Investigations on the antigenic composition of Cryptococcus neoformans

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INVESTIGATIONS ON THE ANTIGENIC COMPOSITION OP CKYPTOCQCCUS NEOFORMANS

A Dissertation Presented to the Faculty of the Graduate School The University of Southern California

In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

hy Ernest Edward Evans, Jr. August 1950

UMI Number: DP29316

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TABLE OF CONTENTS CHAPTER I.

PAGE

THE P R O B L E M ............................... . Statement of the problem

II.

1

. . . . . . .

1

Importance of the study . . . . . . . .

1

REVIEW OF THE L I T E R A T U R E .................

5

A brief review of Cryptococcosis in man

3

History.

• • * « • • • » . . • • • • *

4

Morphologic and cultural character-; istics of Cryptococcus neoformans

5

Literature on immunologic studies of '

Cryptococcus

• • • • • • • • • • • • .

6

Antigenic differences within Bonhamfs Group 3

• • • • . • • • • • • • . •

7

Antibody response to £• neof ormans. • . Chemical studies of the capsular substance III.

MATERIALS AND METHODS Strains of Cryptococcus neoformans

8 9

.

12

• • •

12

The production of anti serum • • • • • • •

12

The preparation of immunizing vaccines

13

Immunization of rabbits

13

• • • • . • •

Serologic methods The agglutination reaction

16 • • • • • •

16

The capsular reaction . • • • • • • • •

17

CHAPTER

PAGE Isolation of the capsularsubstance

*. •

17

The precipitin test

21

Agglutinin absorption • • • • • . . . . •

22

Precipitin absorption • • • • • • • • • .

22

The serologic inhibition t e s t * . . . . *

23

Chemical properties of the capsular substance

. . • • . . • • • • • • • • *

24

Qualitative analysis by filter paper chromatography

• • • • • • • • • • • •

Hydrolysis of capsular polysaccharides Chromatograms

RESULTS

•

* • • • • • • • • • • • *

Other qualitative chemical methods IV*

25

• • •

...........

29 30

* • • • • • • •

30

The agglutination test

* • • • ... • • «

30

The capsular reaction • • • • • . * • • •

43

The precipitin test

* • • * • • • • • •

46

• . • • • • • • • • •

47

Chemical studies of thecapsular substance Preliminary tests

• • • • • • . • • • .

The paper chromatogram

VI.

26

A serologic classification

The inhibition test

V.

25

49 49

* • • • • • • • «

50

D I S C U S S I O N ........................... .......

53

SUMMARY

.....................................

58

....................................

61

BIBLIOGRAPHY

LIST OF TABLES TABLE I.

PAGE Tube Agglutination Titers in Unabsorbed Serum and in Serum Absorbed with Cells of Heterologous Type . . . . . . . . . . .

II;

33

Agglutination Titers in Sera of Types A, B, and C after Absorption with. Cells of Homologous and Heterologous Type • . . . .

III.

Capsular Reactions in Unabsorbed and Absorbed Antisera

IV.

34

• • • • • . • • • • • •

35

Capsular Reactions in Antisera Absorbed

--------- with-Polysaccharides of Homologous and Heterologous Type VV

...............

36

Site of Infection and Results of Typing by Capsular Reaction for Thirty-six Strains of £• neoformans

VI*

* • • • • • • • • • • •

37

Precipitation of Cryptococcus Capsular Polysaccharides of Types A, B, and C in Absorbed and Unabsorbed Antisera . . . . .

VII.

38

Inhibition of Homologous Precipitation in Types A, B, and C by 0.2M Monosaccharide .

VIII*

39

Inhibition of Homologous Precipitation in Type A by D-(+)-Xylose •

............

•

40

vi TABLE IX;

PAGE Inhibition of Homologous Precipitation in Type

X.

B by D - (+)-Glucuronic Acid

•

%> and Rx Values for Chrcmatograms • •• •

.

•41 42

CHAPTER I THE PROBLEM Statement of the problem.

