UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on the cell walls and extracellular material of virulent and avirulent Cryptococcus species Ross, Adrianne 1975

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STUDIES ON THE CELL WALLS AND EXTRACELLULAR MATERIAL OF VIRULENT AND AVIRULENT CRYPTOCOCCUS S'PECIES by ADRIANNE ROSS B.Sc. (Hons.), U n i v e r s i t y o f Swansea, Wales, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f BOTANY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA May, 1975 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date XI -6 i i ABSTRACT R e l a t i o n s h i p s between c e l l envelope b i o c h e m i s t r y , patho-g e n i c i t y and taxonomy o f v i r u l e n t and a v i r u l e n t Cryptococcus s p e c i e s have been s t u d i e d . V a r i a t i o n i n growth medium al l o w e d c o n t r o l l e d produc-t i o n o f c e l l s w i t h o r w ithout e x t e n s i v e capsule s y n t h e s i s . T h i a -mine (10 jig/ml) and a pH o f 7.0 were n e c e s s a r y f o r o p t i m a l growth. Heat treatment was used t o k i l l c e l l s . There was no d e t e c t a b l e damage t o c e l l envelope m a t e r i a l s . P r e p a r a t i o n s o f p u r i f i e d c e l l w a l l and e x t r a c e l l u l a r m a t e r i a l were a n a l y z e d f o r amino a c i d s , amino sugars and n e u t r a l sugars a f t e r s e r i a l h y d r o l y s i s . E x t r a c e l l u l a r m a t e r i a l was a l s o a n a l y z e d f o r phosphorus and u r o n i c a c i d s by c o l o r i m e t r i c methods and f o r 0 - a c e t y l groups by g a s - l i q u i d chromatography. The p r o t e i n p o r t i o n s o f c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l c o n t a i n e d the same amino a c i d s i n d i f f e r e n t p r o p o r t i o n s , s p e c i f i c f o r each s t r a i n . E x t r a c e l l u l a r m a t e r i a l from two s t r a i n s o f C r . neoformans, 365-16 and 365-26, was very s i m i l a r i n amino a c i d c o m p o s i t i o n . Ethanolamine and glucosamine occur-r e d i n a l l p r e p a r a t i o n s , c y s t e i n e / c y s t i n e o n l y i n C r . a l b i d u s and C r . l a u r e n t i i , and galactosamine o n l y i n C r . neoformans w a l l s . Glucose was the predominant c e l l w a l l monomer; x y l o s e , mannose and g a l a c t o s e o c c u r r e d i n s m a l l e r q u a n t i t i e s . E x t r a c e l l u l a r m a t e r i a l c o n t a i n e d l a r g e amounts o f mannose, a t r a c e o f glucose and, i n a d d i t i o n , g l u c u r o n i c a c i d . Only the C r . neoformans s t r a i n s c o n t a i n e d O - a c e t y l groups. E x t r a c e l l u l a r m a t e r i a l and c e l l w a l l composition were s u f f i c i e n t l y d i f f e r e n t to suggest independent s y n t h e s i s . The amount o f capsule s y n t h e s i s and p o l y s a c c h a r i d e composition were e a s i l y a f f e c t e d by growth con-d i t i o n s . E x t r a c e l l u l a r p o l y s a c c h a r i d e produced by the most v i r u -l e n t s t r a i n o f C r . neoformans (365-11), c o n t a i n e d more mannose, g l u c u r o n i c a c i d and O - a c e t y l groups than the l e s s v i r u l e n t s t r a i n s . C e l l w a l l s o f Cr. neoformans 365-26 had more glucose and l e s s glucosamine, mannose and x y l o s e than d i d the w a l l s o f the nonpathogenic Cr. l a u r e n t i i . Both c e l l w a l l and e x t r a c e l l u l a r polymers were s i m i l a r i n composition to those produced by T r e m e l l a mesenterica and other basidiomycetes. Hyphal Cr . neoformans (Coward s t r a i n ) produced septate hyphae and clamp c o n n e c t i o n s . A c l o s e taxo-nomic r e l a t i o n s h i p t o the heterobasidiomycetes i s proposed. i v TABLE OF CONTENTS Page A b s t r a c t . i i Table o f Contents i v L i s t o f Tables .... v i L i s t o f F i g u r e s i x A b b r e v i a t i o n s x Acknowledgements x i Chapter I INTRODUCTION 1 Incidence and D i s t r i b u t i o n 1 Systematics • 3 Pathology and Immunology 4 C e l l W a l l and Capsule B i o c h e m i s t r y 8 II EXPERIMENTAL PROCEDURE 16 1. M a t e r i a l s 16 A. Organisms *• 16 B. Chemicals 16 2. Methods 18 A. K i l l Procedure 18 (1) Chemical Treatments 18 (2) - R a d i a t i o n 18 (3) Heat-Treatment 19 B. D e c a p s u l a t i o n 19 C. Growth C o n d i t i o n s 20 D. Hyphal Growth Forms 22 E. E x t r a c t i o n and P u r i f i c a t i o n o f E x t r a c e l l u l a r M a t e r i a l 23 F. C e l l W a l l E x t r a c t i o n and P u r i f i c a t i o n 24 G. A n a l y t i c a l Procedures 26 (1) Elemental and Ash A n a l y s i s 27 (2) Amino A c i d s 27 (3) Amino Sugars 28 (4) N e u t r a l Sugars 28 (5) U r o n i c A c i d s 31 (6) O-Acetyl 31 V H. E l e c t r o p h o r e s i s and S t a i n i n g 32 (1) Polyacrylamide Gels . 32 (2) C e l l u l o s e A c e t a t e S t r i p s 33 I . Gel Chromatography 34 J . I n f r a r e d S p e c t r a .* 34 K. V i r u l e n c e T e s t i n g w i t h Mice 35 I I I RESULTS 36 1. K i l l Procedure 36 2. D e c a p s u l a t i o n . 40 3. Growth C o n d i t i o n s .. 43 A. Temperature 43 B. pH . . 43 C. Thiamine .... 43 4. C e l l and Capsule Morphology . • 44 5. Growth o f Hyphal Forms 49 6. E x t r a c e l l u l a r M a t e r i a l - E x t r a c t i o n .. 51 7. C e l l W a l l s - E x t r a c t i o n 53 8. A n a l y t i c a l S t u d i e s 53 A. C e l l W a l l A n a l y s i s 53 (1) Amino A c i d s ...... 53 (2) Amino Sugars 54 (3) N e u t r a l Sugars 58 B. E x t r a c e l l u l a r M a t e r i a l A n a l y s i s 58 (1) Amino A c i d s 58 (2) Amino Sugars 62 (3) N e u t r a l Sugars 66 (4) Uronic A c i d s 66 (5) O-Acetyl 66 (6) Elemental and Ash A n a l y s i s 71 (7) Complete A n a l y s i s o f E x t r a c e l l u l a r M a t e r i a l 71 9. Gel E l e c t r o p h o r e s i s 75 A. P o l y a c r y l a m i d e Gels 75 B. C e l l u l o s e A c e t a t e S t r i p s 75 10. Gel Chromatography 79 11. I n f r a r e d Spectroscopy 79 12. V i r u l e n c e T e s t i n g w i t h Mice 86 IV DISCUSSION 89 P r e l i m i n a r y S t u d i e s and Growth C o n d i t i o n s o f Cryptococcus s p e c i e s 89 C e l l Morphology o f Yeast and Hyphal Forms o f C r y p t o -coccus 92 I s o l a t i o n o f the C r y p t o c o c c a l C e l l Envelope 95 Chemical Composition o f the C e l l W a l l s 96 R e l a t i o n o f C e l l W a l l Chemistry to P a t h o g e n i c i t y 101 Chemical Composition o f the E x t r a c e l l u l a r M a t e r i a l .... 102 R e l a t i o n o f Cryptococcus S t r a i n V i r u l e n c e to Chemistry o f the C e l l Envelope ' 114 CONCLUSIONS 119 BIBLIOGRAPHY 123 v i LIST OF TABLES TABLE Page I E f f e c t s o f i n c u b a t i o n f o r d i f f e r e n t times w i t h f o r m a l i n ( f i n a l c o n c e n t r a t i o n 0.5% HCHO) and phenol ( f i n a l c o n c e n t r a t i o n 0.5%) 37 I I Amino a c i d content o f c e l l w a l l s o f C r . l a u r e n t i i 371-1 a f t e r t r e a t i n g the c e l l s w i t h phenol (0V5% f i n a l c o n c e n t r a t i o n ) f o r 10 hr p r i o r to h a r v e s t .... 38 I I I E f f e c t o f temperature on growth o f three s t r a i n s o f C r . neoformans 39 IV Amino a c i d and n e u t r a l sugar content o f e x t r a c e l l u -l a r m a t e r i a l o f C r . neoformans (365-26) a f t e r heat treatment a t 55C f o r 90 min p r i o r to h a r v e s t 41 V E f f e c t o f heat treatment a t 55C on s i x Cryptococcus s t r a i n s growing i n LCM 42 VI P h y s i c a l appearance and y i e l d o f c r y p t o c o c c a l e x t r a -c e l l u l a r m a t e r i a l 52 VII Amino a c i d s i n the c e l l w a l l s o f C r . l a u r e n t i i (371-1) and C r . neoformans (365-26) 55 VI'II Amino a c i d s i n the c e l l w a l l s o f C r . l a u r e n t i i , C r . neoformans. and T r e m e l l a mesenterica 56 IX Amino sugars i n the c e l l w a l l s of C r . l a u r e n t i i (371-1) and C r . neoformans (365-26) 57 X N e u t r a l sugars i n the c e l l w a l l o f C r . l a u r e n t i i (371^1) 59 XI N e u t r a l sugars i n the c e l l w a l l o f Cr. neoformans (365-26) 59 XII Amino a c i d s i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), C r . a l b i d u s (367 and H1354) and Cr. l a u r e n t i i (371-1) (Expressed as jig/mg e x t r a c e l l u l a r m a t e r i a l ) 60 X I I I Amino a c i d s i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), C r . a l b i d u s (367 and H1354) and Cr. l a u r e n t i i (371-1) (expressed as p e r c e n t t o t a l amino a c i d s ) 61 XIV Amino a c i d s i n the e x t r a c e l l u l a r m a t e r i a l and c e l l w a l l s o f C r . neoformans (365-26) and C r . l a u r e n t i i (371-1) 63 v i i XV Amino a c i d r a t i o s i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), Cr. a l -bidus (367 and H1354) and C r . l a u r e n t i i (371-1) and the c e l l w a l l s o f C r . neoformans (365-26) and C r . l a u r e n t i i (371-1) 64 XVI Amino sugars i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), Cr. a l b i d u s (367 and H1354) and C r . l a u r e n t i i (371-1) 65 XVII N e u t r a l sugars i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), C r . a l b i d u s (367 and H1354) and C r . l a u r e n t i i (371-1). (Expres-sed as ug/mg e x t r a c e l l u l a r m a t e r i a l ) .............. 67 XVIII, N e u t r a l sugars i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), Cr. a l b i d u s (367 and H1354) and Cr. l a u r e n t i i (371-1). (Expres-sed as p e r c e n t t o t a l n e u t r a l sugar) 68 XIX U r o n i c a c i d i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), Cr. a l b i d u s (367 and H1354) and Cr. l a u r e n t i i (371-1) 69 XX O - a c e t y l g r o u p s - i n the e x t r a c e l l u l a r m a t e r i a l o f C r . neoformans (365-11, 365-16 and 365-26), Cr. a l b i d u s (367 and H1354) and C r . l a u r e n t i i (371-1) 69 XXI N e u t r a l sugars and u r o n i c a c i d s o f c r y p t o c o c c a l e x t r a c e l l u l a r m a t e r i a l expressed as mole p e r c e n t o f t o t a l nm sugar 70 XXII N e u t r a l sugars, u r o n i c a c i d s and 0 - a c e t y l groups o f e x t r a c e l l u l a r m a t e r i a l from Cr . neoformans (365-11, 365-16 and 365-26) expressed as mole p e r c e n t o f t o t a l nm sugar 70 XXIII Ash, phosphorus and n i t r o g e n a n a l y s i s o f e x t r a c e l l u -l a r m a t e r i a l from Cr . neoformans (365-11, 365-16 and 365-26), Cr. a l b i d u s (367 and H1354) and C r . l a u r e n -t i i (371-1) 72 XXIV D i f f e r e n c e s i n r e c o v e r i e s o f amino a c i d s and amino/ sugars i n two separate p r e p a r a t i o n s o f e x t r a c e l l u l a r m a t e r i a l from Cr . a l b i d u s HI354 73 XXV Complete a n a l y s i s o f e x t r a c e l l u l a r m a t e r i a l from Cr . neoformans (365-11, 365-16 and 365-26), Cr. a l b i d u s (367 and H1354) and C r . l a u r e n t i i (371-1) 74 XXVI Mean Rp va l u e s o f g l y c o p r o t e i n and p r o t e i n bands o f e x t r a c e l l u l a r m a t e r i a l from Cr. a l b i d u s 367 on p o l y -acrylamide g e l s 80 v i i i XXVII Mean Rp valu e s o f g l y c o p r o t e i n and c a r b o x y l i c a c i d bands o f e x t r a c e l l u l a r m a t e r i a l from Cryptococcus s p e c i e s on c e l l u l o s e a c e t a t e s t r i p s 80 XXVIII I n o c u l a t i o n o f mice i n t r a c e r e b r a l l y w i t h v i a b l e c e l l s o f C r . neoformans (365-11, 365-16 and 365-26), C r . a l b i d u s (367 and H1354) and Cr. l a u r e n t i i (371-1) 87 i x LIST OF FIGURES FIGURE Page 1 Proposed s t r u c t u r e of c a p s u l a r p o l y s a c c h a r i d e o f C r . neoformans a c c o r d i n g to Miyazaki (1961) 10 2 E f f e c t o f thiamine on growth and capsule s y n t h e s i s o f C r . a l b i d u s 367 and C r . a l b i d u s H1354 45 3 E f f e c t o f thiamine on growth and capsule s y n t h e s i s o f C r . l a u r e n t i i 371-1 and C r . neoformans '365-16 . 46 4 E f f e c t o f thiamine on growth and capsule s y n t h e s i s o f C r . neoformans 365-11 and 365-26 47 5 D i f f e r e n c e s i n capsule s i z e between c e l l s grown i n 0.1 and 10.0 ug/ml thiamine f o r each Cryptococcus s t r a i n 48 6 Hyphal growth forms i n C r . neoformans 50 7 E l e c t r o p h o r e t i c p a t t e r n o f C r . a l b i d u s 367 e x t r a -c e l l u l a r m a t e r i a l a p p l i e d to p o l y a c r y l a m i d e g e l s 76 8 E l e c t r o p h o r e t i c p a t t e r n o f c r y p t o c o c c a l e x t r a c e l l u -l a r m a t e r i a l a p p l i e d to c e l l u l o s e a c e t a t e s t r i p s . C r . neoformans 365-11 and 365-26 77 9 E l e c t r o p h o r e t i c p a t t e r n o f c r y p t o c o c c a l e x t r a c e l l u -l a r m a t e r i a l a p p l i e d to c e l l u l o s e a c e t a t e s t r i p s . C r . a l b i d u s 367 and Cr . l a u r e n t i i 371-1 78 10 Chromatography o f e x t r a c e l l u l a r p o l y s a c c h a r i d e from Cr . neoformans 365-26 on a DEAE BioGel-A column : 81 11 I n f r a r e d a b s o r p t i o n spectrum o f e x t r a c e l l u l a r m a t e r i a l 82 12 I n f r a r e d a b s o r p t i o n s p e c t r a o f e x t r a c e l l u l a r m a t e r i a l from Cryptococcus s p e c i e s 84 13 I n f r a r e d a b s o r p t i o n s p e c t r a o f c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l from Cr . l a u r e n t i i 371-1 compared w i t h spectrum from glucosamine HC1 85 14 C r . neoformans 365-26 i n b r a i n smears o f Swiss white mice 88 ABBREVIATIONS Symbols used f o r Monomers A l a alanirie) H i s h i s t i d i n e Arg a r g i n i n e Hyp 4-hydroxyproline Asp a s p a r t a t e H e i s o l e u c i n e Asx a s p a r t a t e o r asparagine Leu l e u c i n e (undefined) Lys l y s i n e Cys c y s t e i n e Man mannose Gal g a l a c t o s e Met methionine GalN galactosamine (2-amino-2-deoxy-galactose) Phe p h e n y l a l a n i n e G l c glucose Pro p r o l i n e GlcN glucosamine (2-amino- Ser s e r i n e 2-deoxy-glucose) Thr threonine GlcNx glucosamine or N - a c e t y l -glucosamine (undefined) Tyr - t y r o s i n e Glu glutamate V a l v a l i n e Glx glutamate or glutamine X y l x y l o s e (undefined) Gly g l y c i n e x i ACKNOWLEDGEMENTS T h i s r e s e a r c h p r o j e c t was supported by grants to myself from the Sigma XI S o c i e t y and to Dr. I.E.P. T a y l o r from the N a t i o n a l Research C o u n c i l o f Canada and the U n i v e r s i t y o f B r i t i s h Columbia. C u l t u r e s o f f u n g i were p r o v i d e d by Dr. L. Kapica o f M c G i l l U n i v e r s i t y , Montreal and by Dr. R.J. Bandoni who helpe d w i t h c u l t u r i n g and taxonomic problems. Dr. J . J . Stock i n t r o -duced me to the problem o f c r y p t o c o c c o s i s and much o f the e a r l y work was done i n h i s l a b o r a t o r y . . I am p a r t i c u l a r l y g r a t e f u l t o Dr. Barbara D i l l f o r her advice and encouragement. Drs. C O . Person and G.C. Hughes k i n d l y p r o v i d e d i n c u b a t o r space f o r pathogenic c r y p t o c o c c i . Dr. P.E. Re i d and C h a r l i e Ramey shared t h e i r e x p e r t i s e i n g a s - l i q u i d chromatography and f r a c t i o n a t i o n , and were always a v a i l a b l e f o r c o n s u l t a t i o n . Dr. Don Cameron taught me to be p a t i e n t w i t h the gas chromatograph and amino a c i d a n a l y z e r and was f o r e v e r f u l l o f su g g e s t i o n s . Mel Davies p r o v i d e d prompt t e c h n i c a l a s s i s t a n c e . Most o f a l l I would l i k e t o thank Dr. I a i n T a y l o r f o r making t h i s t h e s i s p o s s i b l e . He p r o v i d e d encouragement, idea s and the necessary i n s t i g a t i o n . I enjoyed b e i n g a member o f h i s l a b o r a t o r y and won't f o r g e t my c o l l e a g u e s . F i n a l l y I want t o thank T r i s h Burnside f o r t y p i n g the t h e s i s and Tim Ames f o r immoral support. 