.H., Ueno, N., Lodoen, M.B., 2018. Toxoplasma gondii disrupts β1 integrin signaling and focal adhesion formation during monocyte hypermotility. J. Biol. Chem. 293, 3374 3385. Available from: https://doi.org/10.1074/ jbc.M117.793281. Coombes, J.L., Charsar, B.A., Han, S.-J., Halkias, J., Chan, S.W., Koshy, A.A., et al., 2013. Motile invaded neutrophils in the small intestine of Toxoplasma gondiiinfected mice reveal a potential mechanism for parasite spread. Proc. Natl. Acad. Sci. U.S.A. 110, E1913 E1922. Available from: https://doi.org/ 10.1073/pnas.1220272110. Corcino, Y.L., Portillo, J.-A.C., Subauste, C.S., 2019. Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii. Sci. Rep. 9, 669. Available from: https://doi.org/10.1038/s41598018-36724-2. Courret, N., Darche, S., Sonigo, P., Milon, G., Buzoni-Gaˆtel, D., Tardieux, I., 2006. CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain. Blood 107, 309 316. Available from: https://doi.org/10.1182/blood-2005-02-0666. Crawford, L.B., Streblow, D.N., Hakki, M., Nelson, J.A., Caposio, P., 2015. Humanized mouse models of human cytomegalovirus infection. Curr. Opin. Virol. 13, 86 92. Available from: https://doi.org/10.1016/j. coviro.2015.06.006. Dai, X.-M., Ryan, G.R., Hapel, A.J., Dominguez, M.G., Russell, R.G., Kapp, S., et al., 2002. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood 99, 111 120. Da¨ubener, W., Pilz, K., Seghrouchni Zennati, S., Bilzer, T., Fischer, H.G., Hadding, U., 1993. Induction of toxoplasmostasis in a human glioblastoma by interferon gamma. J. Neuroimmunol. 43, 31 38.

Da¨ubener, W., Remscheid, C., Nockemann, S., Pilz, K., Seghrouchni, S., Mackenzie, C., et al., 1996. Antiparasitic effector mechanisms in human brain tumor cells: role of interferon-gamma and tumor necrosis factor-alpha. Eur. J. Immunol. 26, 487 492. Available from: https://doi.org/10.1002/eji.1830260231. Davalos, D., Grutzendler, J., Yang, G., Kim, J.V., Zuo, Y., Jung, S., et al., 2005. ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752 758. Available from: https://doi.org/10.1038/ nn1472. David, C.N., Frias, E.S., Szu, J.I., Vieira, P.A., Hubbard, J. A., Lovelace, J., et al., 2016. GLT-1-dependent disruption of CNS glutamate homeostasis and neuronal function by the protozoan parasite Toxoplasma gondii. PLoS Pathog. 12, e1005643. Available from: https://doi.org/ 10.1371/journal.ppat.1005643. Deckert, M., Lu¨tjen, S., Leuker, C.E., Kwok, L.-Y., Strack, A., Mu¨ller, W., et al., 2003. Mice with neonatally induced inactivation of the vascular cell adhesion molecule-1 fail to control the parasite in Toxoplasma encephalitis. Eur. J. Immunol. 33, 1418 1428. Available from: https://doi.org/10.1002/eji.200322826. Deckert-Schlu¨ter, M., Schlu¨ter, D., Hof, H., Wiestler, O.D., Lassmann, H., 1994a. Differential expression of ICAM1, VCAM-1 and their ligands LFA-1, Mac-1, CD43, VLA-4, and MHC class II antigens in murine Toxoplasma encephalitis: a light microscopic and ultrastructural immunohistochemical study. J. Neuropathol. Exp. Neurol. 53, 457 468. Deckert-Schlu¨ter, M., Schlu¨ter, D., Schmidt, D., Schwendemann, G., Wiestler, O.D., Hof, H., 1994b. Toxoplasma encephalitis in congenic B10 and BALB mice: impact of genetic factors on the immune response. Infect. Immun. 62, 221 228. Deckert-Schlu¨ter, M., Bluethmann, H., Kaefer, N., Rang, A., Schlu¨ter, D., 1999. Interferon-gamma receptor-mediated but not tumor necrosis factor receptor type 1- or type 2mediated signaling is crucial for the activation of cerebral blood vessel endothelial cells and microglia in murine Toxoplasma encephalitis. Am. J. Pathol. 154, 1549 1561. Degrandi, D., Kravets, E., Konermann, C., Beuter-Gunia, C., Klu¨mpers, V., Lahme, S., et al., 2013. Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication. Proc. Natl. Acad. Sci. U.S.A. 110, 294 299. Available from: https://doi.org/10.1073/ pnas.1205635110. Dellacasa-Lindberg, I., Hitziger, N., Barragan, A., 2007. Localized recrudescence of Toxoplasma infections in the central nervous system of immunocompromised mice assessed by in vivo bioluminescence imaging. Microbes

Toxoplasma Gondii

References

Infect. 9, 1291 1298. Available from: https://doi.org/ 10.1016/j.micinf.2007.06.003. Denkers, E.Y., Yap, G., Scharton-Kersten, T., Charest, H., Butcher, B.A., Caspar, P., et al., 1997. Perforin-mediated cytolysis plays a limited role in host resistance to Toxoplasma gondii. J. Immunol. 159, 1903 1908. Di Cristina, M., Marocco, D., Galizi, R., Proietti, C., Spaccapelo, R., Crisanti, A., 2008. Temporal and spatial distribution of Toxoplasma gondii differentiation into bradyzoites and tissue cyst formation in vivo. Infect. Immun. 76, 3491 3501. Available from: https://doi. org/10.1128/IAI.00254-08. Dro¨gemu¨ller, K., Helmuth, U., Brunn, A., SakowiczBurkiewicz, M., Gutmann, D.H., Mueller, W., et al., 2008. Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis. J. Immunol. 181, 2683 2693. Dubey, J.P., Ferreira, L.R., Alsaad, M., Verma, S.K., Alves, D.A., Holland, G.N., et al., 2016. Experimental toxoplasmosis in rats induced orally with eleven strains of Toxoplasma gondii of seven genotypes: tissue tropism, tissue cyst size, neural lesions, tissue cyst rupture without reactivation, and ocular lesions. PLoS One 11, e0156255. Available from: https://doi.org/10.1371/ journal.pone.0156255. Dunay, I.R., Damatta, R.A., Fux, B., Presti, R., Greco, S., Colonna, M., et al., 2008. Gr1(1) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii. Immunity 29, 306 317. Available from: https://doi.org/10.1016/j.immuni.2008.05.019. Dupont, C.D., Christian, D.A., Hunter, C.A., 2012. Immune response and immunopathology during toxoplasmosis. Semin. Immunopathol. 34, 793 813. Available from: https://doi.org/10.1007/s00281-012-0339-3. Elmore, M.R.P., Najafi, A.R., Koike, M.A., Dagher, N.N., Spangenberg, E.E., Rice, R.A., et al., 2014. Colonystimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380 397. Available from: https://doi.org/10.1016/j.neuron.2014.02.040. Evans, A.K., Strassmann, P.S., Lee, I.-P., Sapolsky, R.M., 2014. Patterns of Toxoplasma gondii cyst distribution in the forebrain associate with individual variation in predator odor avoidance and anxiety-related behavior in male LongEvans rats. Brain Behav. Immun. 37, 122 133. Available from: https://doi.org/10.1016/j.bbi.2013.11.012. Ferguson, D.J., Hutchison, W.M., 1987a. The host-parasite relationship of Toxoplasma gondii in the brains of chronically infected mice. Virchows Arch., A Pathol. Anat. Histopathol. 411, 39 43. Ferguson, D.J., Hutchison, W.M., 1987b. An ultrastructural study of the early development and tissue cyst

1065

formation of Toxoplasma gondii in the brains of mice. Parasitol. Res. 73, 483 491. Ferguson, D.J., Graham, D.I., Hutchison, W.M., 1991. Pathological changes in the brains of mice infected with Toxoplasma gondii: a histological, immunocytochemical and ultrastructural study. Int. J. Exp. Pathol. 72, 463 474. Fischer, H.G., Bielinsky, A.K., Nitzgen, B., Da¨ubener, W., Hadding, U., 1993. Functional dichotomy of mouse microglia developed in vitro: differential effects of macrophage and granulocyte/macrophage colonystimulating factor on cytokine secretion and antitoxoplasmic activity. J. Neuroimmunol 45, 193 201. Fischer, H.G., Nitzgen, B., Reichmann, G., Gross, U., Hadding, U., 1997. Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain. Parasitol. Res. 83, 637 641. Fleckenstein, M.C., Reese, M.L., Ko¨nen-Waisman, S., Boothroyd, J.C., Howard, J.C., Steinfeldt, T., 2012. A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins. PLoS Biol. 10, e1001358. Available from: https://doi.org/10.1371/journal.pbio.1001358. Flegr, J., Havlı´cek, J., Kodym, P., Maly´, M., Smahel, Z., 2002. Increased risk of traffic accidents in subjects with latent toxoplasmosis: a retrospective case-control study. BMC Infect. Dis. 2, 11. Frenkel, J.K., 1949. Pathogenesis, diagnosis and treatment of human toxoplasmosis. J. Am. Med. Assoc. 140, 369 377. Frickel, E.-M., Sahoo, N., Hopp, J., Gubbels, M.-J., Craver, M.P.J., Knoll, L.J., et al., 2008. Parasite stage-specific recognition of endogenous Toxoplasma gondii-derived CD8 1 T cell epitopes. J. Infect. Dis. 198, 1625 1633. Available from: https://doi.org/10.1086/593019. Fuks, J.M., Arrighi, R.B.G., Weidner, J.M., Kumar Mendu, S., Jin, Z., Wallin, R.P.A., et al., 2012. GABAergic signaling is linked to a hypermigratory phenotype in dendritic cells infected by Toxoplasma gondii. PLoS Pathog. 8, e1003051. Available from: https://doi.org/10.1371/ journal.ppat.1003051. Gale, S.D., Berrett, A.N., Brown, B., Erickson, L.D., Hedges, D.W., 2016. No association between current depression and latent toxoplasmosis in adults. Folia Parasitol. 63. Available from: https://doi.org/10.14411/fp.2016.032. Gaskell, E.A., Smith, J.E., Pinney, J.W., Westhead, D.R., McConkey, G.A., 2009. A unique dual activity amino acid hydroxylase in Toxoplasma gondii. PLoS One 4, e4801. Available from: https://doi.org/10.1371/journal. pone.0004801. Gazzinelli, R., Xu, Y., Hieny, S., Cheever, A., Sher, A., 1992. Simultaneous depletion of CD4 1 and CD8 1 T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii. J. Immunol. 149, 175 180.

Toxoplasma Gondii

1066

24. Cerebral toxoplasmosis

Gazzinelli, R.T., Wysocka, M., Hayashi, S., Denkers, E.Y., Hieny, S., Caspar, P., et al., 1994. Parasite-induced IL-12 stimulates early IFN-gamma synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153, 2533 2543. Geissmann, F., Jung, S., Littman, D.R., 2003. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71 82. Ghatak, N.R., Sawyer, D.R., 1978. A morphologic study of opportunistic cerebral toxoplasmosis. Acta Neuropathol. 42, 217 221. Ghatak, N.R., Zimmerman, H.M., 1973. Fine structure of Toxoplasma in the human brain. Arch Pathol. 95, 276 283. Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., et al., 2010. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841 845. Available from: https:// doi.org/10.1126/science.1194637. Ginhoux, F., Lim, S., Hoeffel, G., Low, D., Huber, T., 2013. Origin and differentiation of microglia. Front. Cell Neurosci. 7, 45. Available from: https://doi.org/ 10.3389/fncel.2013.00045. Glass, W.G., Lim, J.K., Cholera, R., Pletnev, A.G., Gao, J.-L., Murphy, P.M., 2005. Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection. J. Exp. Med. 202, 1087 1098. Available from: https://doi.org/10.1084/jem.20042530. Gonzalez, L.E., Rojnik, B., Urrea, F., Urdaneta, H., Petrosino, P., Colasante, C., et al., 2007. Toxoplasma gondii infection lower anxiety as measured in the plusmaze and social interaction tests in rats A behavioral analysis. Behav. Brain Res. 177, 70 79. Available from: https://doi.org/10.1016/j.bbr.2006.11.012. Graham, D.I., Hay, J., Hutchison, W.M., Siim, J.C., 1984. Encephalitis in mice with congenital ocular toxoplasmosis. J. Pathol. 142, 265 277. Available from: https://doi. org/10.1002/path.1711420405. Gregg, B., Taylor, B.C., John, B., Tait-Wojno, E.D., Girgis, N.M., Miller, N., et al., 2013. Replication and distribution of Toxoplasma gondii in the small intestine after oral infection with tissue cysts. Infect. Immun. 81, 1635 1643. Available from: https://doi.org/10.1128/ IAI.01126-12. Griffin, D.E., Metcalf, T., 2011. Clearance of virus infection from the CNS. Curr. Opin. Virol. 1, 216 221. Available from: https://doi.org/10.1016/j.coviro.2011.05.021. Halle, A., Hornung, V., Petzold, G.C., Stewart, C.R., Monks, B.G., Reinheckel, T., et al., 2008. The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat. Immunol. 9, 857 865. Available from: https://doi.org/10.1038/ni.1636. Halonen, S.K., Lyman, W.D., Chiu, F.C., 1996. Growth and development of Toxoplasma gondii in human neurons and astrocytes. J. Neuropathol. Exp. Neurol. 55, 1150 1156.

Halonen, S.K., Taylor, G.A., Weiss, L.M., 2001. Gamma interferon-induced inhibition of Toxoplasma gondii in astrocytes is mediated by IGTP. Infect. Immun. 69, 5573 5576. Hanamsagar, R., Torres, V., Kielian, T., 2011. Inflammasome activation and IL-1β/IL-18 processing are influenced by distinct pathways in microglia. J. Neurochem. 119, 736 748. Available from: https://doi. org/10.1111/j.1471-4159.2011.07481.x. Ha¨ndel, U., Brunn, A., Dro¨gemu¨ller, K., Mu¨ller, W., Deckert, M., Schlu¨ter, D., 2012. Neuronal gp130 expression is crucial to prevent neuronal loss, hyperinflammation, and lethal course of murine Toxoplasma encephalitis. Am. J. Pathol. 181, 163 173. Available from: https://doi.org/10.1016/j.ajpath.2012.03.029. Harker, K.S., Ueno, N., Wang, T., Bonhomme, C., Liu, W., Lodoen, M.B., 2013. Toxoplasma gondii modulates the dynamics of human monocyte adhesion to vascular endothelium under fluidic shear stress. J. Leukoc. Biol. 93, 789 800. Available from: https://doi.org/10.1189/ jlb.1012517. Harker, K.S., Jivan, E., McWhorter, F.Y., Liu, W.F., Lodoen, M.B., 2014. Shear forces enhance Toxoplasma gondii tachyzoite motility on vascular endothelium. MBio 5, e01111 e01113. Available from: https://doi.org/ 10.1128/mBio.01111-13. Harker, K.S., Ueno, N., Lodoen, M.B., 2015. Toxoplasma gondii dissemination: a parasite’s journey through the infected host. Parasite Immunol. 37, 141 149. Available from: https://doi.org/10.1111/pim.12163. Haroon, F., Ha¨ndel, U., Angenstein, F., Goldschmidt, J., Kreutzmann, P., Lison, H., et al., 2012. Toxoplasma gondii actively inhibits neuronal function in chronically infected mice. PLoS One 7, e35516. Available from: https://doi.org/10.1371/journal.pone.0035516. Harris, T.H., Banigan, E.J., Christian, D.A., Konradt, C., Tait Wojno, E.D., Norose, K., et al., 2012. Generalized Le´vy walks and the role of chemokines in migration of effector CD8 1 T cells. Nature 486, 545 548. Available from: https://doi.org/10.1038/nature11098. Herculano-Houzel, S., 2014. The glia/neuron ratio: how it varies uniformly across brain structures and species and what that means for brain physiology and evolution. Glia 62, 1377 1391. Available from: https://doi. org/10.1002/glia.22683. Hermes, G., Ajioka, J.W., Kelly, K.A., Mui, E., Roberts, F., Kasza, K., et al., 2008. Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection. J. Neuroinflammation 5, 48. Available from: https://doi. org/10.1186/1742-2094-5-48. Hidano, S., Randall, L.M., Dawson, L., Dietrich, H.K., Konradt, C., Klover, P.J., et al., 2016. STAT1 signaling in

Toxoplasma Gondii

References

astrocytes is essential for control of infection in the central nervous system. MBio 7. Available from: https:// doi.org/10.1128/mBio.01881-16. Hirano, A., 2005. The role of electron microscopy in neuropathology. Acta Neuropathol. 109, 115 123. Available from: https://doi.org/10.1007/s00401-004-0960-x. Hunter, C.A., Sibley, L.D., 2012. Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat. Rev. Microbiol. 10, 766 778. Available from: https:// doi.org/10.1038/nrmicro2858. Ihara, F., Nishimura, M., Muroi, Y., Mahmoud, M.E., Yokoyama, N., Nagamune, K., et al., 2016. Toxoplasma gondii infection in mice impairs long-term fear memory consolidation through dysfunction of the cortex and amygdala. Infect. Immun. 84, 2861 2870. Available from: https://doi.org/10.1128/IAI.00217-16. Ingram, W.M., Goodrich, L.M., Robey, E.A., Eisen, M.B., 2013. Mice infected with low-virulence strains of Toxoplasma gondii lose their innate aversion to cat urine, even after extensive parasite clearance. PLoS One 8, e75246. Available from: https://doi.org/10.1371/journal.pone.0075246. John, B., Harris, T.H., Tait, E.D., Wilson, E.H., Gregg, B., Ng, L.G., et al., 2009. Dynamic Imaging of CD8(1) T cells and dendritic cells during infection with Toxoplasma gondii. PLoS Pathog. 5, e1000505. Available from: https://doi.org/10.1371/journal.ppat.1000505. John, B., Ricart, B., Tait Wojno, E.D., Harris, T.H., Randall, L. M., Christian, D.A., et al., 2011. Analysis of behavior and trafficking of dendritic cells within the brain during toxoplasmic encephalitis. PLoS Pathog. 7, e1002246. Available from: https://doi.org/10.1371/journal.ppat.1002246. Johnson, L.L., Sayles, P.C., 2002. Deficient humoral responses underlie susceptibility to Toxoplasma gondii in CD4-deficient mice. Infect. Immun 70, 185 191. Jordan, K.A., Wilson, E.H., Tait, E.D., Fox, B.A., Roos, D.S., Bzik, D.J., et al., 2009. Kinetics and phenotype of vaccine-induced CD8 1 T-cell responses to Toxoplasma gondii. Infect. Immun. 77, 3894 3901. Available from: https://doi.org/10.1128/IAI.00024-09. Kanatani, S., Fuks, J.M., Olafsson, E.B., Westermark, L., Chambers, B., Varas-Godoy, M., et al., 2017. Voltagedependent calcium channel signaling mediates GABAA receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii. PLoS Pathog. 13, e1006739. Available from: https://doi.org/10.1371/ journal.ppat.1006739. Kang, H., Remington, J.S., Suzuki, Y., 2000. Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-gamma, TNF-alpha, and inducible nitric oxide synthase. J. Immunol. 164, 2629 2634. Kaushik, D.K., Gupta, M., Kumawat, K.L., Basu, A., 2012. NLRP3 inflammasome: key mediator of

1067

neuroinflammation in murine Japanese encephalitis. PLoS One 7, e32270. Available from: https://doi. org/10.1371/journal.pone.0032270. Khan, I.A., MacLean, J.A., Lee, F.S., Casciotti, L., DeHaan, E., Schwartzman, J.D., et al., 2000. IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 12, 483 494. Khan, I.A., Murphy, P.M., Casciotti, L., Schwartzman, J.D., Collins, J., Gao, J.L., et al., 2001. Mice lacking the chemokine receptor CCR1 show increased susceptibility to Toxoplasma gondii infection. J. Immunol. 166, 1930 1937. Khan, I.A., Thomas, S.Y., Moretto, M.M., Lee, F.S., Islam, S. A., Combe, C., et al., 2006. CCR5 is essential for NK cell trafficking and host survival following Toxoplasma gondii infection. PLoS Pathog. 2, e49. Available from: https://doi.org/10.1371/journal.ppat.0020049. Kierdorf, K., Erny, D., Goldmann, T., Sander, V., Schulz, C., Perdiguero, E.G., et al., 2013. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat. Neurosci. 16, 273 280. Available from: https://doi.org/10.1038/nn.3318. Konradt, C., Ueno, N., Christian, D.A., Delong, J.H., Pritchard, G.H., Herz, J., et al., 2016. Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system. Nat. Microbiol. 1, 16001. Available from: https://doi.org/10.1038/nmicrobiol.2016.1. Koshy, A.A., Dietrich, H.K., Christian, D.A., Melehani, J.H., Shastri, A.J., Hunter, C.A., et al., 2012. Toxoplasma coopts host cells it does not invade. PLoS Pathog. 8, e1002825. Available from: https://doi.org/10.1371/ journal.ppat.1002825. Ku, C.-C., Besser, J., Abendroth, A., Grose, C., Arvin, A.M., 2005. Varicella-Zoster virus pathogenesis and immunobiology: new concepts emerging from investigations with the SCIDhu mouse model. J. Virol. 79, 2651 2658. Available from: https://doi.org/10.1128/JVI.79.5.2651-2658.2005. Kusbeci, O.Y., Miman, O., Yaman, M., Aktepe, O.C., Yazar, S., 2011. Could Toxoplasma gondii have any role in Alzheimer disease? Alzheimer Dis. Assoc. Disord. 25, 1 3. Available from: https://doi.org/10.1097/ WAD.0b013e3181f73bc2. Lachenmaier, S.M., Deli, M.A., Meissner, M., Liesenfeld, O., 2011. Intracellular transport of Toxoplasma gondii through the blood-brain barrier. J. Neuroimmunol. 232, 119 130. Available from: https://doi.org/10.1016/j. jneuroim.2010.10.029. Lambert, H., Vutova, P.P., Adams, W.C., Lore´, K., Barragan, A., 2009. The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype. Infect. Immun. 77, 1679 1688. Available from: https://doi.org/10.1128/IAI.01289-08. Lang, W., Miklossy, J., Deruaz, J.P., Pizzolato, G.P., Probst, A., Schaffner, T., et al., 1989. Neuropathology of the acquired immune deficiency syndrome (AIDS): a report

Toxoplasma Gondii

1068

24. Cerebral toxoplasmosis

of 135 consecutive autopsy cases from Switzerland. Acta Neuropathol. 77, 379 390. Lee, H.-M., Kang, J., Lee, S.J., Jo, E.-K., 2013. Microglial activation of the NLRP3 inflammasome by the priming signals derived from macrophages infected with mycobacteria. Glia 61, 441 452. Available from: https://doi.org/10.1002/glia.22448. Ley, K., Laudanna, C., Cybulsky, M.I., Nourshargh, S., 2007. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678 689. Available from: https://doi.org/10.1038/ nri2156. Luangsay, S., Kasper, L.H., Rachinel, N., Minns, L.A., Mennechet, F.J.D., Vandewalle, A., et al., 2003. CCR5 mediates specific migration of Toxoplasma gondii-primed CD8 lymphocytes to inflammatory intestinal epithelial cells. Gastroenterology 125, 491 500. Lu¨der, C.G.K., Giraldo-Vela´squez, M., Sendtner, M., Gross, U., 1999. Toxoplasma gondii in primary Rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation. Exp. Parasitol. 93, 23 32. Available from: https://doi.org/10.1006/ expr.1999.4421. Luft, B.J., Remington, J.S., 1992. Toxoplasmic encephalitis in AIDS. Clin. Infect. Dis. 15, 211 222. Mahami-Oskouei, M., Hamidi, F., Talebi, M., Farhoudi, M., Taheraghdam, A.A., Kazemi, T., et al., 2016. Toxoplasmosis and Alzheimer: can Toxoplasma gondii really be introduced as a risk factor in etiology of Alzheimer? Parasitol. Res. 115, 3169 3174. Available from: https://doi.org/10.1007/s00436-016-5075-5. Mammari, N., Vignoles, P., Halabi, M.A., Darde, M.L., Courtioux, B., 2014. In vitro infection of human nervous cells by two strains of Toxoplasma gondii: a kinetic analysis of immune mediators and parasite multiplication. PLoS One 9, e98491. Available from: https://doi.org/ 10.1371/journal.pone.0098491. Martens, S., Parvanova, I., Zerrahn, J., Griffiths, G., Schell, G., Reichmann, G., et al., 2005. Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47resistance GTPases. PLoS Pathog. 1, e24. Available from: https://doi.org/10.1371/journal.ppat.0010024. Mayeux, R., Stern, Y., 2012. Epidemiology of Alzheimer disease. Cold Spring Harb. Perspect. Med. 2. Available from: https://doi.org/10.1101/cshperspect. a006239. McDole, J.R., Danzer, S.C., Pun, R.Y.K., Chen, Y., Johnson, H.L., Pirko, I., et al., 2010. Rapid formation of extended processes and engagement of Theiler’s virus-infected neurons by CNS-infiltrating CD8 T cells. Am. J. Pathol. 177, 1823 1833. Available from: https://doi.org/ 10.2353/ajpath.2010.100231.

McFarland, R., Wang, Z.T., Jouroukhin, Y., Li, Y., Mychko, O., Coppens, I., et al., 2018. AAH2 gene is not required for dopamine-dependent neurochemical and behavioral abnormalities produced by Toxoplasma infection in mouse. Behav. Brain Res. 347, 193 200. Available from: https://doi.org/10.1016/j.bbr.2018.03.023. McKercher, S.R., Torbett, B.E., Anderson, K.L., Henkel, G. W., Vestal, D.J., Baribault, H., et al., 1996. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15, 5647 5658. McLeod, R., Estes, R.G., Mack, D.G., Cohen, H., 1984. Immune response of mice to ingested Toxoplasma gondii: a model of toxoplasma infection acquired by ingestion. J. Infect. Dis. 149, 234 244. Melzer, T.C., Cranston, H.J., Weiss, L.M., Halonen, S.K., 2010. Host cell preference of Toxoplasma gondii cysts in murine brain: a confocal study. J. Neuroparasitol. 1. Available from: https://doi.org/10.4303/jnp/N100505. Mendez, O.A., Potter, C.J., Valdez, M., Bello, T., Trouard, T. P., Koshy, A.A., 2018. Semi-automated quantification and neuroanatomical mapping of heterogeneous cell populations. J. Neurosci. Methods 305, 98 104. Available from: https://doi.org/10.1016/j. jneumeth.2018.05.008. Miller, R.H., Raff, M.C., 1984. Fibrous and protoplasmic astrocytes are biochemically and developmentally distinct. J. Neurosci. 4, 585 592. Mu¨ller, M., Carter, S., Hofer, M.J., Campbell, I.L., 2010. Review: The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 in neuroimmunity–a tale of conflict and conundrum. Neuropathol. Appl. Neurobiol 36, 368 387. Available from: https://doi. org/10.1111/j.1365-2990.2010.01089.x. Mundt, S., Mrdjen, D., Utz, S.G., Greter, M., Schreiner, B., Becher, B., 2019. Conventional DCs sample and present myelin antigens in the healthy CNS and allow parenchymal T cell entry to initiate neuroinflammation. Sci. Immunol. 4. Available from: https://doi.org/10.1126/ sciimmunol.aau8380. Nance, J.P., Vannella, K.M., Worth, D., David, C., Carter, D., Noor, S., et al., 2012. Chitinase dependent control of protozoan cyst burden in the brain. PLoS Pathog. 8, e1002990. Available from: https://doi.org/10.1371/ journal.ppat.1002990. Niedelman, W., Gold, D.A., Rosowski, E.E., Sprokholt, J.K., Lim, D., Farid Arenas, A., et al., 2012. The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferongamma response. PLoS Pathog. 8, e1002784. Available from: https://doi.org/10.1371/journal.ppat.1002784. Niedelman, W., Sprokholt, J.K., Clough, B., Frickel, E.-M., Saeij, J.P.J., 2013. Cell death of gamma interferonstimulated human fibroblasts upon Toxoplasma gondii

Toxoplasma Gondii

References

infection induces early parasite egress and limits parasite replication. Infect. Immun. 81, 4341 4349. Available from: https://doi.org/10.1128/IAI.00416-13. Nimmerjahn, A., Kirchhoff, F., Helmchen, F., 2005. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314 1318. Available from: https://doi.org/10.1126/science.1110647. Oberdo¨rfer, C., Adams, O., MacKenzie, C.R., De Groot, C.J. A., Da¨ubener, W., 2003. Role of IDO activation in antimicrobial defense in human native astrocytes. Adv. Exp. Med. Biol. 527, 15 26. Ochiai, E., Sa, Q., Brogli, M., Kudo, T., Wang, X., Dubey, J.P., et al., 2015. CXCL9 is important for recruiting immune T cells into the brain and inducing an accumulation of the T cells to the areas of tachyzoite proliferation to prevent reactivation of chronic cerebral infection with Toxoplasma gondii. Am. J. Pathol. 185, 314 324. Available from: https://doi.org/10.1016/j.ajpath.2014.10.003. Oldenhove, G., Bouladoux, N., Wohlfert, E.A., Hall, J.A., Chou, D., Dos Santos, L., et al., 2009. Decrease of Foxp3 1 Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity 31, 772 786. Available from: https://doi.org/10.1016/j. immuni.2009.10.001. O’Brien, C.A., Overall, C., Konradt, C., O’Hara Hall, A.C., Hayes, N.W., Wagage, S., et al., 2017. CD11c-expressing cells affect regulatory T cell behavior in the meninges during central nervous system infection. J. Immunol. 198, 4054 4061. Available from: https://doi.org/ 10.4049/jimmunol.1601581. O’Brien, C.A., Batista, S.J., Still, K.M., Harris, T.H., 2019. IL10 and ICOS differentially regulate T cell responses in the brain during chronic Toxoplasma gondii infection. J. Immunol. 202, 1755 1766. Available from: https://doi. org/10.4049/jimmunol.1801229. Pappas, G., Roussos, N., Falagas, M.E., 2009. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int. J. Parasitol. 39, 1385 1394. Available from: https://doi.org/10.1016/j. ijpara.2009.04.003. Parlog, A., Schlu¨ter, D., Dunay, I.R., 2015. Toxoplasma gondii-induced neuronal alterations. Parasite Immunol. 37, 159 170. Available from: https://doi.org/10.1111/ pim.12157. Passeri, E., Jones-Brando, L., Bordo´n, C., Sengupta, S., Wilson, A.M., Primerano, A., et al., 2016. Infection and characterization of Toxoplasma gondii in human induced neurons from patients with brain disorders and healthy controls. Microbes Infect. 18, 153 158. Available from: https://doi.org/10.1016/j.micinf.2015.09.023. Pelloux, H., Pernod, G., Polack, B., Coursange, E., Ricard, J., Verna, J.M., et al., 1996. Influence of cytokines on

1069

Toxoplasma gondii growth in human astrocytomaderived cells. Parasitol. Res. 82, 598 603. Perry, C.E., Gale, S.D., Erickson, L., Wilson, E., Nielsen, B., Kauwe, J., et al., 2016. Seroprevalence and serointensity of latent Toxoplasma gondii in a sample of elderly adults with and without Alzheimer disease. Alzheimer Dis. Assoc. Disord. 30, 123 126. Available from: https:// doi.org/10.1097/WAD.0000000000000108. Peterson, P.K., Gekker, G., Hu, S., Chao, C.C., 1993. Intracellular survival and multiplication of Toxoplasma gondii in astrocytes. J. Infect. Dis. 168, 1472 1478. Peterson, P.K., Gekker, G., Hu, S., Chao, C.C., 1995. Human astrocytes inhibit intracellular multiplication of Toxoplasma gondii by a nitric oxide-mediated mechanism. J. Infect. Dis. 171, 516 518. Ploix, C.C., Noor, S., Crane, J., Masek, K., Carter, W., Lo, D. D., et al., 2011. CNS-derived CCL21 is both sufficient to drive homeostatic CD4 1 T cell proliferation and necessary for efficient CD4 1 T cell migration into the CNS parenchyma following Toxoplasma gondii infection. Brain Behav. Immun. 25, 883 896. Available from: https://doi.org/10.1016/j.bbi.2010.09.014. Pober, J.S., Sessa, W.C., 2007. Evolving functions of endothelial cells in inflammation. Nat. Rev. Immunol. 7, 803 815. Available from: https://doi.org/10.1038/nri2171. Porter, S.B., Sande, M.A., 1992. Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N. Engl. J. Med. 327, 1643 1648. Available from: https://doi.org/10.1056/NEJM199212033272306. Post, M.J., Chan, J.C., Hensley, G.T., Hoffman, T.A., Moskowitz, L.B., Lippmann, S., 1983. Toxoplasma encephalitis in Haitian adults with acquired immunodeficiency syndrome: a clinical-pathologic-CT correlation. AJR Am. J. Roentgenol. 140, 861 868. Available from: https://doi.org/10.2214/ajr.140.5.861. Potente, M., Ma¨kinen, T., 2017. Vascular heterogeneity and specialization in development and disease. Nat. Rev. Mol. Cell Biol. 18, 477 494. Available from: https:// doi.org/10.1038/nrm.2017.36. Qin, A., Lai, D.-H., Liu, Q., Huang, W., Wu, Y.-P., Chen, X., et al., 2017. Guanylate-binding protein 1 (GBP1) contributes to the immunity of human mesenchymal stromal cells against Toxoplasma gondii. Proc. Natl. Acad. Sci. U. S.A. 114, 1365 1370. Available from: https://doi.org/ 10.1073/pnas.1619665114. Reikvam, A., Lorentzen-Styr, A.M., 1976. Virulence of different strains of Toxoplasma gondii and host response in mice. Nature 261, 508 509. Remington, J.S., Cavanaugh, E.N., 1965. Isolation of the encysted form of Toxoplasma gondii from human skeletal muscle and brain. N. Engl. J. Med. 273, 1308 1310. Available from: https://doi.org/10.1056/ NEJM196512092732404.

Toxoplasma Gondii

1070

24. Cerebral toxoplasmosis

Rezaie, P., Male, D., 2002. Mesoglia & microglia—a historical review of the concept of mononuclear phagocytes within the central nervous system. J. Hist. Neurosci. 11, 325 374. Available from: https://doi.org/10.1076/ jhin.11.4.325.8531. Robben, P.M., LaRegina, M., Kuziel, W.A., Sibley, L.D., 2005. Recruitment of Gr-1 1 monocytes is essential for control of acute toxoplasmosis. J. Exp. Med. 201, 1761 1769. Available from: https://doi.org/10.1084/ jem.20050054. Rose, R.W., Vorobyeva, A.G., Skipworth, J.D., Nicolas, E., Rall, G.F., 2007. Altered levels of STAT1 and STAT3 influence the neuronal response to interferon gamma. J. Neuroimmunol. 192, 145 156. Available from: https:// doi.org/10.1016/j.jneuroim.2007.10.007. Rozenfeld, C., Martinez, R., Figueiredo, R.T., Bozza, M.T., Lima, F.R.S., Pires, A.L., et al., 2003. Soluble factors released by Toxoplasma gondii-infected astrocytes downmodulate nitric oxide production by gamma interferonactivated microglia and prevent neuronal degeneration. Infect. Immun. 71, 2047 2057. Sa, Q., Ochiai, E., Sengoku, T., Wilson, M.E., Brogli, M., Crutcher, S., et al., 2014. VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells into the brain during the early stage of reactivation of chronic infection with Toxoplasma gondii to prevent toxoplasmic encephalitis. Infect. Immun. 82, 2826 2839. Available from: https://doi.org/10.1128/IAI.01494-13. Saederup, N., Cardona, A.E., Croft, K., Mizutani, M., Cotleur, A.C., Tsou, C.-L., et al., 2010. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS One 5, e13693. Available from: https://doi.org/ 10.1371/journal.pone.0013693. Salvioni, A., Belloy, M., Lebourg, A., Bassot, E., CantaloubeFerrieu, V., Vasseur, V., et al., 2019. Robust Control of a Brain-Persisting Parasite through MHC I Presentation by Infected Neurons. Cell Rep 27, 3254 3268.e8. Available from: https://doi.org/10.1016/j.celrep.2019.05.051. Schaeffer, M., Han, S.-J., Chtanova, T., Dooren, G.G., van, Herzmark, P., Chen, Y., et al., 2009. Dynamic imaging of T cell-parasite interactions in the brains of mice chronically infected with Toxoplasma gondii. J. Immunol. 182, 6379 6393. Available from: https://doi.org/ 10.4049/jimmunol.0804307. Schaller, M.A., Kallal, L.E., Lukacs, N.W., 2008. A key role for CC chemokine receptor 1 in T-cell-mediated respiratory inflammation. Am. J. Pathol. 172, 386 394. Available from: https://doi.org/10.2353/ajpath.2008.070537. Scharton-Kersten, T.M., Yap, G., Magram, J., Sher, A., 1997. Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplasma gondii. J. Exp. Med. 185, 1261 1273.

Schlu¨ter, D., Kaefer, N., Hof, H., Wiestler, O.D., DeckertSchlu¨ter, M., 1997. Expression pattern and cellular origin of cytokines in the normal and Toxoplasma gondii-infected murine brain. Am. J. Pathol. 150, 1021 1035. Schlu¨ter, D., Deckert-Schlu¨ter, M., Lorenz, E., Meyer, T., Ro¨llinghoff, M., Bogdan, C., 1999. Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does not reactivate the latent disease in T. gondii-resistant BALB/c mice. J. Immunol. 162, 3512 3518. Schlu¨ter, D., Deckert, M., Hof, H., Frei, K., 2001a. Toxoplasma gondii infection of neurons induces neuronal cytokine and chemokine production, but gamma interferon- and tumor necrosis factor-stimulated neurons fail to inhibit the invasion and growth of T. gondii. Infect. Immun. 69, 7889 7893. Available from: https://doi. org/10.1128/IAI.69.12.7889-7893.2001. Schlu¨ter, D., Meyer, T., Strack, A., Reiter, S., Kretschmar, M., Wiestler, O.D., et al., 2001b. Regulation of microglia by CD4 1 and CD8 1 T cells: selective analysis in CD45-congenic normal and Toxoplasma gondii-infected bone marrow chimeras. Brain Pathol 11, 44 55. Schneider, C.A., Figueroa Velez, D.X., Azevedo, R., Hoover, E.M., Tran, C.J., Lo, C., et al., 2019. Imaging the dynamic recruitment of monocytes to the blood-brain barrier and specific brain regions during Toxoplasma gondii infection. Proc. Natl. Acad. Sci. U.S.A . Available from: https://doi.org/10.1073/pnas.1915778116. Serlin, Y., Shelef, I., Knyazer, B., Friedman, A., 2015. Anatomy and physiology of the blood-brain barrier. Semin. Cell Dev. Biol. 38, 2 6. Available from: https:// doi.org/10.1016/j.semcdb.2015.01.002. Shi, C., Pamer, E.G., 2011. Monocyte recruitment during infection and inflammation. Nat. Rev. Immunol. 11, 762 774. Available from: https://doi.org/10.1038/ nri3070. Shi, F., Yang, L., Kouadir, M., Yang, Y., Wang, J., Zhou, X., et al., 2012. The NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation. J. Neuroinflammation 9, 73. Available from: https:// doi.org/10.1186/1742-2094-9-73. Sibley, L.D., Boothroyd, J.C., 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 359, 82 85. Available from: https://doi.org/ 10.1038/359082a0. Silva, N.M., Manzan, R.M., Carneiro, W.P., Milanezi, C.M., Silva, J.S., Ferro, E.A.V., et al., 2010. Toxoplasma gondii: the severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM1 expression in the central nervous system and higher blood-brain barrier permeability. Exp. Parasitol. 126, 167 177. Available from: https://doi.org/10.1016/j. exppara.2010.04.019.