The primary purpose

of this investigation was to determine whether antigenic differences existed among strains of the pathogenic fungus Cryptococcus necformans*

When it was found that

such differences did exist and that they were referable to the capsular antigen, the study was broadened to include a qualitative chemical analysis of the capsular substance* Importance of the study.

Increasing numbers of

case reports on hpman infections with

neof ormans

have made it apparent that cryptococcosis can no longer be considered a rare disease*

In view of the high

mortality rate among those who contract this disease, a method of specific therapy is obviously needed*

Since

antibiotics, sulfonamides, and other chemotherapeutic agents have not generally been successful in the treat­ ment of cryptococcosis (Cox and Tolhurst, 1946; Beck and Voyles, 1946; Kligman and Weidman, 1949), it would t.

appear that the value of antiserum therapy should be investigated*

2 Prerequisite knowledge to the .use of such therapy would include a method for producing antiserum as well as information concerning the existence of antigenic differences among strains of the organism.

The present

investigation has been directed to these ends. With the demon strati on of three serologic types of the organism as well as the development of a method for the production of antiserum a basis for future in vivo studies has been determined. In addition, the identification of a serologically reactive capsular polysaccharide in

neof ormans offers

a new material for studies in immunochemistry.

CHAPTER II REVIEW OF THE LITERATURE I. A BRIEF REVIEW OF CRYPTOCOCCOSIS IN MAN Cryptococcosis is a subacute or chronic infection caused by the encapsulated yeast, Cryptococcus neoformans*

The anatomic sites most frequently involved

are the brain and meninges, and the majority of such cases have terminated fatally (Cox and Tolhurst, 1946). Chronic cases have been reported by Reeves, Butt and Hammack (1941) and Cox and Tolhurst (1946)* In addition to the central nervous system, C« neof ormans has a predilection for the lungs {Reeves, Butt, and Hammack 1941)* affected less frequently*

Other anatomic sites are Cutaneous cryptococcosis has

been described by Rappaport and Kaplan (1926), Weidman (1933) and Mook and Moore (1936)*

Kessel and Holtzwart

(1935) reported a case involving both the knee joint and skin*

Other local sites mentioned in the literature

include the tongue (Berghausen, 1927), nasopharynx (Jones, 1927), an abeess of the pelvic and Inguinal regions (McGehee and Michelson (1926), and a subcutan­ eous tumor (Dienst, 1938)*

History*

Zenker (1861) was the first to report

a case of meningitis probably caused by

neof ormans;

however, this cannot be definitely concluded since no cultural studies were made* Busse (1895) isolated a yeast from a lesion of the tibia, naming the disease saecharomycosis hominis. He referred to the etiologic agent as a Saccharomyces without using a specific name*

A fungus which had been

isolated from fruit by Sanfeliee in 1894 was similar to the one studied by Busse*

Sanfeliee described his yeast

as being pathogenic for animals and named it Sac char omyc e s neof ormans*, Vuillemin (1898); observed that this organism was not a true yeast and named it Cryptococcus hominis ♦

Another synonym was introduced

b y Curtis (1896) who isolated an encapsulated yeast from a myxomatous tumor of the hip and designated it Saccharomyces tumefaciens♦ The first report of central nervous system involvement was that of von Hansemann (1905) and two years later, Turk (1907) described another case of meningitis• A comprehensive study was made of known cases by Stoddard and Cutler in 1916*

They reported two new

cases and performed cultural and animal studies*

Believing that the organism actually dissolved brain tissue, they named the yeast Torula histolytica,

a

name which is still in use by some authors at the present time although Cryptococcus neof or mans is preferred by most authorities in this country (Conant et*al# 1945), since it has priority over other designations# Many cases of cryptococcosis have been reported in medical literature subsequent to the publication of the monograph of Stoddard and Cutler (1916)#

At the

time Freeman1s monograph was written (1931) there were forty-three reported cases of central nervous system involvement, and Cox and Tolhurst (1946) state that there were, 120 cases at the time their monograph was published# Morphologic and cultural characteristics of Cryptococcu8 neoformans;

Yrfhether obtained from culture

or from tissue, C^ neof ormans is a spherical or slightly ovoid yeast 5-15 microns in diameter which reproduces by budding#

A distinguishing characteristic is the mucoid

capsule surrounding each cell#

Capsular size varies from

one strain to another, and with conditions of cultivation. The presence of a capsule is most conveniently demon­ strated by the India ink preparation (Conant, et al# 1945).