1. CHAPTER I INTRODUCTION Incidence and D i s t r i b u t i o n Cryptococcus neoformans ( S a n f e l i c e ) V u i l l e m i n i s the s o l e c a u s a t i v e agent o f c r y p t o c o c c o s i s , an i n f e c t i o u s pulmonary di s e a s e o f man and animals, which f r e q u e n t l y spreads to the cen-t r a l nervous system i n s u s c e p t i b l e p a t i e n t s . The d i s e a s e i s w o r l d wide i n occurrence but i t s a c t u a l i n c i d e n c e i s unknown. Since 1952 the number o f deaths r e p o r t e d due to Cr. neoformans inf*'the U n i t e d S t a t e s has averaged 66 per year ( A j e l l o 1967). Littman and Schneierson (1959) estimated t h a t 5,000-,-- 15,000 cases o f s u b c l i n i c a l or c l i n i c a l pulmonary c r y p t o c o c c o s i s occur a n n u a l l y i n New York C i t y alone. B i r d droppings are thought to c o n s t i t u t e the main r e s e r v o i r s o f i n f e c t i o n ( A j e l l o 1958, Emmons 1954, L i t t m a n and Schneierson 1959) and Cr. neoformans i s found i n f r e q u e n t l y i n n a t u r a l s o i l s and p l a n t sources ( S t a i b and Altmann 1973, F e l t o n e t a l . 1974, S n e l l e r and Swatek 1974). The presence o f t h i s fungus i n the atmosphere and c e r t a i n s o i l s has been e x p l a i n e d by p r i o r exposure to human and animal, p a r -t i c u l a r l y b i r d , e x c r e t a and s e c r e t i o n s ( A j e l l o 1967, G e n t l e s and La Touche 1969). S t a i b (1962, 1963) suggested t h a t the p a r t i c u l a r c l o s e a s s o c i a t i o n o f Cr. neoformans w i t h b i r d drop-pings was r e l a t e d to c r e a t i n i n e , a c o n s t i t u e n t o f b i r d u r i n e , which the y e a s t can u t i l i z e as a s o l e source o f g n i t r o g e n . S t a i b e t a l . (1972) a l s o found t h a t C r . neoformans was able to 2 . c o l o n i z e dead p l a n t s and proposed t h a t decaying p l a n t m a t e r i a l might p r o v i d e an a d d i t i o n a l r e s e r v o i r f o r Cr. neoformans i n n a t u r e . Other members o f the genus Cryptococcus are non-pathogenic and occur u n i v e r s a l l y i n s o i l s , the p h y l l o s p h e r e , i n -c l u d i n g mosses, and the atmosphere (Carmo-Sousa 1969): they have a l s o been i s o l a t e d from 117 d i f f e r e n t s p e c i e s o f p l a n t s ( S t a i b and Altmann 1973). Recent s t u d i e s by S n e l l e r and Swa.tek (1974) have shown a r e l a t i o n s h i p between the average d a i l y tem-p e r a t u r e s and s o i l pH and the s p e c i e s o f Cryptococcus i s o l a t e d from Southern C a l i f o r n i a s o i l s . Cr. -albidus s t r a i n s p r e f e r r e d a temperature range o f 15-22C and a pH o f 5.4-6.6; Cr. d i f f l u e n s was found i n r e g i o n s from 21-26C w i t h pH 6.9-7.9 and Cr. l a u r e n t i i o c c u r r e d at 27-29C and pH 8.1-8.3. Ishaq e t a l . (1967) found t h a t s t e r i l e s o i l s seeded w i t h C r . neoformans supported growth bes t under a l k a l i n e c o n d i t i o n s (pH 7.7 as compared w i t h 7.4 and 6.8) and when humidity was h i g h and temperatures moderate to low. Although Cr. neoformans grows i n mammals at 37C i t does not grow w e l l at 39.4C, and the optimum temperature f o r growth appears to be 29C (Kuhn 1949). T h i s s e n s i t i v i t y to temperatures above 39C may e x p l a i n why b i r d s , which have a h i g h e r body temperature than mammals, are merely c a r r i e r s o f the d i s e a s e . They are seldom i n f e c t e d w i t h the fungus, although Littman e t a l . (1965) found t h a t i n t r a c e r e b r a l i n o c u l a t i o n s o f pigeons caused a f a t a l systemic i n f e c t i o n . Of the o t h e r s p e c i e s o f C r y p t o c o c c u s , o n l y some s t r a i n s o f C r . l a u r e n t i i have the a b i l i t y to grow at 37C. There have been o c c a s i o n a l r e p o r t s of Cr. l a u r e n t i i b e i n g pathogenic to man ( B l a i r e_t a_l. 1970), and more r e c e n t l y Cr. a l b i d u s has been i m p l i c a t e d as the c a u s a l agent o f c r y p t o c o c c o s i s (Da Cunha 1973, Wieser 1973), although t h i s s p e c i e s has a lower optimum growth temperature (Phaff and F e l l 1970). Systematics Phaff and F e l l (1970) based c l a s s i f i c a t i o n o f the genus on (a) the a b i l i t y to a s s i m i l a t e i n o s i t o l as a carbon source f o r growth, (b) the p r o d u c t i o n o f a h e t e r o p o l y s a c c h a r i d e cap-s u l e , (c) the absence o f a w e l l developed pseudomycelium, and (d) the l a c k o f a b i l i t y t o produce sexual stages (ascospores, t e l i o s p o r e s or b a l l i s t o s p o r e s ) or to ferment sugars. However, Bab'yeva and Golubev (1970, c i t e d by Golubev e t _ , a l . 1971) des-c r i b e d c u l t u r e s o f Cryptococcus which do not a s s i m i l a t e i n o s i -t o l but are t y p i c a l i n o t h e r r e s p e c t s . An i n c r e a s i n g number of r e p o r t s have suggested the e x i s t e n c e o f s e x u a l l y a c t i v e spe-c i e s and have proposed a r e l a t i o n s h i p to the lower basidiomy-c e t e s . Van der Walt (1967) observed a type o f endospore forma-t i o n which r e s u l t e d from i n t e r n a l budding a f t e r c o n j u g a t i o n o f h a p l o i d C r . a l b i d u s c e l l s . However, he d i d not c o n c l u s i v e l y demonstrate t h a t the zygote was d i p l o i d . Kurtzman (1973) i s o l a t e d mating s t r a i n s o f C r . l a u r e n t i i . These formed con-j u g a t i o n tubes which gave r i s e to hyphae, 5% o f which were b i n u c l e a t e : the hyphae a l s o produced chlamydospores. The c o n j u g a t i o n tubes were s i m i l a r to those found i n T r e m e l l a spp. (Bandoni 1963) and i n Sporobolomyces odorus Derx (Bandoni ejt 4. a l . 1971). Kurtzman (1973) was unable to o b t a i n any mating response between h a p l o i d forms o f e i t h e r T r e m e l l a a u r a n t i a orj T r e m e l l a encephala and Cr. l a u r e n t i i var l a u r e n t i i although these s t r a i n s had s i m i l a r carbon a s s i m i l a t i o n p a t t e r n s . He r e l a t e d the presence o f h a u s t o r i a - l i k e s t r u c t u r e s i n Cr. l a u r e n t i i to a p o s s i b l e p a r a s i t i c e x i s t e n c e i n n a t u r e . No clamp connections were observed on C r . l a u r e n t i i m y c e l i a (Kurtzman 1973) although Shadomy (1970) i s o l a t e d two s t r a i n s o f C r . neoformans which produced hyphae and formed clamp c o n n e c t i o n s . Pseudomycelium has been r e p o r t e d s e v e r a l times i n the l i t e r a t u r e (Cox and T o l h u r s t 1946, Shadomy and Utz 1966) as w e l l as o c c a s i o n a l r e p o r t s o f sexual r e p r o d u c t i o n (Todd and Hermann 1936, Benham 1955). L u r i e and Shadomy (1971) have demonstrated hyphae i n e l e v e n s t r a i n s o f Cr. neoformans and consequently r e g a r d hyphal forms as other than chance muta-t i o n s . However, sexual stages have y e t to be confirmed i n C r . neoformans. The major d i f f e r e n c e between Cr. neoformans, the type s p e c i e s o f the genus (Phaff and F e l l 1970), and the o t h e r s p e c i e s , i s i t s p a t h o g e n i c i t y i n man and mammals. Pathology and Immunology Cr. neoformans i s thought to enter the human lungs as y a nonencapsulated y e a s t l e s s than 5 pm i n diameter ( F a r h i e t a l . 1970). In a normal i n d i v i d u a l the y e a s t w i l l be e n g u l f e d and k i l l e d by polymorphonuclear l e u k o c y t e s (Tacker e_t a l . 1972) without p r o d u c t i o n o f d i s e a s e (Atkinson and Bennett 1968). K i l l i n g o f microorganisms by l e u k o c y t e s normally r e q u i r e s o p s o n i z a t i o n (Mayer 1973): t h a t i s , r e c o g n i t i o n o f the invader by an antibody, a c t i v a t i o n o f the complement system by t h a t antibody and f i x a t i o n o f the complement on the i n v a d i n g c e l l ' s s u r f a c e . T h i s i s the c l a s s i c a l complement pathway. The a l t e r -nate or p r o p e r d i n pathway does not r e q u i r e antibody to i n i t i a t e i t , but i n s t e a d i n v o l v e s assembly o f p r o p e r d i n enzymes i n r e s -ponse t o m i c r o b i a l p o l y s a c c h a r i d e s (such as y e a s t zymosan), which then a c t i v a t e the l a t e r complement components. E i t h e r way the complement serves to l y s e the i n v a d i n g c e l l or render i t s u s c e p t i b l e to phag o c y t o s i s (Mayer 1973). Most s t u d i e s on c r y p t o c o c c o s i s have suggested t h a t host c e l l u l a r responses, i n c l u d i n g p h a g o c y t o s i s , p l a y the major r o l e i n c o n f e r r i n g r e s i s t a n c e a g a i n s t t h i s i n f e c t i o n (Abrahams e_t a l . 1970), Gadebusch 1972), and i t was thought t h a t humoral a n t i c r y p t o c o c c a l p o l y s a c c h a r i d e d i d not have a p r o t e c t i v e r o l e (Goren and Middlebrook 1967). However, potent a n t i c r y p t o c o c c a l f a c t o r s have been found i n normal human serum (Baum and A r t i s 1963, S z i l a g y i e t a l . 1966) and Bulmer and Tacker (1975) be-l i e v e d t h a t these, along w i t h c a t i o n i c p r o t e i n s r e l e a s e d from ho s t c e l l s , enhance p h a g o c y t o s i s . Baum and A r t i s (1963) con-cluded t h a t the a n t i c r y p t o c o c c a l serum f a c t o r was not r e l a t e d to complement or p r o p e r d i n s i n c e it<>.was heat s t a b l e a t tempera-t u r e s up to 65C. However, Diamond e t a l . (1972) and C l i n e e t a l . (1968) found a requirement f o r a heat l a b i l e f a c t o r i n pha g o c y t o s i s o f c r y p t o c o c c i . Diamond e_t a l . (1974) r e c e n t l y suggested t h a t complement does p l a y a r o l e i n the phag o c y t o s i s o f c r y p t o c o c c i by human p e r i p h e r a l b l o o d l e u k o c y t e s . They were 6. able to demonstrate antibody to c r y p t o c o c c i i n normal serum by a s e n s i t i v e a b s o r p t i o n technique, although i t c o u l d not be i d e n t i f i e d w i t h the u s u a l i n d i r e c t f l u o r e s c e n t antibody t e c h -nique. The c l a s s i c a l pathway appeared to be f u n c t i o n i n g p r i -m a r i l y to a c t i v a t e the a l t e r n a t e pathway, which i n t u r n was r e s p o n s i b l e f o r o p s o n i z a t i o n . S p i n a l f l u i d d i d not opsonize c y p t o c o c c i and no complement was detected on the s u r f a c e of c r y p t o c o c c i o b t a i n e d from the s p i n a l f l u i d o f p a t i e n t s w i t h c r y p t o c o c c a l m e n i n g i t i s . However, the f a c t t h a t s p i n a l f l u i d from p a t i e n t s w i t h the d i s e a s e c o u l d mediate o p s o n i z a t i o n o f c r y p t o c o c c i i n absorbed normal serum suggested t h a t a n t i -c r y p t o c o c c a l antibody and p o s s i b l y p r o p e r d i n were prese n t i n the s p i n a l f l u i d . I g e l and Bolande (1966) were a l s o unable to d e t e c t an a n t i c r y p t o c o c c a l f a c t o r i n s p i n a l f l u i d . A c cord-i n g t o Gadebusch and Johnson (1966) c a t i o n i c p r o t e i n s s t i m u l a t e l e u k o c y t e e m i g r a t i o n and adhesion and i n c r e a s e c a p i l l a r y permea-b i l i t y . Chemotaxis, immune adherence, and r e l e a s e of h i s t a -mine^ (which i n c r e a s e s permeability o f b l o o d c a p i l l a r i e s from l e u k o c y t e s , mast c e l l s and p l a t e l e t s ) are a l l a l s o a c t i v i t i e s o f the l a t e complement components (Mayer 1963). I n d i v i d u a l s who develop c r y p t o c o c c o s i s are thought to have d e f i c i e n t immune systems. The d i s e a s e i s encountered more f r e q u e n t l y i n p a t i e n t s w i t h Hodgkin's d i s e a s e , lymphosarcoma, leukemia, and d i a b e t e s m e l l i t u s and i n those r e c e i v i n g prolonged s t e r o i d therapy (Littman and Walter 1968). F a r h i e t a l . (1970) proposed t h a t i n these people, ph a g o c y t o s i s o f the unencapsula-t e d y e a s t i s delayed s u f f i c i e n t l y . t o a l l o w l a r g e amounts o f 7. c a p s u l a r m a t e r i a l to be produced which then i n h i b i t s phago-c y t o s i s . Borowski et. al_. (1974) found t h a t s u b i n h i b i t o r y con-c e n t r a t i o n s o f amphotericin B i n h i b i t e d capsule formation i n c r y p t o c o c c a l c e