Toxoplasma Gondii

References

Silveira, C., Vallochi, A.L., Rodrigues da Silva, U., Muccioli, C., Holland, G.N., Nussenblatt, R.B., et al., 2011. Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis. Br. J. Ophthalmol. 95, 396 400. Available from: https://doi. org/10.1136/bjo.2008.148205. Sims, T.A., Hay, J., Talbot, I.C., 1988. Host-parasite relationship in the brains of mice with congenital toxoplasmosis. J. Pathol. 156, 255 261. Available from: https://doi. org/10.1002/path.1711560311. Sims, T.A., Hay, J., Talbot, I.C., 1989. An electron microscope and immunohistochemical study of the intracellular location of Toxoplasma tissue cysts within the brains of mice with congenital toxoplasmosis. Br. J. Exp. Pathol. 70, 317 325. Sofroniew, M.V., Vinters, H.V., 2010. Astrocytes: biology and pathology. Acta Neuropathol. 119, 7 35. Available from: https://doi.org/10.1007/s00401-009-0619-8. Song, H.B., Jung, B.-K., Kim, J.H., Lee, Y.-H., Choi, M.-H., Kim, J.H., 2018. Investigation of tissue cysts in the retina in a mouse model of ocular toxoplasmosis: distribution and interaction with glial cells. Parasitol. Res. 117, 2597 2605. Available from: https://doi.org/10.1007/ s00436-018-5950-3. Steinfeldt, T., Ko¨nen-Waisman, S., Tong, L., Pawlowski, N., Lamkemeyer, T., Sibley, L.D., et al., 2010. Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol. 8, e1000576. Available from: https://doi.org/10.1371/journal.pbio.1000576. Stenzel, W., Soltek, S., Schlu¨ter, D., Deckert, M., 2004. The intermediate filament GFAP is important for the control of experimental murine Staphylococcus aureus-induced brain abscess and Toxoplasma encephalitis. J. Neuropathol. Exp. Neurol. 63, 631 640. Stibbs, H.H., 1985. Changes in brain concentrations of catecholamines and indoleamines in Toxoplasma gondii infected mice. Ann. Trop. Med. Parasitol. 79, 153 157. Strack, A., Asensio, V., Campbell, I., Schlu¨ter, D., Deckert, M., 2002a. Chemokines are differentially expressed by astrocytes, microglia and inflammatory leukocytes in Toxoplasma encephalitis and critically regulated by interferon-γ. Acta Neuropathol. 103, 458 468. Available from: https://doi.org/10.1007/s00401-001-0491-7. Strack, A., Schlu¨ter, D., Asensio, V.C., Campbell, I.L., Deckert, M., 2002b. Regulation of the kinetics of intracerebral chemokine gene expression in murine Toxoplasma encephalitis: Impact of host genetic factors. Glia 40, 372 377. Available from: https://doi.org/10.1002/glia.10104. Strittmatter, C., Lang, W., Wiestler, O.D., Kleihues, P., 1992. The changing pattern of human immunodeficiency virus-associated cerebral toxoplasmosis: a study of 46 postmortem cases. Acta Neuropathol. 83, 475 481.

1071

Stumhofer, J.S., Tait, E.D., Quinn, W.J., Hosken, N., Spudy, B., Goenka, R., et al., 2010. A role for IL-27p28 as an antagonist of gp130-mediated signaling. Nat. Immunol. 11, 1119 1126. Available from: https://doi. org/10.1038/ni.1957. Stumhofer, J.S., Silver, J.S., Hunter, C.A., 2013. IL-21 is required for optimal antibody production and T cell responses during chronic Toxoplasma gondii infection. PLoS One 8, e62889. Available from: https://doi.org/ 10.1371/journal.pone.0062889. Sutterland, A.L., Fond, G., Kuin, A., Koeter, M.W.J., Lutter, R., van Gool, T., et al., 2015. Beyond the association. Toxoplasma gondii in schizophrenia, bipolar disorder, and addiction: systematic review and meta-analysis. Acta Psychiatr. Scand. 132, 161 179. Available from: https://doi.org/10.1111/acps.12423. Suzuki, Y., Joh, K., 1994. Effect of the strain of Toxoplasma gondii on the development of toxoplasmic encephalitis in mice treated with antibody to interferon-gamma. Parasitol. Res. 80, 125 130. Suzuki, Y., Conley, F.K., Remington, J.S., 1989a. Differences in virulence and development of encephalitis during chronic infection vary with the strain of Toxoplasma gondii. J. Infect. Dis. 159, 790 794. Suzuki, Y., Conley, F.K., Remington, J.S., 1989b. Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice. J. Immunol. 143, 2045 2050. Suzuki, Y., Wang, X., Jortner, B.S., Payne, L., Ni, Y., Michie, S.A., et al., 2010. Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8 1 T cells. Am. J. Pathol. 176, 1607 1613. Available from: https://doi.org/10.2353/ajpath.2010.090825. Tanaka, N., Ashour, D., Dratz, E., Halonen, S., 2016. Use of human induced pluripotent stem cell-derived neurons as a model for cerebral toxoplasmosis. Microbes Infect. 18, 496 504. Available from: https://doi.org/10.1016/j. micinf.2016.03.012. Tedford, E., McConkey, G., 2017. Neurophysiological changes induced by chronic Toxoplasma gondii infection. Pathogens 6. Available from: https://doi.org/10.3390/ pathogens6020019. Tognetti, F., Galassi, E., Gaist, G., 1982. Neurological toxoplasmosis presenting as a brain tumor. Case report. J. Neurosurg. 56, 716 721. Available from: https://doi. org/10.3171/jns.1982.56.5.0716. Torniainen-Holm, M., Suvisaari, J., Lindgren, M., Ha¨rka¨nen, T., Dickerson, F., Yolken, R.H., 2019. The lack of association between herpes simplex virus 1 or Toxoplasma gondii infection and cognitive decline in the general population: An 11-year follow-up study. Brain Behav. Immun. 76, 159 164. Available from: https:// doi.org/10.1016/j.bbi.2018.11.016.

Toxoplasma Gondii

1072

24. Cerebral toxoplasmosis

Tuladhar, S., Kochanowsky, J.A., Bhaskara, A., Ghotmi, Y., Chandrasekaran, S., Koshy, A.A., 2019. The ROP16IIIdependent early immune response determines the subacute CNS immune response and type III Toxoplasma gondii survival. PLoS Pathog. 15, Available from: https://doi.org/10.1371/journal.ppat.1007856. Ueno, N., Harker, K.S., Clarke, E.V., McWhorter, F.Y., Liu, W.F., Tenner, A.J., et al., 2014. Real-time imaging of Toxoplasma-infected human monocytes under fluidic shear stress reveals rapid translocation of intracellular parasites across endothelial barriers. Cell. Microbiol. 16, 580 595. Available from: https://doi.org/10.1111/ cmi.12239. Umeda, K., Tanaka, S., Ihara, F., Yamagishi, J., Suzuki, Y., Nishikawa, Y., 2017. Transcriptional profiling of Tolllike receptor 2-deficient primary murine brain cells during Toxoplasma gondii infection. PLoS One 12, e0187703. Available from: https://doi.org/10.1371/journal. pone.0187703. van Horssen, J., Singh, S., van der Pol, S., Kipp, M., Lim, J. L., Peferoen, L., et al., 2012. Clusters of activated microglia in normal-appearing white matter show signs of innate immune activation. J. Neuroinflammation 9, 156. Available from: https://doi.org/10.1186/1742-2094-9156. van Os, J., Kapur, S., 2009. Schizophrenia. Lancet 374, 635 645. Available from: https://doi.org/10.1016/ S0140-6736(09)60995-8. Vyas, A., Kim, S.-K., Giacomini, N., Boothroyd, J.C., Sapolsky, R.M., 2007. Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors. Proc. Natl. Acad. Sci. U.S.A. 104, 6442 6447. Available from: https://doi.org/10.1073/ pnas.0608310104. Wang, X., Michie, S.A., Xu, B., Suzuki, Y., 2007. Importance of IFN-gamma-mediated expression of endothelial VCAM-1 on recruitment of CD8 1 T cells into the brain during chronic infection with Toxoplasma gondii. J. Interferon Cytokine Res. 27, 329 338. Available from: https://doi.org/10.1089/jir.2006.0154. Wang, Y., Szretter, K.J., Vermi, W., Gilfillan, S., Rossini, C., Cella, M., et al., 2012. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. Nat. Immunol. 13, 753 760. Available from: https://doi.org/10.1038/ni.2360. Wang, Z.T., Harmon, S., O’Malley, K.L., Sibley, L.D., 2015. Reassessment of the role of aromatic amino acid hydroxylases and the effect of infection by Toxoplasma gondii on host dopamine. Infect. Immun. 83, 1039 1047. Available from: https://doi.org/10.1128/IAI.02465-14. Wegiel, J., Wi´sniewski, H.M., Dziewiatkowski, J., Tarnawski, M., Kozielski, R., Trenkner, E., et al., 1998. Reduced number and altered morphology of microglial

cells in colony stimulating factor-1-deficient osteopetrotic op/op mice. Brain Res. 804, 135 139. Weidner, J.M., Kanatani, S., Herna´ndez-Castan˜eda, M.A., Fuks, J.M., Rethi, B., Wallin, R.P.A., et al., 2013. Rapid cytoskeleton remodelling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype. Cell. Microbiol 15, 1735 1752. Available from: https://doi.org/10.1111/cmi.12145. Weidner, J.M., Kanatani, S., Uchtenhagen, H., VarasGodoy, M., Schulte, T., Engelberg, K., et al., 2016. Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein. Cell. Microbiol. 18, 1537 1550. Available from: https://doi. org/10.1111/cmi.12595. Wilson, D.C., Grotenbreg, G.M., Liu, K., Zhao, Y., Frickel, E.-M., Gubbels, M.-J., et al., 2010. Differential regulation of effector- and central-memory responses to Toxoplasma gondii Infection by IL-12 revealed by tracking of Tgd057-specific CD81 T cells. PLoS Pathog. 6, e1000815. Available from: https://doi.org/10.1371/ journal.ppat.1000815. Wilson, E.H., Wille-Reece, U., Dzierszinski, F., Hunter, C. A., 2005. A critical role for IL-10 in limiting inflammation during toxoplasmic encephalitis. J. Neuroimmunol. 165, 63 74. Available from: https://doi.org/10.1016/j. jneuroim.2005.04.018. Wilson, E.H., Harris, T.H., Mrass, P., John, B., Tait, E.D., Wu, G.F., et al., 2009. Behavior of parasite-specific effector CD8 1 T cells in the brain and visualization of a kinesis-associated system of reticular fibers. Immunity 30, 300 311. Available from: https://doi.org/10.1016/j. immuni.2008.12.013. Worth, A.R., Andrew Thompson, R.C., Lymbery, A.J., 2014. Reevaluating the evidence for Toxoplasma gondiiinduced behavioural changes in rodents. Adv. Parasitol 85, 109 142. Available from: https://doi.org/10.1016/ B978-0-12-800182-0.00003-9. Yap, G., Pesin, M., Sher, A., 2000. Cutting edge: IL-12 is required for the maintenance of IFN-gamma production in T cells mediating chronic resistance to the intracellular pathogen, Toxoplasma gondii. J. Immunol. 165, 628 631. Yona, S., Kim, K.-W., Wolf, Y., Mildner, A., Varol, D., Breker, M., et al., 2013. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79 91. Available from: https://doi.org/10.1016/j.immuni.2012.12.001. Zamanian, J.L., Xu, L., Foo, L.C., Nouri, N., Zhou, L., Giffard, R.G., et al., 2012. Genomic analysis of reactive astrogliosis. J. Neurosci. 32, 6391 6410. Available from: https://doi.org/10.1523/JNEUROSCI.6221-11.2012. Zamora, D.O., Rosenbaum, J.T., Smith, J.R., 2008. Invasion of human retinal vascular endothelial cells

Toxoplasma Gondii

References

by Toxoplasma gondii tachyzoites. Br. J. Ophthalmol. 92, 852 855. Available from: https://doi.org/ 10.1136/bjo.2007.133314. Zerboni, L., Ku, C.-C., Jones, C.D., Zehnder, J.L., Arvin, A. M., 2005. Varicella-zoster virus infection of human dorsal root ganglia in vivo. Proc. Natl. Acad. Sci. U.S.A. 102,

1073

6490 6495. Available from: https://doi.org/10.1073/ pnas.0501045102. Zhao, Z., Nelson, A.R., Betsholtz, C., Zlokovic, B.V., 2015. Establishment and dysfunction of the blood-brain barrier. Cell 163, 1064 1078. Available from: https://doi. org/10.1016/j.cell.2015.10.067.

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C H A P T E R

25 Innate immunity to Toxoplasma gondii Dana G. Mordue1 and Christopher A. Hunter2 1

Department of Microbiology and Immunology, New York Medical College, Valhalla, NY, United States Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States

2

25.1 Introduction The high seroprevalence rates for Toxoplasma gondii in humans in the absence of overt clinical disease indicate that most infections are asymptomatic and resolve with minimal pathology. Nevertheless, the literature on patients with acquired defects in T-cellmediated immunity teaches that while the immune system is critical for parasite control, it fails to achieve sterile immunity and a stable persistent infection results. However, should these individuals develop acquired immune deficiencies associated with reduced T-cell function they are at risk for reactivation. This situation has led to an emphasis on the importance of adaptive immunity, but it belies a literature that describes life-threatening disease associated with acute infection in immune competent individuals (Darde et al., 1998). These observations indicate an important role for innate immunity in resistance to T. gondii and because of the public health consequences of this infection there has been a long-standing interest in understanding the immunological

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00025-6

basis for the ability to control acute toxoplasmosis. Implicit in this topic is the idea that there are innate mechanisms that allow host recognition and control of this organism and which shape the development of parasitespecific T- and B-cell responses. In contrast, the requirement for adaptive immunity to control this organism and the ability of T. gondii to persist indicates the evolution of pathogen strategies to evade innate antimicrobial activities. Because the mouse is a natural host for T gondii it provides a tractable model system to dissect host pathogen interactions that have coevolved to provide innate resistance to an intracellular organism as well as to test the importance of the parasite mechanisms that allow it to persist. Studies on T. gondii over the last 50 years have identified and assigned functions to key players important for innate immunity and dissected mechanisms for antigen presentation and activation of T-cell populations. For example, there is now an appreciation of the host sensors that support dendritic cell (DC) production of the cytokine IL-12 that promotes NK and T cells to produce

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IFN-γ, which is required to activate multiple cell autonomous mechanisms that limit parasite replication. In addition, advances in parasite genetics have provided unprecedented insights into how proteins derived from T. gondii impact on host cell function and influence virulence. This chapter reviews our current understanding of the innate events that underlie the host response to T. gondii and highlights prominent questions in the field about how T. gondii is recognized and restricted and the evasion mechanisms that contribute to parasite success.

25.2 The intimate relationship between Toxoplasma gondii and its host cells As an obligate intracellular parasite, T. gondii pathogenesis and the host immune response to it are impacted by the unique relationship the parasite has with its host cells. Unlike many obligate intracellular pathogens, T. gondii can infect and survive within virtually any type of host cell. T. gondii invades cells through an active invasion process that is dependent on the actin myosin motor in the parasite (Morisaki et al., 1995; Dobrowolski and Sibley, 1996). A nascent parasitophorous vacuole (PV) is formed during invasion by invagination of the host cell plasma membrane (Suss-Toby et al., 1996). The process is fundamentally distinct from phagocytosis and results in minimal host cytoskeleton rearrangement and tyrosine phosphorylation (Mordue and Sibley, 1997; Morisaki et al., 1995). Most host cell membrane proteins are excluded from the nascent PV through a process dependent on physical constraints and lipid portioning (Mordue et al., 1999a; Alexander et al., 2005). This novel mechanism of parasite invasion and PV formation underlies the relatively segregated nonfusogenic nature of the PV from host endocytic and phagocytic processes (Mordue and Sibley, 1997; Mordue et al., 1999b; Joiner et al., 1990).

The PV membrane (PVM) is selectively permeable, allowing 1300 1900 Da molecules to pass through it utilizing a mechanism involving the parasite proteins GRA23 and GRA17 (Gold et al., 2015). The parasite also sequesters host Rab7-associated lipid droplets into the PV to scavenge neutral lipids through a mechanism dependent on the parasite intravacuolar network between the parasite and PVM (Coppens and Romano, 2018). Similarly, T. gondii relies on host-derived sphingolipids it scavenges from Golgi-derived vesicles (Romano et al., 2013). When T. gondii contacts a cell and the invasion process is initiated, there is a carefully choreographed series of events that allow attachment, invasion, and establishment of the PV. Attachment and invasion are coincident with the secretion of microneme proteins, followed by a wave of rhoptry proteins. Once the PV is established there is a second wave of secretion of parasite-derived effectors and multiple proteins derived from the parasite dense granules. One of the earliest events during invasion involves the secretion of microneme and rhoptry proteins that remodel the PV and alter host signaling and antimicrobial effector pathways (Carruthers and Sibley, 1997) (reviewed in Hakimi et al., 2017; Hunter and Sibley, 2012). Parasite dense granules are transported across the PVM via a novel complex that includes parasite MYR1, MYR2, and MYR3 (Franco et al., 2016; Naor et al., 2018). Secreted effectors not only modify the PV and PVM but also alter host cell transcription and signaling to evade host immunity and allow parasite growth (reviewed in Hakimi et al., 2017; Hunter and Sibley, 2012). Global transcriptome sequencing (RNA-seq) of human fibroblast (HF) cells infected with WT or MYR1 KO parasites indicates that a substantial portion of the transcriptional changes in infected cells are dependent on parasite MYR1 (Naor et al., 2018; Franco et al., 2016). Export appears to be dependent on aspartyl protease 5

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25.3 Establishment of infection and mucosal immunity

cleavage of a Toxoplasma export element (TEXEL motif) in proteins that are to be targeted to the MYR1 translocon for secretion (Naor et al., 2018; Marino et al., 2018; Coffey et al., 2015, 2018). The majority of known MYR1-dependent parasite effectors to date are dense granule proteins (GRA), which can be subdivided into those that act close to their site of secretion while the others traffic to the host cell nucleus, discussed in detail later (reviewed in Hakimi et al., 2017; Hunter and Sibley, 2012). More recently T. gondii has also been shown to subvert expression of host cell long noncoding RNAs as well, but whether this is MYR1dependent has not been addressed (Menard et al., 2018). One other illustration of the intimate relationship of T. gondii with its host cells involves the host cell mitochondria. TgMAF1 in certain parasite strains mediates recruitment of host mitochondria to the PVM (Pernas et al., 2014). Deletion of MAF1 in T. gondii results in alterations in the expression of immune genes and in vivo cytokine responses during murine infection. T. gondii also coopts host cell lipophagy to obtain fatty acids important for its replication (Pernas et al., 2018; Nolan et al., 2017). Host mitochondria that fuse around the T. gondii PV appear to siphon fatty acids from the parasite that may play a role in restricting replication (Pernas et al., 2018).

25.3 Establishment of infection and mucosal immunity Although T. gondii can be transmitted congenitally or via organ transplantation, it is most often acquired by ingestion of either tissue cysts through predation or oocysts shed by feline species. Parasites invade enterocytes in the small intestine and can penetrate this polarized epithelial barrier by active invasion without causing extensive damage to the epithelium (reviewed in Buzoni-Gatel et al., 2006; Schulthess et al., 2008). Thereafter, the

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tachyzoite stages access the vascular system and disseminate through the body as free stages in the blood as well as in parasitized cells (Courret et al., 2006; Konradt et al., 2016; Coombes et al., 2013). Because the gut is typically the primary site where the immune system first encounters this organism, the events that occur here have a prominent role in determining the outcome of this infection. Thus after mucosal challenge enterocytes, macrophages and DC and a variety of intraepithelial lymphocytes all contribute to the local response to the parasite (reviewed in BuzoniGatel et al., 2006; Cohen and Denkers, 2015a). Because these primary interactions are rare and difficult to detect there is little known about how epithelial cells in the small intestine respond to initial invasion. One approach to this problem was to challenge isolated rat intestinal epithelial cells with sporozoites of T. gondii to profile the effect of the parasite on the host transcriptome. In these experiments the host response was dominated by expression of genes associated with tumor necrosis factor alpha (TNF-α) and signaling via NFκB (Guiton et al., 2017), pathways associated with recruitment of inflammatory cells and with resistance to T. gondii (discussed later). One way to address the nature of rare host pathogen interactions has been to use intravital microscopy in mice to visualize parasites and host cells that express fluorescent proteins. These approaches have shown that within 2 3 days of oral challenge with T. gondii parasite plaques in individual villi are apparent and free parasites are observed in the lumen that likely contribute to spread of these organisms to neighboring villi (Gregg et al., 2013). These foci are accompanied by a prominent inflammatory response, and parasites are found within macrophages, monocytes, and neutrophils. These foci could be an important reservoir for parasite dissemination and the ability to infect motile DC, monocyte, and neutrophil subsets provides opportunities for the organism to disseminate

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(Courret et al., 2006). Indeed, infected neutrophils have been visualized to migrate across the intestinal epithelium and spread the infection via the intestinal lumen to the tips of villi (Coombes et al., 2013). These processes can quickly become pathological if parasite replication is not controlled. However, inflammation associated with protective immunity can also result in collateral damage to the intestine. The following sections highlight many of the innate events and cell types that contribute to parasite control and how they can lead to immunopathology. Following oral challenge with T. gondii, the recruitment of Gr-11 (Ly6C1) inflammatory monocytes from the blood into local sites of parasite replication in the gut are important for parasite control. These cells differentiate into macrophage populations that express inducible nitric oxide synthase (iNOS) and produce a range of inflammatory cytokines (Mordue and Sibley, 2003; Dunay et al., 2008; Biswas et al., 2015). Nitric oxide has a dichotomas role in T. gondii infection in that it contributes to parasite control (Scharton-Kersten et al., 1997) but can be toxic and lead to tissue necrosis (Khan et al., 1997). In CCr2 KO mice, these monocytes do not exit the bone marrow (Serbina and Pamer, 2006) and their failure to traffic to the intestine results in an inability to control T. gondii (Dunay et al., 2008). The events that lead to monocyte recruitment and activation are complex and the production of the cytokine IL-18 has been linked to the ability of innate lymphoid cells (NKp461 ILCs) resident in the intestinal mucosa to produce the chemokine CCL3 that recruits CCR11ve inflammatory monocytes (Schulthess et al., 2012). While the focus of this chapter is on the innate immune response, CD41 T-cell production of IFN-γ in the lamina propria also contributes to the recruitment of inflammatory monocytes and their expression of antimicrobial effectors (Cohen et al., 2013). However, oral infection with T. gondii can trigger severe CD4-dependent intestinal immunopathology

in certain strains of immune competent mice (Suzuki et al., 2000; Liesenfeld et al., 1999; Schreiner and Liesenfeld, 2009). Damage to the intestinal barrier and microbial dysbiosis is accompanied by bacterial translocation from the lumen that is a key factor in intestinal damage and necrosis (Heimesaat et al., 2006; Liesenfeld et al., 1996). A prominent feature associated with this immune pathology is the loss of Paneth cells, secretory epithelial cells that reside at the base of intestinal crypts that are an important source of antimicrobial peptides. The production of IFN-γ contributes to the loss of these specialized cells (Burger et al., 2018), which results in reduced production of antimicrobial peptides that allows the emergence of a severe bacterial dysbiosis. The escape of commensal gut bacteria from the lumen can stimulate DC production of IL-12 that enhances protection against T. gondii (Benson et al., 2009), but these events also promote the development of long-lived microbiota-specific T cells (Molloy et al., 2013).

25.4 The role of IL-12-dependent IFN-γ production for innate resistance The early events that lead to the control of T. gondii infection are dominated by innate production of IL-12 that promotes the ability of NK and T cells to make IFN-γ (Denkers et al., 1993; Hunter et al., 1994; Scharton-Kersten et al., 1996, 1998; Gazzinelli et al., 1993) (Fig. 25.1). A series of studies that started in the 1980s revealed that the production of IFN-γ by NK or T cells is essential for control of acute and chronic T. gondii infection and mice that lack IFN-γ or IFN-γ-mediated signaling succumb rapidly to toxoplasmosis due to an inability to control parasite replication (Suzuki et al., 1988; Scharton-Kersten et al., 1996; Yap and Sher, 1999). The protective effects of IFN-γ correlate with its ability to activate human and mouse macrophages in vitro to limit parasite

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25.4 The role of IL-12-dependent IFN-γ production for innate resistance

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FIGURE

25.1 Toxoplasma gondii IFN-γ response. IL-12 is produced from diverse immune cells in response to T. gondii infection and drives production of IFN-γ that is important for cell autonomous immune mechanisms in both hematopoietic and nonhematopoietic cells, cell recruitment and antigen presentation.

replication. Genetic support for this model was provided by studies in which expression of a dominant negative IFN-γ receptor on CD681 macrophages results in uncontrolled parasite replication during acute infection even in the presence of IFN-γ (Dunay et al., 2008, 2010; Robben et al., 2005; Mordue and Sibley, 2003) (Fig. 25.1). With the discovery of IL-12 in 1989 (Kobayashi et al., 1989) it was recognized that this cytokine played a key role in driving NKcell production of IFN-γ during toxoplasmosis (Gazzinelli et al., 1993; Khan et al., 1994; Hunter et al., 1994). It should be noted that IL-12 synergizes with other cytokines such as IL-1 and IL-18, TNF-α and signals through CD28 for optimal NK-cell responses during this infection (Hunter et al., 1994, 1995, 1997; Cai et al., 2000). Initial reports highlighted macrophages as the early source of IL-12 (Gazzinelli et al., 1993), but this list has grown to include neutrophils, DCs, and inflammatory monocytes. The relative contribution of each cell type to parasite control remains open to debate and may vary with model. Early studies that utilized various strategies to deplete DC indicated that they play an important early role in immunity to T. gondii through their ability to produce IL-12. For example, because DC expresses the integrin CD11c, the use of a

transgene in which the CD11c promoter drives expression of the diphtheria toxin receptor (CD11c-DTR) provides a strategy for wholesale depletion of DC subsets. When these mice are treated with diptheria toxin, they are highly susceptible to T. gondii challenge associated with reduced Th1 response (Meredith et al., 2012; Liu et al., 2006). We now appreciate that this susceptibility is also a function of the loss of non-DC populations that express CD11c such as NK and T cells and more recent studies raised questions about non-DCs in antigen presentation (Meredith et al., 2012). Nevertheless, a CD8α1 splenic DC subset (DC1) has been shown to be critical for IL-12 production and for priming T. gondii-specific CD81 T cells (Mashayekhi et al., 2011). Challenge with T. gondii resulted in a rapid increase in the number of splenic CD8α1 DCs staining positive for IL-12p40; but unlike other sources of IL-12 during toxoplasmosis, the CD8α1 DCs do not require priming with IFN-γ. Moreover, chimeric mice in which only DC1 was unable to produce IL-12 are highly susceptible to T. gondii, implicating CD8α1 DCs and their production of IL-12 as a major pathway important for IFN-γ-mediated control of T. gondii in vivo (Mashayekhi et al., 2011). The cytokine fms-like tyrosine kinase-3 ligand (Flt3L) is critical for the homeostatic expansion and maintenance of

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DCs. The report that Flt3L KO mice succumb to T. gondii and that susceptibility is associated with impaired IL-12 and NK-cell production of IFN-γ affirms the central role of DC in resistance (Dupont et al., 2015). Similar events appear relevant to humans where blood myeloid DCs can be divided into two broad subpopulations: CD1c1 mDC1 (equivalent to the murine CD8α1 DCs) and CD1411 mDC2 cells. The CD1c1 mDC1 produced IL-12 in response to tachyzoites while the mDC2 population appeared largely nonresponsive (Sher et al., 2017). While there are reports that NK cells are cytotoxic for infected cells (Hauser and Tsai, 1986; Subauste et al., 1992), they are perhaps best studied in toxoplasmosis because they are a prominent early source of IFN-γ required to activate the antimicrobial effectors that control T. gondii. These NK cells also impact on the cellular composition and activation at sites of inflammation. Thus, following challenge with T. gondii, at the local site of infection NK-cellderived IFN-γ drives the recruitment of circulating monocytes that differentiate in situ into inflammatory DCs (MoDCs) and F4/801 macrophages. While DC1 can produce IL-12 in response to T. gondii (independent of IFN-γ), the monocyte-derived CD11b1, CD8α1 DCs required priming by IFN-γ from NK cells to produce IL-12 (Goldszmid et al., 2012), which indicates that NK cells have a role in the amplification of the innate response. With an appreciation that innate lymphoid cells (ILCs) are a heterogeneous group, it is now clear that NK cells are not the only source of innate IFN-γ during infection. Memory phenotype (MP) CD41 T cells are a continuously generated population of innate-like lymphocyte effector cells, which appear to be constitutively stimulated by steady state IL-12 and which contribute to early IFN-γ production in response to T. gondii infection (Kawabe et al., 2017). The expression of the transcription factor T-bet (T box expressed in T cells) is important for the production of IFN-γ and during

infection MP CD41 T-bethigh, but not MP CD41 T-betlow cells produced IFN-γ independent of explicit TCR stimulation. T-bet is also important for NK-cell maturation and NK responses to T. gondii are characterized by increased expression of T-bet. Unexpectedly, T-bet KO mice infected with T. gondii remain capable of developing a strong NK-cell-dependent IFN-γ response that controls parasite replication at the challenge site (Harms Pritchard et al., 2015). While NK cells and ILCs are the major innate sources of IFN-γ, during hematopoietic development neutrophil precursors accumulate IFN-γ in primary granules and can produce IFN-γ in response to T. gondii infection independent of IL-12. Thus although IFN-γ expressing neutrophils are present in the blood and bone marrow, secretion requires a trigger to promote degranulation of primary granules (Sturge et al., 2013, 2015).

25.5 Antigen processing and presentation While the focus of this review is the topic of innate immunity, and DC are a key source of IL-12, it is an inherent property of DC to sample and present foreign antigens that are required for the development of CD41 and CD81 T-cell responses, which are critical to control T. gondii. Following invasion of its host cell, T. gondii resides in a unique PV that does not fuse with the endolysosomal system and there are reports that it also downregulates expression of MHC class II (Luder et al., 1998), which would likely impair activation of CD41 T cells. Nevertheless, infection with T. gondii does lead to the global activation of DC in vivo and deletion of these populations results in decreased T-cell responses (Liu et al., 2006; John et al., 2009). There are data that suggest that the ability of the virulent type I strains to blunt DC expansion compromises the expansion of parasite-specific T-cell responses (Tait et al., 2010). Nevertheless, there are multiple

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25.6 Molecular basis for innate recognition of Toxoplasma gondii

pathways for macrophages and DC to acquire parasite-derived antigens that includes uptake of parasite debris released by lysis of infected cells, or phagocytosis of infected cells going through programmed cell death that may enter the class II processing pathway (Zhao et al., 2009b). There is also experimental evidence that conventional phagolysosomal processing contributes to antigen processing and presentation via MHC class II that includes in vitro studies using opsonized or dead parasites (Goldszmid et al., 2009), the transfer of DC populations pulsed with soluble parasite antigens, which can induce protective T-cell responses (DimierPoisson et al., 2003), and the identification of uninfected plasmacytoid DC that presents a class II restricted model antigen from T. gondii (Pepper et al., 2008). Other studies have concluded that infected host cells, particularly DCs and monocytes, are important in vivo for the activation of parasite-specific CD41 and CD81 T cells rather than those cells that take up the parasite by phagocytosis (Dupont et al., 2014). There is abundant evidence that CD81 T cells can recognize and lyse host cells infected with T. gondii and parasite-specific CD81 T cells have been visualized interacting with infected cells (Chtanova et al., 2009). The fact that recognition is TAP-1 and proteasome dependent indicates the involvement of canonical pathways of antigen presentation (Ishii et al., 2006; Tu et al., 2009; Hakim et al., 1991; Subauste et al., 1991; Dzierszinski et al., 2007). For many years, it was unclear whether parasite-derived antigens were capable of accessing the host cell cytoplasm in order to enter the MHC class I processing pathway, but as noted earlier we now appreciate the events that allow parasite-derived effectors to access the cytosol of the infected cell. In contrast, the ability of DC to sample the environment and cross-present antigen would provide a pathway for antigen presentation removed from the impact of the parasite on the infected cell. Indeed, imaging studies indicate that uninfected

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DCs are involved in T-cell priming (John et al., 2009), which implies that cross-presentation may have a role in priming CD81 T cells. In fact, mice that lack CD8α1 DC, the major subpopulation involved in cross-presentation, are highly susceptible to T. gondii. However, this can also be attributed in part to their ability to produce IL-12 (Mashayekhi et al., 2011). Regardless, it has been difficult to rigorously isolate and distinguish the effects of cross-presentation versus the role of infected cells in the initiation of the CD41 and CD81 T-cell responses.

25.6 Molecular basis for innate recognition of Toxoplasma gondii Given the importance of the innate ability of macrophage and DC populations to produce IL-12 during toxoplasmosis (reviewed in Egan et al., 2009) there has been a long-standing interest in the mechanisms that allow these cells to sense and respond to T. gondii. The use of antigen preparations of T. gondii to stimulate cells in culture or after intravenous injection into mice has been widely used to identify the cells that can respond to the parasite (Reis e Sousa et al., 1997; Gazzinelli et al., 1993). However, others have noted that live parasites are more effective at stimulating macrophage production of IL-12 in vitro than are preparations of parasite antigens (Robben et al., 2004). Fig. 25.2 summarizes innate immune pathways demonstrated to contribute to immune recognition of T. gondii.

25.6.1 Toll-like receptor and MyD88 The initial description of Toll-like receptors (TLRs) and their ability to recognize a wide variety of microbially derived pathogenassociated molecular patterns reinforced the concept that there were hard wired mechanisms to distinguish different classes of pathogens. The availability of mice that lacked TLR

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FIGURE 25.2 Toxoplasma gondii immune recognition. Immune recognition of T. gondii is dominated by the T. gondii profilin-TLR11/12-MyD88 axis in mice while far less is known about the key immune sensors important for recognition of T. gondii in human infection. Stimulation via P2X7R can result in growth suppression of T. gondii and inflammasome activation in mice and humans. Parasite infection of human monocytes results in caspase-dependent secretion of Alarmin S100A11 to drive a chemokine response. This is consistent with a damage response network for recognition of T. gondii in human infection. Surprisingly, T. gondii infection induces the production of ISGs via cGAS, STING, TBK1, and IRF3 that appears to aid parasite replication/survival during infection. ISGs, Interferon stimulated genes.

family members or downstream signaling molecules facilitated these studies for T. gondii and revealed that mice deficient in individual TLRs associated with recognition of cell extrinsic stimuli (TLR1, 2, 4, 6) do not display increased susceptibility to acute infection (Hitziger et al., 2005; Yarovinsky et al., 2005; Debierre-Grockiego et al., 2007; Melo et al., 2010). The endosomal localized TLRs 7 and 9 that can detect microbial RNA and DNA are responsive to T. gondii and stimulate cytokine production but are not required for resistance (Andrade et al., 2013). In contrast, TLR11- and TLR12-deficient mice have an impaired IL-12 response to T. gondii linked to the ability to sense a parasite molecule (Yarovinsky et al., 2005; Andrade et al., 2013). Parasite-derived profilin mediates actin assembly in T. gondii and is important for parasite gliding and host cell invasion (Plattner et al., 2008). TLR11 and TLR12 can function as heterodimers and both contribute to IL-12 production by macrophages, conventional DCs and pDCs and respond to profilin (Raetz et al., 2013; Koblansky et al., 2013). TLR12 is regarded as an endolysosomal sensor linked to UNC93B1 and these KO mice are highly susceptible to T. gondii (Pifer et al., 2011; Melo et al., 2010; Koblansky et al., 2013). DC profilin dependent

IL-12 production through TLR11 and TLR12 is IFN regulatory factor 8 dependent (Raetz et al., 2013). Plasmacytoid DC are a minor population of DC, typically associated with the type I IFN response, but they are activated during toxoplasmosis, present antigen to CD41 T cells, and require TLR11 to produce IL-12 in vitro (Pepper et al., 2008). Subsequent studies showed TLR12-dependent induction of IL-12 by pDC in combination with IFN-α contributes to IFN-γ production by NK cells during infection (Koblansky et al., 2013) (Fig. 25.2). Downstream of TLR is a signaling cascade that includes the central adaptor molecules MyD88, which promotes the activation of NF-κB transcription factors while the adapter TNF-α receptor associated factor 6 (TRAF6) is associated with mitogen-activated protein kinase (MAPK) activation. MyD88 KO mice are highly susceptible to T. gondii associated with decreased production of IL-12 by murine DCs, macrophages and neutrophils in vitro (Scanga et al., 2002; Sukhumavasi et al., 2008). These studies lead to a model in which the murine MyD88-dependent innate immune response is driven predominantly by the recognition of T. gondii profilin by TLR12 and to a lesser extent TLR11. To begin to investigate cell lineage specific functions of MyD88 during

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25.6 Molecular basis for innate recognition of Toxoplasma gondii

toxoplasmosis, mice lacking MyD88 in CD11C cells were generated and shown to be highly susceptible to T. gondii (Hou et al., 2011). However, interpretation of these results is complicated because the cytokines IL-1, IL-18, and IL-33 also utilize MyD88 and are implicated in resistance to T. gondii. Although MyD88 KO mice infected with T. gondii succumb rapidly, have decreased IL-12, and fail to control parasites, the addition of exogenous IL-12 partially restores the Th1 response but does not protect the mice. Surprisingly, chimeric mice in which only T cells lacked expression of MyD88 are more susceptible to T. gondii, a result that further questions its role in innate recognition (LaRosa et al., 2008). Although MyD88 is important in situations when parasite replication and dissemination are dominant, there are MyD88-independent pathways to generate IL-12 that are important for protection. Experiments with the avirulent uracil auxotroph (CPS) mutant of T. gondii that has limited levels of replication indicated that protective immunity could be generated in the absence of MyD88 (Sukhumavasi et al., 2008). There are several possible pathways to IL-12 production that would be independent of Myd88 and infection-induced changes in host cell Ca21 stores result in activation of PKC, MAPK activation, and IL-12 production (Masek et al., 2006, 2007). Bone marrow derived macrophages (BMDM) infected in vitro with T. gondii can produce IL-12 that is dependent on parasite-induced autophosphorylation of p38 MAPK but independent of MyD88 (Kim et al., 2006). Gi protein coupled receptors (GiPCR) appear to contribute to macrophage recognition of T. gondii, resulting in activation of phosphatidylinositol 3-kinase-dependent extracellular-regulated kinase 1 and 2 and protein kinase B (Kim and Denkers, 2006). However, in this case, GiPCR does not regulate IL-12 production but instead contributes to the ability of T. gondii to protect infected cells from apoptosis.