6 CU neof ormans

is not nutritionally fastidious

and can be cultured on any of the common laboratory media*

Blood agar and SabourandTs agar are commonly

employed*

Colonies on Sabourandfs agar are white, mucoid

and glistening, although in older cultures, a light tan pigment may be evident (Smith and Martin, 1948); There is considerable variability in carbohydrate f ermentati on among strains of 0^ neof ormans *

The sugar

most often fermented is glucose, although sane strains appear unable to ferment even this sugar*

If carbo­

hydrates are fermented, acid alone is produced and the fermentation is often slow*

The fact that gas is not

produced is of value in differentiating

neof ormans

fran contaminating yeast (Cox and Tolhurst, 1946)* II*

LITERATURE ON IMMUNOLOGIC STUDIES OP CRYPTOCOCCUS

Certain species of the genus Cryptococcus isolated from man have been divided into four groups by Benham (1935) on the basis of serologic and other differences*

Her collection included strains isolated

from the normal skin and intestinal tract of man as well as cultures of CU neof oimans from clinical cases*

In

her classification all pathogenic strains as well as

some non-pathogenic strains isolated from skin and feces were placed in Group 3*

The present study is

concerned with, the antigenic relationships of strains within the species

neof ormans

(Benham Group 3)*

Species of the genus Cryptococcus contained in Benhamfs Groups 1, 2, and 4 were nan-pathogenic*

Group

1 contained strains isolated from the1 skin and intes­ tinal tract*

Group 2 contained ten non-pathogenic *

strains of which eight were isolated from the foot, one from the tongue and one from feces*

The specific name

C* mucorugoaus was applied to these strains*

Group 4

contained _red_ pigmented strains isolated from skin and feces; Antigenic differences within Benham1s Group 3* The third serologic group of Benham contained all the strains of GU neof ormans in her collection as well as seven strains isolated from skin and two strains from the feces of normal individuals*

Most of the strains

employed by Benham agglutinated to full titer in anti­ sera prepared against four pathogenic strains*

One of

these four, however, did not give reciprocal agglutina­ tion to full titer (strain 11} and strain 13 isolated from feces likewise exhibited a non-reciprocal cross­ reaction.

It agglutinated to full titer in all Group 3

8 antisera, tout an anti serum prepared against it failed to agglutinate other strains in Group 3 beyond 1:10; Aside from these data, there are no reports in the literature which indicate that the species ()• neoformans may toe composed of strains which are antigenically different except for a preliminary report toy the present author (Evans, 1949); Antibody response to C» neof ormans;

In infected

humans,antibodies are either lacking (Flu and Woensdreght, 2918; Shapiro and Heal, 1925; Urtoach and Zach, 1930) or of low titer*

Rappapart and Kaplan (1926) describe

agglutinins In a patient1s serum which titered to 1:40* Varying degrees of success have been attained with antibody production in experimental animals*

Sheppe

(1924) and Kligman (1947) were unable to demonstrate antibodies following the immunization of rabbits* Rappaport and Kaplan (1926) secured antiserum with a titer of 1:80 from rabbits and Benham (1935) reported a titer of 1:160 with her Group 3 antisera*

She further

stated that it was necessary to treat vaccines with 0;05 H HC1 with the purpose of hydrolyzing the capsule; sphere is some doubt as to whether her method of treatment actually accomplished this (Kligman, 1947; Heill et al., 1950)*

Hoff (1942) used mice for immunization and

and reported agglutinins with a titer of 1:280*

Cox

and Tolhurst (1946) obtained antiserum Y/ith an agglutinating titer of 1:128 from rabbits.

Evans (1949)

reported titers of 1:320 against three strains, two of Type A and one