l l s which were then more r e a d i l y p h a g o c y t i z e d . M i t c h e l l and Friedman (1972) a l s o found t h a t the degree o f p h a g o c y t o s i s was p r o p o r t i o n a l to c a p s u l a r t h i c k n e s s . However, the a b i l i t y o f the macrophages to k i l l the y e a s t c e l l s was un-r e l a t e d to capsule t h i c k n e s s and seemed l a r g e l y s t r a i n dependent. Bulmer and Tacker (1975) b e l i e v e d t h a t the capsule i t s e l f was the v i r u l e n c e f a c t o r and they a l s o proposed t h a t myeloperoxi-dase i n normal human s a l i v a may p r o v i d e an a d d i t i o n a l a n t i -c r y p t o c o c c a l f a c t o r . However, i t seems to me t h a t the i n i t i a l f a i l u r e to a c t i v a t e o p s o n i z a t i o n o f the c r y p t o c o c c i i s the c r u c i a l f a c t o r i n the development o f the d i s e a s e and i t s u l t i -mate spread to the c e n t r a l nervous system. T h i s may be dependent upon f a i l u r e o f the i n d i v i d u a l to form a n t i b o d i e s to the unen-c a p s u l a t e d y e a s t or f a i l u r e o f the complement system to adhere to a c t i v a t e d y e a s t c e l l s . The l a t t e r now seems more l i k e l y i n view o f Diamond e t a l . ' s work (1974). Farmer and Komorowski (1973) r e c e n t l y r e p o r t e d a case o f a p a t i e n t i n f e c t e d w i t h a c a p s u l e - d e f i c i e n t s t r a i n of Cr. neoformans. The c a p s u l e - d e f i c i e n t y e a s t invoked an i n t e n s e inflammatory response i n mice which was c h a r a c t e r i z e d by e a r l y suppuration and p h a g o c y t o s i s f o l l o w e d by marked h i s t i o c y t i c and f i b r o b l a s t i c r e a c t i o n , l i m i t i n g the i n f e c t i o n . These f e a -t u r e s were a l s o d e s c r i b e d by Shadomy and L u r i e (1971) working w i t h i n f e c t i o n s caused by the unencapsulated hyphal Coward ' 8. s t r a i n o f C r . neoformans. Thus the c r y p t o c o c c a l antigens may i n v o l v e c e l l w a l l as w e l l as c a p s u l a r components, and normal antiserum may c o n t a i n a n t i b o d i e s to both (Vogel 1966). Most i n v e s t i g a t i o n s have demonstrated t h a t the c a p s u l a r p o l y s a c c h a r i d e i s not a complete a n t i g e n (Cozad e t a_l. 1963, Goren 1966, 1967) and Gadebusch (1960) r e p o r t e d t h a t the a n t i g e n i c m a t e r i a l r e -s i d e s i n the c a p s u l a r p o l y s a c c h a r i d e t h a t i s c l o s e l y bound to the w a l l . D e v l i n (1969) found t h a t z e o l i t e ghost p r e p a r a t i o n s o f whole c e l l s were the most e f f i c i e n t immunizing agents and t h a t the c e l l w a l l a l s o had a n t i g e n i c p r o p e r t i e s . Farmer and Kamorowski (1973) and D e v l i n (1969) have i m p l i e d t h a t the a n t i -g e nic f a c t o r beneath the;-:.capsule may be p r o t e i n a c e o u s . The c e l l w a l l s o f another pathogenic y e a s t , Histoplasma capsulatum, appeared the most a n t i g e n i c a l l y a c t i v e f r a c t i o n i n complement f i x a t i o n t e s t s (Odds e t a l . 1971). Pine and Boone (1968) found l a r g e r q u a n t i t i e s o f both c h i t i n and amino a c i d s i n the c e l l w a l l s o f a n t i g e n i c as compared w i t h n o n a n t i g e n i c s t r a i n s o f Histoplasma capsulatum. v. •. Higher l e v e l s o f c e l l w a l l c h i t i n , p r o t e i n andwphos-p h o l i p i d have a l s o been r e l a t e d to v i r u l e n c e ( f o r mice) i n s t r a i n s o f Blastomyces d e r m a t i t i d i s (Cox and Best 1972 and Di Salvo and Denton 1963). C e l l W a l l and Capsule B i o c h e m i s t r y Only r e c e n t l y has the c e l l w a l l o f Cr. neoformans r e -c e i v e d any a t t e n t i o n , perhaps because the c e l l s are so d i f f i c u l t t o d i s r u p t ( D e v l i n 1969, Erke and Schneidau 1973). Cook e t a l . (1970) compared c e l l w a l l s o f C r . neoformans i s o l a t e d from p a t i e n t s and from s o i l and, i n e s s e n t i a l l y q u a l i t a t i v e work, noted d i f f e r e n c e s i n l i p i d , carbohydrate and hexosamine content No amino a c i d s t u d i e s have been performed except by Uzman et a l . (1956) on somatic p r o t e i n s e x t r a c t e d from a s t r a i n o f Cr. neoformans by treatment o f the c e l l s w i t h hot a l k a l i . The c e l l w a l l s o f nonpathogenic s p e c i e s C r . a l b i d u s and C r . t e r r e u s c o n s i s t e d mainly o f o c - and / i - l i n k e d glucans, whose p r o p o r t i o n s were a f f e c t e d by the composition o f the growth medium (Jones et a l . 1969, Bacon et. a l . 1968). Glucan r e p r e s e n t e d 75% o f the Cr. a l b i d u s c e l l w a l l s (Bacon e_t a l . 1968). The main n i t r o genous c o n s t i t u e n t was glucosamine, p r e s e n t as c h i t i n . Jones et a l . (1969) found t h a t the c h i t i n o u s r e s i d u e , a f t e r a c i d and a l k a l i n e e x t r a c t i o n , was composed o f m i c r o f i b r i l s r a t h e r than b e i n g g r a n u l a r as i n Saccharomyces s p e c i e s . The c a p s u l a r p o l y s a c c h a r i d e s o f Cr. neoformans have a n t i g e n i c a c t i v i t y and have been c l a s s i f i e d i n t o f o u r s e r o l o -g i c a l types; A, B, C and R, d i f f e r e n t i a t e d on the b a s i s o f p r e c i p i t i n , a g g l u t i n a t i o n and " q u e l l i n g " r e a c t i o n s (Evans and K e s s e l 1951). From s t r u c t u r a l and molecular s t u d i e s the cap-s u l e appears to c o n s i s t o f a mannose backbone w i t h branches o f x y l o s e and g l u c u r o n i c a c i d as shown i n F i g . 1 (Miyazaki 1961, Blandamer and Danishefsky 1966). Mannose a l s o o c c u r r e d a s s i d e u n i t s a t t a c h e d by (l-»4) l i n k a g e s . 10. 7 M 44 ^ M -/|4 M n G A 2 M 1 4 4 M 1 4 M I i G A •7 F i g . 1 Proposed s t r u c t u r e o f c a p s u l a r p o l y s a c c h a r i d e o f C r . neoformans a c c o r d i n g to M i y a z a k i (1961). M = ct-D-mannopyranosyl r e s i d u e ; X = c C - D - x y l o p y r a n o s y l r e s i d u e ; GA = ol -D-glucuronopyranosyl r e s i d u e . The s t r u c t u r e and composition o f both a c i d i c and neu-t r a l polymers produced by Cryptococcus s p e c i e s have been ex-t e n s i v e l y reviewed (Phaff 1971; G o r i n and Spencer 1968). Some of the s t u d i e s demonstrate the c l o s e r e l a t i o n s h i p between Cryptococcus s p e c i e s and the heterobasidiomycetous genus, T r e m e l l a ( S l o d k i e t a l . 1966, Helms e t a l . 1969). However, va l u e s f o r the p r o p o r t i o n s o f x y l o s e to mannose r e c o v e r e d from the h e t e r o p o l y s a c c h a r i d e s d i f f e r e d among the v a r i o u s r e p o r t s (Phaff 1971) and not a l l workers t e s t e d f o r , and found, g a l a c -tose and O - a c e t y l groups. Both Evans and T h e r i a u l t (1953) and Rebers e t a l . (1958) b e l i e v e d t h a t C r . neoformans produced two p o l y s a c c h a r i d e s , both types c o n t a i n e d g l u c u r o n i c a c i d but o n l y one c o n t a i n e d g a l a c t o s e . Evans and Mehl (1951) were unable to demonstrate any q u a l i t a t i v e d i f f e r e n c e s i n a c i d h y d r o l y s a t e s o f the three c r y p t o c o c c a l p o l y s a c c h a r i d e a n t i g e n s (A, B and C ) . However, '•' 11. . i n f r a r e d s pectroscopy (Levine e t a l . 1959) r e v e a l e d t h a t d i f f e r -ences i n the s e r o l o g i c a l types may depend .in p a r t on the degree o f a c e t y l a t i o n o f the p o l y s a c c h a r i d e s i d e c h a i n s . K o z e l and C a z i n (1971), compared the c h e m i s t r y o f s o l u b l e p o l y s a c c h a r i d e s from v i r u l e n t and weakly v i r u l e n t s t r a i n s and found the v i r u l e n t s t r a i n had a l a r g e r u r o n i c a c i d content and a l a r g e r m o l e c u l a r s i z e . The t e r m i n a l g l u c u r o n i c a c i d u n i t s have a l s o been i m p l i -c a t e d as a n t i g e n i c agents i n c r o s s - r e a c t i o n s between type I I antipneumococcal serum and C r . l a u r e n t i i and C r . neoformans (Helms e_t a l . 1969). The p o l y s a c c h a r i d e from T r e m e l l a mesen-t e r i c a d i d not c r o s s - r e a c t , and F r a s e r et a l . (1973b) have r e -c e n t l y shown t h i s t o be due t o s t e r i c i n t e r a c t i o n s . X y l o s e u n i t s l o c a t e d i n the v i c i n i t y o f the g l u c u r o n i c a c i d r e s i d u e s may be r e s p o n s i b l e f o r t h i s s t e r i c h i n d r a n c e . In a d d i t i o n , C r . l a u r e n t i i had x y l o s e p r e s e n t o n l y as nonreducing end groups (Miyazaki 1961), whereas T r e m e l l a mesenterica was shown to have 2-0-y3 l i n k e d xylopyranose s i d e c h a i n s , thus forming a much more h i g h l y branched s t r u c t u r e (Fraser et. a l . 1973a). Although the p r o t e i n content o f c r y p t o c o c c a l c a p s u l a r and e x t r a c e l l u l a r m a t e r i a l has not p r e v i o u s l y been i n v e s t i g a t e d , Masler e_t a l . (1966) r e p o r t e d the presence o f immunologically a c t i v e e x t r a c e l l u l a r p o l y s a c c h a r i d e - p r o t e i n complexes i n c e l l - f r e e c u l t u r e f l u i d o f Candida a l b i c a n s . Phaff (1971) suggested t h a t t h i s p r o t e i n may have been l o s t from c e l l s d u r i n g shaking or harvest, r a t h e r than, b e i n g a t r u e e x o c e l l u l a r p r o d u c t . However, M u l l e r and S e t h i (1972) d e s c r i b e d a p r o t e o -l y t i c a c t i v i t y o f Cr. neoformans a g a i n s t human plasma p r o t e i n s , and y e a s t s are known to produce e x t e r n a l i n v e r t a s e and a c i d phosphatases (Lampen 1968). The f a c t o r s r e s p o n s i b l e f o r the v a r i a t i o n s i n compo-s i t i o n o f c r y p t o c o c c a l a c i d i c h e t e r o p o l y s a c c h a r i d e s observed by Phaff (1971) may i n v o l v e d i f f e r e n c e s i n growth media, o r i -g i n and p u r i t y o f the p o l y s a c c h a r i d e s , and d i f f e r e n t methods o f chemical a n a l y s i s . Growth c o n d i t i o n s are known to a f f e c t both composition and p r o d u c t i o n o f e x t r a c e l l u l a r and c a p s u l a r p o l y s a c c h a r i d e ( F a r h i e t a l . 1970, Bulmer and Sans 1968, L i t t -man 1958, S l o d k i e t a l . 1970, Foda e t a l . 1973). Foda e t a l . (1973) found t h a t whereas an a c i d i c p o l y s a c c h a r i d e was synthe-s i z e d by Cr. l a u r e n t i i between pH 3 and 7, a n e u t r a l glucan was produced o n l y when the medium pH dropped to below 3.0 and growth was slowed. They d i d ,not c o n s i d e r this-amylose to be a: t r u e e x t r a c e l l u l a r p o l y s a c c h a r i d e . The oC-D-glue an i s o l a t e d from Cr. l a u r e n t i i i n casamino a c i d s - s a l t s medium a t pH 5 by Abercrombie et. al_. (1960) was not an amylose, and Phaff (1971) proposed t h a t i t may have o r i g i n a t e d from the c e l l w a l l . Bulmer and Sans (1968) found t h a t a low pH i n h i b i t e d the h e t e r o -p o l y s a c c h a r i d e capsule p r o d u c t i o n o f Cr. neoformans whereas h i g h pH s t i m u l a t e d c a p s u l e ^ s y n t h e s i s : d i f f e r e n t carbohydrate carbon sources a l s o a f f e c t e d c a p s u l e - p r o d u c t i o n . F a r h i et. a l . (1970) showed t h a t a d d i t i o n o f s a l t s and water t o s o i l samples c o n t a i n i n g Cr. neoformans i n h i b i t e d capsule p r o d u c t i o n . L i f t -man (1958) s t u d i e d the n u t r i t i o n a l growth f a c t o r s a f f e c t i n g capsule p r o d u c t i o n i n Cr. neoformans and demonstrated a thiamine and sodium glutamate requirement. However, F a r h i (1969) was unable t o e s t a b l i s h a growth s t i m u l a t o r y e f f e c t w i t h thiamine w i t h i n 24 h r . S l o d k i et a_l. (1970) r e p o r t e d t h a t under phos-phate l i m i t i n g c o n d i t i o n s Hansenula h o l s t i i and H. c a p s u l a t a no l o n g e r produced e x o c e l l u l a r phosphomannans, but i n s t e a d r e -l e a s e d non-phosphorylated mannans. The mannans were not o n l y l a c k i n g i n phosphate, but t h e i r s t r u c t u r e was changed: H. cap-s u l a t a r e p l a c e d the normal mixture o f & - a n d ^ - l i n k a g e s w i t h ^ -1inked mannan u n i t s and the H. h o l s t i i mannan was branched r a t h e r than l i n e a r . A galactomannan from Sporobolomyces sp. which under normal growth c o n d i t i o n s had both/.phosphate and 0 - a c e t y l s u b s t i t u e n t s was found to have many o f the phosphate groups r e p l a c e d by 0 - a c e t y l groups when grown under phosphate-l i m i t i n g c o n d i t i o n s . T h i s might e x p l a i n the v a r i a b i l i t y i n r e -covery o f 0 - a c e t y l groups from p o l y s a c c h a r i d e s o f C r . l a u r e n t i i and T r e m e l l a spp. r e p o r t e d by S l o d k i e t a l . (1966). E x t r a c e l l u l a r p o l y s a c c h a r i d e s have been e i t h e r p r e -c i p i t a t e d (with ethanol) from the c e l l f r e e growth medium or e x t r a c t e d d i r e c t l y from the c e l l s , a s adhered m a t e r i a l , by a v a r i e t y o f methods (Gorin and Spencer 1968). In many cases the growth media used were not c h e m i c a l l y d e f i n e d and although the neopeptone was d i a l y z e d by Evans and T h e r i a u l t (1953), there may s t i l l have been amino contaminants from the medium which would have p r e c i p i t a t e d with, e t h a n o l . Most workers d e a l i n g w i t h the pathogen Cr. neoformans have used a chemical method ( f o r example 1% phenol, Evans and K e s s e l 1951) to k i l l the c e l l s b e f o r e h a r v e s t i n g . Treatment w i t h s t r o n g a c i d s or 14. b a s e s , a l c o h o l , o r h e a t may c a u s e d e n a t u r a t i o n c o f p r o t e i n com-p o n e n t s o r h y d r o l y s i s o r o x i d a t i o n o f p o l y s a c c h a r i d e s . Sim-i l a r l y , ' a n a l y t i c a l p r o c e d u r e s w h i c h do n o t a c c o u n t f o r l o s s e s due t o d e g r a d a t i o n o r i n s u f f i c i e n t r e l e a s e o f c e r t a i n compon-... e n t s , w o u l d r g i v e u n d e r e s t i m a t i o n s o f t h e s u g a r c o n s t i t u e n t s o f t h e p o l y s a c c h a r i d e s . None o f t h e s t u d i e s so f a r r e p o r t e d h a v e e m p l o y e d c r i t i c a l q u a n t i t a t i v e a n a l y t i c a l methods; s u g a r s were m e a s u r e d s o l e l y b y p a p e r c h r o m a t o g r a p h y o r c o l o r i m e t r i c methods. S i n c e x y l o s e a p p e a r s t o be an e a s i l y l i b e r a t e d s i d e c h a i n m o i e t y i n c r y p t o c o c c a l and T r e m e l l a p o l y s a c c h a r i d e s ( M i y a z a k i 1961, B l a n d a m e r and D a n i s h e f s k y 1966, Cameron 1973) i t i s l i k e l y t h a t t h e d i s c r e p a n c i e s i n x y l o s e H : mannose r a t i o s were e i t h e r a r e -s u l t o f x y l o s e d e g r a d a t i o n o r an i n c o m p l e t e r e l e a s e o f mannose. An i n v e s t i g a t i o n o f t h e c r y p t o c o c c a l c e l l e n v e l o p e , c o m p r i s i n g c e l l w a l l , c a p s u l e and e x t r a c e l l u l a r m a t e r i a l , w o u l d h a v e s i g n i f i c a n c e f r o m b o t h a t a x o n o m i c and p a t h o g e n i c v i e w p o i n t . The r e s e a r c h d e s c r i b e d i n t h i s t h e s i s h a d t h e f o l l o w i n g a i m s : (1) To d e t e r m i n e t h e optimum c o n d i t i o n s o f t e m p e r a t u r e , pH and n u t r i e n t s f o r g r o w t h and c a p s u l e s y n t h e s i s o f e a c h o f t h r e e s t r a i n s o f C r . n e o f o r m a n s , two o f C r . a l b i d u s , and one o f C r . l a u r e n t i i . (2) To d e v e l o p a method o f k i l l i n g p a t h o g e n i c c e l l s t h a t d o e s n o t a l t e r w a l l o r c a p s u l e c h e m i s t r y . (3) To i s o l a t e and p u r i f y c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l f r o m e a c h o f t h e s e v i r u l e n t and a v i r u l e n t C r y p t o c o c c u s s p e c i e s i n c l u d i n g a p a t h o g e n i c s t r a i n o f C r . a l b i d u s (H1354) and a hyphal s t r a i n o f Cr. neoformans. (4) To analyze the c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l f o r carbohydrates, p r o t e i n and minor c o n s t i t u e n t s such as phos-phorus . (5) To determine the v i r u l e n c e (LD50) o f a l l s t r a i n s a g a i n s t mice. (6) To compare and c o n t r a s t the analyses from d i f f e r e n t s t r a i n s and r e l a t e these to v i r u l e n c e and the taxonomic p o s i t i o n o f the s p e c i e s . (7) To i n v e s t i g a t e the p a t h o g e n i c i t y o f the Cr. a l b i d u s H1354 s t r a i n . (8) To f i n d a medium t h a t w i l l support and e s t a b l i s h mating s t r a i n s o f the Cryptococcus s p e c i e s and to demonstrate sexual stages. CHAPTER I I EXPERIMENTAL PROCEDURE 1. MATERIALS A. Organisms Cryptococcus neoformans s t r a i n s 365-11, 365-16 and 365-26 were i s o l a t e d from sputum and were pathogenic to mice. Cr. l a u r e n t i i 371-1 was' i s o l a t e d from a t r a c h e a l a s p i r a t i o n , and Cr. a l b i d u s 367 from a i r . These f i v e c u l t u r e s were ob-t a i n e d from Dr. L. Kapica, Department o f M i c r o b i o l o g y , M c G i l l U n i v e r s i t y , M o n t r e a l . A pathogenic s t r a i n o f C r . a l b i d u s , H1354, i s o l a t e d from human c e r e b r o s p i n a l f l u i d , was o b t a i n e d from Dr. J3.G. Wieser, Aarau, S w i t z e r l a n d . A l l the c u l t u r e s were maintained a t 4C on Sabouraud dextrose (4%) agar s l a n t s and s u b c u l t u r e d monthly. A hyphal s t r a i n o f C r . neoformans (Coward s t r a i n ) , o b t a i n e d from Dr. H.J. Shadomy, V i r g i n i a Commonwealth U n i v e r s i t y , Richmond, V i r g i n i a , was pathogenic to mice and was maintained i n the hyphal s t a t e on V8 j u i c e agar (Phaff and Mrak 1949). Cr. l a u r e n t i i NRRL Y l 4920 (U.B.C #81114), Cr. a l b i d u s 72539 (U.B.C. #900) and C r . t e r r e u s U.B.C. #8157 were o b t a i n e d from Dr. R.J. Bandoni, Department o f Botany, U n i v e r s i t y o f B r i t i s h Columbia. B. Chemicals Reagents were o b t a i n e d from the s u p p l i e r s as i n d i c a t e d 2-hydroxypyridine ( A l d r i c h Chemical Company, Inc., Milwaukee); Beckman Amino A c i d C a l i b r a t i o n Mixture Type 1 (Beckman I n s t r u -ments, Inc., Spinco D i v i s i o n , Palo A l t o , C a l i f o r n i a ) ; t h i o -d i g l y c o l (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) ; D-galactose, p y r i d i n e AnalaR ACS ( B r i t i s h Drug Houses, L t d . , Poole, England); a l l amino a c i d s (Calbiochem, Los Angeles, C a l i f o r n i a ) ; a l l c u l t u r e media (Di f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n ) ; t r i f l u o r o -a c e t i c a c i d , metaphenyl phenol, a s c o r b i c a c i d (Eastman Kodak Company, Rochester, New Y o r k ) ; A l c i a n Blue 8G-S, benzene, Bromophenol Blue, Coomassie B r i l l i a n t Blue, D-mannose, c y e l o -hexane c e r t i f i e d ACS s p e c t r a n a l y z e d , mannitol r e c r y s t a l l i z e d F i s h e r c e r t i f i e d , N, N-dimethylformamide 99% mole pure ( F i s h e r S c i e n t i f i c Company, F a i r Lawn, New J e r s e y ) ; d i m e t h y l s u l f o x i d e (Koch L i g h t L a b o r a t o r i e s L t d . , Colnbrook, England); m e r t h i o l a t e (E. L i l l y and Company, Canada L t d . , Box 4037, Terminal A, T o r o n t o ) ; D-arabinose, D-glucose, D-xylose ( N u t r i t i o n a l B i o -chemicals C o r p o r a t i o n , C l e v e l a n d , Ohio); p-dimethylaminobenzalde-hyde (MCB, Norwood, Ohio); methyl c e l l o s o l v e , n i n h y d r i n , hexa-m e t h y l d i s i l a z a n e , t r i m e t h y l c h l o r o s i l a n e ( P i e r c e Chemical Company, Rockford, I l l i n o i s ) ; D-glucosamine h y d r o c h l o r i d e , m y o - i n o s i t o l , thiamine h y d r o c h l o r i d e (Sigma Chemical Company, S t . L o u i s , M i s s o u r i ) . A l l othergchemicals were o b t a i n e d l o c a l l y . "Baker Analyzed" grade (J.T. Baker Chemical Company, P h i l l i p s -burg, New Jersey) or e q u i v a l e n t was used when a v a i l a b l e . 18. 2. METHODS A. K i l l Procedure S e v e r a l methods were i n v e s t i g a t e d t o k i l l the c e l l s w ithout a l t e r i n g w a l l or capsule chemistry. The need f o r t h i s study arose from the h e a l t h hazards caused by the p r o -d u c t i o n o f a e r o s o l s d u r i n g h a r v e s t i n g and breakage procedures and from the f a c t t h a t c e l l fragmentation (assumed to be c e l l death) was incomplete. (1) Chemical Treatments Shake c u l t u r e s o f e a c h i ' s t r a i n were prepared i n 125 ml Erlenmeyer f l a s k s c o n t a i n i n g 50 ml Sabouraud dextrose (2%) b r o t h . The f l a s k s were shaken at 125 rev/min i n a R77 Meta-b o l y t e shaker water bath (New Brunswick S c i e n t i f i c Co., Inc., New Brunswick, N.J.) at 25C. After- 36 h r , f o r m a l i n ( f i n a l c o n c e n t r a t i o n 0.5%), phenol ( f i n a l c o n c e n t r a t i o n 0.5%) or KCN ( f i n a l c o n c e n t r a t i o n s between 10~ and 10~ M) was added to a fask o f each s t r a i n and i n c u b a t i o n c o n t i n u e d . Samples (0.1 ml) were withdrawn a t i n t e r v a l s up to 12 h r from the f o r m a l i n and phenol treatments and up to 48 hr from the KCN treatments and spread on Sabouraud dextrose agar p l a t e s which were incubated a t 25C f o r 7 days. P l a t e s were checked d a i l y f o r growth. ( 2 ) # - R a d i a t i o n Dembitzer e t a l . (1972) r e p o r t e d t h a t C r . neoformans c e l l s s u b j e c t e d to 1 x 10^ rads l o s t t h e i r a b i l i t y to reproduce. A 36 hr Cr. l a u r e n t i i c u l t u r e -was exposed f o r 90 min to-a Co 4 source g i v i n g 73.44 x 10 r a d s / h r . Samples were withdrawn and p l a t e d on Sabouraud dextrose agar every 25 min. (3) Heat Treatment Batches (100 ml) of each pathogenic s t r a i n were shaken f o r 36 hr a t 25C. The f l a s k s were then s w i r l e d i n a water bath at 50, 60, 75 and 85C f o r 1 h r . Samples were taken a t i n t e r v a l s , p l a t e d as b e f o r e , and observed f o r 1-2 weeks.. Heat e f f e c t s were a l s o assessed f o r a l l the Crypto-coccus s t r a i n s when grown i n Liftman's Capsule medium (LCM). D u p l i c a t e f l a s k s (100 ml medium) f o r each s t r a i n were t r e a t e d at 55C f o r up to 2 h r i Samples were withdrawn and i n o c u l a t e d as drop c o l o n i e s on Sabouraud agar p l a t e s (two drops per d u p l i -cate f l a s k ) . B. D e c a p s u l a t i o n To o b t a i n pure c e l l w a l l s , a l l c a p s u l a r m a t e r i a l should be removed and c o n v e r s e l y , c a p s u l a r m a t e r i a l must be f r e e from c y t o p l a s m i c or w a l l contaminants. Both w a l l s and capsules should be u n a l t e r e d c h e m i c a l l y by the treatment. A c c o r d i n g to Bulmer and Sans (1968) s o n i c o s c i l l a t i o n removed 80% o f the c a p s u l a r m a t e r i a l without d i s r u p t i n g c e l l s . Heat k i l l e d c e l l s o f Cr. neoformans 365/11 (20g) and 20g g l a s s beads (0.45 mm Braun, Canadian L a b o r a t o r y S u p p l i e s , 14823, 118 Ave., Edmon-ton) were mixed to a t h i c k s l u r r y and were then c o o l e d i n an i c e bath. The suspension was s o n i c o s c i l l a t e d a t 200 watts f o r four 5 min i n t e r v a l s u s i n g a Blackstone U l t r a s o n i c Probe. 20. The c e l l s were kept c h i l l e d i n an e t h a n o l - i c e bath throughout and the probe was immersed i n i c e between treatments. C e l l s were observed by phase c o n t r a s t microscopy a f t e r each t r e a t -ment. The methods o f Goren and Middlebrook (1967) and Vogel (1966) u s i n g d i m e t h y l s u l f o x i d e and 0.5 N HC1 r e s p e c t i v e l y , were a l s o t r i e d as were treatments o f c e l l s w i t h 8 M urea, 5 M sucrose and 5 M NaCl. C. Growth C o n d i t i o n s S e v e r a l authors (Littman 1958, Bulmer and Sans 1968, Foda e_t a l . 1973) showed t h a t extent o f c a p s u l a t i o n i n Cryptococcus s p e c i e s depended on the" growth medium. There-f o r e the optimum c o n d i t i o n s f o r capsule p r o d u c t i o n and f o r s e c r e t i o n o f e x t r a c e l l u l a r m a t e r i a l were determined f o r a l l of the s t r a i n s . L i q u i d media used were Sabouraud dextrose b r o t h (SAB; 1% neopeptone, 2% dextrose) and Littman's modi-f i e d capsule medium (LCM; Littman 1958) w i t h 1.5 mg l i t r e ^ NaNOg r e p l a c i n g 1.5 mg l i t r e - " ' " ^ 2 ^ 0 0 ^ . 211^0 (to a v o i d p r e c i -p i t a t i o n o f the medium) and v a r y i n g c o n c e n t r a t i o n s o f thiamine. E x p o n e n t i a l growth was e s t a b l i s h e d f o r each s t r a i n ' . i n both media at 25 and 37C and pH optima f o r growth and capsule syn-t h e s i s were-Sdetermined. (1) Measurement o f growth and thiamine requirements Samples (50 ml) o f LCM were dispensed i n 125 ml f l a s k s and thiamine HC1 (to give f i n a l c o n c e n t r a t i o n s between 0 and 50 jag/ml) added to h a l f the f l a s k s . F i l t e r s t e r i l i z e d t h i a -mine (the same range o f c o n c e n t r a t i o n s ) was added to the 21. remaining f l a s k s a f t e r a u t o c l a v i n g . S t e r i l e dextrose (1% f i n a l c o n c e n t r a t i o n ) was a l s o added to a l l the f l a s k s a t t h i s time. A suspension o f each s t r a i n was made i n s t e r i l e water from a l o o p f u l o f a 3 day o l d s l a n t c u l t u r e and a g i t a t e d thoroughly w i t h a Vortex mixer. C e l l counts were o b t a i n e d w i t h a haemo-7 cytometer and 1 ml, c o n t a i n i n g approximately 3 x 10 c e l l s , was added to each f l a s k . A s t e r i l e - w a t e r c e l l suspension was l e f t 24 h r a t room temperature and these v i t a m i n d e p l e t e d c e l l s were used to i n o c u l a t e medium c o n t a i n i n g no thiamine. A l l the f l a s k s were incubated at 25C i n a psychrotherm con-t r o l l e d environment r e c i p r o c a l shaker i n c u b a t o r at 100 rev/min. Samples were removed p e r i o d i c a l l y from each f l a s k and growth determined s p e c t r o p h o t o m e t r i c a l l y at 650 mm a g a i n s t a medium blank. Medium wast>also used t o make d i l u t i o n s as c u l t u r e s became too dense to read. When c e l l s had reached s t a t i o n a r y phase the experiment was terminated, and pH measurements taken f o r each;rflask. Ocular micrometer measurements were made o f capsule and c e l l . d i a m e t e r s w i t h f u n g i c i d a l I n d i a i n k mounts (Littman 1958). The mean diameter (microns) o f 50 c e l l s ex-c l u d i n g capsule was s u b t r a c t e d from the mean diameter o f c e l l s i n c l u d i n g c a p s u l e : t h i s f i g u r e , d i v i d e d by two, denoted cap-s u l e t h i c k n e s s (Ishaq, Bulmer and F e l t o n 1968). Photographs were taken under phase c o n t r a s t microscopy to show d i f f e r e n c e s i n c e l l shape and capsule s i z e . S p e c i a l p r e c a u t i o n s were taken duringvgrowth e x p e r i -ments w i t h the pathogenic s t r a i n s . A l l i n o c u l a t i o n and t r a n s -f e r procedures were performed* i n a negative p r e s s u r e m i c r o b i o -22. l o g i c a l hood R 3.5 MP, f i t t e d w i t h u l t r a v i o l e t l i g h t s (Germ Free L a b o r a t o r i e s , Miami, F l o r i d a ) . L a b e l l e d s p e c t r o n i c 20 tubes were f i t t e d w i t h foam stoppers and a u t o c l a v e d immediately readings had been taken. pH measurements were made w i t h the e l e c t r o d e w i t h i n the hood. D. Hyphal Growth Forms Hyphal Cr. neoformans, Coward s t r a i n , was grown i n V8 j u i c e agar at 25C and 30C. Sabouraud agar and corn meal agar d i d not g i v e good filamentous growth. A f t e r 10 days, agar c o n t a i n i n g the submergedsfilaments