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Improved tools to understand how T. gondii interacts with immune populations has revealed a major paradox in that infected cells or those that have phagocytosed the parasite are not the source of IL-12p40 (Christian et al., 2014). Similar observations have been made in other settings and analysis of DC mucosal populations following oral challenge with T. gondii revealed that a population of CD11b1, CD1032 lamina propria DCs contained parasites, but IL-12p40 production was associated with noninfected CD1031 CD11b1 mucosal DCs (Cohen and Denkers, 2015b). Similarly, IL-12 production by inflammatory monocytes recruited to the peritoneum following T. gondii infection was primarily from uninfected cells (Mordue and Sibley, 2003). While this was not anticipated, perhaps it should not be a surprise that T. gondii would limit the ability of infected cells to produce IL-12. However, these observations raise questions about the mechanisms that allow uninfected populations of DC to sense the presence of T. gondii and to then produce IL-12. In humans, innate recognition of T. gondii appears markedly different compared to mice, in part because TLR11 and TLR12 are not expressed in human cells. Thus soluble tachyzoite antigen and profilin are poor stimulators of IL-12p70, TNF-α, and IL-1β from human peripheral blood mononuclear cells (PBMCs) (Andrade et al., 2013). Similarly, human blood mononuclear cells do not have an appreciable response to phagocytosis of dead parasites, but in response to phagocytosis and endocytic processing of live tachyzoites produce TNF-α, IL-12 p40/p70, and a select group of other cytokines and chemokines (Tosh et al., 2016). If human PBMCs are primed with IFN-γ then parasite RNA and DNA can elicit production of IL-12 and TNF-α (Tosh et al., 2016; Andrade et al., 2013). Preliminary studies that used inhibitors of the endosomal TLRs suggest that these endosomal TLRs are not responsible for the cytokine response of human monocytes in

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response to live tachyzoites (Sher et al., 2017). Thus there remain major questions about the TLR-independent pathways in humans and other species that allow the immune system to sense and respond to T. gondii.

25.6.2 Inflammasome-mediated caspase activation The response to diverse cell-intrinsic microbial and host damage signals can lead to the assembly of inflammasomes. These are multiprotein complexes that contain sensor proteins that can lead to the activation of caspases that result in the processing and release of preformed cytokines, including IL-1 and IL-18. Thus while TLRs are associated with the induction of transcriptional responses, the inflammasomes represent a pathway that leads to cell death and release of preformed cytokines or damage-associated molecular patterns. This would be an attractive candidate for innate recognition of T. gondii because it will be independent of any suppressive effects of the parasite on host transcription. For example, human monocytes exposed to T. gondii release the host protein Alarmin S100A11 through a caspase 1 dependent process; S100A11 binds to its receptor RAGE and triggers increased expression of CCL2, a chemokine that recruits monocytes (Safronova et al., 2019). This is an evolving field and there are distinct inflammasomes associated with different stimuli—but the activation of the cytosolic sensors such as NLRP1, NLRP3, NLRC4, and RIG-I leads to oligomerzation and recruitment of the adapter molecule ASC that facilitates the activation of caspase 1, which in turn processes IL-1 and IL-18 and can lead to pyroptosis, an inflammatory form of cell death. Alleles of NLRP1 are associated with susceptibility to congenital toxoplasmosis (Witola et al., 2011) and RNAi depletion of NLRP1 in a human

monocytic cell line resulted in impaired control of parasite replication, decreased production of IL-1β, IL-18, and IL-12, and increased monocyte death (Witola et al., 2011). The course and severity of T. gondii infection in rats is similar to humans. However, Lewis (LEW) rats are particularly resistant to T. gondii. Resistance is associated with inherent cellular oxidative stress in the rat strain (Witola et al., 2017). This refractoriness is intrinsic to bone marrow cells and linked to a genetic locus, Toxo1 (q24 region of rat c10 containing 86 putative genes). This is orthologous to a region in the human genome that contains NLRP1 (Witola et al., 2011; Cavailles et al., 2006; Sergent et al., 2005). Functional studies of resistance related to the Toxo1 locus during in vivo toxoplasmosis revealed a role for intrinsic macrophage suppression of intracellular parasites as well as parasite-induced cell death of macrophages (Cavailles et al., 2014; Cirelli et al., 2014). Death of infected macrophages was associated with features of pyroptosis with reactive oxygen species (ROS) production, activation of caspase 1, and secretion of IL-1β. Thus, the enhanced macrophage-dependent resistance of the LEW rat appears to be dependent on two pathways: one relies on macrophage suppression of parasite replication and the other associated with NLRP1-dependent death of infected macrophages. This may help explain the finding that in mice T. gondii activates both NLRP1 and NLRP3 inflammasomes that contribute to control of infection (Gorfu et al., 2014). There are multiple processes that lead to the engagement of the NLRP3 inflammasome. In current models the binding of ATP, released from damaged cells, binds to the plasma membrane receptor P2X7 (P2X7R), which opens a cation-specific channel in the membrane resulting in alterations in the ionic environment within the cell that in turn activates a number of intracellular pathways that includes the NLRP3 inflammasome (Miller et al., 2011).

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25.7 IFN-γ-dependent cell autonomous immunity

Indeed, ATP-activated macrophages killed T. gondii concurrently with macrophage apoptosis and independent of nitric oxide (Lees et al., 2010) through mechanisms that appeared to be P2X7R dependent (Correa et al., 2010). Human small intestinal epithelial cells produce and secrete IL-1 via a mechanism dependent on P2X7R and NLRP3 that contributes to control of parasite proliferation (Quan et al., 2018). A role for P2XR7 in immunity to T. gondii during human infection was highlighted when three immune competent individuals who presented with clinical toxoplasmosis were found to have polymorphisms that conferred loss of P2X7R function (Lees et al., 2010). Macrophages from these individuals were less able to control T. gondii following stimulation with ATP. Similarly, the P2X7 receptor is required for the ability of ATP to activate macrophages through canonical NLRP3 activation and production of ROS to limit growth of T. gondii (Moreira-Souza et al., 2017). Other studies with the P2X7R KO mice have linked this receptor to the ability to produce IL-1β and IL-12 (Correa et al., 2017). In human monocytes (but not macrophages), T. gondii invasion stimulates the production and secretion of IL-1β through a mechanism dependent on GRA15, potassium influx, and the NLRP3 inflammasome (Gov et al., 2013, 2017). Whether T. gondii or its products directly stimulate inflammasome activation from its location within the PV is currently unknown. Mitochondrial ROS in particular is a regulator of NLRP3 inflammasome activation and may be involved in these events (reviewed in Harijith et al., 2014). Alternatively, the fate of cells productively infected with T. gondii usually ends with cell death as a consequence of parasite replication and egress but also through IFN-γ-mediated events (Martens et al., 2005; Niedelman et al., 2013). Little is known about whether these different forms of cell death (apoptosis, pyroptosis, and necroptosis) intersect with inflammasome activation.

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25.7 IFN-γ-dependent cell autonomous immunity We have known for almost 40 years that T. gondii is a potent stimulator of IFN-γ-dependent cell mediated immunity and yet in vivo this parasite can persist despite an array of relevant antimicrobial mechanisms. Early in infection, T. gondii encounters and invades host cells where it multiplies freely, but as infection proceeds there is local production of IFN-γ, this should result in the activation of the noninfected bystander cells. This makes it likely that when T. gondii egresses it will encounter cells preactivated by IFN-γ and capable of rapid anti T. gondii activity. The finding that inflammatory macrophages or cytotoxic T lymphocytes can trigger premature egress of T. gondii may provide a mechanism to force parasites to invade fully activated bystander host cells where they can be killed (Tomita et al., 2009; Chtanova et al., 2009) (Fig. 25.3). The relative potency and effectiveness of mechanisms of IFN-γ-induced cell autonomous immunity to any intracellular organism depends on many variables: the species- and cell-specific effectiveness of each mediator, the intracellular niche of the pathogen, and the adaptations made by the pathogen to counter antimicrobial effectors. T. gondii is a successful, widespread parasite able to infect any warm-blooded vertebrate or cell type and with the potential to kill its host. Consequently T. gondii will have provided a strong selective pressure for potential host species to evolve appropriate antimicrobial activities. This is a particularly complex evolutionary puzzle for T. gondii that resides in an intracellular niche distinct from those occupied by other organisms, although the IFNγ-mediated antimicrobial mechanisms that operate against T. gondii are relevant to other intracellular pathogens (Figure 25.3). The replication of T. gondii in naı¨ve or resident tissue

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25.7.1 IFN-γ-induced nitrosative and oxidative defense

FIGURE 25.3 Cell autonomous immune mechanisms that contribute to control of Toxoplasma gondii. Optimal activation of cell autonomous immunity to T. gondii relies on IFN-γ to stimulate antimicrobial effectors downstream of STAT1 in concert with TNF-α and or CD40 to activate NFκβ. In mice IRGs/GBPs in concert with noncanonical autophagy proteins and ubiquitin-binding proteins are dominant IFN-γ-dependent effectors important for killing intracellular parasites. Critical mediators of cell autonomous immunity to the parasite in humans are less clear suggesting potential unknown dominant mechanisms important for parasite control in human toxoplasmosis. GBPs, Guanylate-binding proteins; TNF-α, tumor necrosis factor alpha.

macrophages appears largely unrestrained, but stimulation of these cells with IFN-γ (before infection) activates the transcription factor signal transducer and activator of transcription (STAT)1 that induces transcription of host defense pathways critical for control of the parasite (Gavrilescu et al., 2004; Lieberman et al., 2004; MacMicking, 2012; Gazzinelli et al., 2014). However, there are also effects of IFN-γ on nonhematopoietic cells that are important for parasite control. These have been shown using either IFN-γR or lineage-specific deletion of STAT1 (Lykens et al., 2010; Yap and Sher, 1999; Hidano et al., 2016). The aim of this section is to provide a survey of our current knowledge of the cell autonomous immune mechanisms mediated by IFN-γ involved in the control of T. gondii.

The ability of macrophages to produce ROS and reactive nitrogen species (RNS) is an evolutionarily conserved cell autonomous defense mechanism against intracellular pathogens (reviewed in MacMicking, 2012; Fang, 2004; Nathan and Shiloh, 2000). ROS include superoxide radicals, hydrogen peroxide, and hydroxyl radicals that synergize with RNS to produce toxic intermediates, including dinitrogen oxides, compound peroxides, and nitrosothiol adducts. In mammals, there are three classes of cytokine-inducible oxidoreductases that control ROS and RNS production: (1) NADPH oxidases (NOX) directly catalyze the production of O2 2 ; (2) dual oxidases produce H2O2; and (3) nitric oxide synthases, including iNOS (NOS2), produce nitric oxide (NO) during the metabolism of L-arginine to citrulline. NO interacts with heme, nonheme iron and iron sulfur proteins to disrupt microbial enzymes while NO synergizes with ROS to generate additional RNS that interact with protein thiols, tyrosines, lipids, and DNA to cause damage and disrupt function. The phagocytosis of T. gondii results in the production of reactive oxygen intermediates, whereas the traditional view is that T. gondii invasion fails to trigger this response at least in human monocytes (Wilson et al., 1980); but IFN-γ has a major role in enhancing this activity. Thus the NADPH oxidase family of enzymes (NOX1-5) represents the major ROS producers during infection and NADPH oxidase is stimulated in human monocytes and enhanced in IFN-γ-activated murine macrophages and DC both of which exhibit oxygendependent anti T. gondii activity (Wilson et al., 1980; Murray and Cohn, 1979; Murray et al., 1979, 1985). Consistent with a role for ROS in control of T. gondii, monocytes from patients with hereditary myeloperoxidase

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25.7 IFN-γ-dependent cell autonomous immunity

deficiency or chronic granulomatous disease, both due to congenital mutations in NOX2 subunits, have a significant T. gondii cidal defect (Locksley et al., 1982; Murray et al., 1985). However, dual oxidases and nonenzymatic sources of ROS, including those from mitochondrial leakage, also contribute to host defense. NOX4 KO mice showed impaired cell autonomous immunity of murine BMDM to T. gondii and increased susceptibility to T. gondii in vivo. Knockdown of NOX4 in a human epithelial cell line ARPE19 also resulted in impaired cell autonomous immunity to T. gondii associated with increased parasite replication (Zhou et al., 2013). Naı¨ve BMDM and the RAW264.7 macrophage-like cell line (RAW macrophages) both gradually increase ROS production following infection with the “avirulent” CTG strain of T. gondii. Furthermore, mice deficient in NOX1 or NOX2 showed a modest increase in susceptibility during acute infection with the CTG strain of T. gondii (Matta et al., 2018). The observation that T. gondii has an intact antioxidant network (Ding et al., 2004; Sautel et al., 2009), which would provide a mechanism not only to protect its mitochondrion and apicoplast from oxidative stress during respiration (Pino et al., 2007) but may also help protect against antimicrobial effects of host reactive oxygen intermediates (Hunn and Howard, 2010). In mice, activation of murine macrophages with IFN-γ alone is not typically sufficient to limit parasite growth and a second signal such as TNF-α is required to effectively suppress T. gondii replication in vitro via a nitric oxide (NO) dependent mechanism (Sibley et al., 1991; Langermans et al., 1992; Zhao et al., 2008, 2009a; Luder et al., 2003). This activity is associated with an inhibition of replication of T. gondii rather than direct killing and degradation of the parasite (Zhao et al., 2008). In vivo, nitric oxide from hematopoietic cells (most likely macrophages) is critical for the control of chronic T. gondii infection (Khan et al., 1997;

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Scharton-Kersten et al., 1997; Schluter et al., 1999; Yap and Sher, 1999). While STAT1 is the dominant transcription factor associated with IFN-γ-mediated effects, the MAPK phosphatase-2 (MKP-2) also positively regulates iNOS and downregulates arginine expression and MKP2 KO mice are more susceptible to T. gondii (Woods et al., 2013). Nitric oxide may also be important for control of T. gondii in nonhematopoietic cells as the ability of IFN-γ to restrict T. gondii in skeletal muscle is dependent on NO and immunity-related GTPases (IRG) (Takacs et al., 2012). Consistent with a key role for NO in controlling T. gondii, a forward genetics study to identify parasite genes important for survival in activated macrophages uniformly identified genes important for resistance to NO (Mordue et al., 2007; Skariah et al., 2012). T. gondii has also been shown to inhibit NO production through mechanisms that vary with the macrophage population or cell line infected (Cabral et al., 2018). These results suggest that T. gondii has evolved numerous mechanisms to withstand NO/RNS and that novel parasite effectors may act to suppress NO through both iNOSdependent and -independent mechanisms (Cabral et al., 2018; Skariah et al., 2012; Mordue et al., 2007; Tobin and Knoll, 2012).

25.7.2 IFN-γ-induced restriction of nutrients As noted earlier, IFN-γ is also important in nonhematopoietic cells most of which lack the specialized antimicrobial activities utilized by macrophages. However, any intracellular pathogen that scavenges resources from its host cell is vulnerable to metabolic restriction. For T. gondii the best characterized cell autonomous mechanism to limit growth in nonhematopoietic cells is IFN-γ-dependent tryptophan starvation. Indoleamine 2,3-dioxygenases IDO1 and IDO2 are IFN-γ-inducible, heme-containing

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oxidoreductases that degrade L-tryptophan to generate N-formylkynurenine, the initial ratelimiting step of the kynurenine pathway. An antimicrobial role for IFN-γ-induced IDO mediated by restriction of tryptophan was first shown for T. gondii, a tryptophan auxotroph, in HFs (Pfefferkorn, 1984; Pfefferkorn et al., 1986). IDO-mediated tryptophan starvation has since been shown to inhibit T. gondii in a wide variety of cell types in vitro (Daubener et al., 2001; Schwartzman et al., 1990; Dai et al., 1994). Despite the fact that acute toxoplasmosis results in increased expression and activity of IDO, mice deficient in IDO-1 do not appear more susceptible to T. gondii. However, treatment of mice with an inhibitor of both IDO-1 and IDO-2 prior to challenge resulted in a reduced ability to control parasite replication and increased susceptibility during the chronic stage of infection (Divanovic et al., 2012).

25.7.3 IFN-γ-inducible GTPases Genetic studies on mice that utilized iNOS KO mice established the role of this pathway for long-term resistance to T. gondii but also highlighted the presence of an IFN-γ-dependent, iNOS-independent mechanism of parasite restriction critical for control of acute infection (Scharton-Kersten et al., 1997). It has since become apparent that the key mediators of murine control of T. gondii infection are IFNγ-inducible GTPases. These include immunityrelated GTPases (IRGs) and guanylate-binding proteins (GBPs). These GTPases are in large part regulated by autophagy proteins that (in association with ubiquitin-dependent processes) function in noncanonical pathways important for pathogen control. While humans and mice have 7 and 11 GBPs, respectively (Gazzinelli et al., 2014; Hunn et al., 2011), mice possess 23 IRGs, but only one IRG (immunity related GTPase M) is present in humans and it is not inducible by IFN-γ.

25.7.3.1 Immunity-related GTPases (IRGs) Studies with T. gondii led to the first report of a role for IRGs downstream of IFN-γ in the control of specific intracellular pathogens (Collazo et al., 2001; Taylor et al., 2000). Remarkably deletion of a single IRG resulted in mice that were as susceptible to acute T. gondii infection as IFN-γ KO mice. Targeted gene deletions of different IRGs revealed a family of proteins, expression of which was induced by IFN-γ and that had remarkable specificity in their ability to control and kill intracellular pathogens. T. gondii invasion into IFN-γ-stimulated cells was shown to result in trafficking and sequential loading of specific IRGs onto the PVM over a period of 90 minutes (Martens et al., 2005; Khaminets et al., 2010). Observations on the sequential disruption of the PVM in macrophages suggest a mechanism related to extraction of surface area from the PVM by IRG-enforced ruffling and vesiculation resulting in membrane disruption and exposure of the parasite to the cytosol (Howard et al., 2011). The discovery that specific IRGs were important for the differential control of intracellular pathogens resulted in new questions as to their mechanism of action and regulation (reviewed in Tretina et al., 2019; Praefcke, 2017). An important early discovery was that there were regulatory “GMS” IRGs that negatively regulate the function of effector “GKS” IRGs. The effector “GKS” IRGs have a canonical GKS sequence in the P-loop of their GTPase domain while the regulatory “GMS” IRGs have a methionine in that site consistent with their altered function. In the absence of intracellular infection, the regulatory “GMS” within the cell are docked at different endomembrane compartments and prevent the action of “GKS”; in essence they appear to label the endomembrane as “protected self.” As mentioned previously T. gondii actively invades cells and forms a unique PV independent

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of phagocytosis. The “GKS” IRGs recognize the T. gondii PV as foreign (nonself) by its absence of “GMS” IRGs and initiate a complex cascade of events resulting in the destruction of the parasite (reviewed in Saeij and Frickel, 2017). “GKS” IRGs bound to the PVM mediates recruitment of ubiquitin E3 ligases such as TRAF6 and tripartite-motif containing 21 (Foltz et al., 2017; Haldar et al., 2015). The ubiquitylation of proteins on the PVM/GMS creates binding sites for the ubiquitin-binding protein p62/sequestosome. P62 also appears to be a binding protein for mGBP1 and mGBP2 that are recruited to the PVM. GBPs reside in high-molecular-weight complexes and vesicle-like structures that upon binding to the PVM form larger complexes and, after the disruption of the PVM, localize to the parasite membrane. Recruitment of IRGs and GBPs to the PVM also requires specific autophagy proteins that include Atg7, Atg5, Atg6, and Atg12 that act independent of their canonical role in autophagy. In the absence of these core autophagy proteins, the IRGs and GBPs are activated and form aggregates but fail to dock at the PVM. 25.7.3.2 Guanylate-binding protein IFNγ-inducible p65 GTPases Since IRGs are largely absent in humans, there was a concerted search to identify critical effectors that mediate cell autonomous immunity of T. gondii and other intracellular pathogens in humans. The fact that IRGs and GBPs often collaborate in their functions in mice naturally led to an interest in the role of GBPs in IFN-γ-induced cell autonomous immunity to T. gondii. The first proof for the activity of GBPs against T. gondii was made using mouse strains that lacked individual GBPs versus the entire GBP locus on chromosome 3 that contained mGBP1, mGBP2, mGBP3, mGVP5, and mGBP7 (Yamamoto et al., 2012). The generation of mice that lacked GBP1, GBP2, or the GBP chromosome 3 locus revealed that these genes contributed

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to the ability of IFN-γ-stimulated cells to control T. gondii and these mice were more susceptible to T. gondii. GBPs can exert potent anti T. gondii activity in human cells, but their importance in parasite control is highly cell type and parasite-genotype dependent. For example, in IFN-γ-stimulated fibroblasts T. gondii is killed independent of hGPB1 and hGBP2 (Ohshima, 2014). In some human cells hGBP1, but not hGBP2 and hGBP5, contributes to parasite control, but the requirement for hGBP1 recruitment to the PV may differ depending on the cell type (Qin et al., 2017). Activation of human cells with IFN-γ can also result in parasite suppression via diverse modifications of noncanonical autophagy processes that appear to act independent of hGBPs (Selleck et al., 2015; Clough et al., 2016). Thus, it appears clear that in human cells diverse mechanisms of cell autonomous immunity contribute to parasite control some of which require GBPs and others modifications of noncanonical autophagy pathways yet others still unknown mediators. Whether these diverse mechanisms reflect distinct cell type specific antimicrobial mechanisms or overlap and redundancy of mechanisms needs more research. Also, the importance of hGBPs and noncanonical autophagy pathways are yet to be investigated in human phagocytes.

25.7.4 Autophagic processes Autophagic processes are cell-intrinsic mechanisms that allow the removal of internal organelles, often associated with catabolic processes and cellular stress while xenophagy is the use of similar machinery to eliminate intracellular pathogens. T. gondii can induce canonical host cell autophagy in HeLa cells and fibroblasts via a mechanism dependent on the autophagy-related (Atg) protein Atg5 and has been associated with enhanced parasite growth/replication by enabling the parasite to

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compete with the host cell for anabolic resources (Wang et al., 2009). However, canonical host cell autophagy can also result in parasite death and mice deficient in the central autophagy protein Beclin 1 have increased parasite numbers (Portillo et al., 2010). A series of studies have shown that T. gondii activates a FAK-Src-Y845-EGFR-STAT3 signaling axis within mammalian cells that is important for the ability of the parasite to evade autophagy through a mechanism likely dependent on preventing activation of PKR and eIF2α (Portillo et al., 2017). As mentioned previously IRGs, as well as GBPs, work in association with a family of noncanonical autophagy proteins and ubiquitin to target the PV (reviewed in Howard et al., 2011; Hunn et al., 2011; Hakimi et al., 2017; Hunter and Sibley, 2012; Saeij and Frickel, 2017). The Atg7, Atg3, and the Atg12 Atg5 Atg16L1 complex are required to target the IRGs and GBPs to the PVM (Haldar et al., 2014; Selleck et al., 2015; Choi et al., 2014; Clough et al., 2016). Atg3, 5, 7, and 16L1 are additionally needed to deposit ubiquitin and the ubiquitin adaptor protein p62 around the PV (Selleck et al., 2015; Clough et al., 2016). All murine LC3 homologs as well as gamma-aminobutyric acid (GABA) receptor associated proteins (GABARAPL2) are also targeted to the PV. However, only GABARAPL2 (GATE-16) deficiency results in susceptibility to T. gondii in vivo that is comparable to those observed in the absence of IFN-γ-mediated resistance (Sasai et al., 2017). GATE-16 associates with the Golgi-associated ARF1 GTPase and contributes to uniform recruitment/distribution of IRGs and GBPs to the PVM. Thus it appears that noncanonical autophagy pathways may intersect at multiple levels and in different combinations with diverse IFN-γ-inducible GTPases, ubiquitin-binding proteins and cell mechanisms and pathways important for cellular homeostasis in order to control T. gondii infection.

25.7.5 Cofactors for IFN-γ-dependent effector mechanisms While IFN-γ is considered the major mediator of resistance to T. gondii, it does not act in isolation. Rather, IFN-γ is most effective when acting in concert with other stimuli and some avirulent strains of the parasite can be cleared in naı¨ve macrophages through a mechanism that appears independent of IFN-γ. Indeed, there is evidence in humans that loss of function mutations in the IFN-γ receptor is not sufficient to predispose patients to T. gondii (Janssen et al., 2002). As mentioned earlier, TNF-α provides an important second signal to potentiate the effects of IFN-γ while there is also evidence that IL-1 can have a similar role (Jun et al., 1993). This has led to the recognition that the STAT1 and NF-κB pathways synergize to promote macrophage activation. Another member of the TNF-α superfamily that has a role in resistance to T. gondii is CD40. CD40 is expressed on antigen presenting cells as well as on a variety of nonhematopoetic cells. CD40 binds to its ligand CD154 (CD40L) expressed on activated NK and CD4 T cells; cross-linking of CD40 sends a signal important for regulation of multiple aspects of immunity. The first evidence that the interaction between CD40 and CD154 was important for resistance to T. gondii was the finding that patients with X-linked hyper-IgM syndrome, a congenital condition due to loss of functional CD154, have increased susceptibility to T. gondii encephalitis (Leiva et al., 1998; Tsuge et al., 1998). The CD40/CD154 interaction has complex effects on host immunity and is linked to many aspects of cell-mediated immunity, including the production of IL-12 and class switched B-cell responses, all important elements in resistance to T. gondii. Mice deficient in CD154 succumbed to T. gondii encephalitis 4 5 weeks after parasite infection due to an inability to control parasite replication in the brain even in the presence of normal production of IFN-γ

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25.8 Additional immune pathways altered by Toxoplasma gondii

(Reichmann et al., 2000). This discovery along with the finding that IFN-γ and soluble CD154 in combination restores the ability of CD154 KO macrophages to suppress intracellular parasites established an important role for CD40/154 in the control of T. gondii. Subsequent studies established that CD40 signaling in macrophages induces inhibition of T. gondii infection though a mechanism dependent on autophagy but independent of IFN-γ or nitric oxide (Andrade et al., 2005, 2006). That the ability of macrophage CD40dependent killing of T. gondii is dependent on both TNF-α and calcium/calmodulin-dependent kinase-β (CaMKKβ) to activate Beclin-1 and ULK1 mediators of autophagy (Liu et al., 2016) further illustrates the convergence of different killing mechanisms on a limited number of cellular processes that directly control intracellular parasites.

25.8 Additional immune pathways altered by Toxoplasma gondii 25.8.1 Parasite utilization of host cell pathways A basic tenet of host pathogen interactions is illustrated by the ability of T. gondii to utilize/subvert host cell signaling pathways to promote parasite dissemination and survival. DCs, NK cells, and mouse and human monocytes and interneurons have all been shown to display a hypermotility phenotype when infected with T. gondii (Cook et al., 2018; Lambert et al., 2006; Ueno et al., 2015). It has been proposed that this hypermotility of infected cells provides a “Trojan horse” that enhances parasite dissemination (CollantesFernandez et al., 2012; Diana et al., 2004; Lambert et al., 2009). Hypermotility of infected DCs is triggered by GABA/GABAA receptor dependent calcium signaling via the L-type voltage-dependent calcium channel subtype

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Cav1.3 (Kanatani et al., 2017). The 14-3-3 proteins are an evolutionary conserved group of proteins that regulate many aspects of cell signaling and one of the T. gondii family members that localizes to the PVM membrane is associated with DC hypermotility. In addition, a confirmation-independent mechanism regulating the β1 integrin adhesion pathways is associated with hypermotility of infected human monocytes (Cook et al., 2018). IFN regulatory factor 3 (IRF3) is a transcription factor typically associated with the expression of antiviral genes, including type I IFNs and interferon-stimulated genes (ISGs). Surprisingly, it has been reported that T. gondii activates IRF3-dependent transcription that promotes T. gondii survival in human and murine cells through a mechanism independent of type I IFNs but dependent on ISGs (Majumdar et al., 2015). IRF3 activation by T. gondii is independent of TLRs (including the adaptor protein MyD88) and RIG-1-like receptor pathways, but dependent on the cytoplasmic sensors cGAS, STING, and TBK1. These results suggest a role for DNA-triggered signaling and/or endoplasmic reticulum cellular stress that promotes parasite survival or growth (Moretti et al., 2017) and IRF3 KO mice are strikingly resistant to T. gondii infection (Majumdar et al., 2015). However, the absence of IRF3 also resulted in increased production of IL-12 in vivo during infection. Thus, the contribution of IRF3 activation to cell-intrinsic mechanisms of parasite suppression versus increased IL-12 production has not been dissected in vivo to assess their independent effects on T. gondii infection.

25.8.2 Modulation of signal transducer and activator of transcription pathways As noted earlier, STAT1 is required for IFNγ to signal macrophages and other diverse cell types to control T. gondii (Hidano et al., 2016;

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Lieberman et al., 2004), but it has long been appreciated that cells preinfected with T. gondii are less responsive to IFN-γ signaling (Sibley et al., 1985; Luder et al., 2001). For example, type I strains of T. gondii inhibit macrophage iNOS expression in response to IFN-γ and lipopolysaccharide (Luder et al., 2003). Indeed, the ability of T. gondii to inhibit STAT1 signaling protects the intracellular parasite from full activation of IFNγ-induced cell autonomous immune responses. As a result, more than 60% of the IFN-γ-dependent transcripts induced in naı¨ve macrophages are decreased in infected macrophages (Lang et al., 2012). T. gondii inhibitor of STAT transcription (TgIST) is the primary parasite protein responsible for inhibiting IFN-γ-induced signaling. In T. gondii infected cells the parasite dense granule protein TgIST translocates across the PVM and accumulates in the host cell nucleus (Figure 25.4). In the nucleus, TgIST binds to both activated STAT1 Y701-P and chromatin modifying proteins in the nucleosome-remodeling and deacytylase complex and thereby prevents transcription of STAT1-dependent transcripts (Olias et al., 2016; Gay et al., 2016) (Fig. 25.4). The suppressor of cytokine signaling (SOCS) proteins are endogenous host molecules induced by STAT signals that act to terminate cytokine receptor mediated signals. For example, IL-6-mediated activation of STAT3 drives

expression of SOCS3 that binds to the gp130 subunit of the IL-6 receptor complex and so abbreviates IL-6 signaling (reviewed in Kubo et al., 2003). T. gondii infection activates SOCS1 (Zimmermann et al., 2006) and SOCS3 and this led to the idea that it might be used by T. gondii to alter macrophage function (Whitmarsh et al., 2011). However, in macrophages that lacked SOCS3, the ability of T. gondii to suppress inflammatory cytokines was intact. Unexpectedly, mice engineered to lack SOCS3 in macrophages and neutrophils, when challenged with T. gondii had reduced production of IL-12 and were unable to control parasite replication. The basis for this phenotype appears to be due to exaggerated signaling by IL-6 that suppresses IL-12 production. Resistance of these mice is restored by treatment with anti-IL-6 antibody or addition of IL-12. This indicates that SOCS3 is critical during T. gondii infection to limit IL-6 signaling and enable optimal production of IL-12 required to control parasite replication (Whitmarsh et al., 2011). In these studies, it is unclear whether the effects of SOCS proteins are part of a normal regulatory pathway used to limit immune pathology or there are parasite-derived effectors that target this pathway to benefit the parasite. Activation of STAT6 is normally a consequence of IL-4 or IL-13 signaling while the FIGURE 25.4 Toxoplasma gondii effector mechanisms. T. gondii has a diverse array of parasite effectors that alter immune cell signaling cascades and antimicrobial effector functions in infected cells. Many of these are rhoptry proteins secreted during invasion, or dense granule proteins secreted through the “MYR1-translocon.” TgIST1 is a dense granule protein that inhibits IFN-γ signaling via STAT1. ROP5/18/17 can mimic GMS IRGs to prevent parasite destruction mediated by GKS IRGs. Parasite invasion triggers a FAK-Src-STAT3 signaling cascade important for preventing autophagy.

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25.8 Additional immune pathways altered by Toxoplasma gondii

engagement of STAT3 is downstream of IL-6 and IL-10 (reviewed in Denkers et al., 2012; Melo et al., 2011) and in macrophages is associated with an “alternatively activated” or M2 phenotype implicated in tissue repair (reviewed in Mosser and Edwards, 2008). However, M2 macrophages are generally regarded as having a reduced ability to kill intracellular pathogens and IL-4, IL-6, and IL-10 are potent inhibitors of the ability of IFNγ-activated macrophages to limit T. gondii (Oswald et al., 1992; Gazzinelli et al., 1992; Beaman et al., 1994). A series of elegant studies highlighted that the type I and III genotypes of T. gondii utilize the ROP16 kinase to directly phosphorylate STAT3 and STAT6 and thereby suppress IL-12 (Butcher et al., 2005, 2011; Ong et al., 2010; Saeij et al., 2007). T. gondii ROP16 also suppresses plasmacytoid DC activation by mimicking the regulatory effects of IL-10 (Pierog et al., 2018). Thus this ability to pirate STAT3 and STAT6 allows T. gondii to dampen proinflammatory responses. The effects of ROP16 are complex and one of the ROP16 variants is a strong inducer of macrophage arginase (Jensen et al., 2011; Butcher et al., 2011). This has led to the proposal that some strains of Toxoplasma are more likely to induce M2 activation of macrophages, which would allow enhanced parasite replication. A counterintuitive perspective is that a major product of M2 macrophages is arginase, which converts arginine to ornithine (a precursor of polyamines) and since T. gondii is a polyamine auxotroph, increased arginase would deplete arginine levels in the host cell and could limit parasite replication (Woods et al., 2013). Indeed, the report that type I T. gondii ROP16 null parasites have an in vivo growth advantage suggests that arginine may become limited during infection perhaps as a consequence of the host immune response (Butcher et al., 2011). Alternatively, ROP16-mediated STAT6 activation and induction of arginase may also decrease the

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availability of arginine as a substrate for iNOS resulting in decreased nitric oxide (NO) and enhanced parasite replication. This is consistent with the finding that ROP16 gene deleted parasites do not persist in activated astrocytes since NO is a known defense mechanism against T. gondii in the brain (Butcher et al., 2011; Chao et al., 1993; Peterson et al., 1995; Scharton-Kersten et al., 1997; Schluter et al., 1999). Also, mouse astrocytes and microglial cells produce microbicidal NO following activation with IFN-γ and this is suppressed by parasites in a ROP16 dependent manner (Butcher et al., 2011). While ROP16 is perhaps one of the best studied factors from T. gondii that affects host cell function, it is only one member of a large family of secreted effectors and there remains a major knowledge gap in how other rhoptry-derived proteins impact on host cell function (Peixoto et al., 2010). Given the selective pressure that this antimicrobial pathway would exert on T. gondii it is not a surprise that certain strains of T. gondii are resistant to “GKS” IRGs and it appears that there are parasite effectors that are analogous to the “GMS” IRG proteins. Thus ROP17/ ROP18 in concert with ROP5 and GRA7 mediate the phosphorylation-induced deactivation of GKS IRGs (reviewed in Hakimi et al., 2017; Hunter and Sibley, 2012; Saeij and Frickel, 2017). These parasite kinases are secreted during parasite invasion, traffic to the cytosolic face of the PVM and phosphorylate several effector IRGs on two conserved threonine residues in the conformationally important Switch 1 region of the nucleotide-binding region disrupting their enzymatic activity and preventing their loading to the PVM (Steinfeldt et al., 2010; Fentress et al., 2010).

25.8.3 GRA proteins As mentioned previously the majority of known MYR1-dependent parasite effectors are

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GRA proteins. GRA and rhoptry proteins are discussed in detail in other chapters and will only be briefly mentioned here in regards to their effect on immune signaling cascades. GRA16 and GRA24 can cross the PVM and traffic to the nucleus of the infected cells (Bougdour et al., 2013). GRA16 binds two host enyzmes: the deubiquitinase HAUSP and the PP2A phosphatase and positively modulates host genes involved in cell cycle progression and the p53 tumor suppressor pathway. GRA24 triggers noncanonical activation of the host cell p38α MAPK independent of the classical MAPK phosphorylation cascade (Braun et al., 2013). This results in increased expression of the transcription factors Egr-1 and c-Fos. In infected macrophages, GRA24 contributes to alterations in an array of cytokines/chemokines, including increased expression of proinflammatory genes, including IL-12 p40 and chemokines MCP-1/ CCL2, CXCL10/IP-10, and suppression of M2 macrophage transcripts (Braun et al., 2013). GRA15 is secreted but remains localized in association with the PVM where it activates host p50/p65 NF-kβ and induces its translocation to the host cell nucleus. GRA15 activation of NF-kβ is dependent on TRAF6 and the IkB kinase complex but independent of MyD88 and TRIF (Rosowski et al., 2011). These findings explained earlier observations that infection of macrophages with one particular variant of T. gondii (a type II strain) results in substantial translocation of NF-κB and production of IL-12 (Kim et al., 2006; Robben et al., 2004). GRA15 is also responsible for inducing IL-1 production in human monocytes infected with type II genotypes of the parasite (Gov et al., 2013). Likewise, GRA6 is secreted but remains localized to the PV where it activates the host transcription factor nuclear factor of activated T cells (NFAT4) stimulating production of the chemokines Cxcl2 and Ccl2 (Ma et al., 2014). These chemokines promote the recruitment of inflammatory monocytes and neutrophils to the site of infection. Whether

this provides some benefit to the parasite (through enhanced dissemination) or serves to limit parasite replication and allow host survival is unclear.

25.9 Conclusion and perspectives Studies of the innate immune response to T. gondii have been critical for understanding the development of an effective innate immune response to intracellular pathogens in general. Improved methodologies to visualize T. gondii during infection and to genetically modify the parasite and host continue to make this an important model organism to elucidate regulatory and effector activities important in the innate immune response to intracellular pathogens (Koshy et al., 2010, 2012; Christian et al., 2014; Gregg et al., 2013; Coombes et al., 2013). However, important questions remain that include understanding how T. gondii is recognized by and stimulates the innate immune response particularly during human infection. Although MyD88 is critical, the identified parasite and host molecules important for immune stimulation particularly during human infection remain poorly defined. Perhaps, rather than trying to fit T. gondii into existing paradigms of innate recognition, there might be opportunities to utilize genetic approaches to identify other noncanonical pathways that might provide insight into how noninfected cells are capable of sensing and responding to T. gondii. For example, a high-throughput forward genetic screen based on the use of T. gondii-infected cells identified nine host transcription factors, including the orphan nuclear receptor TLX (TLX) as enhancers of STAT1mediated transcription (Beiting et al., 2015). TLX KO mice infected with T. gondii showed impaired DC production of IL-12 and increased parasite burden during chronic infection (Beiting et al., 2015). Nearly 2000 human and mouse IFN-stimulated genes have been

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References

identified to date and the majority remain uncharacterized (Samarajiwa et al., 2009; Hertzog et al., 2011). It seems likely that a portion of these will be directly relevant to the control of T. gondii. The advances in understanding the innate immune response to T. gondii have generated new areas of study and provide an experimental system to understand antimicrobial effector mechanisms and the processes that allow antigen presentation. As discussed earlier, we now appreciate that there are multiple ROP and GRA proteins that are introduced into the host cell that are often associated with subversion of the host responses. What has become apparent is the presence of multiple antimicrobial effectors induced by distinct immune stimuli that vary with the cell type infected, the activation stimuli, the presence and effectiveness of antimicrobial mediators within different host species, and variations in parasite genotype. Perhaps a key point to recognize is that many of these pathways are not redundant, which may reflect the need to protect different cell types and tissue compartments. Further studies are needed to understand the relationship between intracellular effectors, including IRGs, GBPs, canonical and noncanonical autophagy, ubiquitin pathways, inflammasomes, RNS, and oxidative metabolism in effector activity against T. gondii. In addition, the parasite-derived effectors may also represent an Achilles heel as the natural parasite-derived antigens that are presented on class I include the dense granule proteins GRA6 (Blanchard et al., 2008) and GRA4 and the rhoptry protein ROP7 (Frickel et al., 2008). In infected cells there is evidence for “leakage” from the PV (Gubbels et al., 2005) or that fusion of host ER with the PV provides a conduit for antigen into this pathway (Blanchard et al., 2008; Goldszmid et al., 2009). We now recognize that this can also be a part of normal parasite activity and the identification of the MYR-dependent export illustrates advances in this topic (Franco et al., 2016; Naor et al., 2018).