was t r a n s f e r r e d to 10 ml s t e r i l e d i s t i l l e d water and macerated w i t h a g l a s s r o d . T h i s suspension was used to i n o c u l a t e V8 j u i c e b r o t h , Sabouraud b r o t h and Liftman's capsule medium. F l a s k s were incubated a t 25C; e i t h e r shaken very s l o w l y or l e f t as s t i l l c u l t u r e s . Most o f the growth was y e a s t - l i k e and i n s u f f i c i e n t f i l a m e n t o u s growth was o b t a i n e d f o r c e l l w a l l or capsule p r e p a r a t i o n s even a f t e r two weeks. The other y e a s t s t r a i n s , i n c l u d i n g Cr. l a u r e n t i i #811.4, Cr. t e r r e u s #8157 and Cr. a l b i d u s #900 were a l s o t e s t e d f o r hyphal growth and formation o f c o n j u g a t i o n tubes. Media used were corn meal ( D i f c o ) , Sabouraud (2% d e x t r o s e ) , malt e x t r a c t and V8 agars. C u l t u r e s were stre a k e d on the agar p l a t e s and incubated a t 15, 25, 30 and 37C. The two s t r a i n s o f Cr. l a u r e n t i i , 371 and 8114, and o f Cr. a l b i d u s 367 and 900 were t e s t e d f o r mating responses by Kurtzman's method (1973) and inc u b a t e d on malt e x t r a c t .agar and V8 agar at 15C. E. E x t r a c t i o n and P u r i f i c a t i o n o f E x t r a c e l l u l a r M a t e r i a l Inoculum c u l t u r e s o f each s t r a i n were prepared i n 50 ml Littman's capsule medium (Liftman 1958): Dextrose (1% f i n a l c o n c e n t r a t i o n ) was added a f t e r a u t o c l a v i n g . With Cr. a l b i d u s #367, thiamine (10 jig/ml f i n a l c o n c e n t r a t i o n ) was a l s o added a f t e r a u t o c l a v i n g . The c u l t u r e s were shaken a t 100 r e v / min i n the psychrotherm r e c i p r o c a l shaker i n c u b a t o r at 25C u n t i l each s t r a i n reached e x p o n e n t i a l phase. Samples ( 5 ml) of the e x p o n e n t i a l phase c u l t u r e s were t r a n s f e r r e d to 350 ml of the same medium i n 1 l i t r e Erlenmeyer f l a s k s and shaken a t 90 rev/min a t 25C. When the c u l t u r e s reached s t a t i o n a r y phase, as judged by pH and absorbance measurements, the c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 16,000 xg. C e l l s o f pathogenic s t r a i n s were k i l l e d b e f o r e h a r v e s t i n g by h e a t i n g the f l a s k s i n a water bath at 55C f o r 90 min w i t h o c c a s i o n a l s w i r l i n g . To prevent damage to p o l y s a c c h a r i d e c o n s t i t u e n t s , by h e a t i n g i n d i l u t e a c i d ( f i n a l pH o f medium was c a . 3.0) the medium was ad j u s t e d t o ca. pH 6.0 w i t h 0.1N NaOH bef o r e the heat treatment. The , c e l l s were washed twice w i t h water, by re s u s p e n s i o n and c e n t r i -f u g a t i o n , to d i s l o d g e as much adhering c a p s u l a r m a t e r i a l as p o s s i b l e . Washings were combined w i t h the c u l t u r e f l u i d and passed through a m i l l i p o r e f i l t e r (0.45 p pore s i z e ) to? remove whole c e l l and c e l l fragment contaminants. S o l u b l e p o l y s a c c h a r i d e complexes were then e x t r a c t e d a c c o r d i n g to the f o l l o w i n g m o d i f i c a t i o n s o f the procedure o f F a r h i , Bulmer and Tacker (1970). The combined c u l t u r e f l u i d s 24. were co n c e n t r a t e d to 1/10 volume by r o t a r y e v a p o r a t i o n ( F l a s h -Evaporator, Buchler Instruments, F o r t Lee, New Jersey) at 50c i n vacuo and two volumes o f ab s o l u t e e t h a n o l were added to the c o n c e n t r a t e . A f t e r 48 hr a t 4C, the p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a t 300 x g. One volume o f a b s o l u t e ethanol was added to the supernatant and the s o l u t i o n was l e f t f o r a f u r t h e r 48 h r . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a t 300 x g. The two p r e c i p i t a t e s were pooled, washed twice w i t h a b s o l u t e e t h a n o l , c e n t r i f u g e d a t 300 x g and resuspended i n de-i o n i z e d water. The suspension was d i a l y z e d a g a i n s t three changes o f d e i o n i z e d water i n a -20 l i t r e M u l t i p l e D i a l y z e r (Oxford Labora-t o r i e s ) a t 4C. The n o n - d i f f u s i b l e m a t e r i a l was c e n t r i f u g e d at 27,000 x g f o r 60 min a t 2C to remove i n s o l u b l e m a t e r i a l , d i s -pensed i n t o pre-weighed p l a s t i c beakers and l y o p h i l i z e d . The beakers were then reweighed arid the white f l u f f y m a t e r i a l s t o r e d i n screwcap v i a l s a t 4C. At l e a s t two p r e p a r a t i o n s were made from each s t r a i n , grown under i d e n t i c a l c o n d i t i o n s . F. C e l l W a l l E x t r a c t i o n and P u r i f i c a t i o n Seed c u l t u r e s o f Cr. l a u r e n t i i and C r . neoformans 365-26 were prepared from agar s l a n t s by i n o c u l a t i n g 100 ml o f Sabouraud dextrose (2%) b r o t h i n 250 ml f l a s k s and shaking at 25C a t 100 rev/min. A f t e r 36 hr (during the e x p o n e n t i a l growth phase; capsules l e s s than 0.2 p. diameter), 10 ml samples o f each seed c u l t u r e were t r a n s f e r r e d to 2800 ml Fernbach f l a s k s c o n t a i n i n g 1 l i t r e o f b r o t h and were shaken at 25C f o r 36 h r . P r i o r t o h a r v e s t i n g , Cr. neoformans. c e l l s were k i l l e d by h e a t i n g , w i t h constant s w i r l i n g , i n a 65C water bath f o r 25 min. The pH o f the medium a f t e r 36 hr i n c u b a t i o n was about 6.0, t h e r e f o r e no adjustment was necessary to a v o i d a c i d c o n d i t i o n s . The c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 9000 x g f o r 10 min, washed twice w i t h water.to remove adhering e x t r a c e l l u l a r m a t e r i a l , and c o o l e d f o r i l 5 min i n an i c e bath. P-@rtoipri)S>r(;lO>ng) o f washed c e l l s were mixed i n t o a paste w i t h 2 ml i c e - c o l d water and 50 g g l a s s beads (0.45 - 0.50 mm diameter, Braun, Canadian L a b o r a t o r y S u p p l i e s , Edmonton) and d i s r u p t e d i n a Braun MSK homogenizer f o r four to s i x x 2 min p e r i o d s . The process o f breakage was assessed u s i n g phase c o n t r a s t microscopy. Four x 2 mine/periods gave approximately 90% breakage o f C r . l a u r e n t i i c e l l s but s i x x 2 min p e r i o d s were r e q u i r e d to break approximately 70% o f the Cr. neoformans c e l l s . The bead paste was washed 5 x w i t h c o l d d e i o n i z e d water, by r e s u s p e n s i o n and c e n t r i f u g a t i o n , and the supernatants com-bin e d . Whole c e l l s o f . C r . l a u r e n t i i were removed by c e n t r i -f u g a t i o n a t 950 x g f o r 5 min a t 2C. The supernatant was then centrifuged at 27,000 x g f o r 10 min to recover c e l l w a l l f r a g -ments. With Cr . neoformans, however, the combined supernatants from the bead paste had to be shaken w i t h a Vortex mixer to d i s p e r s e aggregates of whole c e l l s and w a l l fragments. The r e -s u l t i n g suspension was c e n t r i f u g e d a t 120 x g f o r 5 min and the p e l l e t o f whole c e l l s and fragments washed 5 times to e x t r a c t w a l l fragments i n t o the supernatant. The supernatants were combined and c e n t r i f u g e d once more to remove remaining whole 26. c e l l s . The c e l l w a l l fragments were then c o l l e c t e d by c e n t r i -f u g a t i o n a t 27,000 x g f o r 10 min. The washing procedure was based on t h a t o f M i t c h e l l and T a y l o r (1969). The c e l l w a l l fragments were'washed 5 times w i t h i c e - c o l d d e i o n i z e d water ( c e n t r i f u g a t i o n a t 27,000 x g f o r 10 min f o r C r . l a u r e n t i i , 12,000 x g f o r C r . neoformans), 5 times w i t h i c e - c o l d 1.0M NaCl s o l u t i o n ( c e n t r i f u g a t i o n at 12,000 x g f o r both p r e p a r a t i o n s ) , 5 times w i t h c o l d water and twice w i t h 8.0 M urea s o l u t i o n ( c e n t r i f u g a t i o n a t 27,000 x g f o r 10 min f o r Cr. l a u r e n t i i , 12,000 x g f o r Cr. neoformans). The fragments were suspended o v e r n i g h t i n 8.0 M urea s o l u t i o n at 4C, then c e n t r i f u g e d and washed a f u r t h e r 3 times w i t h 8M urea s o l u t i o n , 5 times w i t h water, 5 times w i t h 1.0N NH^OH s o l u t i o n ( c e n t r i f u g a t i o n a t 1200 x g f o r 10 min f o r both p r e -p a r a t i o n s ) and 5 times w i t h water, or u n t i l f r e e from c y t o -p l a s m i c contamination (as judged by phase c o n t r a s t m i c r o s c o p y ) . The f i n a l wash was extended f o r 20 min and the c e l l w a l l s were l y o p h i l i z e d and s t o r e d at -20C. The'walls a l s o appeared f r e e from any ad h e r i n g c a p s u l a r m a t e r i a l (as judged by I n d i a i n k s t a i n i n g and phase c o n t r a s t m i c r o s c o p y ) . G. A n a l y t i c a l Procedures Stock s o l u t i o n s c o n t a i n i n g known amounts (approximately 2 mg/ml) o f each dry l y o p h i l i z e d e x t r a c e l l u l a r p r e p a r a t i o n were made i n water. Samples from these stocks were used i n the a n a l y -t i c a l procedures to minimize weighing e r r o r s . Where necessary, the samples were f r e e z e d r i e d i n 13 x 100 mm t e s t tubes b e f o r e use. 27. (1) Elemental and Ash Analyses A n a l y s e s f o r n i t r o g e n were performed by Organic M i c r o -a n a l y s i s , Montreal, Quebec. Phosphorus was determined s p e c t r o -p h o t o m e t r i c a l l y by the method o f Ames (1966) w i t h KH 2P0 4 as standard. Ash was determined g r a v i m e t r i c a l l y . Coors c r u c i b l e s (15 x 9 mm, Canadian Lab o r a t o r y S u p p l i e s L t d . , Edmonton), were weighed on a 6-place balance and approximately 20 mg e x t r a -c e l l u l a r m a t e r i a l weighed i n t o each. They',;were l e f t i n a de-s i c c a t o r over P2°5 o v e r n i g h t , reweighed, heated to constant weight (12 hr) i n a mu f f l e furnace a t 800C, c o o l e d i n a d e s i -c c a t o r and weighed a g a i n . (2) Amino A c i d s C e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l (4-5 mg) were h y d r o l y z e d i n vacuo w i t h 0.5 ml o f 6N HC1 c o n t a i n i n g 1 mg/ml o x a l i c a c i d (James 1972), at HOC f o r 24 and 48 hr ( T r i s t r a m and Smith 1963, Cameron 1973). The h y d r o l y s a t e s were d r i e d i n vacuo over c o n c e n t r a t e d H 2 S 0 4 and KOH p e l l e t s , then r e d i s s o l v e d i n 1.0 ml pH 2.2 sodium c i t r a t e b u f f e r (0.02N). A l i q u o t s o f each s o l u t i o n t ogether w i t h 20 nm of the i n t e r n a l standard s o l u t i o n (o<-amino- fS - g u a n i d o - p r o p i o n i c a c i d f o r b a s i c amino a c i d s and n o r l e u c i n e f o r n e u t r a l and a c i d i c amino a c i d s ) i n pH 2.2 b u f f e r were a p p l i e d t o the a p p r o p r i a t e column o f a Beckman Amino A c i d A n a l y z e r Model 120C. The a n a l y s i s was based on the methods o f Spackman, S t e i n and Moore (1958) and Cameron (1973). B a s i c amino a c i d s were separated on a 16.5 x 0.9 cm 28. column and e l u t e d w i t h pH 5.25 sodium c i t r a t e b u f f e r . A f t e r , a p p l i c a t i o n o f the sample, the space above the r e s i n bed was f i l l e d w i t h pH 3.25 sodium c i t r a t e b u f f e r and the b u f f e r l i n e c o n t a i n i n g pH 5.25 b u f f e r was connected. T h i s procedure allowed s e p a r a t i o n o f l y s i n e from ethanolamine which o c c u r r e d i n most o f the p r e p a r a t i o n s , and a l s o r e s o l v e d p h e n y l a l a n i n e from t y r o s i n e and glucosamine from galactosamine. A c i d i c and n e u t r a l amino a c i d s were separated u s i n g a 58 x 0.9 cm column. Glucosamine was adequately separated from p h e n y l a l a n i n e as lo n g as column l e n g t h was g r e a t e r than 58 cm. H y d r o x y p r o l i n e was determined a f t e r h y d r o l y s i s i n 0.5 ml 6N HC1 i n vacuo f o r 14 hr a t HOC, by the s p e c t r o p h o t o m e t r y method o f Bergman and L o x l e y (1970), usingp-dimethylaminobenzaldehyde. (3) Amino Sugars C e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l (2-4 mg) were h y d r o l y z e d i n vacuo w i t h 0.5 ml o f 2N HC1 (Oates and Schrager 1967) c o n t a i n i n g 1 mg/ml o x a l i c a c i d a t 105C f o r 8, 16, 24, 72 and 96 h r . H y d r o l y s a t e s were d r i e d i n vacuo over concen-t r a t e d H 2 S 0 4 and KOH p e l l e t s , d i s s o l v e d i n 1 ml o f 0.2N sodium c i t r a t e b u f f e r , pH 2.2, and analyzed u s i n g the 16.5 x 0.9 cm column o f the amino a c i d a n a l y z e r . (4) N e u t r a l Sugars N e u t r a l sugars were determined by two separate p r o -cedures. In both cases the f r e e sugars were estimated by g a s - l i q u i d chromatography as t r i m e t h y l s i l y l (TMS) d e r i v a t i v e s . The f i r s t procedure was a m o d i f i c a t i o n o f Cameron's 29. method (1973) based on the procedures o f Albersheim e t a l . (1967) and Sweeley e t a l . (1963). C e l l w a l l s and e x t r a -c e l l u l a r m a t e r i a l (approximately 2 mg) were h y d r o l y z e d i n s e a l e d tubes at 105C w i t h 0.5.ml 2N CFgCOOH, f o r 30, 60, 120 and 240 min to r e l e a s e the n e u t r a l sugars. H y d r o l y s a t e s were d r i e d i n vacuo over KOH p e l l e t s and 0.5 ml o f the i n t e r n a l standard, m y o - i n o s i t o l (1 mg/ml i n d e i o n i z e d water) added. The s o l u t i o n was d r i e d , then d i s s o l v e d i n 1 ml p y r i d i n e and t r e a t e d , s u c c e s s i v e l y , w i t h 0.1 ml h e x a m e t h y l d i s i l a z a n e and 0.05 ml t r i m e t h y l c h l o r o s i l a n e . The mixture was shaken f o r a few seconds, and a f t e r 30 min was, f r e e z e d r i e d to remove the p y r i d i n e . The TMS d e r i v a t i v e s were r e d i s s o l v e d i n 1 ml c y c l o -hexane and a l i q u o t s of t h i s s o l u t i o n i n j e c t e d i n t o a V a r i a n Aerograph dual column gas chromatograph Model 1740, equipped w i t h flame i o n i z a t i o n d e t e c t o r s . The flow r a t e s o f N 2 and were 25 ml/min and a i r 250 ml/min. Columns (4.9m x 