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This knowledge does provide a way to identify potential vulnerabilities of the parasite to host immunity that could be exploited by targeted therapeutics that render these organisms more susceptible to immune-mediated clearance or development of vaccination strategies.

References Alexander, D.L., Mital, J., Ward, G.E., Bradley, P., Boothroyd, J.C., 2005. Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog. 1, e17. Andrade, R.M., Portillo, J.A., Wessendarp, M., Subauste, C. S., 2005. CD40 signaling in macrophages induces activity against an intracellular pathogen independently of gamma interferon and reactive nitrogen intermediates. Infect. Immun. 73, 3115 3123. Andrade, R.M., Wessendarp, M., Gubbels, M.J., Striepen, B., Subauste, C.S., 2006. CD40 induces macrophage antiToxoplasma gondii activity by triggering autophagydependent fusion of pathogen-containing vacuoles and lysosomes. J. Clin. Invest. 116, 2366 2377. Andrade, W.A., Souza Mdo, C., Ramos-Martinez, E., Nagpal, K., Dutra, M.S., Melo, M.B., et al., 2013. Combined action of nucleic acid-sensing Toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice. Cell Host Microbe 13, 42 53. Beaman, M.H., Hunter, C.A., Remington, J.S., 1994. Enhancement of intracellular replication of Toxoplasma gondii by IL-6. Interactions with IFN-gamma and TNFalpha. J. Immunol. 153, 4583 4587. Beiting, D.P., Hidano, S., Baggs, J.E., Geskes, J.M., Fang, Q., Wherry, E.J., et al., 2015. The orphan nuclear receptor TLX is an enhancer of STAT1-mediated transcription and immunity to Toxoplasma gondii. PLoS Biol. 13, e1002200. Benson, A., Pifer, R., Behrendt, C.L., Hooper, L.V., Yarovinsky, F., 2009. Gut commensal bacteria direct a protective immune response against Toxoplasma gondii. Cell Host Microbe 6, 187 196. Biswas, A., Bruder, D., Wolf, S.A., Jeron, A., Mack, M., Heimesaat, M.M., et al., 2015. Ly6C(high) monocytes control cerebral toxoplasmosis. J. Immunol. 194, 3223 3235. Blanchard, N., Gonzalez, F., Schaeffer, M., Joncker, N.T., Cheng, T., Shastri, A.J., et al., 2008. Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum. Nat. Immunol. 9, 937 944.

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1096

25. Innate immunity to Toxoplasma gondii

Bougdour, A., Durandau, E., Brenier-Pinchart, M.P., Ortet, P., Barakat, M., Kieffer, S., et al., 2013. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression. Cell Host Microbe 13, 489 500. Braun, L., Brenier-Pinchart, M.P., Yogavel, M., CurtVaresano, A., Curt-Bertini, R.L., Hussain, T., et al., 2013. A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation. J. Exp. Med. 210, 2071 2086. Burger, E., Araujo, A., Lopez-Yglesias, A., Rajala, M.W., Geng, L., Levine, B., et al., 2018. Loss of paneth cell autophagy causes acute susceptibility to Toxoplasma gondii-mediated inflammation. Cell Host Microbe 23, 177 190 e4. Butcher, B.A., Kim, L., Panopoulos, A.D., Watowich, S.S., Murray, P.J., Denkers, E.Y., 2005. IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages. J. Immunol. 174, 3148 3152. Butcher, B.A., Fox, B.A., Rommereim, L.M., Kim, S.G., Maurer, K.J., Yarovinsky, F., et al., 2011. Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1dependent growth control. PLoS Pathog. 7, e1002236. Buzoni-Gatel, D., Schulthess, J., Menard, L.C., Kasper, L.H., 2006. Mucosal defences against orally acquired protozoan parasites, emphasis on Toxoplasma gondii infections. Cell. Microbiol. 8, 535 544. Cabral, G.R.A., Wang, Z.T., Sibley, L.D., Damatta, R.A., 2018. Inhibition of nitric oxide production in activated macrophages caused by Toxoplasma gondii infection occurs by distinct mechanisms in different mouse macrophage cell lines. Front. Microbiol. 9, 1936. Cai, G., Kastelein, R., Hunter, C.A., 2000. Interleukin-18 (IL-18) enhances innate IL-12-mediated resistance to Toxoplasma gondii. Infect. Immun. 68, 6932 6938. Carruthers, V.B., Sibley, L.D., 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur. J. Cell Biol. 73, 114 123. Cavailles, P., Sergent, V., Bisanz, C., Papapietro, O., Colacios, C., Mas, M., et al., 2006. The rat Toxo1 locus directs toxoplasmosis outcome and controls parasite proliferation and spreading by macrophage-dependent mechanisms. Proc. Natl. Acad. Sci. U.S.A. 103, 744 749. Cavailles, P., Flori, P., Papapietro, O., Bisanz, C., Lagrange, D., Pilloux, L., et al., 2014. A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage. PLoS Pathog. 10, e1004005.

Chao, C.C., Anderson, W.R., Hu, S., Gekker, G., Martella, A., Peterson, P.K., 1993. Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism, Clin. Immunol. Immunopathol., 67. pp. 178 183. Choi, J., Park, S., Biering, S.B., Selleck, E., Liu, C.Y., Zhang, X., et al., 2014. The parasitophorous vacuole membrane of Toxoplasma gondii is targeted for disruption by ubiquitin-like conjugation systems of autophagy. Immunity 40, 924 935. Christian, D.A., Koshy, A.A., Reuter, M.A., Betts, M.R., Boothroyd, J.C., Hunter, C.A., 2014. Use of transgenic parasites and host reporters to dissect events that promote interleukin-12 production during toxoplasmosis. Infect. Immun. 82, 4056 4067. Chtanova, T., Han, S.J., Schaeffer, M., Van Dooren, G.G., Herzmark, P., Striepen, B., et al., 2009. Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node. Immunity 31, 342 355. Cirelli, K.M., Gorfu, G., Hassan, M.A., Printz, M., Crown, D., Leppla, S.H., et al., 2014. Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii. PLoS Pathog. 10, e1003927. Clough, B., Wright, J.D., Pereira, P.M., Hirst, E.M., Johnston, A.C., Henriques, R., et al., 2016. K63-linked ubiquitination targets Toxoplasma gondii for endolysosomal destruction in IFNgamma-stimulated human cells. PLoS Pathog. 12, e1006027. Coffey, M.J., Sleebs, B.E., Uboldi, A.D., Garnham, A., Franco, M., Marino, N.D., et al., 2015. An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell. Elife 4, e10809. Coffey, M.J., Dagley, L.F., Seizova, S., Kapp, E.A., Infusini, G., Roos, D.S., et al., 2018. Aspartyl protease 5 matures dense granule proteins that reside at the host-parasite interface in Toxoplasma gondii. MBio 9, e01796 18. Cohen, S.B., Denkers, E.Y., 2015a. The gut mucosal immune response to Toxoplasma gondii. Parasite Immunol. 37, 108 117. Cohen, S.B., Denkers, E.Y., 2015b. Impact of Toxoplasma gondii on dendritic cell subset function in the intestinal mucosa. J. Immunol. 195, 2754 2762. Cohen, S.B., Maurer, K.J., Egan, C.E., Oghumu, S., Satoskar, A.R., Denkers, E.Y., 2013. CXCR3-dependent CD4(1) T cells are required to activate inflammatory monocytes for defense against intestinal infection. PLoS Pathog. 9, e1003706. Collantes-Fernandez, E., Arrighi, R.B., Alvarez-Garcia, G., Weidner, J.M., Regidor-Cerrillo, J., Boothroyd, J.C., et al., 2012. Infected dendritic cells facilitate systemic dissemination and transplacental passage of the obligate intracellular parasite Neospora caninum in mice. PLoS One 7, e32123.

Toxoplasma Gondii

References

Collazo, C.M., Yap, G.S., Sempowski, G.D., Lusby, K.C., Tessarollo, L., Woude, G.F., et al., 2001. Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogenspecific roles in resistance to infection. J. Exp. Med. 194, 181 188. Cook, J.H., Ueno, N., Lodoen, M.B., 2018. Toxoplasma gondii disrupts beta1 integrin signaling and focal adhesion formation during monocyte hypermotility. J. Biol. Chem. 293, 3374 3385. Coombes, J.L., Charsar, B.A., Han, S.J., Halkias, J., Chan, S. W., Koshy, A.A., et al., 2013. Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread. Proc. Natl. Acad. Sci. U.S.A. 110, E1913 E1922. Coppens, I., Romano, J.D., 2018. Hostile intruder: Toxoplasma holds host organelles captive. PLoS Pathog. 14, e1006893. Correa, G., Marques Da Silva, C., De Abreu Moreira-Souza, A.C., Vommaro, R.C., Coutinho-Silva, R., 2010. Activation of the P2X(7) receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages. Microbes Infect. 12, 497 504. Correa, G., Almeida Lindenberg, C., Moreira-Souza, A.C., Savio, L.E., Takiya, C.M., Marques-Da-Silva, C., et al., 2017. Inflammatory early events associated to the role of P2X7 receptor in acute murine toxoplasmosis. Immunobiology 222, 676 683. Courret, N., Darche, S., Sonigo, P., Milon, G., Buzoni-Gatel, D., Tardieux, I., 2006. CD11c and CD11b expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain. Blood 107, 309 316. Dai, W., Pan, H., Kwok, O., Dubey, J.P., 1994. Human indoleamine 2,3-dioxygenase inhibits Toxoplasma gondii growth in fibroblast cells. J. Interferon Res. 14, 313 317. Darde, M.L., Villena, I., Pinon, J.M., Beguinot, I., 1998. Severe toxoplasmosis caused by a Toxoplasma gondii strain with a new isoenzyme type acquired in French Guyana. J. Clin. Microbiol. 36, 324. Daubener, W., Spors, B., Hucke, C., Adam, R., Stins, M., Kim, K.S., et al., 2001. Restriction of Toxoplasma gondii growth in human brain microvascular endothelial cells by activation of indoleamine 2,3-dioxygenase. Infect. Immun. 69, 6527 6531. Debierre-Grockiego, F., Campos, M.A., Azzouz, N., Schmidt, J., Bieker, U., Resende, M.G., et al., 2007. Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii. J. Immunol. 179, 1129 1137. Denkers, E.Y., Gazzinelli, R.T., Martin, D., Sher, A., 1993. Emergence of NK1.1 1 cells as effectors of IFN-gamma dependent immunity to Toxoplasma gondii in MHC class I-deficient mice. J. Exp. Med. 178, 1465 1472.

1097

Denkers, E.Y., Bzik, D.J., Fox, B.A., Butcher, B.A., 2012. An inside job: hacking into janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii. Infect. Immun. 80, 476 482. Diana, J., Persat, F., Staquet, M.J., Assossou, O., Ferrandiz, J., Gariazzo, M.J., et al., 2004. Migration and maturation of human dendritic cells infected with Toxoplasma gondii depend on parasite strain type. FEMS Immunol. Med. Microbiol. 42, 321 331. Dimier-Poisson, I., Aline, F., Mevelec, M.N., Beauvillain, C., Buzoni-Gatel, D., Bout, D., 2003. Protective mucosal Th2 immune response against Toxoplasma gondii by murine mesenteric lymph node dendritic cells. Infect. Immun. 71, 5254 5265. Ding, M., Kwok, L.Y., Schluter, D., Clayton, C., Soldati, D., 2004. The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury. Mol. Microbiol. 51, 47 61. Divanovic, S., Sawtell, N.M., Trompette, A., Warning, J.I., Dias, A., Cooper, A.M., et al., 2012. Opposing biological functions of tryptophan catabolizing enzymes during intracellular infection. J. Infect. Dis. 205, 152 161. Dobrowolski, J.M., Sibley, L.D., 1996. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933 939. Dunay, I.R., Damatta, R.A., Fux, B., Presti, R., Greco, S., Colonna, M., et al., 2008. Gr1(1) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii. Immunity 29, 306 317. Dunay, I.R., Fuchs, A., Sibley, L.D., 2010. Inflammatory monocytes but not neutrophils are necessary to control infection with Toxoplasma gondii in mice. Infect. Immun. 78, 1564 1570. Dupont, C.D., Christian, D.A., Selleck, E.M., Pepper, M., Leney-Greene, M., Harms Pritchard, G., et al., 2014. Parasite fate and involvement of infected cells in the induction of CD4 1 and CD8 1 T cell responses to Toxoplasma gondii. PLoS Pathog. 10, e1004047. Dupont, C.D., Harms Pritchard, G., Hidano, S., Christian, D.A., Wagage, S., Muallem, G., et al., 2015. Flt3 ligand is essential for survival and protective immune responses during toxoplasmosis. J. Immunol. 195, 4369 4377. Dzierszinski, F., Pepper, M., Stumhofer, J.S., Larosa, D.F., Wilson, E.H., Turka, L.A., et al., 2007. Presentation of Toxoplasma gondii antigens via the endogenous major histocompatibility complex class I pathway in nonprofessional and professional antigen-presenting cells. Infect. Immun. 75, 5200 5209. Egan, C.E., Sukhumavasi, W., Butcher, B.A., Denkers, E.Y., 2009. Functional aspects of Toll-like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii. Clin. Exp. Immunol. 156, 17 24.

Toxoplasma Gondii

1098

25. Innate immunity to Toxoplasma gondii

Fang, F.C., 2004. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2, 820 832. Fentress, S.J., Behnke, M.S., Dunay, I.R., Mashayekhi, M., Rommereim, L.M., Fox, B.A., et al., 2010. Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence. Cell Host Microbe 8, 484 495. Foltz, C., Napolitano, A., Khan, R., Clough, B., Hirst, E.M., Frickel, E.M., 2017. TRIM21 is critical for survival of Toxoplasma gondii infection and localises to GBP-positive parasite vacuoles. Sci. Rep. 7, 5209. Franco, M., Panas, M.W., Marino, N.D., Lee, M.C., Buchholz, K.R., Kelly, F.D., et al., 2016. A novel secreted protein, MYR1, is central to Toxoplasma’s manipulation of host cells. MBio 7, e02231 15. Frickel, E.M., Sahoo, N., Hopp, J., Gubbels, M.J., Craver, M. P., Knoll, L.J., et al., 2008. Parasite stage-specific recognition of endogenous Toxoplasma gondii-derived CD8 1 T cell epitopes. J. Infect. Dis. 198, 1625 1633. Gavrilescu, L.C., Butcher, B.A., Del Rio, L., Taylor, G.A., Denkers, E.Y., 2004. STAT1 is essential for antimicrobial effector function but dispensable for gamma interferon production during Toxoplasma gondii infection. Infect. Immun. 72, 1257 1264. Gay, G., Braun, L., Brenier-Pinchart, M.P., Vollaire, J., Josserand, V., Bertini, R.L., et al., 2016. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-gammamediated host defenses. J. Exp. Med. 213, 1779 1798. Gazzinelli, R.T., Oswald, I.P., James, S.L., Sher, A., 1992. IL10 inhibits parasite killing and nitrogen oxide production by IFN-gamma-activated macrophages. J. Immunol. 148, 1792 1796. Gazzinelli, R.T., Hieny, S., Wynn, T.A., Wolf, S., Sher, A., 1993. Interleukin 12 is required for the T-lymphocyteindependent induction of interferon g by an intracellular parasite and induces resistance in T-cell deficient hosts. Proc. Natl. Acad. Sci. U.S.A. 90, 6115 6119. Gazzinelli, R.T., Mendonca-Neto, R., Lilue, J., Howard, J., Sher, A., 2014. Innate resistance against Toxoplasma gondii: an evolutionary tale of mice, cats, and men. Cell Host Microbe 15, 132 138. Gold, D.A., Kaplan, A.D., Lis, A., Bett, G.C., Rosowski, E.E., Cirelli, K.M., et al., 2015. The Toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole, Cell Host Microbe, 17. pp. 642 652. Goldszmid, R.S., Coppens, I., Lev, A., Caspar, P., Mellman, I., Sher, A., 2009. Host ER-parasitophorous vacuole interaction provides a route of entry for antigen crosspresentation in Toxoplasma gondii-infected dendritic cells. J. Exp. Med. 206, 399 410.

Goldszmid, R.S., Caspar, P., Rivollier, A., White, S., Dzutsev, A., Hieny, S., et al., 2012. NK cell-derived interferon-gamma orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection. Immunity 36, 1047 1059. Gorfu, G., Cirelli, K.M., Melo, M.B., Mayer-Barber, K., Crown, D., Koller, B.H., et al., 2014. Dual role for inflammasome sensors NLRP1 and NLRP3 in murine resistance to Toxoplasma gondii. MBio 5, e01117 13. Gov, L., Karimzadeh, A., Ueno, N., Lodoen, M.B., 2013. Human innate immunity to Toxoplasma gondii is mediated by host caspase-1 and ASC and parasite GRA15. mBio 4. Gov, L., Schneider, C.A., Lima, T.S., Pandori, W., Lodoen, M.B., 2017. NLRP3 and potassium efflux drive rapid IL1beta release from primary human monocytes during Toxoplasma gondii infection. J. Immunol. 199, 2855 2864. Gregg, B., Taylor, B.C., John, B., Tait-Wojno, E.D., Girgis, N.M., Miller, N., et al., 2013. Replication and distribution of Toxoplasma gondii in the small intestine after oral infection with tissue cysts. Infect. Immun. 81, 1635 1643. Gubbels, M.J., Streipen, B., Shastri, N., Turkoz, M., Robey, E.A., 2005. Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii. Infect. Immun. 73, 703 711. Guiton, P.S., Sagawa, J.M., Fritz, H.M., Boothroyd, J.C., 2017. An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-kappaB-like signature in infected host cells. PLoS One 12, e0173018. Hakim, F.T., Gazzinelli, R.T., Denkers, E., Hieny, S., Shearer, G.M., Sher, A., 1991. CD8 1 T cells from mice vaccinated against Toxoplasma gondii are cytotoxic for parasite-infected or antigen-pulsed host cells. J. Immunol. 147, 2310 2316. Hakimi, M.A., Olias, P., Sibley, L.D., 2017. Toxoplasma effectors targeting host signaling and transcription. Clin. Microbiol. Rev. 30, 615 645. Haldar, A.K., Piro, A.S., Pilla, D.M., Yamamoto, M., Coers, J., 2014. The E2-like conjugation enzyme Atg3 promotes binding of IRG and Gbp proteins to Chlamydia- and Toxoplasma-containing vacuoles and host resistance. PLoS One 9, e86684. Haldar, A.K., Foltz, C., Finethy, R., Piro, A.S., Feeley, E.M., Pilla-Moffett, D.M., et al., 2015. Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins. Proc. Natl. Acad. Sci. U.S.A. 112, E5628 E5637. Harijith, A., Ebenezer, D.L., Natarajan, V., 2014. Reactive oxygen species at the crossroads of inflammasome and inflammation. Front. Physiol. 5, 352.

Toxoplasma Gondii

References

Harms Pritchard, G., Hall, A.O., Christian, D.A., Wagage, S., Fang, Q., Muallem, G., et al., 2015. Diverse roles for T-bet in the effector responses required for resistance to infection. J. Immunol. 194, 1131 1140. Hauser, W.E., Tsai, V., 1986. Acute Toxoplasma infection of mice induces spleen NK cells that are cytotoxic for T. gondii in vitro. J. Immunol. 136, 313 319. Heimesaat, M.M., Bereswill, S., Fischer, A., Fuchs, D., Struck, D., Niebergall, J., et al., 2006. Gram-negative bacteria aggravate murine small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii. J. Immunol. 177, 8785 8795. Hertzog, P., Forster, S., Samarajiwa, S., 2011. Systems biology of interferon responses. J. Interferon Cytokine Res. 31, 5 11. Hidano, S., Randall, L.M., Dawson, L., Dietrich, H.K., Konradt, C., Klover, P.J., et al., 2016. STAT1 signaling in astrocytes is essential for control of infection in the central nervous system. MBio 7, e01881 16. Hitziger, N., Dellacasa, I., Albiger, B., Barragan, A., 2005. Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging. Cell. Microbiol. 7, 837 848. Hou, B., Benson, A., Kuzmich, L., Defranco, A.L., Yarovinsky, F., 2011. Critical coordination of innate immune defense against Toxoplasma gondii by dendritic cells responding via their Toll-like receptors. Proc. Natl. Acad. Sci. U.S.A. 108, 278 283. Howard, J.C., Hunn, J.P., Steinfeldt, T., 2011. The IRG protein-based resistance mechanism in mice and its relation to virulence in Toxoplasma gondii. Curr. Opin. Microbiol. 14, 414 421. Hunn, J.P., Howard, J.C., 2010. The mouse resistance protein Irgm1 (LRG-47): a regulator or an effector of pathogen defense? PLoS Pathog. 6, e1001008. Hunn, J.P., Feng, C.G., Sher, A., Howard, J.C., 2011. The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens. Mamm. Genome 22, 43 54. Hunter, C.A., Sibley, L.D., 2012. Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat. Rev. Microbiol. 10, 766 778. Hunter, C.A., Subauste, C.S., Van Cleave, V.H., Remington, J.S., 1994. Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha. Infect. Immun. 62, 2818 2824. Hunter, C.A., Chizzonite, R., Remington, J.S., 1995. Interleukin 1ß is required for the ability of IL-12 to induce production of IFN-γ by NK cells: a role for IL-1ß in the T cell independent mechanism of resistance against intracellular pathogens. J. Immunol. 155, 4347 4354.

1099

Hunter, C.A., Ellis-Neyer, L., Gabriel, K.E., Kennedy, M.K., Grabstein, K.H., Linsley, P.S., et al., 1997. The role of the CD28/B7 interaction in the regulation of NK cell responses during infection with Toxoplasma gondii. J. Immunol. 158, 2285 2293. Ishii, K., Hisaeda, H., Duan, X., Imai, T., Sakai, T., Fehling, H.J., et al., 2006. The involvement of immunoproteasomes in induction of MHC class I-restricted immunity targeting Toxoplasma SAG1. Microbes Infect. 8, 1045 1053. Janssen, R., Van Wengen, A., Verhard, E., De Boer, T., Zomerdijk, T., Ottenhoff, T.H., et al., 2002. Divergent role for TNF-alpha in IFN-gamma-induced killing of Toxoplasma gondii and Salmonella typhimurium contributes to selective susceptibility of patients with partial IFN-gamma receptor 1 deficiency. J. Immunol. 169, 3900 3907. Jensen, K.D., Wang, Y., Wojno, E.D., Shastri, A.J., Hu, K., Cornel, L., et al., 2011. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation. Cell Host Microbe 9, 472 483. John, B., Harris, T.H., Tait, E.D., Wilson, E.H., Gregg, B., Ng, L.G., et al., 2009. Dynamic Imaging of CD8(1) T cells and dendritic cells during infection with Toxoplasma gondii. PLoS Pathog. 5, e1000505. Joiner, K.A., Fuhrman, S.A., Miettinen, H.M., Kasper, L.H., Mellman, I., 1990. Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science 249, 641 646. Jun, C.D., Kim, S.H., Soh, C.T., Kang, S.S., Chung, H.T., 1993. Nitric oxide mediates the toxoplasmastatic activity of murine microglial cells in vitro. Immunol. Invest. 22, 487 501. Kanatani, S., Fuks, J.M., Olafsson, E.B., Westermark, L., Chambers, B., Varas-Godoy, M., et al., 2017. Voltagedependent calcium channel signaling mediates GABAA receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii. PLoS Pathog. 13, e1006739. Kawabe, T., Jankovic, D., Kawabe, S., Huang, Y., Lee, P.H., Yamane, H., et al., 2017. Memory-phenotype CD4(1) T cells spontaneously generated under steady-state conditions exert innate TH1-like effector function. Sci. Immunol. 2, eaam9304. Khaminets, A., Hunn, J.P., Konen-Waisman, S., Zhao, Y.O., Preukschat, D., Coers, J., et al., 2010. Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole. Cell. Microbiol. 12, 939 961. Khan, I.A., Matsuura, T., Kasper, L.H., 1994. Interleukin-12 enhances murine survival against acute Toxoplasmosis. Infect. Immun. 62, 1639 1642.

Toxoplasma Gondii

1100

25. Innate immunity to Toxoplasma gondii

Khan, I.A., Schwartzman, J.D., Matsuura, T., Kasper, L.H., 1997. A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice. Proc. Natl. Acad. Sci. U.S.A. 94, 13955 13960. Kim, L., Denkers, E.Y., 2006. Toxoplasma gondii triggers Gi-dependent PI 3-kinase signaling required for inhibition of host cell apoptosis. J. Cell Sci. 119, 2119 2126. Kim, L., Butcher, B.A., Lee, C.W., Uematsu, S., Akira, S., Denkers, E.Y., 2006. Toxoplasma gondii genotype determines MyD88-dependent signaling in infected macrophages. J. Immunol. 177, 2584 2591. Kobayashi, M., Fitz, L., Ryan, M., Hewick, R.M., Clark, S.C., Chan, S., et al., 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170, 827 845. Koblansky, A.A., Jankovic, D., Oh, H., Hieny, S., Sungnak, W., Mathur, R., et al., 2013. Recognition of profilin by Toll-like receptor 12 is critical for host resistance to Toxoplasma gondii. Immunity 38, 119 130. Konradt, C., Ueno, N., Christian, D.A., Delong, J.H., Pritchard, G.H., Herz, J., et al., 2016. Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system. Nat. Microbiol. 1, 16001. Koshy, A.A., Fouts, A.E., Lodoen, M.B., Alkan, O., Blau, H. M., Boothroyd, J.C., 2010. Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions. Nat. Methods 7, 307 309. Koshy, A.A., Dietrich, H.K., Christian, D.A., Melehani, J.H., Shastri, A.J., Hunter, C.A., et al., 2012. Toxoplasma coopts host cells it does not invade. PLoS Pathog. 8, e1002825. Kubo, M., Hanada, T., Yoshimura, A., 2003. Suppressors of cytokine signaling and immunity. Nat. Immunol. 4, 1169 1176. Lambert, H., Hitziger, N., Dellacasa, I., Svensson, M., Barragan, A., 2006. Induction of dendritic cell migration upon Toxoplasma gondii infection potentiates parasite dissemination. Cell. Microbiol. 8, 1611 1623. Lambert, H., Vutova, P.P., Adams, W.C., Lore, K., Barragan, A., 2009. The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype. Infect. Immun. 77, 1679 1688. Lang, C., Hildebrandt, A., Brand, F., Opitz, L., Dihazi, H., Luder, C.G., 2012. Impaired chromatin remodelling at STAT1-regulated promoters leads to global unresponsiveness of Toxoplasma gondii-infected macrophages to IFN-gamma. PLoS Pathog. 8, e1002483. Langermans, J.A., Van Der Hulst, M.E., Nibbering, P.H., Hiemstra, P.S., Fransen, L., Van Furth, R., 1992. IFNgamma-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated

by endogenous tumor necrosis factor-alpha. J. Immunol. 148, 568 574. Larosa, D.F., Stumhofer, J.S., Gelman, A.E., Rahman, A.H., Taylor, D.K., Hunter, C.A., et al., 2008. T cell expression of MyD88 is required for resistance to Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 105, 3855 3860. Lees, M.P., Fuller, S.J., Mcleod, R., Boulter, N.R., Miller, C. M., Zakrzewski, A.M., et al., 2010. P2X7 receptormediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages. J. Immunol. 184, 7040 7046. Leiva, L.E., Junprasert, J., Hollenbaugh, D., Sorensen, R.U., 1998. Central nervous system toxoplasmosis with an increased proportion of circulating gd T cells in a patient with hyper-IgM syndrome. J. Clin. Immunol. 18, 283 290. Lieberman, L.A., Banica, M., Reiner, S.L., Hunter, C.A., 2004. STAT1 plays a critical role in the regulation of antimicrobial effector mechanisms, but not in the development of Th1-type responses during toxoplasmosis. J. Immunol. 172, 457 463. Liesenfeld, O., Kosek, J., Remington, J.S., Suzuki, Y., 1996. Association of CD4 1 T cell-dependent, interferongamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii. J. Exp. Med. 184, 597 607. Liesenfeld, O., Kang, H., Park, D., Nguyen, T.A., Parkhe, C.V., Watanabe, H., et al., 1999. TNF-alpha, nitric oxide and IFN-gamma are all critical for development of necrosis in the small intestine and early mortality in genetically susceptible mice infected perorally with Toxoplasma gondii. Parasite Immunol. 21, 365 376. Liu, C.H., Fan, Y.T., Dias, A., Esper, L., Corn, R.A., Bafica, A., et al., 2006. Cutting edge: dendritic cells are essential for in vivo IL-12 production and development of resistance against Toxoplasma gondii infection in mice. J. Immunol. 177, 31 35. Liu, E., Lopez Corcino, Y., Portillo, J.A., Miao, Y., Subauste, C.S., 2016. Identification of signaling pathways by which CD40 stimulates autophagy and antimicrobial activity against Toxoplasma gondii in macrophages. Infect. Immun. 84, 2616 2626. Locksley, R.M., Wilson, C.B., Klebanoff, S.J., 1982. Role for endogenous and acquired peroxidase in the toxoplasmacidal activity of murine and human mononuclear phagocytes. J. Clin. Invest. 69, 1099 1111. Luder, C.G., Lang, T., Beuerle, B., Gross, U., 1998. Downregulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii. Clin. Exp. Immunol. 112, 308 316.

Toxoplasma Gondii

References

Luder, C.G., Walter, W., Beuerle, B., Maeurer, M.J., Gross, U., 2001. Toxoplasma gondii down-regulates MHC class II gene expression and antigen presentation by murine macrophages via interference with nuclear translocation of STAT1alpha. Eur. J. Immunol. 31, 1475 1484. Luder, C.G., Algner, M., Lang, C., Bleicher, N., Gross, U., 2003. Reduced expression of the inducible nitric oxide synthase after infection with Toxoplasma gondii facilitates parasite replication in activated murine macrophages. Int. J. Parasitol. 33, 833 844. Lykens, J.E., Terrell, C.E., Zoller, E.E., Divanovic, S., Trompette, A., Karp, C.L., et al., 2010. Mice with a selective impairment of IFN-gamma signaling in macrophage lineage cells demonstrate the critical role of IFN-gammaactivated macrophages for the control of protozoan parasitic infections in vivo. J. Immunol. 184, 877 885. Ma, J.S., Sasai, M., Ohshima, J., Lee, Y., Bando, H., Takeda, K., et al., 2014. Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6. J. Exp. Med. 211, 2013 2032. Macmicking, J.D., 2012. Interferon-inducible effector mechanisms in cell-autonomous immunity. Nat. Rev. Immunol. 12, 367 382. Majumdar, T., Chattopadhyay, S., Ozhegov, E., Dhar, J., Goswami, R., Sen, G.C., et al., 2015. Induction of interferon-stimulated genes by IRF3 promotes replication of Toxoplasma gondii. PLoS Pathog. 11, e1004779. Marino, N.D., Panas, M.W., Franco, M., Theisen, T.C., Naor, A., Rastogi, S., et al., 2018. Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii. PLoS Pathog. 14, e1006828. Martens, S., Parvanova, I., Zerrahn, J., Griffiths, G., Schell, G., Reichmann, G., et al., 2005. Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47resistance GTPases. PLoS Pathog. 1, e24. Masek, K.S., Fiore, J., Leitges, M., Yan, S.F., Freedman, B.D., Hunter, C.A., 2006. Host cell Ca21 and protein kinase C regulate innate recognition of Toxoplasma gondii. J. Cell Sci. 119, 4565 4573. Masek, K.S., Zhu, P., Freedman, B.D., Hunter, C.A., 2007. Toxoplasma gondii induces changes in intracellular calcium in macrophages. Parasitology 134, 1973 1979. Mashayekhi, M., Sandau, M.M., Dunay, I.R., Frickel, E.M., Khan, A., Goldszmid, R.S., et al., 2011. CD8alpha(1) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites. Immunity 35, 249 259. Matta, S.K., Patten, K., Wang, Q., Kim, B.H., Macmicking, J.D., Sibley, L.D., 2018. NADPH oxidase and guanylate binding protein 5 restrict survival of avirulent type III strains of Toxoplasma gondii in naive macrophages. MBio 9, e01393 18.

1101

Melo, M.B., Kasperkovitz, P., Cerny, A., Konen-Waisman, S., Kurt-Jones, E.A., Lien, E., et al., 2010. UNC93B1 mediates host resistance to infection with Toxoplasma gondii. PLoS Pathog. 6, e1001071. Melo, M.B., Jensen, K.D., Saeij, J.P., 2011. Toxoplasma gondii effectors are master regulators of the inflammatory response. Trends Parasitol. 27, 487 495. Menard, K.L., Haskins, B.E., Colombo, A.P., Denkers, E.Y., 2018. Toxoplasma gondii manipulates expression of host long noncoding RNA during intracellular infection. Sci. Rep. 8, 15017. Meredith, M.M., Liu, K., Darrasse-Jeze, G., Kamphorst, A. O., Schreiber, H.A., Guermonprez, P., et al., 2012. Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage. J. Exp. Med. 209, 1153 1165. Miller, C.M., Boulter, N.R., Fuller, S.J., Zakrzewski, A.M., Lees, M.P., Saunders, B.M., et al., 2011. The role of the P2X(7) receptor in infectious diseases. PLoS Pathog. 7, e1002212. Molloy, M.J., Grainger, J.R., Bouladoux, N., Hand, T.W., Koo, L.Y., Naik, S., et al., 2013. Intraluminal containment of commensal outgrowth in the gut during infection-induced dysbiosis. Cell Host Microbe 14, 318 328. Mordue, D.G., Sibley, L.D., 1997. Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry. J. Immunol. 159, 4452 4459. Mordue, D.G., Sibley, L.D., 2003. A novel population of Gr-1 1 -activated macrophages induced during acute toxoplasmosis. J. Leukoc. Biol. 74, 1015 1025. Mordue, D.G., Desai, N., Dustin, M., Sibley, L.D., 1999a. Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring. J. Exp. Med. 190, 1783 1792. Mordue, D.G., Hakansson, S., Niesman, I., Sibley, L.D., 1999b. Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways. Exp. Parasitol. 92, 87 99. Mordue, D.G., Scott-Weathers, C.F., Tobin, C.M., Knoll, L.J., 2007. A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages. Mol. Microbiol. 63, 482 496. Moreira-Souza, A.C.A., Almeida-Da-Silva, C.L.C., Rangel, T.P., Rocha, G.D.C., Bellio, M., Zamboni, D.S., et al., 2017. The P2X7 receptor mediates Toxoplasma gondii control in macrophages through canonical NLRP3 inflammasome activation and reactive oxygen species production. Front. Immunol. 8, 1257.

Toxoplasma Gondii

1102

25. Innate immunity to Toxoplasma gondii

Moretti, J., Roy, S., Bozec, D., Martinez, J., Chapman, J.R., Ueberheide, B., et al., 2017. STING senses microbial viability to orchestrate stress-mediated autophagy of the endoplasmic reticulum. Cell 171, 809 823.e13. Morisaki, J.H., Heuser, J.E., Sibley, L.D., 1995. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J. Cell Sci. 108 (Pt 6), 2457 2464. Mosser, D.M., Edwards, J.P., 2008. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958 969. Murray, H.W., Cohn, Z.A., 1979. Macrophage oxygendependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii to oxygen intermediates. J. Exp. Med. 150, 938 949. Murray, H.W., Juangbhanich, C.W., Nathan, C.F., Cohn, Z.A., 1979. Macrophage oxygen-dependent antimicrobial activity. II. The role of oxygen intermediates. J. Exp. Med. 150, 950 964. Murray, H.W., Rubin, B.Y., Carriero, S.M., Harris, A.M., Jaffee, E.A., 1985. Human mononuclear phagocyte antiprotozoal mechanisms: oxygen-dependent vs oxygenindependent activity against intracellular Toxoplasma gondii. J. Immunol. 134, 1982 1988. Naor, A., Panas, M.W., Marino, N., Coffey, M.J., Tonkin, C.J., Boothroyd, J.C., 2018. MYR1-dependent effectors are the major drivers of a host cell’s early response to Toxoplasma, including counteracting MYR1-independent effects. MBio 9, e02401 17. Nathan, C., Shiloh, M.U., 2000. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl. Acad. Sci. U.S.A. 97, 8841 8848. Niedelman, W., Sprokholt, J.K., Clough, B., Frickel, E.M., Saeij, J.P., 2013. Cell death of gamma interferonstimulated human fibroblasts upon Toxoplasma gondii infection induces early parasite egress and limits parasite replication. Infect. Immun. 81, 4341 4349. Nolan, S.J., Romano, J.D., Coppens, I., 2017. Host lipid droplets: an important source of lipids salvaged by the intracellular parasite Toxoplasma gondii. PLoS Pathog. 13, e1006362. Olias, P., Etheridge, R.D., Zhang, Y., Holtzman, M.J., Sibley, L.D., 2016. Toxoplasma effector recruits the Mi-2/ NuRD complex to repress STAT1 transcription and block IFN-gamma-dependent gene expression. Cell Host Microbe 20, 72 82. Ohshima, J., Lee, Y., Sasai, M., Saitoh, T., Su Ma, J., Kamiyama, N., et al., 2014. Role of mouse and human proteins in IFN-gamma-induced cell autonomous responses against Toxoplasma gondii. J. Immunol. 192, 3328 3335. Ong, Y.C., Reese, M.L., Boothroyd, J.C., 2010. Toxoplasma rhoptry protein 16 (ROP16) subverts host function by

direct tyrosine phosphorylation of STAT6. J. Biol. Chem. 285, 28731 28740. Oswald, I.P., Gazzinelli, R.T., Sher, A., James, S.L., 1992. IL10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity. J. Immunol. 148, 3578 3582. Peixoto, L., Chen, F., Harb, O.S., Davis, P.H., Beiting, D.P., Brownback, C.S., et al., 2010. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses. Cell Host Microbe 8, 208 218. Pepper, M., Dzierszinski, F., Wilson, E., Tait, E., Fang, Q., Yarovinsky, F., et al., 2008. Plasmacytoid dendritic cells are activated by Toxoplasma gondii to present antigen and produce cytokines. J. Immunol. 180, 6229 6236. Pernas, L., Adomako-Ankomah, Y., Shastri, A.J., Ewald, S.E., Treeck, M., Boyle, J.P., et al., 2014. Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS Biol. 12, e1001845. Pernas, L., Bean, C., Boothroyd, J.C., Scorrano, L., 2018. Mitochondria restrict growth of the intracellular parasite Toxoplasma gondii by limiting its uptake of fatty acids. Cell Metab. 27, 886 897 e4. Peterson, P.K., Gekker, G., Hu, S., Chao, C.C., 1995. Human astrocytes inhibit intracellular multiplication of Toxoplasma gondii by a nitric oxide-mediated mechanism. J. Infect. Dis. 171, 516 518. Pfefferkorn, E.R., 1984. Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan. Proc. Natl. Acad. Sci. U.S.A. 81, 908 912. Pfefferkorn, E.R., Eckel, M., Rebhun, S., 1986. Interferongamma suppresses the growth of Toxoplasma gondii in human fibroblasts through starvation for tryptophan. Mol. Biochem. Parasitol. 20, 215 224. Pierog, P.L., Zhao, Y., Singh, S., Dai, J., Yap, G.S., Fitzgerald-Bocarsly, P., 2018. Toxoplasma gondii inactivates human plasmacytoid dendritic cells by functional mimicry of IL-10. J. Immunol. 200, 186 195. Pifer, R., Benson, A., Sturge, C.R., Yarovinsky, F., 2011. UNC93B1 is essential for TLR11 activation and IL-12dependent host resistance to Toxoplasma gondii. J. Biol. Chem. 286, 3307 3314. Pino, P., Foth, B.J., Kwok, L.Y., Sheiner, L., Schepers, R., Soldati, T., et al., 2007. Dual targeting of antioxidant and metabolic enzymes to the mitochondrion and the apicoplast of Toxoplasma gondii. PLoS Pathog. 3, e115. Plattner, F., Yarovinsky, F., Romero, S., Didry, D., Carlier, M.F., Sher, A., et al., 2008. Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response. Cell Host Microbe 3, 77 87.