3mm) con-t a i n i n g 10% s i l i c o n e f l u i d SF 96 on a c i d washed DMCS t r e a t e d 60/80 mesh f l u x - c a l c i n e d d i a t o m i t e (Chromosorb W, Chromato-gr a p h i c S p e c i a l i t i e s , B r o c k v i l l e , Ontario) were used. They were l i n e a r l y temperature programmed from 130C (at i n j e c t i o n ) to 230C at 2 degrees/min. Peak areas were measured w i t h a V a r i a n Aerograph 477 i n t e g r a t o r . Only one i n t e r n a l standard, m y o - i n o s i t o l , was employed s i n c e the i n t e g r a t o r gave accurate readings when the g a s - l i q u i d chromatograph peaks were o f f - s c a l e . The second procedure i n v o l v e d f r e e i n g the n e u t r a l sugars by r e s i n h y d r o l y s i s a c c o r d i n g to the method o f Lehn-h a r d t and Wdnzler (1968). Approximately 1 mg o f e x t r a c e l l u l a r m a t e r i a l (0.5 ml stock s o l u t i o n ) was.hydrolyzed w i t h an equal volume o f 40% w/v AG 50 x 2 (H+) 200/400 mesh r e s i n (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) i n 0.02N HC1 i n s e a l e d t e s t tubes a t 100C f o r 24, 48 and 72 h r (Niedermeier 1971). Samples were c o o l e d , the s e a l was broken, 0.4 mg o f the i n -t e r n a l standard (mannitol) was added and the s o l u t i o n was mixed immediately w i t h a Vortex mixer. The mixture was t r a n s f e r -r e d to a Pasteur p i p e t t e column plugged w i t h g l a s s wool and the h y d r o l y s a t e s c o l l e c t e d i n Erlenmeyer f l a s k s . The h y d r o l y -s i s tubes were washed s e v e r a l times w i t h d e i o n i z e d water and the washings poured through the p i p e t t e column. A t o t a l o f 30 ml d e i o n i z e d water was siphoned through the r e s i n and g l a s s wool and c o l l e c t e d i n the Erlenmeyer f l a s k s . The h y d r o l y s a t e s p l u s washings were passed through a second Pasteur p i p e t t e column c o n t a i n i n g 1 ml o f a 20% W/V suspension o f AG1-X8 (HC0 3~) 200/400 mesh r e s i n (Bio-Rad) i n water. The E r l e n -meyer f l a s k s were r i n s e d s e v e r a l times w i t h a 50% v/v s o l u -t i o n o f methanol-water and the washings siphoned through the columns. The e l u a t e s were c o l l e c t e d i n round bottom f l a s k s and c o n c e n t r a t e d to a sma l l volume w i t h a r o t a r y e v a p o r a t o r . The c o n c e n t r a t e s were then t r a n s f e r r e d to 10 ml round bottom f l a s k s and d r i e d w i t h the r o t a r y e v a p o r a t o r . The samples were then t r e a t e d w i t h 0.5 ml N,N-dimethylformamide c o n t a i n i n g 0.1 M 2-hydroxypyridine and allowed t o mutarotate o v e r n i g h t a t 40C (Reid e t a l . 1970). The sugars were now a t m u t a r o t a t i o n e q u i l i b r i u m and were s i l y l a t e d by shaking f o r 30 min w i t h 0.5 ml h e x a m e t h y l d i s i l a z a n e and 0.25ml t r i m e t h y l c h l o r o s i l a n e . 31. Samples (2 p i ) o f the mixtures were i n j e c t e d i n t o a Hewlett Packard 7610A dual column gas chromatograph f i t t e d w i t h flame i o n i z a t i o n d e t e c t o r s and d i r e c t on-column i n j e c t i o n . The flow r a t e o f He ( c a r r i e r gas) was 60 ml/min, 35 ml/min, and a i r 500 ml/min. Dual copper columns (2.4 x 6 mm) con-t a i n i n g 10% (w/w) SE-52 on 80/100 mesh d i a p o r t S were used, and the chromatograph was operated i s o t h e r m a l l y a t 190C w i t h a f l a s h h e a t e r temperature o f 260C and a d e t e c t o r temperature o f 270C. Peak areas were measured w i t h an e l e c t r o n i c i n t e -g r a t o r . Molar response f a c t o r s f o r each sugar r e l a t i v e ; to. mannitol (Dutton e_t a l . 1968), and the percentage composition o f each of the sugars at m u t o r o t a t i o n e q u i l i b r i u m , had been p r e v i o u s l y determined. (5) U r o n i c A c i d s U r o n i c a c i d s were estimated c o l o r i m e t r i c a l l y w i t h meta-phenylphenol a c c o r d i n g to the method o f Blumenkrantz and Asboe-Hansen (1973). The authors claimed t h i s method to be both more s e n s i t i v e and more s p e c i f i c f o r u r o n i c a c i d s than the e a r l i e r methods d e s c r i b e d by Dische (1947) and Brown (1946). Glucuronolactone was used as the standard. (6) O-Acetyl The method o f 0 - a c e t y l d e t e r m i n a t i o n i n v o l v e d t r a n s -e s t e r i f i c a t i o n w i t h sodium methoxide (0.1 M i n a b s o l u t e methanol) at 0C f o r 30 min (Whistler and Jeanes 1943). The r e s u l t i n g methyl a c e t a t e was estimated by gas chromatography a c c o r d i n g to the method of Reid e t a l . (personal communication). 30 p i 32. benzene standard (89.9 mg/ml) were added to 10 mg e x t r a c e l l u -l a r m a t e r i a l b e f o r e treatment w i t h 0.1M sodium methoxide and 2 was- i n j e c t e d i n t o a Hewlett Packard F and M 402 gas chromato-graph f i t t e d w i t h flame i o n i z a t i o n d e t e c t o r s . The flow r a t e o f He was 25 ml/min, B.^ 40 ml/min and a i r 400 ml/min. Dual copper columns (1.8m x 3mm) o f Chromosorb 101 were l i n e a r l y temperature programmed from 100 (at i n j e c t i o n ) to 150C a t 4 degrees/min. H. E l e c t r o p h o r e s i s and S t a i n i n g (1) Polyacrylamide g e l s Approximately 25 mg o f Cryptococcus a l b i d u s e x t r a -c e l l u l a r m a t e r i a l was d i s s o l v e d i n 1.5 ml d e i o n i z e d water by degassing i n a small b e l l j a r . Sample (0.05 - 0.2 ml) was a p p l i e d t o 0.5 x 6.0 cm p o l y a c r y l a m i d e g e l columns (pH 9.0) prepared by the method o f O r n s t e i n and Davis (1962) as modi-f i e d by Fox, Thurman and B o u l t e r (1964) except t h a t the l a r g e pore g e l was omitted. Gels were run a t 3 ma per tube u n t i l the marker dye reached the end o f each tube. P r o t e i n was de-t e c t e d i n the g e l s by s t a i n i n g w i t h 1% w/v amido b l a c k i n 1% w/v a c e t i c a c i d f o r one hour. D e s t a i n i n g was conducted f o r one to three days i n 7% a c e t i c a c i d . Carbohydrate was de-t e c t e d by a m o d i f i e d p e r i o d i c a c i d S c h i f f s t a i n (Page and Stock 1974). Band m o b i l i t y (Rp) was expressed as a f r a c t i o n o f the m o b i l i t y o f the bromophenol blue f r o n t - t r a c k i n g dye. Distance was measured from the middle o f each band. (2) C e l l u l o s e a c e t a t e s t r i p s Samples (2 mg) o f e x t r a c e l l u l a r m a t e r i a l were d i s -s o l v e d i n 500 u l t r i s - b a r b i t a l - s o d i u m - b a r b i t a l b u f f e r , pH 8.8, 0.05 M (Gelman High R e s o l u t i o n B a r b i t a l b u f f e r , Gelman Instrument Company, Ann Arbor, M i c h i g a n ) . Samples (5 p i ) were a p p l i e d t o Gelman Sepraphore 111 c e l l u l o s e a c e t a t e s t r i p s (2.5 cm x 15 cm). E l e c t r o p h o r e s i s was c a r r i e d out i n a Gelman e l e c t r o p h o r e s i s u n i t at 3 ma per s t r i p f o r 40 min. To d i f f e r e n t i a t e and demonstrate both g l y c o p r o t e i n and a c i d i c p o l y s a c c h a r i d e , the s t r i p s were cut i n h a l f w i t h a r a z o r blade and e i t h e r s t a i n e d f o r g l y c o p r o t e i n by the A l c i a n blue method o f Wardi and A l l e n (1972) or f o r c a r b o x y l groups w i t h A l c i a n blue (1% i n 2.5% a c e t i c a c i d ) alone. The l a t t e r s t r i p s were d e s t a i n e d i n two changes o f 1:1 (v/v) 0.1 M c i t r a t e b u f f e r (pH 3.0): a b s o l u t e e t h a n o l , and c l e a r e d w i t h two changes of ab s o l u t e methanol (60 sec each) and one r i n s e 10% a c e t i c a c i d v/v i n a b s o l u t e methanol (60 s e c ) . Separate s t r i p s were s t a i n e d f o r p r o t e i n w i t h 0.25% w/v Coomassie b r i l l i a n t blue i n 7% w/v a c e t i c a c i d f o r 15 min (Dulaney and T o u s e r l 9 7 0 ) . These s t r i p s were d e s t a i n e d i n fo u r changes 5% aqueous a c e t i c a c i d and c l e a r e d as d e s c r i b e d above. The c l e a r e d s t r i p s were p l a c e d on a grease f r e e g l a s s p l a t e and d r i e d i n an oven a t 60C f o r 15 min. Band m o b i l i t y was expressed as a f r a c t i o n o f the m o b i l i t y o f a standard a p p l i e d to separate s t r i p s : h e p a r i n f o r the A l c i a n blue s t a i n and h y l a n d c o n t r o l serum f o r Coomassie b l u e . 34. I . Gel Chromatography Approximately 15 mg o f C r . neoformans #365-26 e x t r a -c e l l u l a r p o l y s a c c h a r i d e were suspended i n 10.0 ml o f 0.02 M p y r i d i n e - H C l b u f f e r (pH 5.5) and degassed to d i s s o l v e . The s o l u t i o n was d i a l y z e d a t 4C a g a i n s t three changes o f the same b u f f e r and chromatographed on a column o f DEAE B i o Gel-A (100/200 mesh, Bio-Rad) which had been e q u i l i b r a t e d w i t h p y r i -dine-HCl b u f f e r pH 5.5. The flow r a t e w a s i^lO ml per hr and the v o i d volume was 250 ml. A f t e r e l u t i o n o f the column w i t h 400 ml o f the b u f f e r , a convex g r a d i e n t from 0-3.0 M NaCl (700 ml) i n 0.02 M p y r i d i n e - H C l b u f f e r (700 ml) was a p p l i e d . F r a c t i o n s o f 10 ml were c o l l e c t e d and p o r t i o n s (2 ml) assayed-f o r carbohydrate by the p h e n o l s u l p h u r i c a c i d method,, (Dubois e t a l . 1956), and f o r p r o t e i n by u l t r a v i o l e t a b s o r p t i o n a t 280 nm w i t h a Beckman DU spectrophotometer. C o n d u c t i v i t y was measured u s i n g a c o n d u c t i v i t y b r i d g e model RC 216B2 and was p r o p o r t i o n a l to the m o l a r i t y o f NaCl. J . I n f r a r e d Spectra Approximately 1.5 mg dry, l y o p h i l i z e d c e l l w a l l or e x t r a c e l l u l a r m a t e r i a l were ground w i t h 200 mg dry KBr i n an agate mortar. The dry, f i n e l y ground powder was scraped i n t o an evacuable d i e and p r e s s e d i n t o a c l e a r p e l l e t w i t h a h y d r a u l i c p r e s s a t 18 tons. The 13 mm d i s c s were scanned u s i n g a Unicam SP 200G spectrophotometer. 35. K. V i r u l e n c e T e s t i n g w i t h Mice The v i r u l e n c e o f each s t r a i n - used was t e s t e d by i n o -c u l a t i n g 12 Swiss white mice per s t r a i n w i t h 0.02 - 0.04 ml o f a 10^ c e l l suspension i n s t e r i l e s a l i n e . The suspensions were prepared from t h r e e - d a y - o l d c u l t u r e s o f each s t r a i n and i n j e c t e d i n t r a c e r e b r a l l y w i t h a 26 gauge needle. C e l l counts were made w i t h a haemocytometer and s e r i a l d i l u t i o n s p l a t e d out on Sabouraud dextrose agar to check the v i a b l e count. Deaths began to occur a f t e r four days and the experiments were terminated a f t e r four weeks. M o r t a l i t y r a t e was expressed as the time taken to k i l l 50% of the mice. A u t o p s i e s were per-formed, agar s l a n t s i n o c u l a t e d w i t h b r a i n t i s s u e and smears examined w i t h f u n g i c i d a l I n d i a i n k ubiylephasie.seonbriasfesmicroscopy. CHAPTER I I I RESULTS 1. K i l l Procedures 6 2 Potassium cyanide (10~ - ,10~ M) f a i l e d t o k i l l any o f the Cryptococcus s t r a i n s t e s t e d even a f t e r 48 h r s incuba-t i o n . Capsule s y n t h e s i s , as judged by average capsule s i z e , was not a f f e c t e d . Exposure to 10^ rads from a C o ^ source slowed the growth but f a i l e d to k i l l C r . l a u r e n t i i 371-1. There was no d e t e c t a b l e l o s s o f c a p s u l e . L i t t l e m o r p h o l o g i c a l change was observed a f t e r exposure to lower doses o f r a d i a t i o n , but a f t e r 10^ rads some c e l l s appeared d i s t o r t e d and were budding ab-normally. I n c u b a t i o n w i t h f o r m a l i n or phenol a t 25C f o r 2 and 6 h r , r e s p e c t i v e l y , k i l l e d a l l the C r . neoformans s t r a i n s (Table 1 ) . However, pretreatment w i t h f o r m a l i n l e d to e x t e n s i v e f l o c c u l a t i o n o f w a l l fragments w i t h whole c e l l s d u r i n g the breakage procedure. Treatment o f Cr. l a u r e n t i i c e l l s w i t h phenol f o r 10 h r p r i o r to h a r v e s t was a l s o an u n s u i t a b l e method of k i l l i n g the c e l l s s i n c e the subsequent r e c o v e r y o f amino a c i d s from the c e l l w a l l s was s u b s t a n t i a l l y reduced (Table I I ) . The e f f e c t o f h i g h temperatures f o r up to 1 hr on the three Cr. neoformans s t r a i n s i s shown i n Table I I I . A l l the s t r a i n s were s e n s i t i v e t o h i g h temperatures. Heat treatment TABLE I EFFECTS OF INCUBATION, FOR DIFFERENT TIMES, WITH FORMALIN (FINAL CONCENTRATION 0.5% HCHO) AND PHENOL (FINAL CONCENTRA-TION 0.5%) ON GROWTH OF CR. NEOFORMANS Inc u b a t i o n Growth o f S t r a i n s at 25C a time i n h r Phenol F o r m a l i n 365-11 365-16 365-26 365-11 365-16 365-26 0 +++ +++ +++ +++ +++ +++ 1 +++ +++ +++ + ++ + 2 ++ ++ + - - -3 6 + ++ — — — — 8 10 — — 12 - - - - - -Scale o f growth from - = no growth t o +++ -- maximum growth o f c o l o n i e s on Sabouraud dextrose agar p l a t e s i n o c u l a t e d w i t h 0.1 ml t r e a t e d c e l l s and incubated a t 25C f o r 7 days. R e s u l t s are averages o f growth recorded on two p l a t e s / t r e a t m e n t / time. 38. TABLE I I AMINO ACID CONTENT OF CELL WALLS OF CR. LAURENTII 371-1 AFTER TREATING THE CELLS WITH PHENOL (0.5% FINAL CONCEN-TRATION) FOR 10 HR PRIOR TO HARVEST ' (ug anhydro amino a c i d recovered/mg c e l l w a l l 9 p r e p a r a t i o n ) 24 hr h y d r o l y s i s w i t h 6N HC1 Amino a c i d Untreated c e l l s Phenol t r e a t e d c e l l s % decrease T b Lys 1.3 1.3 0.0 His 0.6 0.5 16.7 Arg 0.6 0.4 33.3 Asx 1.9 1.3 31.6 Thr 1.1 1.0 9.1 Ser 1.7 1.5 11.8 Glx 2.4 1.3 45.8 Pro 0.9 0.5 44.4 Gly 1.0 0.9 10.0 A l a . 1-3 0.9 30.8 Cys 0.0 0.0 0.0 V a l 0.9 0.6 33.3 Met 0.2 0.1 50.0 H e 0.8 0.4 50.0 Leu 1.3 0.8 38.5 Tyr 0.7 0.6 14.3 Phe 0.8 0.6 25.0 T o t a l Recovery 17.2 12.7 a p a r t i a l l y p u r i f i e d w a l l p r e p a r a t i o n s were used ^ L y s i n e p o o r l y r e s o l v e d 39. TABLE I I I EFFECT OF TEMPERATURE ON GROWTH OF THREE STRAINS OF CR. NEOFORMANS Growth o f S t r a i n s a t 25C Incub a t i o n -time i n min. 365-11 365-16 365-26 50 Treatment 60 . 