Toxoplasma Gondii

References

Portillo, J.A., Okenka, G., Reed, E., Subauste, A., Van Grol, J., Gentil, K., et al., 2010. The CD40-autophagy pathway is needed for host protection despite IFN-gammadependent immunity and CD40 induces autophagy via control of P21 levels. PLoS One 5, e14472. Portillo, J.C., Muniz-Feliciano, L., Lopez Corcino, Y., Lee, S.J., Van Grol, J., Parsons, S.J., et al., 2017. Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy. PLoS Pathog. 13, e1006671. Praefcke, G.J.K., 2018. Regulation of innate immune functions by guanylate-binding proteins. Int. J. Med. Microbiol. 308, 237 245. Qin, A., Lai, D.H., Liu, Q., Huang, W., Wu, Y.P., Chen, X., et al., 2017. Guanylate-binding protein 1 (GBP1) contributes to the immunity of human mesenchymal stromal cells against Toxoplasma gondii. Proc. Natl. Acad. Sci. U. S.A. 114, 1365 1370. Quan, J.H., Huang, R., Wang, Z., Huang, S., Choi, I.W., Zhou, Y., et al., 2018. P2X7 receptor mediates NLRP3dependent IL-1beta secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells. Parasit Vectors 11, 1. Raetz, M., Kibardin, A., Sturge, C.R., Pifer, R., Li, H., Burstein, E., et al., 2013. Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin. J. Immunol. 191, 4818 4827. Reichmann, G., Walker, W., Villegas, E.N., Craig, L., Cai, G., Alexander, J., et al., 2000. The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis. Infect. Immun. 68, 1312 1318. Reis e Sousa, C., Hieny, S., Scharton-Kersten, T., Jankovic, D., Charest, H., Germain, R.N., et al., 1997. In vivo microbial stimulation induces rapid CD40 ligandindependent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J. Exp. Med. 186, 1819 1829. Robben, P.M., Mordue, D.G., Truscott, S.M., Takeda, K., Akira, S., Sibley, L.D., 2004. Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype. J. Immunol. 172, 3686 3694. Robben, P.M., Laregina, M., Kuziel, W.A., Sibley, L.D., 2005. Recruitment of Gr-1 1 monocytes is essential for control of acute toxoplasmosis. J. Exp. Med. 201, 1761 1769. Romano, J.D., Sonda, S., Bergbower, E., Smith, M.E., Coppens, I., 2013. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole. Mol. Biol. Cell 24, 1974 1995. Rosowski, E.E., Lu, D., Julien, L., Rodda, L., Gaiser, R.A., Jensen, K.D., et al., 2011. Strain-specific activation of the

1103

NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208, 195 212. Saeij, J.P., Frickel, E.M., 2017. Exposing Toxoplasma gondii hiding inside the vacuole: a role for GBPs, autophagy and host cell death. Curr. Opin. Microbiol. 40, 72 80. Saeij, J.P., Coller, S., Boyle, J.P., Jerome, M.E., White, M.W., Boothroyd, J.C., 2007. Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445, 324 327. Safronova, A., Araujo, A., Camanzo, E.T., Moon, T.J., Elliott, M.R., Beiting, D.P., et al., 2019. Alarmin S100A11 initiates a chemokine response to the human pathogen Toxoplasma gondii. Nat. Immunol. 20, 64 72. Samarajiwa, S.A., Forster, S., Auchettl, K., Hertzog, P.J., 2009. Interferome: the database of interferon regulated genes. Nucleic Acids Res. 37, D852 D857. Sasai, M., Sakaguchi, N., Ma, J.S., Nakamura, S., Kawabata, T., Bando, H., et al., 2017. Essential role for GABARAP autophagy proteins in interferon-inducible GTPasemediated host defense. Nat. Immunol. 18, 899 910. Sautel, C.F., Ortet, P., Saksouk, N., Kieffer, S., Garin, J., Bastien, O., et al., 2009. The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences. Mol. Microbiol. 71, 212 226. Scanga, C.A., Aliberti, J., Jankovic, D., Tilloy, F., Bennouna, S., Denkers, E.Y., et al., 2002. Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J. Immunol. 168, 5997 6001. Scharton-Kersten, T., Nakajima, H., Yap, G., Sher, A., Leonard, W.J., 1998. Infection of mice lacking the common cytokine receptor gamma-chain (gamma(c)) reveals an unexpected role for CD4 1 T lymphocytes in early IFN-gamma-dependent resistance to Toxoplasma gondii. J. Immunol. 160, 2565 2569. Scharton-Kersten, T.M., Wynn, T.A., Denkers, E.Y., Bala, S., Grunvald, E., Hieny, S., et al., 1996. In the absence of endogenous IFN-gamma, mice develop unimpaired IL12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157, 4045 4054. Scharton-Kersten, T.M., Yap, G., Magram, J., Sher, A., 1997. Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplasma gondii. J. Exp. Med. 185, 1261 1273. Schluter, D., Deckert-Schluter, M., Lorenz, E., Meyer, T., Rollinghoff, M., Bogdan, C., 1999. Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/ 6 mice but does not reactivate the latent disease in T. gondii-resistant BALB/c mice. J. Immunol. 162, 3512 3518.

Toxoplasma Gondii

1104

25. Innate immunity to Toxoplasma gondii

Schreiner, M., Liesenfeld, O., 2009. Small intestinal inflammation following oral infection with Toxoplasma gondii does not occur exclusively in C57BL/6 mice: review of 70 reports from the literature. Mem. Inst. Oswaldo Cruz 104, 221 233. Schulthess, J., Fourreau, D., Darche, S., Meresse, B., Kasper, L., Cerf-Bensussan, N., et al., 2008. Mucosal immunity in Toxoplasma gondii infection. Parasite 15, 389 395. Schulthess, J., Meresse, B., Ramiro-Puig, E., Montcuquet, N., Darche, S., Begue, B., et al., 2012. Interleukin-15dependent NKp46 1 innate lymphoid cells control intestinal inflammation by recruiting inflammatory monocytes. Immunity 37, 108 121. Schwartzman, J.D., Gonias, S.L., Pfefferkorn, E.R., 1990. Murine gamma interferon fails to inhibit Toxoplasma gondii growth in murine fibroblasts. Infect. Immun. 58, 833 834. Selleck, E.M., Orchard, R.C., Lassen, K.G., Beatty, W.L., Xavier, R.J., Levine, B., et al., 2015. A noncanonical autophagy pathway restricts Toxoplasma gondii growth in a strain-specific manner in IFN-gamma-activated human cells. MBio 6, e01157 15. Serbina, N.V., Pamer, E.G., 2006. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat. Immunol. 7, 311 317. Sergent, V., Cautain, B., Khalife, J., Deslee, D., Bastien, P., Dao, A., et al., 2005. Innate refractoriness of the Lewis rat to toxoplasmosis is a dominant trait that is intrinsic to bone marrow-derived cells. Infect. Immun. 73, 6990 6997. Sher, A., Tosh, K., Jankovic, D., 2017. Innate recognition of Toxoplasma gondii in humans involves a mechanism distinct from that utilized by rodents. Cell. Mol. Immunol. 14, 36 42. Sibley, L.D., Weidner, E., Krahenbuhl, J.L., 1985. Phagosome acidification blocked by intracellular Toxoplasma gondii. Nature 315, 416 419. Sibley, L.D., Adams, L.B., Fukutomi, Y., Krahenbuhl, J.L., 1991. Tumor necrosis factor-alpha triggers antitoxoplasmal activity of IFN-gamma primed macrophages. J. Immunol. 147, 2340 2345. Skariah, S., Bednarczyk, R.B., Mcintyre, M.K., Taylor, G.A., Mordue, D.G., 2012. Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates. J. Immunol. 188, 3404 3415. Steinfeldt, T., Konen-Waisman, S., Tong, L., Pawlowski, N., Lamkemeyer, T., Sibley, L.D., et al., 2010. Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol. 8, e1000576.

Sturge, C.R., Benson, A., Raetz, M., Wilhelm, C.L., Mirpuri, J., Vitetta, E.S., et al., 2013. TLR-independent neutrophil-derived IFN-gamma is important for host resistance to intracellular pathogens. Proc. Natl. Acad. Sci. U.S.A. 110, 10711 10716. Sturge, C.R., Burger, E., Raetz, M., Hooper, L.V., Yarovinsky, F., 2015. Cutting edge: developmental regulation of IFN-gamma production by mouse neutrophil precursor cells. J. Immunol. 195, 36 40. Subauste, C.S., Koniaris, A.H., Remington, J.S., 1991. Murine CD8 1 cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells. J. Immunol. 147, 3955 3959. Subauste, C.S., Dawson, L., Remington, J.S., 1992. Human lymphokine-activated killer cells are cytotoxic against cells infected with Toxoplasma gondii. J. Exp. Med. 176, 1511 1519. Sukhumavasi, W., Egan, C.E., Warren, A.L., Taylor, G.A., Fox, B.A., Bzik, D.J., et al., 2008. TLR adaptor MyD88 is essential for pathogen control during oral Toxoplasma gondii infection but not adaptive immunity induced by a vaccine strain of the parasite. J. Immunol. 181, 3464 3473. Suss-Toby, E., Zimmerberg, J., Ward, G.E., 1996. Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore. Proc. Natl. Acad. Sci. U.S.A. 93, 8413 8418. Suzuki, Y., Orellana, M.A., Schreiber, R.D., Remington, J.S., 1988. Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science 240, 516 518. Suzuki, Y., Sher, A., Yap, G., Park, D., Neyer, L.E., Liesenfeld, O., et al., 2000. IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii. J. Immunol. 164, 5375 5382. Tait, E.D., Jordan, K.A., Dupont, C.D., Harris, T.H., Gregg, B., Wilson, E.H., et al., 2010. Virulence of Toxoplasma gondii is associated with distinct dendritic cell responses and reduced numbers of activated CD8 1 T cells. J. Immunol. 185, 1502 1512. Takacs, A.C., Swierzy, I.J., Luder, C.G., 2012. Interferongamma restricts Toxoplasma gondii development in murine skeletal muscle cells via nitric oxide production and immunity-related GTPases. PLoS One 7, e45440. Taylor, G.A., Collazo, C.M., Yap, G.S., Nguyen, K., Gregorio, T.A., Taylor, L.S., et al., 2000. Pathogenspecific loss of host resistance in mice lacking the IFNgamma-inducible gene IGTP. Proc. Natl. Acad. Sci. U.S.A. 97, 751 755. Tobin, C.M., Knoll, L.J., 2012. A patatin-like protein protects Toxoplasma gondii from degradation in a nitric oxide-dependent manner. Infect. Immun. 80, 55 61.

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References

Tomita, T., Yamada, T., Weiss, L.M., Orlofsky, A., 2009. Externally triggered egress is the major fate of Toxoplasma gondii during acute infection. J. Immunol. 183, 6667 6680. Tosh, K.W., Mittereder, L., Bonne-Annee, S., Hieny, S., Nutman, T.B., Singer, S.M., et al., 2016. The IL-12 response of primary human dendritic cells and monocytes to Toxoplasma gondii is stimulated by phagocytosis of live parasites rather than host cell invasion. J. Immunol. 196, 345 356. Tretina, K., Park, E.S., Maminska, A., Macmicking, J.D., 2019. Interferon-induced guanylate-binding proteins: guardians of host defense in health and disease. J. Exp. Med. 216, 482 500. Tsuge, I., Matsuoka, H., Nakagawa, A., Kamachi, Y., Aso, K., Negoro, T., et al., 1998. Necrotizing toxoplasmic encephalitis in a child with the X-linked hyper-IgM syndrome. Eur. J. Pediatr. 157, 735 737. Tu, L., Moriya, C., Imai, T., Ishida, H., Tetsutani, K., Duan, X., et al., 2009. Critical role for the immunoproteasome subunit LMP7 in the resistance of mice to Toxoplasma gondii infection. Eur. J. Immunol. 39, 3385 3394. Ueno, N., Lodoen, M.B., Hickey, G.L., Robey, E.A., Coombes, J.L., 2015. Toxoplasma gondii-infected natural killer cells display a hypermotility phenotype in vivo. Immunol. Cell Biol. 93, 508 513. Wang, Y., Weiss, L.M., Orlofsky, A., 2009. Host cell autophagy is induced by Toxoplasma gondii and contributes to parasite growth. J. Biol. Chem. 284, 1694 1701. Whitmarsh, R.J., Gray, C.M., Gregg, B., Christian, D.A., May, M.J., Murray, P.J., et al., 2011. A critical role for SOCS3 in innate resistance to Toxoplasma gondii. Cell Host Microbe 10, 224 236. Wilson, C.B., Tsai, V., Remington, J.S., 1980. Failure to trigger the oxidative metabolic burst by normal macrophages: possible mechanism for survival of intracellular pathogens. J. Exp. Med. 151, 328 346. Witola, W.H., Mui, E., Hargrave, A., Liu, S., Hypolite, M., Montpetit, A., et al., 2011. NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells. Infect. Immun. 79, 756 766. Witola, W.H., Kim, C.Y., Zhang, X., 2017. Inherent oxidative stress in the Lewis rat is associated with resistance to toxoplasmosis. Infect. Immun. 85, 756 766.

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Woods, S., Schroeder, J., Mcgachy, H.A., Plevin, R., Roberts, C.W., Alexander, J., 2013. MAP kinase phosphatase-2 plays a key role in the control of infection with Toxoplasma gondii by modulating iNOS and arginase-1 activities in mice. PLoS Pathog. 9, e1003535. Yamamoto, M., Okuyama, M., Ma, J.S., Kimura, T., Kamiyama, N., Saiga, H., et al., 2012. A cluster of interferon-gamma-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii. Immunity 37, 302 313. Yap, G.S., Sher, A., 1999. Effector cells of both nonhemopoietic and hemopoietic origin are required for interferon (IFN)-gamma- and tumor necrosis factor (TNF)alpha-dependent host resistance to the intracellular pathogen, Toxoplasma gondii. J. Exp. Med. 189, 1083 1092. Yarovinsky, F., Zhang, D., Andersen, J.F., Bannenberg, G. L., Serhan, C.N., Hayden, M.S., et al., 2005. TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 308, 1626 1629. Zhao, Y., Ferguson, D.J., Wilson, D.C., Howard, J.C., Sibley, L.D., Yap, G.S., 2009a. Virulent Toxoplasma gondii evade immunity-related GTPase-mediated parasite vacuole disruption within primed macrophages. J. Immunol. 182, 3775 3781. Zhao, Y.O., Khaminets, A., Hunn, J.P., Howard, J.C., 2009b. Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death. PLoS Pathog. 5, e1000288. Zhao, Z., Fux, B., Goodwin, M., Dunay, I.R., Strong, D., Miller, B.C., et al., 2008. Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens, Cell Host Microbe, 4. pp. 458 469. Zhou, W., Quan, J.H., Lee, Y.H., Shin, D.W., Cha, G.H., 2013. Toxoplasma gondii proliferation require downregulation of host Nox4 expression via activation of PI3 kinase/Akt signaling pathway. PLoS One 8, e66306. Zimmermann, S., Murray, P.J., Heeg, K., Dalpke, A.H., 2006. Induction of suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling. J. Immunol. 176, 1840 1847.

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C H A P T E R

26 Adaptive immunity Nicolas Blanchard1, Anna Salvioni1 and Ellen A. Robey2 1

Center for Pathophysiology Toulouse-Purpan (CPTP), INSERM, CNRS, University of Toulouse, Toulouse, France 2Department of Molecular and Cell Biology, University of California, Berkeley, CA, United States

26.1 Introduction While innate immunity limits Toxoplasma gondii growth during the first hours and days after infection, the adaptive (or acquired) immune system plays a crucial role in the days and weeks that follow. Indeed, T. gondii is able to persist long term in its mammalian host thanks to the adaptive immune system, which keeps parasite growth in check and allows the host to survive, providing a long-term niche for the parasite that ultimately facilitates transmission to other hosts. Proper balance of the adaptive immune response is the key for the survival of both parasite and host, since inadequate immune responses (as in immunocompromised or fetal hosts) lead to uncontrolled parasite replication and severe disease, whereas excessive immune responses lead to immune-mediated pathology. In contrast to innate immunity (Chapter 25 “Innate immunity to Toxoplasma gondii”) which is relatively nonspecific and rapid, adaptive immunity is highly specific for a particular pathogen, and it develops over a period of days

Toxoplasma Gondii DOI: https://doi.org/10.1016/B978-0-12-815041-2.00026-8

or weeks. While innate immunity is largely mediated by cells of bone marrow origin (termed myeloid cells) such as macrophages and dendritic cells (DC), adaptive immunity is mediated by highly specialized classes of white blood cells termed lymphocytes, including T cells and B cells (Fig. 26.1A). Finally, while innate immunity uses preformed receptors with broad specificity for particular classes of pathogens (termed pattern recognition receptors), the adaptive immune system uses clonotypically variable antigen receptors encoded by gene segments that undergo somatic gene rearrangement during development (antibodies and T cell antigen receptors or TCRs). This chapter summarizes our current understanding of how the adaptive immune system recognizes and responds to T. gondii infection. The chapter begins with a brief overview of the mammalian adaptive immune system, emphasizing those components which are most relevant in the setting of T. gondii infection. The discussion then progresses providing a more detailed description of the adaptive immune response to T. gondii. Since most of the adaptive immune

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FIGURE 26.1 An overview of the mammalian adaptive immune system. (A) Specialized lymphoid cells derived from a common lymphoid progenitor carry out the recognition and effector functions of adaptive immunity. Cell types are shown in gray, and immune protective “effector” mechanisms are indicated in purple. Within the αβ T cell lineage, CD8 1 T cells play a key protective role during Toxoplasma gondii infection by producing the immune-protective cytokine IFN-γ. CD4 1 T cells help to regulate the immune response, in part through the action of regulatory T cells (Treg) which produce immune-suppressive cytokines IL-10 and TGF-β. B cells contribute to protection by producing antibodies that bind specifically to the parasite. γδ T cells are an alternative thymus-derived T cell population whose function during T. gondii infection is not well understood. NK cells have features of both adaptive and innate immunity and, along with CD8 and CD4 T cells, produce the key protective cytokine IFN-γ. (B) T cells detect specific pathogens via their clonotypic TCR, which recognizes pathogen-derived peptides displayed on the surface of APCs bound to MHC proteins. In this example, a CD8 1 T cell recognizes a peptide presented by MHC class I (MHC I), resulting in the release of cytokines and/or the killing of the pathogen-infected cell. APCs, Antigen presenting cells; IFN-γ, interferon-γ; IL-10, interleukin 10; MHC, major histocompatibility complex; NK, natural killer; TCR, T cell antigen receptor; TGF-β, transforming growth factor β.

mechanisms during toxoplasmosis have been identified and dissected using mouse models, this chapter mostly refers to data generated in those models.

26.1.1 αβ T cells While all major components of adaptive immunity are activated during T. gondii infection, the most prominent and protective response is from αβ T cells, particularly CD8 1 T cells. Specific recognition of pathogens by αβ T cells is mediated by clonally variable TCRs which bind to pathogen-derived peptides displayed on the surface of antigen-presenting cells (APCs) bound to major histocompatibility

complex (MHC) encoded proteins (Fig. 26.1B). MHC proteins are highly polymorphic, and different allelic forms have distinct specificity for peptides, contributing to differences in T cell responses to pathogens between different individual humans or different inbred strains of mice. T cells develop in the thymus, where T cell progenitors rearrange and express their TCR genes and where they undergo a stringent selection process. As a result, the mature T cells that emerge from the thymus are not overtly reactive to self-MHC but have the potential to respond to pathogen-derived peptides presented by MHC molecules. After mature T cells exit the thymus, they circulate between secondary lymphoid organs

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26.1 Introduction

such as spleen and lymph nodes, waiting to be called into action. Meanwhile, when pathogens, such as T. gondii, enter the body, they activate tissue-resident DC (via the innate signaling pathways discussed in Chapter 25 “Innate immunity to Toxoplasma gondii”). Some of these DC also acquire pathogen-derived peptides and migrate from tissues to lymph nodes or spleen where they can engage with T cells of the appropriate specificity. Approximately 1/106 naı¨ve T cells will possess such specificity, due to the particular form of the TCR that they express. These T cells can form a tight cell cell contact with the peptide-loaded DC, triggering the “priming” (i.e., clonal expansion and further maturation) of the T cell. After T cell priming, pathogen-specific T cells are found at a higher frequency. They acquire effector functions, such as the ability to produce cytokines or kill target cells. They also acquire the ability to enter infected tissues, where they encounter APC displaying the same pathogen-derived peptide that induced their priming, allowing them to deliver effector molecules at sites of active infection throughout the body. αβ T cells are composed of two major functional subsets, CD8 1 T cells that recognize pathogen-derived peptides presented by MHC class I molecules (MHC I) and CD4 1 T cells that recognize peptides presented by MHC class II molecules (MHC II). CD8 and CD4 expression on T cells also correlates with functional potential. CD4 1 T cells differentiate into a variety of “helper” T cells, which are specialized to regulate other immune cells, including B cells and CD8 1 T cells. On the other hand, CD8 1 T cells give rise to cytotoxic or “killer” T cells upon effector differentiation and are specialized to defend against intracellular pathogens. Effector CD8 1 T cells can directly kill host cells harboring intracellular pathogens, thus disrupting their replicative niche. Effector CD8 1 T cells can also secrete the immune-protective cytokines interferon-γ

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(IFN-γ) and tumor necrosis factor-α (TNF-α), which act on neighboring cells to enhance their ability to control pathogen replication (Fig. 26.1B). These two cytokines are particularly important for the immune control of T. gondii. While CD8 1 T cells have a relative limited range of effector activities, CD4 1 T cells can differentiate into a variety of different functional helper T cell subsets that produce distinct cytokine profiles. Most relevant for T. gondii infection is a subset known as T helper 1 (Th1) that, such as CD8 effector cells, produces the important immune-protective cytokines, IFN-γ and TNF-α. T cells can also give rise to alternative helper subsets, such as Th2 cells that produce IL-4 and IL-13, Th17 cells that produce IL17, and follicular helper T cells (Tfh) that produce IL-21 and are essential for the formation of germinal centers and promote more effective humoral responses. T cells can also differentiate into regulatory T cells (Treg) that secrete the immunosuppressive cytokines IL-10 and TGF-β, and play a key role in maintaining homeostasis by suppressing excessive immune responses. This is pivotal in situations of infection when exacerbated responses can be deleterious for the host and cause immune pathology. Following initial activation, one additional outcome of T cell differentiation is the formation of memory T cell populations, which either recirculate from secondary lymphoid organs to tissues or reside within tissues without recirculating. Memory T cells are typically able to mount faster and stronger responses toward the invading pathogen; they are thus at the heart of long-lasting immune protection. In keeping with their role in presenting peptides from intracellular pathogens to CD8 1 T cells, MHC I proteins generally acquire their peptides from cytosolic or nuclear proteins via the classical MHC I presentation pathway (termed the endogenous pathway). In contrast, MHC II proteins acquire their peptides from

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proteins that are taken up via phagocytosis or endocytosis (termed the exogenous pathway). There are also important exceptions to this dichotomy between the MHC I/endogenous and MHC II/exogenous antigen presentation pathways, as will be discussed later in this chapter. Interestingly, while T. gondii is an intracellular pathogen, it resides within the host cell in a specialized parasitophorous vacuole (PV) that limits the ability of parasite proteins to access the classical MHC I presentation pathway. The mechanisms by which parasite antigens are presented by MHC molecules will be discussed in more details in a later section.

26.1.2 Other adaptive cell types While T cells possess only a cell surface form of the antigen receptor and require MHC and a presenting cell for antigen recognition, B cells produce a secreted version of their antigen receptor (termed antibody or immunoglobulin) which can bind directly to antigen. Like T cells, B cells assemble their antigen receptor genes during development by somatic DNA recombination, resulting in a diverse B cell population with each B cell or clone expressing a unique antigen specificity. Also like T cells, B cells initially exist in a quiescent or “naı¨ve” form and require activation by antigen encounter to trigger clonal expansion and differentiation into antibody secreting cells. Activated B cells secrete antibody into extracellular spaces in tissues and mucosal sites. Antibodies can then bind directly to pathogens, marking them for destruction by other components of the immune system. The presence of T. gondii specific antibodies is often used to identify infected individuals, although their protective role appears to be secondary to that of αβ T cells in mouse models of toxoplasmosis. In addition to the more prominent and wellcharacterized αβ T cells, there is also a subset of thymus-derived T cells that expresses an

alternative γδ TCR, termed γδ T cells. γδ T cells can secrete many of the same cytokines as αβ T cells, but they appear to play a more modest role during T. gondii infection than αβ T cells. Finally, natural killer (NK) cells are lymphocytes that have some characteristics of both adaptive and innate immune cells. They do not express clonally diverse antigen receptors but instead are triggered by changes in the balance of activating and inhibiting receptors on target cells. Like CD8 1 and CD4 1 T cells, NK cells can produce the protective cytokine IFN-γ. NK cells exhibit increased activation during T. gondii infection and also appear to play a secondary protective role relative to CD8 1 T cells.

26.1.3 Dendritic cells: innate sentinels that initiate and shape adaptive immunity Although DC are not adaptive immune cells, they play a decisive role in triggering, polarizing and sustaining adaptive responses. As covered in Chapter 25 “Innate immunity to Toxoplasma gondii”, DC are sentinels that excel at sensing a variety of environmental cues to which they respond by maturing into highly efficient APCs. In this process, DC uptake, process, and present antigens via MHC I and MHC II. DC are superior to other cells for activating naı¨ve T cells, thus they are considered as the most potent APC for priming T cell responses. Depending on the subset of DC and the type of ligands that were sensed, DC shape the functional outcome of the adaptive immune response, for example, by promoting the differentiation of T cells into a particular Th profile. At steady-state, there are two main categories of DC, the plasmacytoid DC (pDC) and the conventional DC (cDC). cDC can be further subdivided into type I (cDC1) and type II (cDC2). In a context of infection or inflammation, monocytes can give rise to an additional category called inflammatory DC. Different DC

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26.2 How is Toxoplasma gondii “seen” by the adaptive immune system?

subsets exhibit specialized functions. pDC are well adapted to respond to viral infection by producing type I IFN, but pDC are also capable of antigen presentation, including in the context of T. gondii infection. The cDC1 subset comprises lymphoid tissue-resident CD8α 1 DC as well as migratory CD103 1 DC that are found in non-lymphoid tissues and can migrate to the draining lymph nodes. cDC1 are best equipped for initiating CD8 1 T cell responses toward exogenous antigens and for promoting Th1 responses, which is particularly relevant in the context of T. gondii infection. cDC2 are important for MHC II presentation, in particular for the differentiation of Tfh.

26.2 How is Toxoplasma gondii “seen” by the adaptive immune system? 26.2.1 Antigen presentation by major histocompatibility complex molecules to T cells It was initially discovered that MHC I molecules are loaded with peptides produced endogenously by the protein synthesis machinery of the APC, whereas MHC II molecules display peptides derived from antigens acquired from the extracellular space (Blum et al., 2013; Guermonprez et al., 2002). It is now well established that inverse situations exist and that they play major functions in controlling T cell responses. In brief, MHC II molecules can present endogenous antigens, such as those contained in autophagosomes (Jurewicz and Stern, 2019; Valecka et al., 2018). Moreover, the MHC I pathway is able to present peptides derived from exogenous, internalized products, including those originating from intracellular pathogens. The pathway by which APC present antigens that have not been synthesized within them is coined MHC I cross-presentation (Blander, 2018; Embgenbroich and Burgdorf, 2018).

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While virtually all nucleated cells express MHC I and thus have the ability to present antigens to CD8 1 T cells via the “classical” MHC I pathway, MHC I cross-presentation appears to be most effective in specific subsets of DC. Notably, however, in certain contexts, other cell types, for example, brain endothelial cells and liver sinusoid endothelial cells, are also endowed with the ability to perform cross-presentation. Whereas MHC II presentation naturally occurs in “professional” APC, comprising tissueresident macrophages (including microglia in the brain), DC and B lymphocytes, “non-professional” APC, for example, high endothelial venule and blood endothelial cells, stromal cells, hepatocytes, can also present MHC II peptides, in particular when the MHC II has been upregulated by an inflammatory environment.

26.2.2 Major histocompatibility complex class I presentation 26.2.2.1 The classical major histocompatibility complex I presentation pathway Final products of the classical MHC I pathway are typically 8 10 residue peptides that are loaded in the antigen-binding groove of the MHC I heavy chain, complexed with the “light chain” β-2-microglobulin (β2m). Depending on the MHC I allele, antigenic peptides share varying consensus motifs that allow anchoring of the peptide within the peptide-binding groove. The classical pathway is the merger of two independent pathways, joining at the level of the transporter associated with antigen processing (TAP) molecule. One pathway generates peptide-receptive MHC I molecules in the endoplasmic reticulum (ER), thanks to housekeeping molecules, which preserve correct folding of MHC I prior to peptide loading (Adiko et al., 2015; Peaper and Cresswell, 2008). By degrading antigenic proteins, the other pathway shapes the final peptides that will be loaded onto the MHC molecules.

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FIGURE 26.2 The classical MHC I processing pathway generates MHC I ligands from endogenous antigens for CD8 1 T cell recognition. In all nucleated cells, cytosolic antigens are degraded by cytosolic proteases, including the proteasome, into shorter polypeptides. These antigenic precursors are imported into the endoplasmic reticulum via the TAP transporter. Amino-terminal processing can further degrade the proteolytic intermediates into final peptides of appropriate size and consensus motifs that, with the help of chaperone molecules within the peptide-loading complex, bind to nascent MHC I heavy chains. Stable peptide-MHC I heavy chain-β2 microglobulin complexes can then traffic to the cell surface. MHC, Major histocompatibility complex; TAP, transporter associated with antigen processing.

Source antigens in the cytoplasm are fragmented into smaller polypeptides by the proteasome, with the assistance of other proteases and chaperones. The exact nature of the source antigens is still debated, but it seems clear that both end-of-life proteins as well as newly synthesized, possibly defective, products are relevant antigenic sources (Anton and Yewdell, 2014; Rock et al., 2016). Following transport of chaperone-protected proteolytic intermediates into the ER by TAP, proteolysis continues in the ER through N-terminal trimming by ERlocalized aminopeptidases (Blanchard and

Shastri, 2008; Weimershaus et al., 2013). Stable complexes formed between MHC I and optimally sized peptides leave the ER on their way to the cell surface, to be ultimately surveyed by patrolling CD8 1 T cells (Fig. 26.2). 26.2.2.2 The major histocompatibility complex I cross-presentation (or exogenous) pathways Contrary to the classical pathway that displays peptides synthesized within the APC, the cross-presentation pathway allows for the presentation of antigens that are internalized from

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26.2 How is Toxoplasma gondii “seen” by the adaptive immune system?

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FIGURE 26.3 The MHC I cross-presentation pathways generate MHC I ligands from exogenous antigens for CD8 1 T cell recognition. Exogenous antigens internalized may be processed and presented by MHC I on professional APC by to two distinct pathways. (A) In the “cytosolic” pathway, antigens are translocated from the phago/endosome to the cytosol through a Sec61-based transport machinery that is imported from the ER into the phago/endosomal membrane. Either the last steps resemble that of the classical MHC I pathway and the peptides are loaded onto MHC I following import into the ER (1), or the peptides are transported back into the intracellular organelle for loading onto recycling MHC I molecules (2). (B) In the “vacuolar” pathway, both antigen fragmentation and loading occur within the intracellular organelle that contains the antigen. APCs, Antigen presenting cells; MHC, major histocompatibility complex.

the surrounding environment, whether it be via macropinocytosis, phagocytosis, receptormediated endocytosis, or active parasite invasion. Two major scenarios of cross-presentation have been described: the “cytosolic” and the “vacuolar” pathways. The most common scenario is the “cytosolic” pathway, also known as phagosome-tocytosol pathway. This pathway involves uptake of antigens into intracellular organelles, followed by their translocation into the cytosol. This translocation relies on a protein

machinery that is normally used for ERassociated degradation (ERAD), a qualitycontrol process during which misfolded proteins are retrotranslocated from the ER to the cytosol. Sec61, a major effector of ERAD, plays a prominent role in cytosol escape of phagosome- and endosome-contained antigens. This mechanism implies that components of the ER, and/or of the ER-Golgi intermediate compartment (ERGIC), fuse with the antigencontaining organelle. This results in the import of the Sec61-based ERAD machinery onto the

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antigen-containing organelle (Fig. 26.3A). Efficient cross-presentation requires limited antigen degradation within the phagosome before cytosol export. The low degradative nature of early endocytic compartments in DC compared to macrophages partially explains why they are superior in cross-presentation (Blander, 2018; Embgenbroich and Burgdorf, 2018). Two possibilities exist once the antigens have been shuttled to the cytosol and processed by the proteasome: either the proteolytic intermediates are transported into the ER or they are transported back to the intracellular organelle of origin. In the latter case, final loading of MHC I occurs in the endocytic compartment and involves recycling MHC I molecules (Blander, 2018; Cebrian et al., 2016; Nair-Gupta et al., 2014). In the less frequently observed “vacuolar” pathway, particulate antigens taken up during phagocytosis or endocytosis are degraded within the endocytic compartment itself. In this context, peptide loading onto MHC I likely occurs in the same endocytic compartment. This process is typically TAP-independent and resistant to proteasome inhibition (Blander, 2018; Embgenbroich and Burgdorf, 2018) (Fig. 26.3B). Such a mechanism applies in the case of Leishmania parasites but not T. gondii (Bertholet et al., 2006).

26.2.3 Major histocompatibility complex I presentation of Toxoplasma gondii antigens 26.2.3.1 The role of secretion Active secretion of antigens from the parasite into the host cell is a general requirement for efficient MHC I presentation of T. gondii antigens. Using transgenic type II parasites expressing a secreted versus non-secreted version of the heterologous β-galactosidase (β-gal) model antigen (see Table 26.1 for a description of T. gondii endogenous and model T cell

antigens), it was reported that only β-gal-secreting tachyzoites are competent to prime β-gal specific CD8 1 T cells (Kwok et al., 2003). The fact that initiation of CD8 1 T cell responses requires active protein secretion by T. gondii rather than the degradation of phagocytosed parasites and/or parasite products was confirmed with the ovalbumin (OVA) model antigen, which is presented only when secreted in the vacuolar space or to a lower extent when GPI-anchored at the parasite membrane but not when confined within the parasite cytosol, mitochondria, or inner membrane complex (Gregg et al., 2011). Beyond the necessity of being released, both the mode of secretion and the trafficking properties of secreted antigens strongly influence MHC I presentation. First, the type of source organelle releasing the antigen (e.g., rhoptries that are injected once into host cells during invasion or dense granules that are constitutively secreted into the PV), affects the efficacy of parasite-specific CD8 1 T cell responses. Secretion of the endogenous ROP5 antigen through dense granules enhances ROP5specific CD8 1 T cell responses, possibly thanks to the antigen building up within infected cells (Grover et al., 2014). Second, as shown by manipulating the localization of the endogenous GRA6 antigen, targeting that antigen to the PV limiting membrane provides superior MHC I processing efficiency than when forced to have a vacuolar localization (Lopez et al., 2015). The topology of membrane insertion also plays a critical role since inverting the orientation of the epitope (i.e., swapping it from cytosol-exposed to vacuole-exposed) reduces its access to the MHC I pathway (Buaillon et al., 2017). 26.2.3.2 The role of actively infected cells In line with the need for the antigen to be secreted into the host cell, the induction of CD8 1 T cell responses relies on DC that are actively infected. Ex vivo presentation of the

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26.2 How is Toxoplasma gondii “seen” by the adaptive immune system?

TABLE 26.1 Natural and model antigens used to study T cell responses to Toxoplasma gondii in mice. Endogenous/natural antigens Name of protein

ToxoDB ID (TGME49_xxx)

MHC restriction

Peptide sequence

Localization

References

GRA4

310780

H-2k1

SVSTEDSGLTGVKD

iTVN

Chardes et al. (1993)

SAG1

233460

H-2d1

Not characterized

Parasite surface

Khan et al. (1994)

d

GRA6

275440

H-2 L

HPGSVNEFDF

PV limiting membrane iTVN

Blanchard et al. (2008)

GRA4

310780

H-2 Ld

SPMNGGYYM

iTVN

Frickel et al. (2008)

ROP7

295110

H-2 Ld

IPAAAGRFF

Unknown

Frickel et al. (2008)

Tgd057

015980

H-2 Kb

SVLAFRRL

PV lumen

Wilson et al. (2010)

ROP5

308090

b

H-2 D

YAVANYFFL

PV limiting membrane iTVN

Grover et al. (2014)

Profilin

293690

H-2 I-Ab

Not characterized

Cytoskeleton

Yarovinsky et al., (2006)

28m (GRA32) 212300

H-2 I-Ab

AVEIHRPVPGTAPPS Unknown

Grover et al. (2012)

Model antigens Name of model antigen β-Galactosidase

Details on protein MHC sequence restriction d

Peptide sequence

Localization Parasite stage expression

References

Escherichia coli β-gal

H-2 L

SAG1-OVA (vacOVA)

SAG1ΔGPI-OVA [140-386]

H-2 Kb

SIINFEKL

PV lumen (vacuolar space)

Gubbels et al. (2005)

OVA with different subcellular localizations

OVA[140-386]

H-2 Kb

SIINFEKL

PV lumen

Gregg et al. (2011)

TPHPARIGL Parasite cytosol or PV lumen Tachyzoite-restricted or bradyzoite-restricted

Plasma membrane

Kwok et al. (2003)

Cytosolic Mitochondria IMC GRA6-OVA

GRA6(II)-LEQLESIINFEKL

b

H-2 K

SIINFEKL

PV limiting membrane and iTVN

Salvioni et al. (2019)

Both stages or tachyzoiterestricted 1 Restricting MHC I molecule unknown. Columns show MHC restriction, subcellular localization of the source antigen, stage expression and the initial reference describing each antigen. iTVN, intravacuolar tubulovesicular network; MHC, major histocompatibility complex; OVA, ovalbumin; PV, parasitophorous vacuole; IMC, inner membrane complex.

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vacuolar SAG1-OVA model antigen to reporter CD8 1 T cell hybridomas occurs primarily in actively infected DC rather than bystander DC (Gubbels et al., 2005). The critical role of actively infected cells in initiating T. gondii specific CD4 1 and CD8 1 T cell responses has been established using a cytometry-based approach enabling to discriminate between active invasion and phagocytic uptake (Dupont et al., 2014). Phagocytosis of heat-killed or invasion-blocked parasites is not sufficient to induce T cell responses, but transfer of DC or macrophages actively infected with a replicationattenuated strain (and not those that phagocytosed the parasite) potently induces T cell responses, conferring protection against challenge with virulent T. gondii (Dupont et al., 2014). 26.2.3.3 The impact of antigen biochemical properties and trafficking Luminal versus membrane-bound antigens seem to follow processing mechanisms that are partly similar and partly distinct (Fig. 26.4). For both types of antigens, TAP and the proteasome are involved, suggesting that antigenic precursors exit the PV and transit via the host cytosol before MHC I loading (Bertholet et al., 2006; Blanchard et al., 2008; Gubbels et al., 2005). For luminal antigens, as exemplified with the SAG1-OVA model antigen, entry into the cytosol of DC is facilitated by fusion of vesicles from the host ERGIC onto the PV (Goldszmid et al., 2009) through a mechanism that is dependent on the soluble NSF attachment protein receptor (SNARE) protein Sec22b (Cebrian et al., 2011) (Fig. 26.4A). This process, which recruits components of the ERAD machinery onto the PV membrane, is largely reminiscent of the “cytosolic” mode of crosspresentation (see Fig. 26.3A). It is thought to allow retrotranslocation of luminal antigens to the host cytosol. In IFN-γ-primed APC, including macrophages but not DC (Dzierszinski et al., 2007),

an additional gateway to the MHC I pathway exists for luminal antigens. In a process that is dependent on certain immunity-related GTPases (Irgm1 and Irgm3) and autophagy proteins, the PV limiting membrane is decorated with poly-ubiquitin chains and is recognized by the P62 sequestosome (SQSTM1). The accumulation of p62/Sqstm1 on T. gondii vacuoles after damage by IFN-γ-inducible GTPases contributes to the cytosolic release of luminal antigens and the activation of CD8 1 T cells specific for vacuolar antigens (Lee et al., 2015) (Fig. 26.4B). Finally, similar to what has been suggested for phagosomal antigens (Blander, 2018; NairGupta et al., 2014), the pool of recycling MHC I molecules also plays a major role in T. gondii presentation by DC. Rab22a, a protein widely distributed in the endocytic network of DC, controls MHC I intracellular distribution, recycling, and trafficking. Rab22a is recruited to the T. gondii PV and is required for efficient presentation of luminal antigens by infected DC, suggesting that whole or part of the MHC I loading may occur outside the ER, in the PV or in endocytic compartments (Cebrian et al., 2016) (Fig. 26.4A). Remarkably, a number of features differ concerning the processing mechanisms of membrane-bound antigens. While production of the GRA6-derived immunodominant peptide, which is necessary to mount protective CD8 1 T cell-mediated responses, requires aminopeptidase trimming in the ER (Blanchard et al., 2008), processing of that antigen is independent from Sec22b-mediated ER-PV interactions (Buaillon et al., 2017). Instead, optimal processing requires the antigenic epitope to be located at the C-terminus of the GRA6 source antigen. Indeed, displacing the C-terminal epitope reduces its immunogenicity, and inserting a subdominant epitope to the C-terminal position ameliorates the response (Feliu et al., 2013) (Fig. 26.4C).

Toxoplasma Gondii

FIGURE 26.4 MHC I antigen presentation pathways for luminal and membrane-bound T. gondii derived antigens. Depending on the biochemical properties of the antigen [i.e., luminal (A, B) or membrane-bound (C, D)], various mechanisms explain how T. gondii secreted antigens are degraded and presented onto MHC I molecules by infected DC or macrophages. (A, B) In the case of luminal antigens (e.g., SAG1-OVA), two non-mutually exclusive scenarios have been described. (A) Antigenic proteins are exported to the host cytosol thanks to the Sec22b-mediated recruitment of a host ER Sec61-based translocation machinery (Cebrian et al., 2011; Goldszmid et al., 2009). Antigens are then further processed via the “classical” MHC I pathway, including peptide loading in the ER. Based on the importance of Rab22a-controlled recycling of MHC I molecules in T. gondii antigen presentation (Cebrian et al., 2016), a fraction of peptides may be loaded onto MHC I outside of the ER, possibly in ERC. (B) Upon IFN-γ stimulation, in a pathway dependent on the autophagy machinery and the IRG system, the PV membrane is decorated with poly-ubiquitin chains and recognized by the P62 sequestosome. P62 may bind to and target vacuolar antigens to the proteasome although the nature of the ubiquitinated targets remains unclear (Jensen, 2016; Lee et al., 2015). Antigens may cross the PV membrane due to complete or partial disruption of the latter. (C, D) In the case of membrane-bound antigens (i.e., the T. gondii dense granule GRA6 protein or variants thereof, that are integrated within the PV membrane and the membranous iTVN), ER PV interactions do not seem to be important for MHC I presentation (Buaillon et al., 2017). (C) Based on the topology of the C-terminal domain of GRA6 that protrudes in the host cytosol (Buaillon et al., 2017), a yet unknown protease is thought to liberate a fragment that then enters the ER through TAP and is further processed by ER aminopeptidases before loading onto MHC I (Blanchard et al., 2008). (D) When the iTVN is prevented from forming in the PV (e.g., in GRA2-deficient parasites), GRA6 preferentially localizes to the PV membrane and more readily enters the MHC I antigen presentation pathway (Lopez et al., 2015), suggesting that this structure exerts a modulatory function on CD8 1 T cell recognition of T. gondii. ERC, Endocytic recycling compartments; IRG, immunity-related GTPases; iTVN, intravacuolar tubulo-vesicular network; MHC, major histocompatibility complex; PV, parasitophorous vacuole; TAP, transporter associated with antigen processing.

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26.2.4 Modulation of the major histocompatibility complex I presentation pathway by Toxoplasma gondii While it is likely that T. gondii cysts interfere with the MHC I presentation to escape CD8 1 T cell recognition and to persist in the brain and muscle, the molecular mechanisms behind this evasion remain unknown. A scenario of immune modulation affecting MHC I presentation has been described in tachyzoite-infected DC and macrophages. In this context, presentation of the GRA6 antigen is negatively regulated by a membranous intravacuolar tubulovesicular network (iTVN) generated by the parasite itself in a GRA2-dependent manner. In the absence of iTVN, GRA6 is redistributed at the PV limiting membrane and is presented at higher levels by MHC I both in vitro and in vivo, which leads to enhanced CD8 1 T cell responses in a vaccination context (Lopez et al., 2015) (Fig. 26.4D).

26.3 Initiation (priming) of T cell responses by dendritic cells Using type II parasites secreting the OVA model antigen, it was found early on that spleen cells from infected mice that are enriched in CD11c1 cDC are more potent than unsorted or DC-depleted cells to present the OVA epitope to reporter T cell hybridomas (Gubbels et al., 2005). Among cDC, the important role of cDC1 was revealed using Batf3 KO mice, which selectively lack lymphoid-resident CD8α 1 DC (and the related peripheral CD103 1 DC) at steady-state. Eight days postinfection with type II parasites, Batf3 KO animals exhibit a reduced IFN-γ production by GRA4 and GRA6-specific CD8 1 T cells in the spleen and peritoneum, indicating that cDC1 are essential for CD8 1 T cell priming (Mashayekhi et al., 2011). Of note, CD4 1 T cell responses and thus CD4 1 T cell help to

CD8 1 T cells is also perturbed in these mice, which may contribute to this finding. Intriguingly, absence of the cDC1-specific major regulator of cross-presentation WDFY4 does not alter survival upon T. gondii infection (Theisen et al., 2018), suggesting either that cross-priming of T. gondii specific CD8 1 T cells is not dependent on WDFY4 and/or that other DC subsets can compensate in the absence of cross presentation-competent cDC1.

26.4 Major histocompatibility complex class II presentation MHC II α and β chains are synthesized in the ER, where they associate with the invariant chain, called Ii or CD74, a nonpolymorphic chaperone that occupies the MHC II peptide-binding groove and prevents further peptide loading in the biosynthetic pathway. Within late endosomal compartments, aspartic and cysteine proteases (e.g., cathepsins), which auto-catalytically activate in acidic environment, degrade proteins into smaller antigenic peptides and cleave CD74 to yield a class II associated invariant-chain peptide (CLIP) that remains lodged into the MHC II groove. Thereafter, the peptide editor H-2 DM displaces CLIP, which promotes binding of higher affinity peptides onto mature MHC II molecules. The peptideMHC II complex is finally transported to the surface for presentation to CD4 1 T cells (Fig. 26.5A)

26.4.1 Major histocompatibility complex II presentation of Toxoplasma gondii antigens As of now, only one natural T. gondii MHC II ligand presented by I-Ab in C57BL/6 mice has been described (see Table 26.1) and the exact processing pathways leading to T. gondii

Toxoplasma Gondii

26.4 Major histocompatibility complex class II presentation

1119

FIGURE 26.5 MHC II presentation pathway for exogenously acquired antigens and consequences of T. gondii infection. (A) Internalized antigens are processed into smaller fragments by lysosomal proteases (1). In parallel, heterodimers of MHC II α and β chains assemble in the ER along with a nonpolymorphic chaperone called invariant chain, or Ii. Upon proteolytic cleavage of Ii, a peptide (CLIP) remains bound in the MHC II peptide-binding groove when the MHC II molecules move forward in the secretory pathway (2). The “editing” function of HLA/H-2 DM is needed to replace the CLIP peptide by an antigenic peptide, which may be further adjusted by proteases while associated with the MHC II peptide-binding groove. (B) The mechanisms of T. gondii MHC II presentation remain poorly studied. However, infection is known to have direct modulatory effects on the MHC II pathway by enhancing the expression of CD74 and reducing the expression of DM (Leroux et al., 2015b). T. gondii infection also indirectly affects the MHC II pathway by impeding the IFN-γ-mediated upregulation of MHC II (Lang et al., 2012; Luder et al., 2003). CLIP, Class II associated invariant-chain peptide; MHC, major histocompatibility complex.

MHC II presentation have yet to be elucidated. Using parasites expressing a secreted versus non-secreted version of the OVA model antigen, it was reported that antigen secretion is important for CD4 1 T cell proliferation and IFN-γ production (Pepper et al., 2004). Interestingly, the natural MHC II antigen (28m) contains a predicted signal sequence, suggesting that it is a secretory protein. However,

robust presentation of the 28m-derived AS15 peptide is observed even when DC are fed heat-killed parasites (Draheim et al., 2017; Grover et al., 2012), in line with the notion that both secreted and non-secreted parasite antigens have access to the MHC II antigen degradative compartments, most likely via phagocytosis of whole parasites or infected cell fragments.

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26. Adaptive immunity

26.4.2 Modulation of the major histocompatibility complex II presentation pathway by Toxoplasma gondii Several lines of evidence show that T. gondii tachyzoites manipulate MHC II presentation, both at steady-state and following stimulation of APC with IFN-γ. First, independently from IFN-γ, T. gondii infection enhances the expression of CD74, which accumulates in the ER of infected cells. Combined with the reduced transcription and expression of H2-DM, these two phenomena converge to inhibit MHC II presentation of parasite-derived antigens (Leroux et al., 2015b) (Fig. 26.5B). Second, IFN-γ typically induces transcriptional activation of MHC II and other related genes, but active T. gondii infection is known to impede IFN-γ-mediated upregulation of MHC II. In cell types such as human glioblastoma cells, primary rat astrocytes, microglia (Luder et al., 2003), and murine macrophage cell lines (Lang et al., 2006), T. gondii infection indeed disturbs chromatin remodeling at the class II transactivator CIITA locus (Lang et al., 2012; Luder et al., 2003). The dysregulation of IFN-γ-inducible gene expression is linked to a blockade of the transcriptional activity of STAT1, a transcription factor involved in activation of the CIITA promoter (Kim et al., 2007; MuhlethalerMottet et al., 1998). The parasite effector(s) responsible for this effect was initially reported to be a rhoptry and/or dense granule-derived soluble protein (Leroux et al., 2015a). Notably, a parasite secreted effector that translocates to the host cell nucleus and represses STAT1dependent promoters (T. gondii inhibitor of STAT1-dependent transcription, TgIST) (Gay et al., 2016; Olias et al., 2016) was recently identified, making it likely that the inhibition of IFN-γ-mediated MHC II upregulation is linked to TgIST injection into infected APC. Functionally, the consequence of preventing upregulation of MHC II on the surface of infected DC is to make them refractory to

subsequent activation by TLR ligands or CD40 signaling and to impair antigen presentation, leading to altered priming and activation of CD4 1 T cells in vivo (McKee et al., 2004).

26.5 Adaptive immune responses in the intestinal mucosa and associated lymphoid tissues Beside congenital transmission a common infection route for T. gondii is through the gastrointestinal tract, whether it be by ingestion of raw food soiled with feline-shed oocysts or ingestion of tissue cysts (from undercooked meat in the case of humans). Rupture of the cyst or oocyst structure within the intestinal lumen liberates parasites, which invade the surrounding host cells, convert into fast-replicating tachyzoites and disseminate throughout the lamina propria, gut-associated lymphoid tissues before reaching more distant tissues.

26.5.1 Early dissemination in the small intestine In the intestinal villi, the parasite infects goblet cells and enterocytes (Coombes et al., 2013; Dubey, 1997). These cells can produce chemokines, such as CCL2 which is selectively implicated in the recruitment of Gr11 inflammatory monocytes to the ileum villi, providing a first line of defense that controls the initial parasite replication (Dunay et al., 2008). Other chemokines such as MCP-1, MIP-1α, CXCL2, and CXCL10 (Bonnart et al., 2017; Buzoni-Gatel et al., 2001; Gopal et al., 2011; Mennechet et al., 2002) contribute to the recruitment of a variety of innate immune cells (neutrophils/granulocytes, macrophages, monocytes, and DC) as well as T cells. To migrate through the epithelium and reach the lamina propria, the parasite uses various mechanisms involving lysis of infected enterocytes (Chardes et al., 1994),

Toxoplasma Gondii

26.5 Adaptive immune responses in the intestinal mucosa and associated lymphoid tissues

1121

infection of CD11c1 cells via “sensing” processes that extend into the intestinal lumen (Courret et al., 2006) or parasite motility-based paracellular migration (Barragan and Sibley, 2002). After entering the lamina propria, the parasite invades leukocytes and gains access to the lymphatic and blood system. While parasite replication in the intestine is observed within the first week of infection (Bonnart et al., 2017; Coombes et al., 2013; Courret et al., 2006), parasites can be detected by PCR as early as 24 hours postingestion in gut-associated lymphoid tissues, such as Peyer’s patches and mesenteric lymph nodes (Dubey, 1997).

infection may have a global and possibly negative impact on gut homeostasis. In practice, C57BL/6 mice infected per os with .20 type II T. gondii cysts have provided a useful model to analyze the molecular and cellular bases of an exacerbated form of T. gondii driven gut pathology. Mice develop massive necrosis of the villi and severe ileitis, which cause death of the animals within 10 days. This disease is aggravated by Gram-negative bacteria (Heimesaat et al., 2006) and is associated with the loss of Paneth cells and their antimicrobial peptides, as well as with a profound dysbiosis characterized by the expansion of Enterobacteriaceae bacteria (Raetz et al., 2013).

26.5.2 Intestinal humoral responses to Toxoplasma gondii

26.5.4 Th1/Th17 CD4 1 T cells are main effectors of intestinal pathology

The lamina propria is populated with numerous B cells that can differentiate into immunoglobulin A (IgA) plasmacytoid cells. This switch is naturally favored by the presence of TGF-β in the intestine. Early studies have indicated that acute intestinal T. gondii infection is associated with the production of secretory IgA, beginning at 2 weeks postinfection in serum and milk, and beginning at 3 weeks postinfection in intestinal secretions (Chardes et al., 1990). These antibodies react to a variety of T. gondii proteins, including the SAG1 surface protein and the GRA4 dense granule antigen (Chardes et al., 1993; Mevelec et al., 1998). Although the protective capacity of these IgA responses remains illdefined, eliciting them may represent an attractive mucosal vaccination strategy.

26.5.3 Toxoplasma gondii acute ileitis: a T cell mediated immune pathology Several mammalian species, including nonhuman primates, have been reported to display small intestine inflammation upon ingestion of T. gondii (Schreiner and Liesenfeld, 2009), suggesting that in the animal kingdom, T. gondii

Paradoxically, T cells, and more specifically Th1 CD4 1 T cells, are central mediators of intestinal pathology. Athymic C57BL/6 mice, which lack T cells, do not display necrosis of the ileum and succumb significantly later than control C57BL/6 mice (Liesenfeld et al., 1996). Mice deficient in αβ T cells or CD4 1 T cells do not develop ileitis, whereas wild-type control mice and mice deficient in γδ T cells or CD8 1 T cells do. Furthermore, antibody neutralization of IFNγ or TNF-α before the onset of illness prolong time to death and prevent ileitis (Liesenfeld et al., 1996, 1999), suggesting that Th1 CD4 1 T cells are pivotal effectors of this disease. Th1 CD4 1 T cells contribute to the disease by also promoting IFN-γ-dependent Paneth cell death in conjunction with uncontrolled expansion of Gram-negative bacteria of the Enterobacteriaceae family (Raetz et al., 2013). Of note, the ability of CD4 1 T cells to acquire the Th1 effector phenotype and to mediate immunopathological responses is not dependent on the T-bet transcription factor, which is typically highly expressed in Th1 CD4 1 T cells. The analysis of T. gondii infected WT and Tbx21 2 / 2 mice revealed that T-bet-deficient Th1 cells are fully

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26. Adaptive immunity

capable to induce IFN-γ-mediated intestinal pathology and sufficient for the parasitemediated Paneth cell loss (Lopez-Yglesias et al., 2018). The deleterious implication of Th1 is corroborated by results showing that both IL-12, a major cytokine for Th1 polarization, and IL-18 contribute to the development of intestinal immunopathology following T. gondii oral infection (Vossenkamper et al., 2004). In addition to Th1 cells, Th17 CD4 1 T cells also seem to favor T. gondii mediated gut inflammatory pathology. Indeed, IL-17RA deficient C57BL/6 mice and C57BL/6 mice receiving IL-17A neutralizing antibody present reduced inflammatory scores and less tissue damage in the ileum (Guiton et al., 2010). While this is in line with the finding that IL-23 is essential in the development of small intestinal immunopathology by locally upregulating MMP-2 and IL-22 production by ileal CD4 1 T cells, this is contrasting with the down-regulation of IL-17 production observed in this context (Munoz et al., 2009). While IFN-γ and TNF-α are clearly required for the immunopathology, they do not appear to be sufficient. Whereas mice deficient for protease-activated receptor 2 (PAR-2), a G protein-coupled receptor that is activated through proteolytic cleavage and is involved in the host inflammatory response, produce similarly high levels of both cytokines as WT mice, these PAR2 KO mice develop less severe T. gondii induced gut inflammation (Bonnart et al., 2017). It is likely that the secretion of proinflammatory chemokines and cytokines that are produced by synergistic interactions of CD4 1 T cells with enterocytes contribute to immunopathogenesis (Mennechet et al., 2002).

26.5.5 Treg and intraepithelial lymphocytes protect the host from gut pathology A subset of CD4 1 T cells that express the Foxp3 transcription factor (Treg) normally play

a key role in maintaining immune homeostasis by suppressing excessive immune responses. As might be expected, CD4 1 Treg cells are beneficial in preventing the pathology and the Treg compartment is globally perturbed in this context. First, the abundance of these cells is reduced during pathology. T. gondii oral infection is accompanied by a dramatic reduction in the number and percentage of Foxp3 1 Treg cells in the lamina propria, which may be due to the blunted production of IL-2 by effector CD4 1 T cells (Oldenhove et al., 2009). Moreover, retinoic acid production, an important cue for induced Treg differentiation, is downregulated in mucosal DC upon T. gondii infection (Cohen and Denkers, 2015). The drop in intestinal Treg likely contributes to ileitis since adoptive transfer of Foxp3 1 CD4 1 Treg cells alleviates intestinal pathology at day 7 postinfection, with reduced IFN-γ and TNF-α detected in the ileum (Olguin et al., 2015). In addition to reduced numbers, Treg functions may also be impaired. Following infection, Treg cells in the intestine display reduced expression of the ectonucleotidase CD73, which regulates inflammatory responses by sequentially degrading extracellular ATP and releasing immunosuppressive adenosine. Consistently, administration of an adenosine receptor agonist improves immunopathology in the gut (Francois et al., 2015). Oral infection also triggers a phenotype shift of Treg toward Th1, with environmental cues provided by both local DC and effector T cells inducing the expression of T-bet and IFN-γ by Tregs (Oldenhove et al., 2009). Besides Tregs, other T cell populations such as T. gondii primed intraepithelial TCRαβ CD8αβ lymphocytes (IEL) exert a major beneficial regulatory function (Buzoni-Gatel et al., 1997, 2001). Being located at the interface with the outside environment, IEL are critical for providing protective immunity while safeguarding the integrity of the epithelial barrier. Following adoptive transfer, T. gondii primed IEL are able to modulate the pathogenic

Toxoplasma Gondii

26.6 Lymphoid system

activity of lamina propria CD4 1 T cells and prevent tissue damage in a CD154 (CD40L)-, CD95L (FasL)-, and IL-10-independent fashion, possibly through elevated secretion of TGF-β (Mennechet et al., 2004).

26.5.6 Intestinal adaptive immunity in chronic phase Of note, oral T. gondii infection has been reported to induce transient bacterial translocation, which is sufficient to result in the formation of microbiota-specific memory T cells. These T cells are functional, and they can be reactivated by subsequent gastrointestinal tract infection (Hand et al., 2012). Along these lines, once the inflammatory pathology has resolved, it is probable that local memory parasite-specific T cells and humoral responses develop and later provide important lines of defense upon T. gondii reinfection. However, the adaptive immune responses in the intestinal environment have remained poorly investigated during the chronic phase.

26.6 Lymphoid system 26.6.1 The pivotal role of the IL-12/IFN-γ axis Once the parasite exits the gut tissue and reaches the blood circulation and lymphatics, a systemic type I immune response is triggered. Type I cytokines, in particular IFN-γ and TNF-α, play a central role in controlling the parasite by activating cell-autonomous parasiticidal mechanisms, such as induction of reactive oxygen and nitrogen species, induction of immunity-related GTPases (IRG) and guanylate-binding proteins, and starvation of important amino acids such as tryptophan. The cytokine IL-12 is the major inducer of type I immunity, promoting the production of IFN-γ from CD8 1 T cells, and promoting the CD4 Th1 fate (see Table 26.2 and Fig. 26.6).

1123

A major source of IL-12 for polarizing the T cell response appears to be a subset of DC called cDC1 which expresses the transcription factor Batf3. Batf3 2 / 2 mice display a loss of the cDC1 population and, upon T. gondii infection, show impaired IL-12 production and increased mortality. This susceptibility is reversed by the administration of IL-12 and mice in which IL-12 production is ablated only from cDC1 fail to control infection, underscoring the critical and exclusive function of these cells (Mashayekhi et al., 2011). Moreover the numbers of IFN-γ producing CD4 1 and CD8 1 T cells are reduced in Batf3 2 / 2 mice infected with T. gondii. This may be the result of both the impact of cDC1derived IL-12 in polarizing the T cell response, as well as a role for this APC population in T cell priming during infection. While cDC1 are major IL-12 producers during T. gondii infection, other cellular sources have been identified. For example, in a model of intraperitoneal infection, inflammatory DC differentiated from monocytes that infiltrate the site of infection are able to produce this cytokine (Goldszmid et al., 2012). Neutrophils (Bliss et al., 2000), macrophages (Robben et al., 2004), and pDC (Pepper et al., 2008) are also capable of IL-12 production during T. gondii infection. Paradoxically, while IL-12 promotes CD8 1 T cell effector differentiation, it also impairs long-term CD8 memory responses. This is illustrated in the context of immunization with a replication-attenuated type I CPS strain. While IL-12-induced T-bet promoted the differentiation of effector T cells (identified as CD62Llow KLRG11) in the spleen and peritoneum (Wilson et al., 2008), IL-12 had a negative impact on the formation of the central memory T cell compartment. These data suggest that excessive exposure to IL-12 during CD8 1 T cell priming is detrimental to long-term protective immunity through the decreased fitness of central memory CTL responses (Wilson et al., 2010).

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TABLE 26.2 Major cytokines implicated in Toxoplasma gondii infection. Cytokine Producing cells

Action

References

IL-12

cDC1, along with pDC, neutrophils

Induces IFN-γ and other type I cytokines in CD8 1 , CD4 1 T cells and NK cells

Mashayekhi et al. (2011)

IFN-γ

CD8 and CD4 T cells and Activates parasite killing activity in a variety of cell NK cells types. Positively regulates its own expression, thus reinforcing type I immunity

Suzuki et al. (1988)

TNF-α

CD8 and CD4 T cells and Activates parasite killing activity in a variety of cell NK cells types

Gazzinelli et al. (1993)

IL-2

T cells, especially CD4 T cells

Promotes survival and function of Tregs and T effector cells. Competition for IL-2 helps to regulate the balance between Teff and Treg during infection

Oldenhove et al. (2009) and Benson et al. (2012)

IL-21

Tfh cells

Promotes immunoglobulin production and supports CD8 T cell function

Moretto et al. (2017)

IL-10

Treg cells and a variety of other cell types

Suppresses immune responses and limits immunopathology

Gazzinelli et al. (1996), Hall et al. (2012) and Jankovic et al. (2007)

IL-27

A variety of cells upon exposure to inflammatory stimuli

Binds to its receptor WSX-1 and limits IFN-γ by T cells

Villarino et al. (2003)

cDC, Conventional dendritic cell; NK, natural killer; pDC, plasmacytoid dendritic cell.

FIGURE 26.6 Cytokines implicated in T. gondii infection. Some of the major cytokines and cell types involved are depicted here. A more comprehensive list is provided in Table 26.2.

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IFN-γ is fundamental for survival during acute infection, as mice in which this cytokine is neutralized with antibody injection all succumb during acute stage (Norose et al., 2001; Suzuki et al., 1988; Suzuki and Remington, 1990). In addition to CD4 1 and CD8 1 T cells, which are major producers of IFN-γ and will be described in more detail below, NK cells secrete IFN-γ and contribute to survival throughout acute infection. The important role of NK-derived IFN-γ was particularly highlighted in contexts of T cell deficiency such as SCID mice (Hunter et al., 1994; Sher et al., 1993) or β2m KO (CD8 1 T cell-deficient) mice (Denkers et al., 1993). Neutrophils can also produce IFN-γ in a TLR11-independent manner, as was reported in mice depleted of NK and T cells (Sturge et al., 2013). In this TLR11 KO context, selective elimination of neutrophils resulted in acute susceptibility to T. gondii, similar to that observed in IFN-γ-deficient mice. In addition to producing IFN-γ, CD8 1 T cells can also provide protection by direct killing of invaded host cells. For example, the pore-forming protein perforin, which plays a key role in CD8 1 T cell killing, is dispensable during the acute phase but contributes to protection 2 months postinfection in TE-susceptible mice (Denkers et al., 1997). In humans a human-specific antimicrobial peptide, granulysin, which selectively destroys microbial membranes and is contained in NK and CD8 1 T cell cytotoxic granules, likely plays a major function in the containment of T. gondii. Granulysin-transgenic C57BL/6 and BALB/c mice exhibit improved resistance to T. gondii throughout acute stage (Dotiwala et al., 2016).

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beneficial for parasite control and deleterious through the excessive production of proinflammatory cytokines that can cause tissue damage. This is exemplified in situations of infection with type I (with a LD100 of 1 tachyzoite) or high doses of type II strain, which lead to early mouse death during acute phase (but not related to intestinal pathology). These lethal outcomes are accompanied by extreme high levels of cytokines in the serum, including IFN-γ, TNF-α, IL-12, and IL-18; extensive liver damage; and TNF-dependent splenic lymphoid cell apoptosis. IL-18 plays a key detrimental role since neutralization of IL-18, but not TNF-α or IFN-γ, slightly increases time to death following lethal type I infection (Mordue et al., 2001). These processes underscore the essentiality of regulatory mechanisms that allow the host to limit tissue damage and acute mortality.

26.6.3 IL-27 IL-27 is a heterodimeric cytokine composed of EBI3 and IL-27p28, produced by accessory cells following exposure to inflammatory stimuli. The receptor for IL-27 (IL-27R) is composed of WSX-1, a receptor homologous to the IL-12 receptor, and gp130, the common receptor chain employed by several cytokines, including IL-6. Using WSX-1-deficient mice, it was established that signaling through IL-27R is critical for limiting the intensity and duration of T cell activity, in particular the excessive production of IFN-γ, thereby alleviating immunopathology during acute toxoplasmosis (Villarino et al., 2003).

26.6.4 IL-10

26.6.2 Immunoregulation during Toxoplasma gondii infection In acute systemic toxoplasmosis, T cell responses play dual roles by being both

IL-10 is a major regulatory cytokine produced in large quantities during T. gondii infection by Treg cells, as well as several cell types including NK, T, and B cells (Khan et al., 1995).

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As shown using infected IL-10 KO mice, IL-10 synthesis during acute toxoplasmosis is crucial to dampen CD4 1 T cell dependent IFNγ-mediated early mortality and liver immunopathology, independently from parasite load control (Gazzinelli et al., 1996). Taking advantage of IL-27 KO mice, which succumb from immunopathology during the acute phase (Villarino et al., 2003), a requirement for IL-10 production by Treg was discovered. Indeed, transfer of WT Treg in IL-27 KO mice is protective, while transfer of IL-10 KO Treg is not (Hall et al., 2012). In addition, the analysis of the source of IL-10 revealed that other, FoxP3negative, CD4 1 T cells are important producers of IL-10. Foxp3-negative IL-10 2 producing CD4 1 T cells simultaneously secrete IFN-γ. Moreover, IL-102 IFN-γ1 CD4 1 T cells from infected mice are able to secrete IL-10 upon further stimulation in vitro. These findings indicate that IL-10 production can also originate from regular Th1 cells as part of the effector response (Jankovic et al., 2007).

26.6.5 Glucocorticoids and antiinflammatory lipids Additional molecules beyond regulatory cytokines can alleviate the pathogenicity of Th1 responses and contribute to hamper immunopathology triggered during T. gondii acute phase. For example, endogenous glucocorticoids (GC) are induced during acute T. gondii infection and are pivotal to negatively regulate Th1 cell responses and prevent collateral tissue damage. Indeed, mice in which the GC receptor has been selectively ablated in T cells rapidly succumb to infection due to immunopathology and exacerbated Th1 cytokine production, despite producing similar levels of IL-12 and IL-27 and controlling the parasite equally well (Kugler et al., 2013). Furthermore, particular classes of lipid mediators involved in antagonizing or in resolving

inflammation are also involved in helping the host to “tolerate” the parasite and the associated inflammatory responses and thus to survive throughout acute phase. For instance, T. gondii infection markedly activates the biosynthesis of lipoxin A4 (LXA4), a potent antiinflammatory compound, which is proposed to elicit a state of paralysis of DC associated with the suppression of IL-12 production (Aliberti et al., 2002; Bannenberg et al., 2004).

26.6.6 CD8 1 T cells 26.6.6.1 CD8 1 T cells play a prominent role in controlling Toxoplasma gondii The protective roles of CD8 1 T cells after primary infection and in a vaccination context have been established in a variety of different mouse strains. For example, transfer of immune splenocytes from BALB/c (H-2d) mice vaccinated with an attenuated ts-4 strain protects the recipient mice from lethal challenge. Depletion of CD8 1 T cells from these immune cells before transfer completely abrogates protection, while depletion of CD4 1 T cells has a partial effect (Suzuki and Remington, 1988). The beneficial role of immune CD8 1 T cells against lethal challenge was confirmed in a subsequent study, this time isolating CD8 1 T cells from chronically infected T. gondii infected BALB/c (Parker et al., 1991). In primarily infected JCL-ICR mice, CD8 1 T cells are the predominant subset contributing to the early IFN-γ response and to resistance to acute infection (Shirahata et al., 1994). A comparison of CD4 KO and CD8 KO C57BL/6 mice infected with type II parasites showed that survival throughout acute phase is strongly dependent on the presence of CD8 1 T cells, whereas the loss of CD4 1 T cells only has a minor effect (Schaeffer et al., 2009). CD8 1 T cells also play a critical protective role upon T. gondii secondary infection. This has been addressed in C57BL/6 mice that were

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first exposed to a virulent type III parasites to establish chronic infection and then challenged with virulent strains. While WT mice all survive a secondary infection with RH, depletion of CD8 1 T cells, but not depletion of CD4 1 T cells, completely abolishes the protection (Splitt et al., 2018). 26.6.6.2 CD8 1 T cell response in susceptible and resistant mouse strains The course of T. gondii infection is highly dependent on the genetic background of the host animal. While in the rat, genetic differences in innate immune response lead to dramatic differences in resistance to initial infection (Chapter 7 “Toxoplasma animal models and therapeutics"), in mice, a difference in the ability to control infection during the chronic phase has been characterized. Specifically, resistant BALB/c mice develop an asymptomatic persistent infection and have a normal lifespan, much like immunocompetent humans. On the other hand, many mouse strains, including C57BL/6, initially control acute infection but then develop chronic, progressing disease, eventually succumbing to toxoplasmic encephalitis (TE). Early studies showed that resistance to TE could be transferred by introducing an MHC I gene encoding Ld from BALB/c mice, into the C57BL/6 background, implicating the Ld-restricted CD8 1 T cell response as the key difference (Brown and McLeod, 1990). The majority of mouse infection studies have been performed using the C57BL/6 strain, which is the background strain for most available mutations, and which provides a useful model for progressing chronic infection and T cell dysfunction (see “T cell exhaustion” section about T cell exhaustion in the TE context). Infection of Ld-expressing mice provides an alternative model that is valuable for studies of effective control of persistent infection by CD8 1 T cells (see Fig. 26.7). Initial studies tracking specific T cell responses during T. gondii infection relied on

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parasite strains engineered to express model antigens, such as chicken OVA (Gubbels et al., 2005) and LacZ (Kwok et al., 2003). More recently, natural parasite antigens have been identified, including those recognized by T cells in C57BL/6 and BALB/c infected mice (Table 26.1). Both approaches make it possible to study specific T cell responses in vivo using (1) peptide-MHC tetramers as flow cytometry staining reagents that selectively bind to T cells carrying a TCR that is specific for the peptide loaded on the MHC tetramer and (2) engineered mouse strains expressing TCR specific for defined parasite antigens. Studies of CD8 1 T cell responses in TEresistant mice led to the identification of a CD8 1 T cell epitope derived from the dense granule-secreted protein GRA6, which is presented by the MHC I Ld molecule and induces immunodominant CD8 1 T cell responses following infection with type II parasites (Blanchard et al., 2008). GRA6-specific CD8 1 T cells make up the majority of parasitespecific CD8 1 T cells in Ld1 infected mice, whereas T cells directed against other Ldrestricted proteins (dense granule protein GRA4 and the rhoptry protein ROP7) (Frickel et al., 2008) represent ,10% of the response. Either thanks to the endogenous GRA6-specific CD8 1 T cell compartment (Feliu et al., 2013) or following transfer of naı¨ve or activated transnuclear GRA6-specific T cells (Sanecka et al., 2016), clearance of the parasite in the periphery during acute phase is ameliorated by CD8 1 T cells specific for the GRA6 peptide. Moreover, H-2d mice immunized with GRA6 peptide-pulsed DC are able to survive a lethal challenge with type II parasites in a CD8 1 T cell-dependent manner (Blanchard et al., 2008). As detailed in Section 26.7, these same CD8 1 T cells have a critical function in the resistance of H-2d mice to encephalitis. Studies of GRA6-specific T cell response in chronically infected TE-resistant mice revealed a division of labor that helps to explain the

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FIGURE 26.7

Characteristics of T. gondii encephalitis (TE) and mouse models to study chronic toxoplasmosis. (A) Hallmark features of T. gondii encephalitis. (B) Representative brain cortical sections from a TE-susceptible mouse (top) versus a control uninfected mouse (bottom), stained for Iba1, a marker expressed by microglia at steady-state. Red signal indicates T. gondii tachyzoites surrounded by a cluster of activated Iba1 1 cells that display a less ramified, more amoeboid morphology. White arrows point to clusters of activated Iba1 1 cells. (C) Mouse/parasite combinations useful to study different contexts of CNS inflammation, depending on the MHC haplotype expressed by the mouse strain or the CD8 T cell antigen expressed by the parasite strain. CNS, Central nervous system; MHC, major histocompatibility complex. Source: (B) Images by M. Belloy.

long-lasting protective effect of this T cell response. By longitudinally tracking endogenous CD8 1 T cells specific for GRA6 as well as other subdominant responses in the course of infection in TE-resistant H-2bxd F1 mice, it was observed that the GRA6-specific CD8 1 T

cell response expanded much more rapidly than subdominant responses, lacked a typical contraction phase, and did not show signs of functional exhaustion (Chu et al., 2016). The use of TCR-transgenic mice specific for the GRA6 decameric antigenic peptide (TG6)

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revealed that this persistent effective response was sustained by a division of labor in which a relatively quiescent and long-lived memory population gave rise to a proliferative “intermediate” CXCR31 KLRG11 Tint population, which drove the production of large numbers of short-lived CXCR32 KLRG11 cells with high effector potential. The Tint population was less represented in subdominant responses such as those targeting the GRA4 and ROP5 antigens (Chu et al., 2016).

50% of parasitized cells in the blood are T cells, and blocking T cell egress from the lymph nodes resulted in reduced spreading to the spleen and nondraining lymph nodes (Chtanova et al., 2009). Thus, while the predominant function of T cells is protection of the host, they may also be exploited by the parasites to aid in their spread through the body.

26.6.6.3 T cell dynamics during infection in vivo The T. gondii mouse infection model provides a useful system to investigate the in vivo dynamics of T cell responses during infection [reviewed in (Coombes and Robey, 2010)]. Most studies on the in situ dynamics of antigenspecific T cells in the context of T. gondii infection have been performed using OT-I CD8 1 T cells, which express a transgenic TCR specific for the OVA-derived SIINFEKL model epitope, and a parasite strain engineered to express a secreted luminal form of OVA (Schaeffer et al., 2009). Using an experimental setting in which CD8 1 T cells make a recall response to intradermal T. gondii challenge, it was observed that T cells migrate to foci of infection at the subscapular region of draining lymph nodes, where they form stable, antigen-dependent contacts with subscapular macrophages harboring parasites, as well as uninfected DC (Chtanova et al., 2009). Similar observations were made in mesenteric lymph nodes following intraperitoneal infection (John et al., 2009). Infection also profoundly disrupts the lymph node’s architecture, with increased collagen structures and loss of separation between the B and T cell zones (John et al., 2009). Intriguingly, stable contacts with parasitized target cells exposed T cells to direct invasion by parasites, which could be observed passing from the target cell to the T cells upon target cell lysis. Moreover, at 1 week after infection,

CD8 1 T cells are prominent mediators of primary acute resistance to T. gondii but CD4 1 T cells also contribute to resistance. Antibody depletion of CD4 1 T cells before and throughout infection with type II T. gondii leads to an increase in acute mortality in CBA, C57BL/6, and BALB/c mouse backgrounds (Araujo, 1991). CD4 1 T cells are also essential in a vaccination context. Immunization of C57BL/6 mice with DC pulsed with the I-Abrestricted AS15 peptide provides significant protection against subsequent type II parasite challenge, resulting in lower parasite burden in the brain (Grover et al., 2012). CD4 1 T cells play a synergistic role in vaccine-immunity induced by the attenuated ts-4 strain (Gazzinelli et al., 1991).