75 85 o Temperatures 50 60 75 85 C 50 60 75 85 0 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ " 5 » +++ ++ + - +++ ++ + - +++ +++ ++ -10 +++ - - +++ + - +++ + -15 +++ - - +++ - - +++ - -30 +++ - - +++ - - +++ - -45 +++ - - +++ - - +++ - -60 + - ++ - - ++'+ - -S c a l e o f growth from - = no growth to +++ = maximum growth on Sabouraud dextrose agar p l a t e s i n o c u l a t e d w i t h 0.1 ml t r e a t e d c e l l s and incubated a t 25C f o r 7 days. R e s u l t s are averages o f growth recorded on two p l a t e s / t r e a t m e n t / t i m e . 40. d i d not appear to a f f e c t p r o t e i n or p o l y s a c c h a r i d e composition of e x t r a c e l l u l a r m a t e r i a l (Table I V ) . In a l l the f o l l o w i n g experiments c e l l s were k i l l e d by heat treatment a t 65C f o r 25 m i n . ( p r i o r t o h a r v e s t ) f o r c e l l w a l l e x t r a c t i o n and a t 55C f o r 90 min ( p r i o r to h a r v e s t ) f o r e x t r a c e l l u l a r m a t e r i a l prep-a r a t i o n . Table V shows d i f f e r e n c e s i n r e s i s t a n c e to heat t r e a t -ment at 55C among the s i x Cryptococcus s t r a i n s growing i n LCM. The two Cr. a l b i d u s s t r a i n s were the l e a s t r e s i s t a n t , and Cr. l a u r e n t i i 371-1 the most r e s i s t a n t t o heat. There was l i t t l e d i f f e r e n c e i n r e s i s t a n c e among the Cr. neoformans s t r a i n s : s t r a i n 365-16 seemed the most r e s i s t a n t to heat. 2. D e c a p s u l a t i o n None o f the methods t e s t e d gave complete d e c a p s u l a t i o n . Short p e r i o d s o f s o n i c o s c i l l a t i o n (1-5 min) removed l i t t l e or no c a p s u l a r m a t e r i a l and, i n c o n t r a s t to Bulmer and Sans' work (1968),.periods o f above 5 min r u p t u r e d some o f the c e l l s . Extended treatment (overnight) w i t h d i m e t h y l s u l f o x i d e (Goren and Middlebrook, 1967) removed some of the capsules but even l a r g e volumes o f s o l v e n t (500 ml/40 ml c e l l s ) f a i l e d to com-p l e t e l y d ecapsulate a l l the c e l l s . Consequently c e l l s f o r w a l l p r e p a r a t i o n s were grown i n SAB f o r 36 hr (minimum capsule p r o d u c t i o n ) and f o r e x t r a c e l l u l a r m a t e r i a l LCM was used (5 days growth gave maximum capsule p r o d u c t i o n ) . Adhering c a p s u l a r m a t e r i a l was not e x t r a c t e d and analyzed i n t h i s study because of these d i f f i c u l t i e s . 41. TABLE IV AMINO ACID AND NEUTRAL SUGAR CONTENT OF EXTRACELLULAR MATERIAL OF CR. NEOFORMANS 365-26 AFTER HEAT TREATMENT AT 55C FOR 90 MIN PRIOR TO HARVEST pg anhydro amino a c i d recovered/mg | ug anhydro n e u t r a l sugar e x t r a c e l l u l a r m a t e r i a l . 24 hr hydro- recovered/mg e x t r a c e l l u -l y s i s w i t h 6N HC1C l a r m a t e r i a l . 1 hr hydro-l y s i s w i t h 2NiTFA b Untreated c e l l s Heat t r e a t e d c e l l s U n t reated Heat c e l l s t r e a t e d c e l l s Lys H i s Arg Asx Thr Ser Glx Pro G l y A l a Cys V a l Met H e Leu Tyr Phe T o t a l Recovery 0.7 0.2 0.3 4.8 2.9 4.5 2.4 4.3 0.8 3.9 0.0 1.9 0.0 2.1 0.9 0.1 2.5 0.8 0.2 0. 3 4.8 2.8 4.6 2.5 4.2 0.8 3.8 0.0 1.9 0.0 2.0 0.9 0.1 2.4 X y l 175.64 Man 239.50 Gal 54.21 Glc 10.82 T o t a l 480.17 Recovery 174.74 241.99 51.43 11.55 479.71 32.3 32.1 Mean o f d u p l i c a t e h y d r o l y s i s 3Mean o f d u p l i c a t e h y d r o l y s e s . Sugars estimated as TMS d e r i -v a t i v e s by gas chromatography (Cameron 1973). For d e t a i l s o f procedure see Chapter I I , S e c t i o n 2 G(4). TABLE V EFFECT OF HEAT TREATMENT OF 55C ON SIX CRYPTOCOCCUS STRAINS GROWING I N LCM 9 C r . n e o f o r m a n s C r . ne o f o r m a n s C r . neoformans C r . l a u r e n t i i C r . a l b i d u s C r . a l b i d u s 365-11 365-16 365-26 371-1 367 ~ H1354 Time Mi n Drop 1 2 ho. 3 b 4 Drop 1 2 no. 3 4 Drop 1 2 no. 3 4 Drop 1 2 no. 3 4 1 Drop ho. 2 3 4 1 Drop no. 2 3 4 0 +++ +++ '+'++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 15 +++ +++! +++ +++ +++ +++ +++ +++ '++ ++ + + +++ +++ +++ +++ ++ ++ ++ ++ ++ +++ ++ +++ 20 +++ ++ ++ +++ +++ +++ +++ +++ ++ ++ ++ - +++ +++ +++ + + + - - - ++ + — 30 ++ + - r + ++ ++ +++ + + ++ + ++ ++ ++ +++ - - - • ++ - _ -40 + - - - + + ++ +++ - - - + + +++ + ' 60 + - - - + + + - - - + + + 80 - - - - - - • - - — . — — + + 90 110 120 a: L i t t m a n ' s C a p s u l e Medium c o n t a i n i n g 10 ug/ml t h i a m i n e HC1 b: 4 d r o p s / s t r a i n (2 f r o m e a c h o f d u p l i c a t e f l a s k s ) , were p l a c e d on Sabouraud a g a r p l a t e s (1 s t r a i n p e r p l a t e ) and i n c u b a t e d a t 25C f o r 1 week. - = no g r o w t h o f d r o p +++ = maximum g r o w t h o f d r o p c: . p l a t e c o n t a m i n a t e d , c o l o n y a t y p i c a l o f C r . neoformans • 43. 3. Growth C o n d i t i o n s A. Temperature A l l the s t r a i n s grew w e l l a t 25C i n SAB and LCM. Expon-e n t i a l phase was e s t a b l i s h e d as 36 hr i n both media f o r f i v e o f the s t r a i n s . Cr. a l b i d u s 367 was the e x c e p t i o n : l o g a r i t h m i c phase was reached a f t e r 72 h r i n LCM and a f t e r 65 hr i n SAB. Cr. l a u r e n t i i 371-1 and a l l the Cr. neoformans s t r a i n s grew w e l l a t 37C but Cr. a l b i d u s 367 grew p o o r l y a t 33C and not at a l l at 37C. C r . a l b i d u s H1354 grew w e l l at 33C but a l s o showed no — _ _ — _ _ _ _ _ _ _ — _ _ ^ growth a t 37C. B. pH The optimum pH f o r capsule p r o d u c t i o n o f a l l s i x s t r a i n s was between pH 6.5 and pH 7.0. T h i s confirmed the work o f L i f t -man (1958) and Bulmer and Sans (1968). Above pH 7.0 both growth and capsule s y n t h e s i s were much reduced: C r . a l b i d u s s t r a i n s 367 and H1354 e x h i b i t e d an extended l a g phase o f 48 h r f o l l o w e d by scanty growth. In c o n t r a s t Bulmer and Sans (1968) r e p o r t e d maximum capsule s i z e a t pH 7.5 f o r a s t r a i n o f Cr. neoformans, CIA. A l l the s t r a i n s produced much s m a l l e r capsules i n SAB (0.2 - lp. capsule t h i c k n e s s ) than i n LCM (1.5 - 4.0u). The percentage o f encapsulated c e l l s was a l s o lower i n SAB than LCM and l o g a r i t h m i c phase c e l l s i n SAB produced few c a p s u l e s , mostly l e s s than 0.5u i n width. C. Thiamine Li f t m a n (1958) r e p o r t e d a s t i m u l a t o r y e f f e c t o f thiamine on capsule s y n t h e s i s and growth over a range o f c o n c e n t r a t i o n s 44. from 0.0001 to 0.1 pg/ml. F i g u r e s 2 to 4 compare the e f f e c t s o f a u t o c l a v e d and unautoclaved thiamine on c e l l growth (measured as i n c r e a s e d absorbance at,650 nm) and capsule t h i c k n e s s . Only Cr. a l b i d u s 367 gave poor growth w i t h a u t o c l a v e d thiamine: the other s t r a i n s c o u l d use heat separated thiamine ( i n t o t h i a z o l e and p y r i m i d i n e , d u r i n g a u t o c l a v i n g , F r i e s 1965) as e a s i l y as they c o u l d the i n t a c t molecule. Although L i t t m a n r e p o r t e d a r e d u c t i o n i n capsule width a t thiamine c o n c e n t r a t i o n s above 0.1 pg/ml, a l l o f the s t r a i n s here showed i n c r e a s e s up to 10 pg/ml; C r . a l b i d u s 367 and Cr. neoformans 365-26 gave l a r g e s t v a l u e s at 50 pg/ml. F i g u r e 5 shows the d i f f e r e n c e s i n capsule s i z e between c e l l s grown i n 0.1 and 10.0 pg/ml thiamine f o r each s t r a i n . Cr. l a u r e n t i i 371-1 showed l i t t l e d i f f e r e n c e i n capsule s i z e between the two c o n c e n t r a t i o n s o f thiamine. C e l l s o f Cr. neoformans s t r a i n s 365-26 and 365-16 were l a r g e r when grown i n 10 pg/ml thiamine. The histograms i n F i g u r e s 2-4 i n d i -c ate no s u b s t a n t i a l d i f f e r e n c e s i n capsule t h i c k n e s s between c e l l s grown i n a u t o c l a v e d and those i n unautoclaved thiamine. As a r e s u l t of these experiments, 10 pg/ml thiamine was added to l a r g e s c a l e c u l t u r e s grown f o r capsule e x t r a c t i o n b e f o r e a u t o c l a v i n g except f o r Cr. a l b i d u s 367 to which i t was added a s e p t i c a l l y a f t e r w a r d s . 4. C e l l and Capsule Morphology The photographs (Figure 5) show the d i f f e r e n c e s i n c e l l s i z e and shape among the s t r a i n s . Cr. a l b i d u s 367 has o v a l c e l l s which e x h i b i t p o l a r budding. T h i s s t r a i n was p i n k i n l i q u i d 45 a FIGURE 2 E f f e c t o f thiamine on growth and capsule s y n t h e s i s o f Cr. a l b i d u s 367 and Cr. a l b i d u s H1354 i n BSM measured a f t e r 120 hr (Cr. a l b i d u s 367) and 72 hr (Cr. a l b i d u s HI354) i n c u b a t i o n at 25C, shake c u l t u r e ' A - - A , ______ r e p r e s e n t unautoclaved thiamine A--A } 1 1 r e p r e s e n t a u t o c l a v e d thiamine a: f i r s t r e a d i n g f o r v i t a m i n - d e p l e t e d c e l l c o n t r o l c a p s u l e t h i c k n e s s expressed a s : diameter o f c e l l + capsule - c e l l diameter 2 45 b E c o m CO u Z < ca ae O t o 00 < 6 5 4 3 2 1 , - A r - - - 4 . 3 Cryptococcus o'bic/us 367 5 4 2 1 0.1 1.0 10.0 50 THIAMINE CONCENTRATION H9/ml 6 5 Ul z U a . < 4 Cryptococcus a/bidus H1354 3 2 1 iao o THIAMINE CONCENTRATION pg/ml FIG. 2 46 a FIGURE 3 ' E f f e c t o f thiamine on growth and capsule s y n t h e s i s o f Cr. l a u r e n t i i 371-1 and Cr. neoformans 365-16 i n BSM measured a f t e r 72 h r i n c u b a t i o n a t 25C, shake c u l t u r e . • - - - A Ytutua r e p r e s e n t unautoclaved thiamine A--A i "I r e p r e s e n t a u t o c l a v e d thiamine Capsule t h i c k n e s s expressed as: diameter o f c e l l + c a p s u l e - c e l l diameter 2 46 b Cryptococcus laurentii 4 371-1 f 2 10J0 50 THIAMINE CONCENTRATION pg/ ml E . a. 12 u X «— ui _i < Cryptococcus neoformans 365-16 iao so THIAMINE CONCENTRATION pg/ ml FIG. 3 47 a FIGURE 4 E f f e c t o f thiamine on growth and c a p s u l e s y n t h e s i s o f Cr. neoformans s t r a i n s 365-11 and 365-26 i n BSM measured a f t e r 72 h r i n c u b a t i o n a t 25C, shake c u l t u r e . A - - A t i v y ^ r e p r e s e n t unautoclaved thiamine A--A , I -) r e p r e s e n t a u t o c l a v e d thiamine Capsule t h i c k n e s s expressed a s : diameter o f c e l l + capsule - c e l l diameter 2 47 b A . — f 6 T 5 T 4 Cryptococcus 365-11 neoformans 0,1 1.0 mo so T H I A M I N E C O N C E N T R A T I O N p g / m l T 5 E . a_. to to tu Z U UJ _j Z> in a. < Cryptococcus neoformans 4 365-26 T H I A M I N E C O N C E N T R A T I O N p g / m l F I G . 4 4 8 a FIGURE 5 DIFFERENCES IN CAPSULE SIZE BETWEEN CELLS GROWN IN 0.1 AND 10.0 yg/ml THIAMINE FOR EACH CRYPTOCOCCUS STRAIN (I n d i a i n k p r e p a r a t i o n s , I 1 = 15.3 jim\ With 0.1 yg/ml thiamine. With 10 yg/ml thiamine a. Cr. l a u r e n t i i 371^1 b. Cr. l a u r e n t i i 371-1 c. Cr. a l b i d u s H1354 d. Cr. a l b i d u s Hi354 e. Cr. a l b i d u s 36 7 f. Cr. a l b i d u s 36 7 g- Cr. neoformans 36 5-11 h. Cr. neoformans 365-11 i . Cr. neoformans365- 16 j • Cr. neoformans 36 5-16 k. Cr. neoformans 365-26 1. Cr. neoformans 365-26 49 c u l t u r e and very mucilaginous i n appearance on s l a n t s . Cr. a l b i d u s H1354 was p a l e r pink and a l s o mucilaginous. Cr. l a u r e n t i i 371-1 had s m a l l e r , s p h e r i c a l c e l l s and c u l t u r e s were y e l l o w i s h , c o l o n i e s b e i n g d r i e r and somewhat w r i n k l e d . A l l three s t r a i n s o f Cr. neoformans had s p h e r i c a l c e l l s , were white i n gross appearance and growth was sl i m y and muc i l a g i n o u s . C e l l s o f C r . neoformans 365-11 appeared s m a l l e r than the other two s t r a i n s . The capsules appear (see F i g u r e 5) to be d i v i d e d i n t o a b r i g h t v i s c o u s i n n e r zone and a l e s s v i s c o u s outer mucous h a l o . A l l the C r . neoformans s t r a i n s examined here and Cr. a l b i d u s 367 e x h i b i t e d an outer mucous h a l o . C r . l a u r e n t i i 371-1 showed o n l y a s l i g h t h a l o and i t was not v i s i b l e i n Cr. a l b i d u s H1354. I t seemed t h a t i n c r e a s e i n capsule width c o r -responded t o i n c r e a s e i n the outer zones i n the C r . neoformans s t r a i n s and Cr. a l b i d u s 367. 5. Growth o f Hyphal Forms Cr. neoformans, Coward s t r a i n , grew w e l l on V8 j u i c e agar a t 25 and 30C, produ c i n g branching septate f i l a m e n t s as submerged growth. The s u r f a c e c o l o n i e s were l a r g e l y y e a s t - l i k e and mucoid. F i g u r e 6 shows clamp connections but i t was not p o s s i b l e to see i f the f i l a m e n t s were d i k a r y o t i c . The y e a s t -l i k e c e l l s had small c a p s u l e s . I t was not p o s s i b l e to o b t a i n s u f f i c i e n t f ilamentous growth f o r c e l l w a l l or capsule e x t r a c -t i o n . None o f the ot h e r s t r a i n s showed hyphal growth or conjuga-t i o n tubes on the s p o r u l a t i o n media t e s t e d . However, Cr. a l b i d u s 367 and Cr. t e r r e u s 8157 developed many ciga r - s h a p e d c e l l s and 50. FIGURE 6. HYPHAL GROWTH FORMS IN CR. NEOFORMANS (Unstained p r e p a r a t i o n s examined under phase c o n t r a s t micro-scopy a l l photographs x 200) l a C r . neoformans, Coward s t r a i n , clamp connections l b C r . neoformans, Coward s t r a i n , s e ptate hyphae 2a Cr. neoformans, 365-26 abnormally budding c e l l a f t e r heat treatment. 2b Hyphal p r o t r u s i o n from Cr. neoformans 365-26 51. hyphal bodies when growing i n LCM or SAB a f t e r i n c u b a t i o n p e r i o d s o f seven days or more. One o f the C r . neoformans s t r a i n s , 365-26, produced hyphal outgrowths (Figure 6) and p o s s i b l e c o n j u g a t i o n tubes a f t e r heat treatment (10 min at 60C). A l l the s t r a i n s which I examined, except the Coward s t r a i n , appear to produce hyphal forms o n l y i n response to adverse c o n d i t i o n s such as heat treatment or n u t r i e n t d e f i c i e n c y . 