26.6.7 CD4 1 T cells

26.6.7.1 Help for CD8 1 T cells In humans, the emergence of severe toxoplasmosis upon HIV infection is concomitant with a sharp decline in CD4 1 T cell numbers. Interestingly, however, most cases of toxoplasmic encephalitis in HIV patients occur during the advanced stage of AIDS, when a deficiency in CD8 1 T cells is also evident. This supports the notion that while CD8 1 T cells play an important effector role during chronic T. gondii infection, CD4 1 T cells likely provide an essential help needed for their maintenance. In mice, one piece of evidence for this notion is the fact that following per os infection with a type II strain, CD4 KO mice exhibit a decreased

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long-term CD8 1 T cell response and a progressive mortality associated with increased brain parasite burden (Casciotti et al., 2002). Moreover, the gradual loss of CD8 1 T cell function during chronic infection of the sensitive C57BL/6 mouse strain has been attributed to CD4 1 T cell exhaustion and inadequate help for CD8 1 T cells. Similar to CD8 1 T cells, both parasite-specific and total CD4 1 T cells display increased expression of inhibitory receptors (e.g., PD-1, 2B4, and LAG-3) during chronic infection. The Blimp-1 transcription factor is involved in the dysfunction of CD4 1 T cells since conditional deletion of Blimp-1 in CD4 1 T cells restores their functionality, reverses CD8 1 T cell exhaustion, and leads to enhanced parasite control. Among the various functionally altered CD4 1 T cell subsets during chronic T. gondii infection, Tfh cells may play a prominent role (Moretto et al., 2017). This subset is strongly induced upon infection, and it displays evidence of exhaustion during late (7 weeks) chronic infection, manifested by increased the expression of the inhibitory receptors such as LAG-3 and 2B4. Given that loss of IL-21R signaling correlates with increased CD8 1 T cell exhaustion and that IL21R signaling seems to play a role in T. gondii reactivation, it is proposed that Tfh cells and IL-21 are important regulators of CD8 1 T cell exhaustion during chronic toxoplasmosis (Moretto et al., 2017). 26.6.7.2 Immunosuppression and regulatory CD4 1 T cells (Treg) Contrasting evidence has been published regarding the importance of Treg cells during acute T. gondii infection. In C57BL/6, depletion of Treg cells using anti-CD25 antibody administration has no effect on survival throughout acute phase (Jankovic et al., 2007), while in BALB/c, anti-CD25 treatment prior to infection increases mortality during acute stage (Tenorio et al., 2010). These disparate results may be due to the mouse genetic background and the

use of anti-CD25 antibody, which also affects effector T cell populations. A natural collapse of Treg cells is associated with higher virulence of certain parasite strains and with lethal outcome. In accordance the enhancement of Treg cell survival and stability with IL-2/anti-IL-2 complexes protects mice from T. gondii induced liver immunopathology and mortality (Oldenhove et al., 2009). It is proposed that the reduction in frequency and absolute number of Treg cells is due to IL-2 deprivation by effector CD4 1 T cells, and this it is essential for the initiation of potent Th1 responses and effective control of parasite burden (Benson et al., 2012), highlighting the need for a fine balance between Treg and effector T cells to achieve optimal parasite control while preserving tissue integrity. 26.6.7.3 Antibody production and T follicular helper cells While B cell produced immunoglobulins are dispensable during acute phase, they are essential for survival throughout the chronic infection phase. μMT mice, which are deficient in B cells, survive the acute phase of infection but die at 3 4 weeks postinfection with large areas of necrosis associated with numerous tachyzoites in the brain (Kang et al., 2000). Based on the tight relationship between CD4 1 T cell and B cell responses (i.e., a cross-talk between Tfh CD4 1 cells and B cells is key for mounting higher affinity and longer lasting antibody responses), some of the defects observed in T. gondii infected CD4 KO mice likely arise from a perturbed B cell compartment. The survival of CD4 KO mice chronically infected with type II parasites is indeed prolonged by the administration of immune serum, which illustrates the important role of CD4 1 T cells as helper cells for humoral responses (Johnson and Sayles, 2002). CD4 1 T cells also promote protective humoral responses following vaccination. CD4 KO mice vaccinated with attenuated ts-4 and

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challenged with RH display a shorter survival than vaccinated C57BL/6 mice, and the survival of CD4 KO is improved by the administration of T. gondii immune serum (Johnson and Sayles, 2002). In line with an important role of antibodies during chronic phase, the deficiency in IL-21, a cytokine produced by Tfh cells that promotes immunoglobulin production and B cell memory formation, leads to a defect in IgG production after infection, correlating with a decrease in germinal center B cell numbers. In parallel, IL-21 KO infected mice exhibit an increase in parasite burden. This is consistent with a role for antibody responses in controlling the parasite during chronic phase, although interpretations are complicated by the fact that IL-21 is also clearly involved in maintaining optimal CD4 1 and CD8 1 T cell effector function in the brain (Stumhofer et al., 2013). T. gondii bradyzoites/cysts exhibit a preferential tropism for postmitotic neuronal and muscular cells. As a result, the parasite has the ability to persist for years in the muscle and central nervous system (CNS), two tissues with a peculiar immunological status. So far, most of what is known has been uncovered in the CNS.

26.7 Adaptive immunity in the brain To investigate the functions of adaptive immune cells and effector molecules in the brain during chronic T. gondii infection, two types of models have been used: (1) immunocompetent mice that are chronically infected and that exhibit a varying susceptibility to T. gondii encephalitis according to their genotype (e.g., KO or congenic mice with different MHC) or to the experimental conditions (e.g., injected with depleting/neutralizing antibody), (2) immunocompromised mice (e.g., SCID, nude) undergoing temporary treatment with sulfadiazine, which is discontinued to elicit parasite reactivation. Parasite reactivation during

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chronic phase or suboptimal control of T. gondii during a primary infection can both lead to the development of T. gondii encephalitis (TE). TE is characterized by high cyst burden, foci of tachyzoite replication throughout the CNS, massive immune cell influx, activation of recruited, and local myeloid cells and cerebral tissue damage (Blanchard et al., 2015; Schluter and Barragan, 2019) (Fig. 26.7A, B). In agreement with the parasite reactivation and the cerebral clinical symptoms that occur in chronically infected human individuals with acquired immune deficiency affecting T cells, examination of the mouse models have confirmed that T lymphocytes play a prominent role in the control of CNS parasites and in the limitation of CNS inflammation, and that antibody responses have important, though secondary functions.

26.7.1 T cell entry and behavior in the Toxoplasma gondii infected brain Following activation within secondary lymphoid tissues, T cells undergo changes in the expression of various integrins, selectins, and chemokine receptors, which endow them with the ability to migrate to non-lymphoid organs in order to exert their effector functions. Depending on the combination of homing receptors (homing “signature”), T cells preferentially become equipped to enter a selective tissue. 26.7.1.1 Three ways to enter the brain During pathological conditions, T cells may access the brain by crossing three distinct barriers: the “blood brain barrier” (BBB), the blood-cerebrospinal fluid barrier within the choroid plexus, and the blood leptomeningeal barrier (Engelhardt and Ransohoff, 2012). The BBB represents an interface between the blood circulation and the neural tissue. The BBB tightly controls the passage of cells, molecules, and ions in order to both deliver

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nutrients according to neuronal needs and protect the brain from toxins and pathogens. BBB crossing by T cells is a multistep process that begins with circulating T cells making a first contact with endothelial cells and then rolling onto the endothelial surface until they firmly arrest and crawl until they find a permissive site for diapedesis. This process is mediated by integrins (i.e., VCAM1 and ICAM1), selectins (P-selectin), and chemokine receptors and it enables T cells to gain access to the perivascular space. To enter the brain parenchyma, T cells then secrete matrix metalloproteases, allowing them to cross the glia limitans, a structure formed of processes from astrocytic end feet (Engelhardt and Ransohoff, 2012). A second barrier is found at the level of the choroid plexus, a structure responsible for the synthesis of the cerebrospinal fluid. The choroid plexus parenchyma is delineated by a cuboid epithelium that presents tight junctions and constitutes the blood-CSF barrier. Adhesion molecules such as ICAM1 and VCAM1 are constitutively expressed on the surface of the epithelium in this compartment and are upregulated in the context of injury or infection (Meeker et al., 2012). However, these molecules are expressed on the apical surface of epithelial cells and are therefore hidden from the immune cells within the choroid plexus parenchyma. The transmigration through this epithelium is tightly regulated although the mechanisms are still not fully uncovered. The third access to the brain parenchyma is via the leptomeningeal barrier, which constitutes a blood CSF barrier in the microvessels of the meninges of the brain and spinal cord. This structure consists of endothelial cells and is morphologically distinct from the other two. In particular, the blood leptomeningeal barrier is devoid of astrocytic end feet, making it a more permissive site of infiltration (Meyer et al., 2017). T cell infiltration through this barrier depends on P-selectin, which is constitutively

expressed by the endothelial cells of this barrier (Engelhardt and Ransohoff, 2012). 26.7.1.2 T cell entry in the Toxoplasma gondii infected brain So far, the exact mechanism of T cell entry in the T. gondii infected CNS with regard to the three possible entry gates mentioned above has not been precisely documented. Yet, T. gondii infection has been known for a long time to trigger the upregulation of the cell adhesion molecules ICAM1/CD54 and VCAM1/CD106 on cerebral blood vessel endothelial cells in an IFN-γ-dependent fashion (Deckert-Schluter et al., 1999; Sa et al., 2014; Wang et al., 2007). These molecules are essential for T cell recruitment into the brain parenchyma. In the infected mouse, VCAM1 blocking heavily reduces infiltration of both CD4 1 and CD8 1 T cells in the CNS and, in agreement with the expression of α4β1 (VLA-4, an integrin ligand of VCAM1) by activated T cells, treatment with an anti-VLA4 antibody reduces T cell infiltration in the brain (Sa et al., 2014; Wilson et al., 2009). These data show that the transmigration of T cells in T. gondii infected brains relies on VCAM1/α4β1 integrin interaction. Surprisingly, however, it was shown that mice with neonatal deletion of VCAM1 have normal brain T cell infiltration but fail to control infection during chronic phase (Deckert et al., 2003). This discrepancy may be linked to a compensatory effect of other adhesion molecules or to residual VCAM 1 expression on the choroid plexus of these mice and a potential entry of T cells through this structure. Alternatively, it may just reflect a global disruption of the BBB during acute infection (Estato et al., 2018) and the possibility for T cells to enter the tissue independently from specific integrin cues. In addition to adhesion molecules, chemokines and chemokine receptors regulate the entry of T lymphocytes in the CNS. This is the case for CXCR3, a receptor expressed by

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activated and memory T cells and associated with Th1 responses, and for its ligands, CXCL9 and CXCL10, which are expressed in the T. gondii invaded brain. Blockade of CXCL9 and CXCL10 indeed decreases the accumulation of CD4 1 and CD8 1 T cells in infected brain (Harris et al., 2012; Ochiai et al., 2015). 26.7.1.3 Dynamics of Toxoplasma gondii specific T cells in brain Chemokines and chemokine receptors also regulate the movements of T lymphocytes once inside the T. gondii infected brain parenchyma. Within the CNS, CXCL10 enhances the velocity of CD8 1 T cells and is required for CD8 1 T cells to adopt a Levy walk search mode, a strategy that is considered optimal to find rare targets (Harris et al., 2012). CCL21, the ligand of CCR7, is also upregulated upon infection, and it plays a role in CD4 1 T cell dynamics within the brain. The trajectories of infiltrating CD4 1 T cells are closely associated with an infection-induced CCL21-decorated reticular system of fibers that guide lymphocyte migration (Wilson et al., 2009). Regarding the interactions with APC, the dynamical behavior of CD8 1 T cells in the brain has been evaluated mostly in a context of poorly controlled parasite load, using transgenic parasites expressing the SAG1-OVA model antigen. While transfer of OT-I in these infected mice confers a certain degree of protection during acute phase (Schaeffer et al., 2009), the mice do not clear the parasite and they still develop substantial CNS inflammation (Schaeffer et al., 2009; Wilson et al., 2009). In this context, OVA-specific CD8 1 T cells are found predominantly surrounding foci of tachyzoite replication. While they completely ignore cysts, they make contacts with CD11b 1 CD11c 1 granuloma-like structures containing isolated tachyzoites (Schaeffer et al., 2009). Parasite-specific T cells are also able to establish prolonged interactions with CD11c 1 cells that are not infected (John et al., 2011), suggesting that MHC I cross-presentation of parasite

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debris by bystander APC can occur in the brain (see Section 26.6.6.2). 26.7.1.4 T cell recirculation in the chronically infected brain In a context of active TE, continuous refueling of T cells from the periphery most likely takes place due to BBB permeability. Upon transfer 3 or 4 weeks following infection, OVAspecific OT-I CD8 T cells enter the brain within a few days, whether or not the parasites express their cognate antigen. Yet they persist in situ only if parasites express the cognate antigen (Schaeffer et al., 2009; Wilson et al., 2009). During chronic TE, the presence of CD8 1 T cells in the CNS is largely a consequence of recruitment of T cells from the periphery and not local expansion (Wilson et al., 2009). In TE-resistant H-2d mice, brain infiltration of T cells is also observed during acute phase (between day 8 and 14 postinfection) (Blanchard et al., 2008; Schluter et al., 2002; Salvioni et al, 2019). However, once chronic infection is established, the recruitment of T cells from the periphery is severely blunted, as shown by the absence of effect of anti-CD4 and anti-CD8 depletion on the intracerebral T cell populations (Schluter et al., 2002). Furthermore, these persisting T cells lack a proliferative capacity and undergo a certain degree of apoptosis, potentially explaining their contraction over time in chronically infected TE-resistant mice (Schluter et al., 2002).

26.7.2 Th1 cytokines and cytotoxicity are essential for parasite control in the central nervous system As in the periphery, IFN-γ plays a pivotal role for parasite restriction in the brain. Upon administration of anti-IFN-γ mAb in TEsusceptible CBA or C57BL/6 mice, progression of TE is markedly accelerated. The higher leukocyte infiltration and more abundant

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inflammatory foci result in death of the animals by 9 12 days posttreatment (Gazzinelli et al., 1993; Suzuki et al., 1989). The number of cysts and of tachyzoites in the brain of antiIFN-γ-treated mice is elevated, while no effect on T. gondii specific antibody titer is detected (Gazzinelli et al., 1992; Suzuki et al., 1989). Genetic IFN-γ deficiency recapitulates a similar phenotype. IFN-γ KO mice of the H-2d haplotype that are maintained alive throughout chronic phase thanks to sulfadiazine treatment, die after discontinuation of sulfadiazine with an elevated number of cysts in their brains (Suzuki et al., 2000). Diverse sources of IFN-γ have been reported in the CNS. As expected, a major producer is the T cell compartment. Indeed, upon discontinuation of the sulfadiazine treatment in athymic (T cell-deficient) nude mice, transfer of wild-type but not IFN-γ KO T cells prevents TE development (Wang et al., 2004). Nevertheless, IFN-γ KO mice transferred with spleen cells from immunized mice do not survive upon sulfadiazine discontinuation (Kang and Suzuki, 2001), meaning that IFN-γ production by T cells is of utmost importance, but it is not sufficient. Other sources of IFN-γ may be NK cells, which are fundamental IFN-γ producers during the early phase of infection. However, they were found not to be required as a source of IFN-γ in a TE reactivation model in SCID mice (Kang and Suzuki, 2001). Microglia (Wang and Suzuki, 2007) and neutrophils (Biswas et al., 2017) constitute additional relevant sources of IFN-γ in infected brains. Beside IFN-γ, TNF-α and perforin are central players for parasite control during chronic phase. Neutralization of TNF-α in chronically infected C57BL/6 triggers massive parasite reactivation and exacerbates TE (Gazzinelli et al., 1993). Type II-infected perforin KO C57BL/6 mice exhibit impaired survival at late chronic phase with increased brain parasite load. Since IFN-γ production was unaltered in the periphery, the increased susceptibility of

these mice is not due to impaired production of the latter cytokine (Denkers et al., 1997). The data have been less consistent in TE reactivation settings analyzed with sulfadiazinetreated immunocompromised H-2d mice. On the one hand, transfer of T cells from BALB/c perforin KO mice into BALB/c SCID mice failed to control parasite load in the brain upon the withdrawal of sulfadiazine treatment (Suzuki et al., 2010), also suggesting that perforin plays an important role. On the other hand, transfer of T cells from BALB/c perforin KO mice into BALB/c nude mice achieved efficient brain parasite control, suggesting little need for perforin (Wang et al., 2004). Whether the discrepancy is linked to the type of immunodeficient mice used (SCID vs nude) remains to be investigated. Regardless, the overall trend is that IFN-γ and T cell cytotoxicity are important drivers of parasite control. In keeping with the inhibitory function of IL-4 on Th1 cytokines, which are needed for parasite control in this case, IL-4 KO mice show reduced number of cysts and reduced foci of inflammation (Roberts et al., 1996). IL-17 signaling is also detrimental for protection since chronically infected IL-17RA-deficient mice display lower cyst load and inflammation in the brain (Guiton et al., 2010). CD28 is not important for the development of an efficient intracerebral T cell response, and control of T. gondii in the brain is independent of CD28. Yet CD28 is involved in the development of immune-mediated pathology during TE (Reichmann et al., 1999). Consistent with immunosuppressive cytokines being important for preventing lethal immunopathology during T. gondii infection (see Section 26.6.6.2), antibody-mediated blockade of IL-10 signaling led to severe tissue destruction in the brain, associated with an elevated inflammatory response, including increased APC activation, expansion of CD4 1 T cells, and neutrophil recruitment to the brain (O’Brien et al., 2019).

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26.7 Adaptive immunity in the brain

26.7.3 Roles of CD4 1 and CD8 1 T cells in infected brain Akin to what is reported in the periphery, CD8 1 T cells are the predominant subset involved in parasite control in the brain. While antibody depletion of both CD4 1 and CD8 1 T cells at the same time during chronic phase leads to large areas of parenchymal necrosis with foci of leukocyte infiltration, and is lethal, depletion of only CD8 1 T cells or use of CD8 KO mice results in increased mortality compared to the control group. In contrast, treatment with anti-CD4 antibody alone or use of CD4 KO mice has little or no impact on mortality during chronic phase (Gazzinelli et al., 1992; Schaeffer et al., 2009; Splitt et al., 2018). Using parasites expressing the β-gal model antigen (Table 26.1), depletion of CD4 1 T cells prior to infection was found not to affect frequencies of parasite-specific CD8 1 T cells but to result in a strong reduction of intracerebral production of IFN-γ and cytotoxic responses upon restimulation with that model antigen (Lutjen et al., 2006). This indicates a supportive role of CD4 1 T cells in the optimization of the functional activity of CD8 1 T cells. Conversely, conventional CD4 1 T cells can also contribute to the development of pathology. Discontinuation of treatment with anti-CD4 antibody results in increased inflammatory brain damage and in the elevated mortality observed during late chronic phase (Gazzinelli et al., 1992; Israelski et al., 1989). The detrimental role of CD4 1 T cells is particularly underscored in IL-10 KO mice, in which the severe and destructive inflammatory pathology observed during chronic infection is prevented by removal of CD4 1 T cells (Wilson et al., 2005). The fact that a rebound of CD4 1 T cells in the T. gondii infected brain causes life-threatening encephalitis is reminiscent of the pathogenesis of IRIS, the immune reconstitution inflammation syndrome (MartinBlondel et al., 2012).

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As of now, there is contrasting evidence regarding the function of CD4 1 Treg during chronic phase. Although one study reported that CD25 antibody administration (which depletes Treg as well as potentially other effector cells) has no effect on survival during chronic phase (Jankovic et al., 2007), another study that transferred Treg 2 days postinfection found increased survival during chronic phase (Olguin et al., 2015). What is clear is that Treg accumulate in the brain of infected C57BL/6 mice, make up to 10% of the total CD4 1 T cells, and produce IL-10 with a high frequency. In contrast to conventional T cells that are found predominantly in the brain parenchyma, a peculiarity of Treg is to localize mostly in the meninges and the perivascular cuffs, where they colocalize and interact with CD11c 1 cells (O’Brien et al., 2017).

26.7.4 Resistance to encephalitis is mediated by CD8 1 T cells that efficiently recognize tachyzoite-infected neurons Over the years the analysis of mouse and parasite combinations leading to varying degrees of parasite control and brain inflammation has allowed to shed light on how CD8 1 T cells efficiently control parasite in the brain and limit TE. As mentioned above (see Section 26.6.6.2), by comparing parasite chronic cyst burden in various mouse genetic backgrounds and MHCcongenic animals, the resistance to TE was first mapped to the H-2d MHC haplotype (Brown and McLeod, 1990) and was a few years later pinpointed to the H-2 Ld MHC I allele (Brown et al., 1995). Consequently, H-2k (e.g., CBA) and H-2b (e.g., C57BL/6) mice are susceptible to TE, while H-2d mice (e.g., BALB/c or congenic C57BL/6.H-2d) are TE-resistant, meaning that they display decreased brain parasite

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burden, reduce immune influx, and lower the activation of local and recruited myeloid cells at chronic phase (Fig. 26.7C). This H-2 Ldrelated protection is due to CD8 1 T cell responses targeting an immunodominant epitope (HPGSVNEFDF or HF10) that is processed from the C-terminus of the dense granule-secreted GRA6 protein in type II parasites and is presented by MHC I Ld (Blanchard et al., 2008). Because the equivalent C-terminal sequence in type I and type III GRA6 contains four nonsynonymous single-nucleotide polymorphisms, including one that changes the Cterminal Ld anchor residue (HPERVNVFDY), type I and type III strains represent natural “HF10-null” strains. Accordingly, type I/III parasites do not elicit GRA6/HF10-specific Ldrestricted CD8 1 T cell responses, which explains why the protective effect of Ld is observed only with type II parasites (Feliu et al., 2013; Johnson et al., 2002). Several lines of evidence have since confirmed the critical role of GRA6-specific CD8 1 T cell populations in the control of cerebral parasite burden during chronic phase. First, introduction in type III parasites of a version of GRA6 that contains the HF10 peptide elicits HF10-specific CD8 1 T cells that are able to strongly decrease brain cyst load in H-2d mice (Feliu et al., 2013). Second, C57BL/6 mice infected with transgenic type II parasites expressing the OVA SIINFEKL epitope at the C-terminus of GRA6 (GRA6-OVA) develop strong OVA-specific CD8 1 T cells responses that provide effective parasite control and dampen encephalitis up to 2 months postinfection (Fig. 26.8A). Expression of the GRA6-OVA model antigen by T. gondii remains protective even when restricted to the tachyzoite stage (and thus is no longer expressed by bradyzoites). As a corollary, GRA6-OVA-expressing T. gondii are useful parasites to study TE resistance in C57BL/6 mice. The use of mice specifically deleted from Ld in CNS neurons revealed the critical

importance of MHC I presentation by neurons to restrict brain parasite burden in the chronic phase (Salvioni et al., 2019) (Fig. 26.8B).

26.7.5 T cell exhaustion Following an initial encounter with their cognate antigen and the provision of costimulatory (signal 2) and cytokine (signal 3) signals, pathogen-specific T cells typically proliferate and give rise to effector and memory precursor cells. If the pathogen is cleared, a phase of contraction normally leaves behind only a small number of central and effector memory T cells that provide protective immunity against reinfection. Yet, if the pathogen establishes chronic infection, and if this chronic phase is accompanied by a systemic inflammatory condition such as in HIV infection, persistence of the antigen and chronic inflammation may force the T cells into a dysfunctional status called “exhaustion.” This phenomenon has been observed in the CNS of weakly protected T. gondii infected mice (TE context). Upon infection of C57BL/6 mice, parasite-specific CD8 1 T cells initially expand and control acute infection, but they eventually show a graded increase in the expression of the inhibitory receptor PD-1, a marker of dysfunction/exhaustion that is typically upregulated in chronic viral infections. PD-1 was found upregulated in total CD8 1 T cells from the spleen, liver, and brain of mice developing TE (Bhadra et al., 2011b). The deleterious function of PD-1 preferentially affects poly-functional memory CD8 1 T cells, leading to loss of functionality and higher susceptibility to apoptosis (Bhadra et al., 2012). In vivo blockade of PD-1 interaction with its receptor PD-L1 rescues exhausted PD-1-expressing CD8 1 T cells in an intrinsic CD40/CD40L-dependent manner (Bhadra et al., 2011a). Importantly, this leads to reinvigoration of CD8 1 T cell responses, preventing TE-associated mortality (Bhadra et al., 2011b). Interestingly, the ability

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26.7 Adaptive immunity in the brain

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FIGURE 26.8 CD8 1 T cell recognition of tachyzoite-derived antigens presented by CNS neurons determines efficiency of parasite control during chronic phase. During acute phase, the brain parenchyma is invaded by Toxoplasma gondii tachyzoites, which can infect many cell types including neurons. (A) Depending on the strength of parasite antigen presentation, tachyzoite-infected neurons are more or less efficiently recognized by CD8 1 T cells. In the presence of an efficiently processed parasite antigen, CD8 1 T cells hamper the development of cysts and provide efficient parasite control thanks to mechanisms that likely involve IFN-γ/TNF-α production and/or cytotoxicity toward the infected cell (Denkers et al., 1997; Suzuki et al., 2010; Wang et al., 2004). This process takes place regardless of whether the antigen is expressed in bradyzoites, meaning that, in a context of TE resistance, CD8 1 T cell surveillance is mostly achieved with tachyzoite-infected cells (Salvioni et al, 2019). (B) Lack of neuronal MHC I expression does not affect the abundance of CNS CD8 1 T cells, which likely accumulate via MHC I presentation by nonneuron antigen-presenting cells. Yet, absence of MHC I on neurons results in impaired parasite control and thus in larger cyst burden (Salvioni et al., 2019). CNS, Central nervous system; MHC, major histocompatibility complex.

of PD-1 blockade to restore immune protection differs depending upon the infection model. In chronically infected mice exposed secondarily to hypervirulent strains (e.g., MAS, GUY-DOS, and GTA1), several inhibitory receptors such as PD-1, TIM-3, 4-1BB, and CTLA-4 are upregulated, yet antibody-mediated blockade therapy of these inhibitory receptors could not restore immune protection following challenge (Splitt et al., 2018).

Parasite-specific CD4 1 T cells (defined in this case using the activation markers CD11a CD49d) also lose their functionality. They express PD-1 plus other inhibitory receptors typical of dysfunction, as well as the transcription factor B lymphocytes induced maturation protein-1 (Blimp-1) (Hwang et al., 2016), which has been associated with functional exhaustion of CD8 1 T cells (Shin et al., 2009). It was reported that the exhaustion of CD8 1 T cells in

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the TE brain is regulated by CD4 1 T cells since CD4 1 T cell-intrinsic deletion of Blimp-1 reverses CD8 1 T cell dysfunction and results in improved pathogen control (Hwang et al., 2016). So far, little evidence exists on the expression of exhaustion markers and their functional importance in the TE-resistant situation. PD-1 has been detected on ROP7-specific T cells in chronically infected BALB/c mice. Yet, comparing T cells with a fixed TCR of varying affinity for the MHC-peptide, no difference was observed between T cell populations that either contract or are sustained for longer periods in the CNS (Sanecka et al., 2018).

26.7.6 Tissue-resident memory T cells Beside exhaustion, a possible fate of antigenexperienced memory T cells is to acquire a phenotype of tissue residency. Tissue-resident memory T cells (Trm) were first described for barrier tissues such as the skin and gut, but it is now clear that they populate most nonlymphoid tissues. In contrast to other memory T cell subsets, a hallmark of Trm is that they do not recirculate. In accordance, they show low or no expression of CCR7 and CD62L, two lymphoid tissue homing molecules, and they lack the expression of sphingosine-1-phosphate-receptor 1, which is required for tissue egress. Their defining surface markers are usually CD69 and CD103, but CD103-negative or CD69-negative cells with Trm features have been described in some contexts. The main roles ascribed to this subset are to ensure protection from a secondary challenge [i.e., in the case of viral CNS infection (Steinbach et al., 2016)] and to trigger tissuewide alerts that mobilize innate and adaptive effector cells (Mueller and Mackay, 2016; Topham and Reilly, 2018). A pioneer analysis of CD103 1 CD8 1 T cells from chronically infected brains of TE mice 4 weeks postinfection compared their transcriptomic signature with that of CD103 2 CD8 1 T cells from T. gondii infected brains,

and that of Trm from a viral infection. This confirmed that CD103 1 CD8 1 T cells of TE brains express a core set of genes associated with tissue residency. Genes such as Klf2 and S1pr1 are downregulated, whereas several genes associated with extracellular adhesion and migration are upregulated. In T. gondii infected brains, Trm comprise up to 40% of the total CNS CD8 1 T cells, and they are not confined to a certain area of the brain or specifically localized to regions of infection. In terms of function, CD103 1 CD8 T cells produce a significantly greater percentage of TNF-α and IFN-γ, but their role in parasite control remains unclear. In a context of TE resistance the TCRMHC affinity has been found to dictate the efficiency of Trm formation in the brain. T cells with higher affinity for the ROP7-Ld complex indeed differentiate more frequently into CD103 1 CD8 1 T cells (Sanecka et al., 2018). Again the functional relevance of these cells in this context remains to be investigated.

26.8 Adaptive immunity in the muscle Overall, little is known about the role of adaptive immune cells in muscle. One study analyzed the role of muscle Treg cells in C57BL/6 mice orally infected with type II cysts. On day 30 postinfection, these mice exhibit extensive inflammatory infiltrates in muscle with a marked Th1 profile, correlating with significant muscle weakness compared to uninfected control mice. This myositis is associated with sustained accumulation of inflammatory monocytes and macrophages. Using Treg depletion and transfer experiments, this study unexpectedly found that Treg cells inhibited the switch of macrophages toward a repair-prone phenotype, thereby preventing muscle tissue regeneration (Jin et al., 2017). This is in contrast to the pro-repair function of tissue Treg cells in other settings (Burzyn et al., 2013).

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References

26.9 Conclusion Adaptive immunity and the genes that specify this response and the innate immune response (Chapter 25 “Innate immunity to Toxoplasma gondii") and their interplay are critical to the outcome of T. gondii infection. Understanding these immune responses and parasite manipulation of the host immune response is critical for understanding the pathogenesis of infection and outcome of infection. These responses are driven by the genetic diversity of both the host and the parasite. Insights into these genetic variations are beginning to provide explanations for the varying disease manifestations that can occur following infection. All of these factors provide the foundation for the development of protective immunity and inform therapeutic strategies for disease prevention.

References Adiko, A.C., Babdor, J., Gutierrez-Martinez, E., Guermonprez, P., Saveanu, L., 2015. Intracellular transport routes for MHC I and their relevance for antigen cross-presentation. Front. Immunol. 6, 335. Aliberti, J., Hieny, S., Reis e Sousa, C., Serhan, C.N., Sher, A., 2002. Lipoxin-mediated inhibition of IL-12 production by DCs: a mechanism for regulation of microbial immunity. Nat. Immunol. 3, 76 82. Anton, L.C., Yewdell, J.W., 2014. Translating DRiPs: MHC class I immunosurveillance of pathogens and tumors. J. Leukoc. Biol. 95, 551 562. Araujo, F.G., 1991. Depletion of L3T4 1 (CD4 1 ) T lymphocytes prevents development of resistance to Toxoplasma gondii in mice. Infect. Immun. 59, 1614 1619. Bannenberg, G.L., Aliberti, J., Hong, S., Sher, A., Serhan, C., 2004. Exogenous pathogen and plant 15-lipoxygenase initiate endogenous lipoxin A4 biosynthesis. J. Exp. Med. 199, 515 523. Barragan, A., Sibley, L.D., 2002. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. J. Exp. Med. 195, 1625 1633. Benson, A., Murray, S., Divakar, P., Burnaevskiy, N., Pifer, R., Forman, J., et al., 2012. Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism. J. Immunol. 188, 800 810.

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Bertholet, S., Goldszmid, R., Morrot, A., Debrabant, A., Afrin, F., Collazo-Custodio, C., et al., 2006. Leishmania antigens are presented to CD8 1 T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo. J. Immunol. 177, 3525 3533. Bhadra, R., Gigley, J.P., Khan, I.A., 2011a. Cutting edge: CD40-CD40 ligand pathway plays a critical CD8intrinsic and -extrinsic role during rescue of exhausted CD8 T cells. J. Immunol. 187, 4421 4425. Bhadra, R., Gigley, J.P., Weiss, L.M., Khan, I.A., 2011b. Control of Toxoplasma reactivation by rescue of dysfunctional CD8 1 T-cell response via PD-1-PDL-1 blockade. Proc. Natl. Acad. Sci. U.S.A. 108, 9196 9201. Bhadra, R., Gigley, J.P., Khan, I.A., 2012. PD-1-mediated attrition of polyfunctional memory CD8 1 T cells in chronic toxoplasma infection. J. Infect. Dis. 206, 125 134. Biswas, A., French, T., Dusedau, H.P., Mueller, N., RiekBurchardt, M., Dudeck, A., et al., 2017. Behavior of neutrophil granulocytes during Toxoplasma gondii infection in the central nervous system. Front. Cell. Infect. Microbiol. 7, 259. Blanchard, N., Shastri, N., 2008. Coping with loss of perfection in the MHC class I peptide repertoire. Curr. Opin. Immunol. 20, 82 88. Blanchard, N., Gonzalez, F., Schaeffer, M., Joncker, N.T., Cheng, T., Shastri, A.J., et al., 2008. Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum. Nat. Immunol. 9, 937 944. Blanchard, N., Dunay, I.R., Schluter, D., 2015. Persistence of Toxoplasma gondii in the central nervous system: a fine-tuned balance between the parasite, the brain and the immune system. Parasite Immunol. 37, 150 158. Blander, J.M., 2018. Regulation of the cell biology of antigen cross-presentation. Annu. Rev. Immunol. 36, 717 753. Bliss, S.K., Butcher, B.A., Denkers, E.Y., 2000. Rapid recruitment of neutrophils containing prestored IL-12 during microbial infection. J. Immunol. 165, 4515 4521. Blum, J.S., Wearsch, P.A., Cresswell, P., 2013. Pathways of antigen processing. Annu. Rev. Immunol. 31, 443 473. Bonnart, C., Feuillet, G., Vasseur, V., Cenac, N., Vergnolle, N., Blanchard, N., 2017. Protease-activated receptor 2 contributes to Toxoplasma gondii-mediated gut inflammation. Parasite Immunol. 39. Available from: https:// doi.org/10.1111/pim.12489. Brown, C.R., McLeod, R., 1990. Class I MHC genes and CD8 1 T cells determine cyst number in Toxoplasma gondii infection. J. Immunol. 145, 3438 3441. Brown, C.R., Hunter, C.A., Estes, R.G., Beckmann, E., Forman, J., David, C., et al., 1995. Definitive identification of a gene that confers resistance against Toxoplasma cyst burden and encephalitis. Immunology 85, 419 428.

Toxoplasma Gondii

1140

26. Adaptive immunity

Buaillon, C., Guerrero, N.A., Cebrian, I., Blanie, S., Lopez, J., Bassot, E., et al., 2017. MHC I presentation of Toxoplasma gondii immunodominant antigen does not require Sec22b and is regulated by antigen orientation at the vacuole membrane. Eur. J. Immunol. 47, 1160 1170. Burzyn, D., Benoist, C., Mathis, D., 2013. Regulatory T cells in nonlymphoid tissues. Nat. Immunol. 14, 1007 1013. Buzoni-Gatel, D., Lepage, A.C., Dimier-Poisson, I.H., Bout, D.T., Kasper, L.H., 1997. Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii. J. Immunol. 158, 5883 5889. Buzoni-Gatel, D., Debbabi, H., Mennechet, F.J., Martin, V., Lepage, A.C., Schwartzman, J.D., et al., 2001. Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes. Gastroenterology 120, 914 924. Casciotti, L., Ely, K.H., Williams, M.E., Khan, I.A., 2002. CD8(1)-T-cell immunity against Toxoplasma gondii can be induced but not maintained in mice lacking conventional CD4(1) T cells. Infect. Immun. 70, 434 443. Cebrian, I., Visentin, G., Blanchard, N., Jouve, M., Bobard, A., Moita, C., et al., 2011. Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells. Cell 147, 1355 1368. Cebrian, I., Croce, C., Guerrero, N.A., Blanchard, N., Mayorga, L.S., 2016. Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells. EMBO Rep. 17, 1753 1765. Chardes, T., Bourguin, I., Mevelec, M.N., Dubremetz, J.F., Bout, D., 1990. Antibody responses to Toxoplasma gondii in sera, intestinal secretions, and milk from orally infected mice and characterization of target antigens. Infect. Immun. 58, 1240 1246. Chardes, T., Velge-Roussel, F., Mevelec, P., Mevelec, M.N., Buzoni-Gatel, D., Bout, D., 1993. Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice. Immunology 78, 421 429. Chardes, T., Buzoni-Gatel, D., Lepage, A., Bernard, F., Bout, D., 1994. Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta 1 Thy-1 1 gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes. J. Immunol. 153, 4596 4603. Chtanova, T., Han, S.J., Schaeffer, M., van Dooren, G.G., Herzmark, P., Striepen, B., et al., 2009. Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node. Immunity 31, 342 355. Chu, H.H., Chan, S.W., Gosling, J.P., Blanchard, N., Tsitsiklis, A., Lythe, G., et al., 2016. Continuous effector CD8(1) T cell production in a controlled persistent infection is sustained by a proliferative intermediate population. Immunity 45, 159 171.

Cohen, S.B., Denkers, E.Y., 2015. Impact of Toxoplasma gondii on dendritic cell subset function in the intestinal mucosa. J. Immunol. 195, 2754 2762. Coombes, J.L., Robey, E.A., 2010. Dynamic imaging of hostpathogen interactions in vivo. Nat. Rev. Immunol. 10, 353 364. Coombes, J.L., Charsar, B.A., Han, S.J., Halkias, J., Chan, S. W., Koshy, A.A., et al., 2013. Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread. Proc. Natl. Acad. Sci. U.S.A. 110, E1913 1922. Courret, N., Darche, S., Sonigo, P., Milon, G., Buzoni-Gatel, D., Tardieux, I., 2006. CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain. Blood 107, 309 316. Deckert, M., Lutjen, S., Leuker, C.E., Kwok, L.Y., Strack, A., Muller, W., et al., 2003. Mice with neonatally induced inactivation of the vascular cell adhesion molecule-1 fail to control the parasite in Toxoplasma encephalitis. Eur. J. Immunol. 33, 1418 1428. Deckert-Schluter, M., Bluethmann, H., Kaefer, N., Rang, A., Schluter, D., 1999. Interferon-gamma receptor-mediated but not tumor necrosis factor receptor type 1- or type 2mediated signaling is crucial for the activation of cerebral blood vessel endothelial cells and microglia in murine Toxoplasma encephalitis. Am. J. Pathol. 154, 1549 1561. Denkers, E.Y., Gazzinelli, R.T., Martin, D., Sher, A., 1993. Emergence of NK1.1 1 cells as effectors of IFN-gamma dependent immunity to Toxoplasma gondii in MHC class I-deficient mice. J. Exp. Med. 178, 1465 1472. Denkers, E.Y., Yap, G., Scharton-Kersten, T., Charest, H., Butcher, B.A., Caspar, P., et al., 1997. Perforin-mediated cytolysis plays a limited role in host resistance to Toxoplasma gondii. J. Immunol. 159, 1903 1908. Dotiwala, F., Mulik, S., Polidoro, R.B., Ansara, J.A., Burleigh, B.A., Walch, M., et al., 2016. Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites. Nat. Med. 22, 210 216. Draheim, M., Wlodarczyk, M.F., Crozat, K., Saliou, J.M., Alayi, T.D., Tomavo, S., et al., 2017. Profiling MHC II immunopeptidome of blood-stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells. EMBO Mol. Med. 9, 1605 1621. Dubey, J.P., 1997. Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii. J. Eukaryot. Microbiol. 44, 592 602. Dunay, I.R., Damatta, R.A., Fux, B., Presti, R., Greco, S., Colonna, M., et al., 2008. Gr1(1) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii. Immunity 29, 306 317.

Toxoplasma Gondii

References

Dupont, C.D., Christian, D.A., Selleck, E.M., Pepper, M., Leney-Greene, M., Harms Pritchard, G., et al., 2014. Parasite fate and involvement of infected cells in the induction of CD4 1 and CD8 1 T cell responses to Toxoplasma gondii. PLoS Pathog. 10, e1004047. Dzierszinski, F., Pepper, M., Stumhofer, J.S., LaRosa, D.F., Wilson, E.H., Turka, L.A., et al., 2007. Presentation of Toxoplasma gondii antigens via the endogenous major histocompatibility complex class I pathway in nonprofessional and professional antigen-presenting cells. Infect. Immun. 75, 5200 5209. Embgenbroich, M., Burgdorf, S., 2018. Current concepts of antigen cross-presentation. Front. Immunol. 9, 1643. Engelhardt, B., Ransohoff, R.M., 2012. Capture, crawl, cross: the T cell code to breach the blood-brain barriers. Trends Immunol. 33, 579 589. Estato, V., Stipursky, J., Gomes, F., Mergener, T.C., FrazaoTeixeira, E., Allodi, S., et al., 2018. The neurotropic parasite Toxoplasma gondii induces sustained neuroinflammation with microvascular dysfunction in infected mice. Am. J. Pathol. 188, 2674 2687. Feliu, V., Vasseur, V., Grover, H.S., Chu, H.H., Brown, M.J., Wang, J., et al., 2013. Location of the CD8 T cell epitope within the antigenic precursor determines immunogenicity and protection against the Toxoplasma gondii parasite. PLoS Pathog. 9, e1003449. Francois, V., Shehade, H., Acolty, V., Preyat, N., Delree, P., Moser, M., et al., 2015. Intestinal immunopathology is associated with decreased CD73-generated adenosine during lethal infection. Mucosal Immunol. 8, 773 784. Frickel, E.M., Sahoo, N., Hopp, J., Gubbels, M.J., Craver, M. P., Knoll, L.J., et al., 2008. Parasite stage-specific recognition of endogenous Toxoplasma gondii-derived CD8 1 T cell epitopes. J. Infect. Dis. 198, 1625 1633. Gay, G., Braun, L., Brenier-Pinchart, M.P., Vollaire, J., Josserand, V., Bertini, R.L., et al., 2016. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFNgamma-mediated host defenses. J. Exp. Med. 213, 1779 1798. Gazzinelli, R.T., Hakim, F.T., Hieny, S., Shearer, G.M., Sher, A., 1991. Synergistic role of CD4 1 and CD8 1 T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J. Immunol. 146, 286 292. Gazzinelli, R., Xu, Y., Hieny, S., Cheever, A., Sher, A., 1992. Simultaneous depletion of CD4 1 and CD8 1 T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii. J. Immunol. 149, 175 180. Gazzinelli, R.T., Eltoum, I., Wynn, T.A., Sher, A., 1993. Acute cerebral toxoplasmosis is induced by in vivo neutralization of TNF-alpha and correlates with the downregulated expression of inducible nitric oxide synthase

1141

and other markers of macrophage activation. J. Immunol. 151, 3672 3681. Gazzinelli, R.T., Wysocka, M., Hieny, S., Scharton-Kersten, T., Cheever, A., Kuhn, R., et al., 1996. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4 1 T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha. J. Immunol. 157, 798 805. Goldszmid, R.S., Coppens, I., Lev, A., Caspar, P., Mellman, I., Sher, A., 2009. Host ER-parasitophorous vacuole interaction provides a route of entry for antigen crosspresentation in Toxoplasma gondii-infected dendritic cells. J. Exp. Med. 206, 399 410. Goldszmid, R.S., Caspar, P., Rivollier, A., White, S., Dzutsev, A., Hieny, S., et al., 2012. NK cell-derived interferon-gamma orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection. Immunity 36, 1047 1059. Gopal, R., Birdsell, D., Monroy, F.P., 2011. Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection. Parasite Immunol. 33, 12 24. Gregg, B., Dzierszinski, F., Tait, E., Jordan, K.A., Hunter, C. A., Roos, D.S., 2011. Subcellular antigen location influences T-cell activation during acute infection with Toxoplasma gondii. PLoS One 6, e22936. Grover, H.S., Blanchard, N., Gonzalez, F., Chan, S., Robey, E.A., Shastri, N., 2012. The Toxoplasma gondii peptide AS15 elicits CD4 T cells that can control parasite burden. Infect. Immun. 80, 3279 3288. Grover, H.S., Chu, H.H., Kelly, F.D., Yang, S.J., Reese, M.L., Blanchard, N., et al., 2014. Impact of regulated secretion on antiparasitic CD8 T cell responses. Cell Rep. 7, 1716 1728. Gubbels, M.J., Striepen, B., Shastri, N., Turkoz, M., Robey, E.A., 2005. Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii. Infect. Immun. 73, 703 711. Guermonprez, P., Valladeau, J., Zitvogel, L., Thery, C., Amigorena, S., 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20, 621 667. Epub 2001 Oct 2004. Guiton, R., Vasseur, V., Charron, S., Arias, M.T., Van Langendonck, N., Buzoni-Gatel, D., et al., 2010. Interleukin 17 receptor signaling is deleterious during Toxoplasma gondii infection in susceptible BL6 mice. J. Infect. Dis. 202, 427 435. Hall, A.O., Beiting, D.P., Tato, C., John, B., Oldenhove, G., Lombana, C.G., et al., 2012. The cytokines interleukin 27 and interferon-gamma promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 37, 511 523.

Toxoplasma Gondii

1142

26. Adaptive immunity

Hand, T.W., Dos Santos, L.M., Bouladoux, N., Molloy, M.J., Pagan, A.J., Pepper, M., et al., 2012. Acute gastrointestinal infection induces long-lived microbiota-specific T cell responses. Science 337, 1553 1556. Harris, T.H., Banigan, E.J., Christian, D.A., Konradt, C., Tait Wojno, E.D., Norose, K., et al., 2012. Generalized Levy walks and the role of chemokines in migration of effector CD8 1 T cells. Nature 486, 545 548. Heimesaat, M.M., Bereswill, S., Fischer, A., Fuchs, D., Struck, D., Niebergall, J., et al., 2006. Gram-negative bacteria aggravate murine small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii. J. Immunol. 177, 8785 8795. Hunter, C.A., Subauste, C.S., Van Cleave, V.H., Remington, J.S., 1994. Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha. Infect. Immun. 62, 2818 2824. Hwang, S., Cobb, D.A., Bhadra, R., Youngblood, B., Khan, I.A., 2016. Blimp-1-mediated CD4 T cell exhaustion causes CD8 T cell dysfunction during chronic toxoplasmosis. J. Exp. Med. 213, 1799 1818. Israelski, D.M., Araujo, F.G., Conley, F.K., Suzuki, Y., Sharma, S., Remington, J.S., 1989. Treatment with antiL3T4 (CD4) monoclonal antibody reduces the inflammatory response in toxoplasmic encephalitis. J. Immunol. 142, 954 958. Jankovic, D., Kullberg, M.C., Feng, C.G., Goldszmid, R.S., Collazo, C.M., Wilson, M., et al., 2007. Conventional Tbet(1)Foxp3(2) Th1 cells are the major source of hostprotective regulatory IL-10 during intracellular protozoan infection. J. Exp. Med. 204, 273 283. Jensen, K.D.C., 2016. Antigen presentation of vacuolated apicomplexans—two gateways to a vaccine antigen. Trends Parasitol. 32, 88 90. Jin, R.M., Blair, S.J., Warunek, J., Heffner, R.R., Blader, I.J., Wohlfert, E.A., 2017. Regulatory T cells promote myositis and muscle damage in Toxoplasma gondii infection. J. Immunol. 198, 352 362. John, B., Harris, T.H., Tait, E.D., Wilson, E.H., Gregg, B., Ng, L.G., et al., 2009. Dynamic Imaging of CD8(1) T cells and dendritic cells during infection with Toxoplasma gondii. PLoS Pathog. 5, e1000505. John, B., Ricart, B., Tait Wojno, E.D., Harris, T.H., Randall, L.M., Christian, D.A., et al., 2011. Analysis of behavior and trafficking of dendritic cells within the brain during toxoplasmic encephalitis. PLoS Pathog. 7, e1002246. Johnson, L.L., Sayles, P.C., 2002. Deficient humoral responses underlie susceptibility to Toxoplasma gondii in CD4-deficient mice. Infect. Immun. 70, 185 191. Johnson, J.J., Roberts, C.W., Pope, C., Roberts, F., Kirisits, M.J., Estes, R., et al., 2002. In vitro correlates of Ldrestricted resistance to toxoplasmic encephalitis and

their critical dependence on parasite strain. J. Immunol. 169, 966 973. Jurewicz, M.M., Stern, L.J., 2019. Class II MHC antigen processing in immune tolerance and inflammation. Immunogenetics 71, 171 187. Kang, H., Suzuki, Y., 2001. Requirement of non-T cells that produce gamma interferon for prevention of reactivation of Toxoplasma gondii infection in the brain. Infect. Immun. 69, 2920 2927. Kang, H., Remington, J.S., Suzuki, Y., 2000. Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-gamma, TNF-alpha, and inducible nitric oxide synthase. J. Immunol. 164, 2629 2634. Khan, I.A., Ely, K.H., Kasper, L.H., 1994. Antigen-specific CD8 1 T cell clone protects against acute Toxoplasma gondii infection in mice. J. Immunol. 152, 1856 1860. Khan, I.A., Matsuura, T., Kasper, L.H., 1995. IL-10 mediates immunosuppression following primary infection with Toxoplasma gondii in mice. Parasite Immunol. 17, 185 195. Kim, S.K., Fouts, A.E., Boothroyd, J.C., 2007. Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: insights from a genome-wide transcriptional profiling. J. Immunol. 178, 5154 5165. Kugler, D.G., Mittelstadt, P.R., Ashwell, J.D., Sher, A., Jankovic, D., 2013. CD4 1 T cells are trigger and target of the glucocorticoid response that prevents lethal immunopathology in toxoplasma infection. J. Exp. Med. 210, 1919 1927. Kwok, L.Y., Lutjen, S., Soltek, S., Soldati, D., Busch, D., Deckert, M., et al., 2003. The induction and kinetics of antigen-specific CD8 T cells are defined by the stage specificity and compartmentalization of the antigen in murine toxoplasmosis. J. Immunol. 170, 1949 1957. Lang, C., Algner, M., Beinert, N., Gross, U., Luder, C.G., 2006. Diverse mechanisms employed by Toxoplasma gondii to inhibit IFN-gamma-induced major histocompatibility complex class II gene expression. Microbes Infect. 8, 1994 2005. Lang, C., Hildebrandt, A., Brand, F., Opitz, L., Dihazi, H., Luder, C.G., 2012. Impaired chromatin remodelling at STAT1-regulated promoters leads to global unresponsiveness of Toxoplasma gondii-infected macrophages to IFN-gamma. PLoS Pathog. 8, e1002483. Lee, Y., Sasai, M., Ma, J.S., Sakaguchi, N., Ohshima, J., Bando, H., et al., 2015. p62 plays a specific role in interferon-gamma-induced presentation of a toxoplasma vacuolar antigen. Cell Rep. 13, 223 233. Leroux, L.P., Dasanayake, D., Rommereim, L.M., Fox, B.A., Bzik, D.J., Jardim, A., et al., 2015a. Secreted Toxoplasma gondii molecules interfere with expression of MHC-II in interferon gamma-activated macrophages. Int. J. Parasitol. 45, 319 332.

Toxoplasma Gondii

References

Leroux, L.P., Nishi, M., El-Hage, S., Fox, B.A., Bzik, D.J., Dzierszinski, F.S., 2015b. Parasite manipulation of the invariant chain and the peptide editor H2-DM affects major histocompatibility complex class II antigen presentation during Toxoplasma gondii infection. Infect. Immun. 83, 3865 3880. Liesenfeld, O., Kosek, J., Remington, J.S., Suzuki, Y., 1996. Association of CD4 1 T cell-dependent, interferongamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii. J. Exp. Med. 184, 597 607. Liesenfeld, O., Kang, H., Park, D., Nguyen, T.A., Parkhe, C.V., Watanabe, H., et al., 1999. TNF-alpha, nitric oxide and IFN-gamma are all critical for development of necrosis in the small intestine and early mortality in genetically susceptible mice infected perorally with Toxoplasma gondii. Parasite Immunol. 21, 365 376. Lopez, J., Bittame, A., Massera, C., Vasseur, V., Effantin, G., Valat, A., et al., 2015. Intravacuolar membranes regulate CD8 T cell recognition of membrane-bound Toxoplasma gondii protective antigen. Cell Rep. 13, 2273 2286. Lopez-Yglesias, A.H., Burger, E., Araujo, A., Martin, A.T., Yarovinsky, F., 2018. T-bet-independent Th1 response induces intestinal immunopathology during Toxoplasma gondii infection. Mucosal Immunol. 11, 921 931. Luder, C.G., Lang, C., Giraldo-Velasquez, M., Algner, M., Gerdes, J., Gross, U., 2003. Toxoplasma gondii inhibits MHC class II expression in neural antigen-presenting cells by down-regulating the class II transactivator CIITA. J. Neuroimmunol. 134, 12 24. Lutjen, S., Soltek, S., Virna, S., Deckert, M., Schluter, D., 2006. Organ- and disease-stage-specific regulation of Toxoplasma gondii-specific CD8-T-cell responses by CD4 T cells. Infect. Immun. 74, 5790 5801. Martin-Blondel, G., Mars, L.T., Liblau, R.S., 2012. Pathogenesis of the immune reconstitution inflammatory syndrome in HIV-infected patients. Curr. Opin. Infect. Dis. 25, 312 320. Mashayekhi, M., Sandau, M.M., Dunay, I.R., Frickel, E.M., Khan, A., Goldszmid, R.S., et al., 2011. CD8alpha(1) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites. Immunity 35, 249 259. McKee, A.S., Dzierszinski, F., Boes, M., Roos, D.S., Pearce, E.J., 2004. Functional inactivation of immature dendritic cells by the intracellular parasite Toxoplasma gondii. J. Immunol. 173, 2632 2640. Meeker, R.B., Williams, K., Killebrew, D.A., Hudson, L.C., 2012. Cell trafficking through the choroid plexus. Cell Adhes. Migration 6, 390 396. Mennechet, F.J., Kasper, L.H., Rachinel, N., Li, W., Vandewalle, A., Buzoni-Gatel, D., 2002. Lamina propria

1143

CD4 1 T lymphocytes synergize with murine intestinal epithelial cells to enhance proinflammatory response against an intracellular pathogen. J. Immunol. 168, 2988 2996. Mennechet, F.J., Kasper, L.H., Rachinel, N., Minns, L.A., Luangsay, S., Vandewalle, A., et al., 2004. Intestinal intraepithelial lymphocytes prevent pathogen-driven inflammation and regulate the Smad/T-bet pathway of lamina propria CD4 1 T cells. Eur. J. Immunol. 34, 1059 1067. Mevelec, M.N., Mercereau-Puijalon, O., Buzoni-Gatel, D., Bourguin, I., Chardes, T., Dubremetz, J.F., et al., 1998. Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route. Parasite Immunol. 20, 183 195. Meyer, C., Martin-Blondel, G., Liblau, R.S., 2017. Endothelial cells and lymphatics at the interface between the immune and central nervous systems: implications for multiple sclerosis. Curr. Opin. Neurol. 30, 222 230. Mordue, D.G., Monroy, F., La Regina, M., Dinarello, C.A., Sibley, L.D., 2001. Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines. J. Immunol. 167, 4574 4584. Moretto, M.M., Hwang, S., Khan, I.A., 2017. Downregulated IL-21 response and T follicular helper cell exhaustion correlate with compromised CD8 T cell immunity during chronic toxoplasmosis. Front. Immunol. 8, 1436. Mueller, S.N., Mackay, L.K., 2016. Tissue-resident memory T cells: local specialists in immune defence. Nat. Rev. Immunol. 16, 79 89. Muhlethaler-Mottet, A., Di Berardino, W., Otten, L.A., Mach, B., 1998. Activation of the MHC class II transactivator CIITA by interferon-gamma requires cooperative interaction between Stat1 and USF-1. Immunity 8, 157 166. Munoz, M., Heimesaat, M.M., Danker, K., Struck, D., Lohmann, U., Plickert, R., et al., 2009. Interleukin (IL)-23 mediates Toxoplasma gondii-induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17. J. Exp. Med. 206, 3047 3059. Nair-Gupta, P., Baccarini, A., Tung, N., Seyffer, F., Florey, O., Huang, Y., et al., 2014. TLR Signals Induce Phagosomal MHC-I Delivery from the Endosomal Recycling Compartment to Allow Cross-Presentation. Cell 158, 506 521. Norose, K., Mun, H.S., Aosai, F., Chen, M., Hata, H., Tagawa, Y., et al., 2001. Organ infectivity of Toxoplasma gondii in interferon-gamma knockout mice. J. Parasitol. 87, 447 452.

Toxoplasma Gondii

1144

26. Adaptive immunity

O’Brien, C.A., Overall, C., Konradt, C., O’Hara Hall, A.C., Hayes, N.W., Wagage, S., et al., 2017. CD11c-expressing cells affect regulatory T cell behavior in the meninges during central nervous system infection. J. Immunol. 198, 4054 4061. O’Brien, C.A., Batista, S.J., Still, K.M., Harris, T.H., 2019. IL10 and ICOS differentially regulate T cell responses in the brain during chronic Toxoplasma gondii infection. J. Immunol. 202, 1755 1766. Ochiai, E., SA, Q., Brogi, M., Kudo, T., Wang, X., Dubey, J.P., et al., 2015. CXCL9 is important for recruiting immune T cells into the brain and inducing an accumulation of the T cells to the areas of tachyzoite proliferation to prevent reactivation of chronic cerebral infection with Toxoplasma gondii. Am J Pathol 185, 314 324. Oldenhove, G., Bouladoux, N., Wohlfert, E.A., Hall, J.A., Chou, D., Dos Santos, L., et al., 2009. Decrease of Foxp3 1 Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity 31, 772 786. Olguin, J.E., Fernandez, J., Salinas, N., Juarez, I., Rodriguez-Sosa, M., Campuzano, J., et al., 2015. Adoptive transfer of CD4(1)Foxp3(1) regulatory T cells to C57BL/6J mice during acute infection with Toxoplasma gondii down modulates the exacerbated Th1 immune response. Microbes Infect. 17, 586 595. Olias, P., Etheridge, R.D., Zhang, Y., Holtzman, M.J., Sibley, L.D., 2016. Toxoplasma effector recruits the Mi2/NuRD complex to repress STAT1 transcription and block IFN-gamma-dependent gene expression. Cell Host Microbe 20, 72 82. Parker, S.J., Roberts, C.W., Alexander, J., 1991. CD8 1 T cells are the major lymphocyte subpopulation involved in the protective immune response to Toxoplasma gondii in mice. Clin. Exp. Immunol. 84, 207 212. Peaper, D.R., Cresswell, P., 2008. Regulation of MHC class I assembly and peptide binding. Annu. Rev. Cell. Dev. Biol. 24, 343 368. Pepper, M., Dzierszinski, F., Crawford, A., Hunter, C.A., Roos, D., 2004. Development of a system to study CD4 1 -T-cell responses to transgenic ovalbuminexpressing Toxoplasma gondii during toxoplasmosis. Infect. Immun. 72, 7240 7246. Pepper, M., Dzierszinski, F., Wilson, E., Tait, E., Fang, Q., Yarovinsky, F., et al., 2008. Plasmacytoid dendritic cells are activated by Toxoplasma gondii to present antigen and produce cytokines. J. Immunol. 180, 6229 6236. Raetz, M., Hwang, S.H., Wilhelm, C.L., Kirkland, D., Benson, A., Sturge, C.R., et al., 2013. Parasite-induced TH1 cells and intestinal dysbiosis cooperate in IFNgamma-dependent elimination of Paneth cells. Nat. Immunol. 14, 136 142. Reichmann, G., Villegas, E.N., Craig, L., Peach, R., Hunter, C.A., 1999. The CD28/B7 interaction is not required for resistance to Toxoplasma gondii in the brain but

contributes to the development of immunopathology. J. Immunol. 163, 3354 3362. Robben, P.M., Mordue, D.G., Truscott, S.M., Takeda, K., Akira, S., Sibley, L.D., 2004. Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype. J. Immunol. 172, 3686 3694. Roberts, C.W., Ferguson, D.J., Jebbari, H., Satoskar, A., Bluethmann, H., Alexander, J., 1996. Different roles for interleukin-4 during the course of Toxoplasma gondii infection. Infect. Immun. 64, 897 904. Rock, K.L., Reits, E., Neefjes, J., 2016. Present yourself! By MHC class I and MHC class II molecules. Trends Immunol. 37, 724 737. Sa, Q., Ochiai, E., Sengoku, T., Wilson, M.E., Brogli, M., Crutcher, S., et al., 2014. VCAM-1/alpha4beta1 integrin interaction is crucial for prompt recruitment of immune T cells into the brain during the early stage of reactivation of chronic infection with Toxoplasma gondii to prevent toxoplasmic encephalitis. Infect. Immun. 82, 2826 2839. Salvoni, A., Belloy, M., Lebourg, A., Bassot, E., CantaloubeFerrieu, V., Vasseur, V., et al., 2019. Robust Control of a Brain-Persisting Parasite through MHC I Presentation by Infected Neurons. Cell Rep 27, 3254 3268 e8. Sanecka, A., Yoshida, N., Dougan, S.K., Jackson, J., Shastri, N., Ploegh, H., et al., 2016. Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute-phase Toxoplasma gondii burden. Immunology 149, 270 279. Sanecka, A., Yoshida, N., Kolawole, E.M., Patel, H., Evavold, B.D., Frickel, E.M., 2018. T cell receptor-major histocompatibility complex interaction strength defines trafficking and CD103(1) memory status of CD8 T cells in the brain. Front. Immunol. 9, 1290. Schaeffer, M., Han, S.J., Chtanova, T., van Dooren, G.G., Herzmark, P., Chen, Y., et al., 2009. Dynamic imaging of T cell-parasite interactions in the brains of mice chronically infected with Toxoplasma gondii. J. Immunol. 182, 6379 6393. Schluter, D., Barragan, A., 2019. Advances and challenges in understanding cerebral toxoplasmosis. Front. Immunol. 10, 242. Schluter, D., Meyer, T., Kwok, L.Y., Montesinos-Rongen, M., Lutjen, S., Strack, A., et al., 2002. Phenotype and regulation of persistent intracerebral T cells in murine Toxoplasma encephalitis. J. Immunol. 169, 315 322. Schreiner, M., Liesenfeld, O., 2009. Small intestinal inflammation following oral infection with Toxoplasma gondii does not occur exclusively in C57BL/6 mice: review of 70 reports from the literature. Mem. Inst. Oswaldo. Cruz. 104, 221 233. Sher, A., Oswald, I.P., Hieny, S., Gazzinelli, R.T., 1993. Toxoplasma gondii induces a T-independent IFN-gamma

Toxoplasma Gondii

References

response in natural killer cells that requires both adherent accessory cells and tumor necrosis factor-alpha. J. Immunol. 150, 3982 3989. Shin, H., Blackburn, S.D., Intlekofer, A.M., Kao, C., Angelosanto, J.M., Reiner, S.L., et al., 2009. A role for the transcriptional repressor Blimp-1 in CD8(1) T cell exhaustion during chronic viral infection. Immunity 31, 309 320. Shirahata, T., Yamashita, T., Ohta, C., Goto, H., Nakane, A., 1994. CD8 1 T lymphocytes are the major cell population involved in the early gamma interferon response and resistance to acute primary Toxoplasma gondii infection in mice. Microbiol. Immunol. 38, 789 796. Splitt, S.D., Souza, S.P., Valentine, K.M., Castellanos, B.E., Curd, A.B., Hoyer, K.K., et al., 2018. PD-L1, TIM-3, and CTLA-4 blockade fails to promote resistance to secondary infection with virulent strains of Toxoplasma gondii. Infect. Immun. 86, e00459 18. Steinbach, K., Vincenti, I., Kreutzfeldt, M., Page, N., Muschaweckh, A., Wagner, I., et al., 2016. Brainresident memory T cells represent an autonomous cytotoxic barrier to viral infection. J. Exp. Med. 213, 1571 1587. Stumhofer, J.S., Silver, J.S., Hunter, C.A., 2013. IL-21 is required for optimal antibody production and T cell responses during chronic Toxoplasma gondii infection. PLoS One 8, e62889. Sturge, C.R., Benson, A., Raetz, M., Wilhelm, C.L., Mirpuri, J., Vitetta, E.S., et al., 2013. TLR-independent neutrophil-derived IFN-gamma is important for host resistance to intracellular pathogens. Proc. Natl. Acad. Sci. U.S.A. 110, 10711 10716. Suzuki, Y., Remington, J.S., 1988. Dual regulation of resistance against Toxoplasma gondii infection by Lyt-2 1 and Lyt1 1 , L3T4 1 T cells in mice. J. Immunol. 140, 3943 3946. Suzuki, Y., Remington, J.S., 1990. The effect of anti-IFNgamma antibody on the protective effect of Lyt-2 1 immune T cells against toxoplasmosis in mice. J. Immunol. 144, 1954 1956. Suzuki, Y., Orellana, M.A., Schreiber, R.D., Remington, J.S., 1988. Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science 240, 516 518. Suzuki, Y., Conley, F.K., Remington, J.S., 1989. Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice. J. Immunol. 143, 2045 2050. Suzuki, Y., Kang, H., Parmley, S., Lim, S., Park, D., 2000. Induction of tumor necrosis factor-alpha and inducible nitric oxide synthase fails to prevent toxoplasmic encephalitis in the absence of interferon-gamma in genetically resistant BALB/c mice. Microbes Infect. 2, 455 462. Suzuki, Y., Wang, X., Jortner, B.S., Payne, L., Ni, Y., Michie, S.A., et al., 2010. Removal of Toxoplasma gondii cysts

1145

from the brain by perforin-mediated activity of CD8 1 T cells. Am. J. Pathol. 176, 1607 1613. Tenorio, E.P., Olguin, J.E., Fernandez, J., Vieyra, P., Saavedra, R., 2010. Reduction of Foxp3 1 cells by depletion with the PC61 mAb induces mortality in resistant BALB/c mice infected with Toxoplasma gondii. J. Biomed. Biotechnol. 2010, 786078. Theisen, D.J., Davidson, J.Tt, Briseno, C.G., Gargaro, M., Lauron, E.J., Wang, Q., et al., 2018. WDFY4 is required for cross-presentation in response to viral and tumor antigens. Science 362, 694 699. Topham, D.J., Reilly, E.C., 2018. Tissue-resident memory CD8(1) T cells: from phenotype to function. Front. Immunol. 9, 515. Valecka, J., Almeida, C.R., Su, B., Pierre, P., Gatti, E., 2018. Autophagy and MHC-restricted antigen presentation. Mol. Immunol. 99, 163 170. Villarino, A., Hibbert, L., Lieberman, L., Wilson, E., Mak, T., Yoshida, H., et al., 2003. The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity 19, 645 655. Vossenkamper, A., Struck, D., Alvarado-Esquivel, C., Went, T., Takeda, K., Akira, S., et al., 2004. Both IL-12 and IL-18 contribute to small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii, but IL-12 is dominant over IL-18 in parasite control. Eur. J. Immunol. 34, 3197 3207. Wang, X., Suzuki, Y., 2007. Microglia produce IFN-gamma independently from T cells during acute toxoplasmosis in the brain. J. Interferon Cytokine Res. 27, 599 605. Wang, X., Kang, H., Kikuchi, T., Suzuki, Y., 2004. Gamma interferon production, but not perforin-mediated cytolytic activity, of T cells is required for prevention of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease. Infect. Immun. 72, 4432 4438. Wang, X., Michie, S.A., Xu, B., Suzuki, Y., 2007. Importance of IFN-gamma-mediated expression of endothelial VCAM-1 on recruitment of CD8 1 T cells into the brain during chronic infection with Toxoplasma gondii. J. Interferon Cytokine Res. 27, 329 338. Weimershaus, M., Evnouchidou, I., Saveanu, L., van Endert, P., 2013. Peptidases trimming MHC class I ligands. Curr. Opin. Immunol. 25, 90 96. Wilson, E.H., Wille-Reece, U., Dzierszinski, F., Hunter, C. A., 2005. A critical role for IL-10 in limiting inflammation during toxoplasmic encephalitis. J. Neuroimmunol. 165, 63 74. Wilson, D.C., Matthews, S., Yap, G.S., 2008. IL-12 signaling drives CD8 1 T cell IFN-gamma production and differentiation of KLRG1 1 effector subpopulations during Toxoplasma gondii Infection. J. Immunol. 180, 5935 5945.

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26. Adaptive immunity

Wilson, E.H., Harris, T.H., Mrass, P., John, B., Tait, E.D., Wu, G.F., et al., 2009. Behavior of parasite-specific effector CD8 1 T cells in the brain and visualization of a kinesis-associated system of reticular fibers. Immunity 30, 300 311. Wilson, D.C., Grotenbreg, G.M., Liu, K., Zhao, Y., Frickel, E. M., Gubbels, M.J., et al., 2010. Differential regulation of

effector- and central-memory responses to Toxoplasma gondii Infection by IL-12 revealed by tracking of Tgd057specific CD8 1 T cells. PLoS Pathog. 6, e1000815. Yarovinsky, F., Kanzler, H., Hieny, S., Coffman, R.L., Sher, A., 2006. Toll-like receptor recognition regulates immunodominance in an antimicrobial CD4(1) T cell response. Immunity 25, 655 664.

A P P E N D I X

A The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection Craig W. Roberts and Stuart Woods University of Strathclyde, Glasgow, United Kingdom

TABLE A.1 The effect of interleukin (IL) or IL receptor gene deficiency on murine Toxoplasma gondii infection. Gene Mouse Parasite deficiency background strain

Route of infection

Effect on parasite

Effect on pathology

Effect on survival

Immunological effects

Reference

Not reported

Reduced

Reduced serum IL-18

Gorfu et al. (2014)

Reduced IL-12p40 and IFN-γ

Villegas et al. (2002a)

IL-1R

C57BL/6

76K i/p GFP-LUC

Increased parasite burden by IVIS

IL-2

C57BL/6

Me49

i/p

No difference in None parasite number reported in brain

Reduced

IL-4

C57BL/6

Beverley

Oral

No difference in Decreased intestine necrosis in intestine

Enhanced Increased plasma IFN-γ and IL-12

IL-4

129/J

Me49

Oral

Increased tachyzoites and cysts in brain

Increased areas of focal inflammation

Nickdel et al. (2004)

Increased IL-10 transcripts in intestine Reduced

No difference in Suzuki et al. transcripts for IFN- (1996) γ, TNF-α, IL-6, and IL-10 Reduced IFN-γ production by antigen stimulated splenocytes

(Continued)

1147

1148

Appendix A: The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection

TABLE A.1 (Continued) Gene Mouse Parasite deficiency background strain

Route of infection

Effect on parasite

Effect on pathology

Effect on survival

Immunological effects

Reference

IL-4

Oral

Reduced cysts in brain

Decreased encephalitis

Reduced

Increased IFN-γ (day 7)

Roberts et al. (1996)

129/ Beverley SvxC57BL/6

Reduced IL-10 (day 28)

Decreased necrotic lesions IL-5

C57BL/6

Me49

i/p

Not reported Increased parasite number in brain

Reduced

Reduced splenocyte IL-12 production

IL-5

C57BL/6

Beverley

Oral

No difference in Decreased intestine necrosis in intestine

Enhanced Increased plasma IFN-γ and IL-12

Zhang and Denkers (1999) Nickdel et al. (2001)

Reduced eosinophilia

IL-6

129/Sv x C57BL/6

Me49

i/p

Increased percentage of infected peritoneal cells

Increased necrosis in brain

Not reported

Reduced transcripts for IFN-γ and increased transcripts for IL-10 in brain

Suzuki et al. (1997)

IL-6

129/SvJ

Beverley

Oral

Increased cyst number and uncontrolled tachyzoites replication in brain

Increased brain pathology

Reduced

Reduced serum IL-6 levels

Jebbari et al. (1998)

IL-6

129/SVJ

Beverley

i/p

Increased tachyzoite number

Increased inflammation in the eye

Not reported

Decreased ocular IL-1 mRNA transcripts

Lyons et al. (2001)

Increased ocular TNF-α mRNA transcripts IL-6

C57BL/6

Me49

i/p

Not reported

Not reported

Not reported

Increased serum levels of IL-17

Passos et al. (2010)

Decrease in frequency of IL-17producing CD31 and NK1.11 cells GP130 (neurons)

C57BL/6

DX

i/p

Increased number of cysts and tachyzoites in brain

Increased necrosis in brain

Reduced

Increased IL-17 and IFN-γ producing CD4 and CD8 T cells, reduce intracerebral TGF-β and IL-127

Handel et al. (2012)

(Continued)

Toxoplasma Gondii

Appendix A: The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection

1149

TABLE A.1 (Continued) Gene Mouse Parasite deficiency background strain

Route of infection

Effect on parasite

Effect on pathology

Effect on survival

Immunological effects

IL-10

C57BL/6

Me49

Oral

Not reported

Increased necrosis in ileum

Reduced

Increased IFN-γ mRNA transcripts in

Suzuki et al. (2000)

IL-10

BALB/c

Me49

Oral

Not reported

Focal necrosis Reduced in ileum

Not reported

Suzuki et al. (2000)

IL-10

C57BL/6

Me49

i/p

None observed in brain

Increase inflammation and necrosis in brain

Reduced

Increased CD41 T cells in brain

Wilson et al. (2005)

Reference

Partial depletion of CD41 T cells decreased inflammation and increased survival

IL-10

C57BL/6

Me49

Oral

Reduced number (brain)

Increased liver pathology

Increased Increased serum IL-12(p70), IL-12p (40) and IFN-γ

Gazzinelli et al. (1996)

IL-10

C57BL/6

Me49

i/p

Increased numbers (intestine and liver)

Not reported

Reduced

Not reported

Wille et al. (2004)

IL-10

BALB/c

RH

i/p

Not reported

Not examined

None

No difference in serum IL-12p40 or IFN-γ levels

Wille et al. (2001)

IL-10

C57BL/6

RH

Intraocular None reported

Increased necrosis and inflammation in the eye

Not reported

Increased serum IFN-γ

Lu et al. (2003)

IL-10

BALB/c

RH

Intraocular None reported

Increased necrosis and inflammation in the eye

Not reported

Increased serum IFN-γ

Lu et al. (2003)

p40 (IL-12/ IL-23)

C57BL/6

Ts-4

i/p

Not reported

Not reported

Reduced

Surviving mice have increased resistance to increased doses of ts-4 or infection with 76K strain of T. gondii

Ely et al. (1999)

p40 (IL-12/ IL-23)

BALB/c

Ts-4

i/p

Not reported

Not reported

Reduced

Reduced serum IFN-γ

Lieberman et al. (2004b)

p35 (IL-12)

BALB/c

Ts-4

i/p

Increased numbers (peritoneal exudate)

Not reported

Reduced

Reduced serum IFN-γ

Lieberman et al. (2004b)

(Continued)

Toxoplasma Gondii

1150

Appendix A: The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection

TABLE A.1 (Continued) Gene Mouse Parasite deficiency background strain

Route of infection

Effect on parasite

p40 (IL12/IL-23)

C57BL/6

Me49

i/p

p40 (IL12/IL-23)

C57BL/6

Me49

p40 (IL12/IL-23)

C57BL/6

p40 (IL12/IL-23)

Effect on pathology

Effect on survival

Immunological effects

Not reported Increased number in brain and lung

Reduced

Not reported

Villarino et al. (2003)

i/p

Not reported

Not reported

Reduced

Exogenous IL-12 restores resistance but reactivation occurs on withdrawal

Yap et al. (2000)

Me49

i/p

Increased percentage of infected peritoneal cells

Not reported

Reduced

Not examined

SchartonKersten et al. (1997a)

C57BL/6

Me49

i/p

Increased numbers (peritoneal exudate)

Not reported

Reduced

None reported

Lieberman et al. (2004b)

p35 (IL12)

C57BL/6

Me49

i/p

None

Not reported

Reduced

None reported

Lieberman et al. (2004b)

IL-15

C57BL/6

Me49

i/p

Not reported

None

None

Similar levels of IFN-γ production and T-cell activation

Lieberman et al. (2004c)

IL-15

C57BL/6

Me49

Oral

No difference in Reduced intestinal Intestine or pathology MLN

Reduced

Reduced IL-1b, TNF-α, and IL-6 by LP cells

Schulthess et al. (2012)

IL-15

C57BL/6

76K

i/p

Not reported

Not reported

Increased Not reported

Oral

Increased parasite levels in gut, spleen liver, and brain

Reduced intestinal and liver pathology

Reduced IFN-γ producing cells in MLN and spleen. Reduced IFN-γ transcripts in gut Increased CD41 IFNγ1

IL-17A

C57BL/6

Fukaya

Oral

No difference in parasite numbers in mLNs and ileum

Reduced neutrophil infiltration in ileal mucosa

Reduced

IL-17RA

C57BL/6

76K

Oral

Reduced cysts in brain

Reduced brain and acute intestinal inflammation

Increased Increased Th1 cytokine response in particular IFN-γ and IL-2 levels

Reference

Combe et al. (2006)

Moroda et al. (2017)

Guiton et al. (2010)

Reduced systemic inflammation

(Continued)

Toxoplasma Gondii

Appendix A: The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection

1151

TABLE A.1 (Continued) Gene Mouse Parasite deficiency background strain

Route of infection

Effect on parasite

IL-18

Oral

No difference in Reduced lung necrosis in intestine and Increased liver number in liver

C57BL/6

Me49

Effect on pathology

Effect on survival

Immunological effects

Increased Reduced serum IFN-γ

Reference Vossenkamper et al. (2004)

Reduced intestinal IFN-γ production

Reduced number in intestine IL-18

C57BL/6

76K GFP- i/p LUC

Increased parasite burden by IVIS

Not reported

Reduced

Reduced serum IL-18

Gorfu et al. (2014)

IL-18R

C57BL/6

76K GFP- i/p LUC

Increased parasite burden by IVIS

Not reported

Reduced

Reduced serum IL-18

Gorfu et al. (2014)

p19 (IL23