6. E x t r a c e l l u l a r M a t e r i a l - E x t r a c t i o n I n i t i a l p r e p a r a t i o n s were brown or y e l l o w i s h presumably because the dextrose i n the medium ch a r r e d d u r i n g a u t o c l a v i n g . T h i s was r e s o l v e d by a u t o c l a v i n g dextrose and medium s e p a r a t e l y and then adding the dextrose a s p e c t i c a l l y . The p h y s i c a l appear-ance o f the e t h a n o l p r e c i p i t a t e from each s t r a i n and the y i e l d o f pure e x t r a c e l l u l a r m a t e r i a l are g i v e n i n Table VI. P r e c i p i -t a t e s from the Cr. neoformans s t r a i n s were extremely v i s c o u s and appeared h i g h l y polymerized, y i e l d i n g l a r g e amounts o f e x t r a -c e l l u l a r m a t e r i a l . A p p a r e n t l y the o l d e r the c u l t u r e , the lower the pH became and the h i g h e r was the y i e l d o f p o l y s a c c h a r i d e . Fode e t . a l . (1973)•showed t h a t a s t r a i n o f Cr. l a u r e n t i i p r o -duced h e t e r o p o l y s a c c h a r i d e capsule i n the pH range from 3 to 7 and s y n t h e s i z e d amylose when pH dropped below 3. The e t h a n o l p r e c i p i t a t e o f Cr. l a u r e n t i i 371-1 and both Cr. a l b i d u s s t r a i n s c o n t a i n e d a water i n s o l u b l e f r a c t i o n which may have been s t a r c h -l i k e although produced at a pH o f above 3, arid showing n o " r e a c t i o n w i t h i o d i n e . Very small amounts o f i n s o l u b l e m a t e r i a l were r e -covered from the Cr. neoformans p r e p a r a t i o n s . 52 TABLE V I PHYSICAL APPEARANCE AND YIELD OF EXTRACELLULAR MATERIAL FROM CRYPTOCOCCUS SPECIES Y i e l d o f S t r a i n A p p e a r a n c e o f Pur e Mat-Age o f P i n a l pH E t h a n p l p r e c i p i - e r i a l mg/ Ash and C u l t u r e o f t a t e i n c o n c e n - 100 ml c o n t e n t B a t c h No. H r . c u l t u r e t r a t e d c u l t u r e c u l t u r e % d r y f l u i d f l u i d f l u i d w t . C r . a l b . 3 6 7 1 120 4.63 3 y e l l o w s u s p e n s i o n 45.00 ND C r . a l b . 3 6 7 2 144 4.01 n o n - f i b r o u s , w h i t e 16.43 4.64 C r . a l b . 367? 120 3.09 s l i g h t l y f i b r o u s .43.00 6.88 C r . a l b . H 1354 1 144 . 6.0 8 b s l i g h t l y f i b r o u s 12.00 ND C r . a l b . H 1 354 2 120 3.33 f i b r o u s 14.50 8.52 C r . al b . ' H 1 3 5 4 3 120 ND f i b r o u s 29.60 3.61 C r . l a u r . 3 7 1 - 1 1 120 3.15 n o n - f i b r o u s s u s - 43.04 43.00 3 7 1 - 1 2 p e n s i o n C r . l a u r . 120 3.19 n o n - f i b r o u s 14.87 11.63 C r . l a u r . 3 7 1 - 1 3 120 ND n o n - f i b r o u s 30.26 5.71 C r . n e o f . 3 6 5 - 1 1 1 108 3.75 f i b r o u s 22.00 9.55 C r . n e o f . 3 6 5 - 1 1 2 120 3.10 v i s c o u s , f i b r o u s 70.30 4.54 C r . n e o f . 365-16 1 120 ND v e r y f i b r o u s 58.67 2.27 C r . n e o f . 3 6 5 - 1 6 2 110 3.40 V i s c o u s , f i b r o u s 50.15 2.12 C r . n e o f . 365-26 1 110 3.32 v i s c o u s , f i b r o u s 56.00 2.19 C r . n e o f . 3 6 5-26 2 112 3.18 v i s c o u s , f i b r o u s 59.00 2.25 d e x t r o s e a u t o c l a v e d w i t h m e d i u m - c h a r r i n g o c c u r r e d - p r e c i p i t a t e t o o k on c o l o u r o f medium ^ h i g h i n i t i a l pH may have i n h i b i t e d g r o w t h 7. C e l l W a l l s - E x t r a c t i o n Numerous methods were t r i e d t o break the c e l l s o f Cryptococcus s p e c i e s . Each s p e c i e s (even each s t r a i n ) needed d i f f e r e n t c o n d i t i o n s t o break the w a l l . Even though c e l l s were grown i n medium f o r minimal capsule p r o d u c t i o n , some s t r a i n s had more capsule adhering and were t h e r e f o r e more r e s i l i e n t . Cr. l a u r e n t i i c e l l s proved the most e a s i l y broken and the c e l l s i n 61 medium y i e l d e d 80 mg c e l l w a l l s a f t e r breakage w i t h a Braun homogenizer. Only 60 mg/6 1 were ob-t a i n e d from Cr. neoformans 365-26 a f t e r l a b o r i o u s s e p a r a t i o n procedures. As found by many oth e r workers, Cr. neoformans w a l l s , whatever the method o f breakage, have a tendency to aggregate w i t h remaining whole c e l l s and c y t o p l a s m i c d e b r i s . Attempts t o separate these c o n s t i t u e n t s by sucrose g r a d i e n t s , d i f f e r e n t i a l c e n t r i f u g a t i o n and s o n i c a t i o n , y i e l d e d i n s u f f i -c i e n t amounts o f pure w a l l s f o r complete a n a l y s i s . The c e l l w a l l p r e p a r a t i o n s (three from Cr. l a u r e n t i i 371-1, i n c l u d i n g one from phenol t r e a t e d c e l l s , and one from Cr. neoformans 365-26) showed no evidence o f c y t o p l a s m i c contamination by phase c o n t r a s t l i g h t microscopy. No c a p s u l a r m a t e r i a l was d e t e c t e d w i t h the I n d i a ink t e s t . 8. A n a l y t i c a l S t u d i e s A. C e l l W a l l A n a l y s i s (1) Amino A c i d s D u p l i c a t e s o f each w a l l p r e p a r a t i o n were h y d r o l y z e d i n vacuo i n 6N HC1 at HOC f o r 24 h r . S e r i a l h y d r o l y s e s were not performed because o f the l i m i t e d amounts o f c e l l w a l l s a v a i l a b l e Table VII shows the amounts o f amino a c i d s recovered/mg pure, dry c e l l w a l l p r e p a r a t i o n from Cr. l a u r e n t i i and Cr. neoformans and T r e m e l l a mesenterica (Cameron 1973). There were l a r g e d i f f e r e n c e s i n t o t a l r e c o v e r y but, as can be seen from Table V I I I , there were d i f f e r e n c e s between the p r o t e i n s when the percentages o f t o t a l amino a c i d s were compared. In view o f the proposed taxonomic r e l a t i o n s h i p between Cryptococcus and T r e -m e l l a ( S l o d k i , Wickerham and Bandoni 1966), i t i s i n t e r e s t i n g to compare the amino a c i d compositions o f the two s p e c i e s . H i s t i d i n e and s e r i n e are lower i n T r e m e l l a than i n Cryptococcus s p e c i e s whereas glutamic a c i d and methionine are h i g h e r . N e i -t h e r Cryptococcus s p e c i e s possessed h y d r o x y p r o l i n e and c y s t e i n e / c y s t i n e was d e t e c t e d i n Cr. l a u r e n t i i o n l y . The two Crypto-coccus s p e c i e s were very s i m i l a r i n amino a c i d composition ex-cept t h a t Cr. neoformans had s u b s t a n t i a l l y more l y s i n e . (2) Amino Sugars Glucosamine was found i n the c e l l w a l l s o f both s p e c i e s and a t r a c e o f galactosamine was d e t e c t e d i n Cr. neoformans 365-26. The r e c o v e r i e s o f glucosamine a f t e r h y d r o l y s i s o f the c e l l w a l l p r e p a r a t i o n s w i t h 2N and 6N HC1 are pr e s e n t e d i n Table IX. Cameron (1973) r e p o r t e d massive d e g r a d a t i o n o f glucosamine w i t h 6N HC1 and ob t a i n e d the b e s t estimate f o r Tre m e l l a (23.9 pg/mg c e l l w a l l ) a f t e r 72 h r i n 2N HC1. A l l the h y d r o l y s e s here were performed i n the presence o f 1 mg/ml o x a l i c a c i d (James 1972); t h i s appeared to reduce d e g r a d a t i o n of glucosamine i n 6N H C l . The maximum value f o r C r . neoformans was o b t a i n e d a f t e r 9 hr i n 6N HCl and f o r Cr. l a u r e n t i i , a f t e r 55. TABLE VII AMINO ACIDS IN THE CELL WALLS OF CR. LAURENTII 371-1, CR. NEOFORMANS 365-26 AND TR. MESENTERICA pg anhydro amino a c i d recovered/mg c e l l w a l l p r e p a r a t i o n ' Cr. neoformans''3 Cr. l a u r e n t i i ' ' 3 T r . Mesente Lys 1.32 1.79 3.3 H i s 0.47 1.36 1.2 Arg 0.85 1.70 3.5 Asx 1.07 3.20 5.0 Thr 0.63 1.66 2.5 Ser 0.79 1.75 1.8 Glx 1.40 3.91 7.8 Pro 0.64 1.55 2.6 Gly 0.66 1.30 2.3 A l a 0.81 2.16 3.3 Cys 0.00 0.20 0.0 V a l 0.65 1.71 2.8 Met 0.21 0.35 1.3 H e 0.43 1.27 2.5 Leu 0.76 2.23 4.3 Tyr 0.50 1.32 2.0 Phe 0.49 1.59 2.2 Hyp. d 0.00 0.00 0.4 T o t a l Recovery 11.68 29.05 48.9 a: h y d r o l y z e d w i t h 6N HC1 f o r 24 hr b: average o f t r i p l i c a t e a n a l y s es (Cr. l a u r e n t i i ) a n d d u p l i -c a t e s (Cr. neoformans) c: r e s u l t s expressed as b e s t estimate (maximum value) o f s e r i a l h y d r o l y s e s w i t h 6N HC1 performed by D.S.Cameron (1973) d: determined s p e c t r o p h o t o m e t r i c a l l y (Bergman and L o x l e y 1970) 56. TABLE V I I I AMINO ACIDS IN THE CELL WALLS OF CR. LAURENTII, CR. NEOFORMANS AND TREMELLA MESENTERICA (Amino A c i d s as per c e n t o f t o t a l pg anhydro amino a c i d s recovered) Amino Cr. neoformans Cr. l a u r e n t i i T r e m e l l a M e s e n t e r i c a A c i d Lys 11.3 6.2 6.8 Hi s 4.0 4.7 2.5 Arg 7.3 5.9 7.2 Asx 9.2 11.0 10.3 Thr 5.4 5.7 5.1 Ser 6.8 6.0 3.7 Glx 12.0 13.5 16.0 Pro 5.5 5.3 5.3 Gly 5.7 4.5 4.7 A l a 6.9 7.4 6.8 Cys 0.0 0.7 0.0 Va l 5.6 5.9 5.7 Met 1.8 1.2 2.7 H e 3.7 4.4 5.1 Leu 6.5 7.7 8.8 Tyr 4.3 4.5 4.1 Phe 4.2 5.5 4.5 Hyp. 0.0 0.0 0.8 57. TABLE IX AMINO SUGARS IN THE CELL WALLS OF CR. LAURENTII 371-1 AND CR. NEOFORMANS 365-26 (pg anhydro GlcNx/mg c e l l w a l l p r e p a r a t i o n ) D u r a t i o n o f H y d r o l y s i s w i t h 2N HCl 8 hr 16 h r 32 hr 72 h r 96 h r Cr. l a u r . 6.2 9.1 14.3 28.4 36.1 b Cr. neof. ND a 22.4 0.2' Du r a t i o n o f H y d r o l y s i s w i t h 6N HCl 7 hr 8 h r 9 h r 24 h r Best Estimate  Cr. l a u r . ND 39.71 ND 40.0 40.0 Cr. neof. 26.0 ND 30.6 20.7 30.6 c a: ND = not determined b: 0.2 pg/mg anhydro galactosamine d e t e c t e d i n Cr. neoformans c: maximum value 58. 24 hr i n 6N HC1. The r e s u l t s show t h a t r e l e a s e and degrada-t i o n o f glucosamine from each s p e c i e s ' c e l l w a l l p r e p a r a t i o n may be d i f f e r e n t and r e q u i r e s s e r i a l h y d r o l y s e s , w i t h both 2N and 6N HC1, i n the presence o f o x a l i c a c i d . (3) N e u t r a l Sugars Xy l o s e , mannose, g a l a c t o s e and glucose were i d e n t i f i e d i n the c e l l w a l l s o f both Cryptococcus s p e c i e s . The r e s u l t s are p r e s e n t e d i n Tables X and XI. The c e l l w a l l s o f Cr. neo-formans co n t a i n e d a much l a r g e r percentage o f glucose w i t h small amounts o f the o t h e r sugars. T r e m e l l a c e l l w a l l s con-t a i n e d l a r g e r amounts o f x y l o s e and s m a l l e r amounts o f g l u -cose: they a l s o c o n t a i n e d small q u a n t i t i e s o f ara b i n o s e , fucose and rhamnose. B. E x t r a c e l l u l a r M a t e r i a l A n a l y s i s (1) Amino A c i d s The r e s u l t s p r e s e n t e d i n Tables XII and X I I I show t h a t a l l the u s u a l p r o t e i n amino a c i d s were pr e s e n t i n the p r e p a r a -t i o n s from a l l the s t r a i n s t e s t e d . C y s t e i n e / c y s t i n e was de-t e c t e d i n both s t r a i n s o f C r . a l b i d u s , i n Cr. l a u r e n t i i , a small amount o c c u r r e d i n C r . neoformans 365-11 and t r a c e amounts o n l y were pr e s e n t i n Cr. neoformans 365-16 and 365-26. The p r o t e i n h y d r o l y s a t e s o f a l l s t r a i n s c o n t a i n e d l a r g e quan-t i t i e s o f s e r i n e , t h r e o n i n e , a s p a r t i c and glutamic a c i d s . Cr. neoformans 365-16 and 365-26 had l a r g e amounts o f p r o l i n e and showed s u b s t a n t i a l d i f f e r e n c e s i n the r a t i o s o f G l y / A l a , I l e / L e u and Tyr/Phe from the other s t r a i n s (Table XV). Cr. TABLE X NEUTRAL SUGARS IN THE CELL WALL OF CR. LAURENTII 371-1 (ug anhydro sugar/mg c e l l w a l l p r e p a r a t i o n ) D u r a t i o n o f H y d r o l y s i s w i t h 2N CF^COOH 3 11.1 12.0 3.0 74.0 1 hr 2 hr 3 hr best estimate X y l 57.5 56.9 49.9 57.5 Man 47.6 62.5 61.9 62.5 Gal 13.6 15.4 11.3 15.4 Glc 321.1 384.1 382.0 384.1 T o t a l Recovery 519.5 a: sugars d e t e c t e d as TMS d e r i v a t e s by : (1973) b: maximum value TABLE XI NEUTRAL SUGARS IN THE CELL WALL OF CR. NEOFORMANS 365-26 (ug anhydro sugar/mg c e l l w a l l p r e p a r a t i o n ) D u r a t i o n o f H y d r o l y s i s w i t h 2N CF-COOH 1 hr 2 h r 3 h r best estimate % t o t a l uq X y l 11.3 10.5 9.0 11.3 2.4 Man 15.1 18.2 18.3 18.3 3.9 Gal 2.6 2.8 2.8 2.8 0.6 Glc 360.0 432.0 435.0 435.0 93.1 T o t a l Recovery 467.4 60. TABLE XII AMINO ACIDS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) D u p l i c a t e samples ( 4 - 5 mg/ml) were h y d r o l y z e d w i t h 6N HCl c o n t a i n i n g 1 mg/ml o x a l i c a c i d f o r 24, 48 and 72 h r (pg anhydro amino a c i d recovered/mg e x t r a c e l l u l a r m a t e r i a l ) C r . A l b . H1354 #2 C r . A l b . 367 Cr.Laur. 371-1 Cr.Neof. 365-11 Cr.Neof. 365-16 Cr. Neof. 365-26 Lys 0.84 0.35 1.03 0.73 0.34 0.80 His 0.78 0.29 2.26 0.51 0.21 0.20 Arg 1.28 0.65 1.30 0.64 0.28 0.35 Asx 4.12 2.53 5.77 3.96 2.86 4.88 Thr 4.72 3.24 4.42 3.16 1.85 2.95 Ser 5.12 3.52 5.10 4.07 3.19 4.57 Glx 7.44 3.25 6.78 3.79 1.85 2.49 Pro 2.60 1.33 3.75 1.40 2.06 4.51 Gly 3.55 1.48 2.82 1.38 0.79 0.90 A l a 4.81 2.88 5.29 2.51 2.51 3.94 Cys 2.11 0.34 0.91 0.23 0.00 0.00 V a l 2.86 1.54 2.07 1.40 1.22 1.86 Met 0.20 0.08 0.31 0.28 0.04 0.04 H e 1.42 0.74 1.58 0.86 1.40 2.12 Leu 1.77 0.78 2.33 1.26 0.65 0.91 Tyr 1.23 0.63 1.64 1.08 0.27 0.14 Phe 1.12 0.53 1.67 0.71 1.33 2.48 Hyp' ' 0.00 0.00 0.00 0.00 0.00 0.00 T o t a l scovery 45.97 24.16 49.03 27.97 20.85 33.14 a: Values r e p r e s e n t the mean o f value) from two p r e p a r a t i o n s the b e s t o f each estimates s t r a i n (maximum b: determined c o l o r i m e t r i c a l l y (Bergman and L o x l e y 1970) 61. TABLE X I I I AMINO ACIDS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) D u p l i c a t e samples (4-5 mg/ml)were h y r o l y z e d w i t h 6N HC1 con-t a i n i n g 1 mg/ml o x a l i c a c i d f o r 24, 48 and 72 hr (Amino a c i d s as per c e n t o f t o t a l ug anhydro amino a c i d s recovered) C r . A l b . H1354 #2 C r . A l b . 367 Cr.Laur. 371-1 Cr.Neof. 365-11 Cr.Neof. 365-16 Cr.Neof. 365-26 Lys 1.8 1.5 2.1 2.6 1.6 2.4 Hi s 1.7 1.2 4.6 1.8 1.0 0.6 Arg 2.8 2.7 2.7 2.3 1.3 1.1 Asx 9.0 10.5 11.8 14.2 13.7 14.7 Thr 10.3 13.4 9.0 11.3 8.9 8.9 Ser 11.1 14.6 10.4 14.6 15.3 13.8 Glx 16.2 13.5 13.8 13.6 8.9 7.5 Pro 5.7 5.5 7.7 5.0 9.9 13.6 Gly 7.7 6.1 5.8 4.9 3.8 2.7 A l a 10.5 11.9 10.8 9.0 12.0 11.9 Cys 4.6 1.4 1.9 0.8 0.0 0.0 V a l 6.2 6.4 4.2 5.0 5.9 5.6 Met 0.4 0.3 0.6 1.0 0.2 0.1 H e 3.1 3.1 3.2 3.1 6.7 6.4 Leu 3.9 3.2 4.8 4.5 3.1 2.7 Tyr 2.7 2.6 3.3 3.9 1.3 0.4 Phe 2.4 2.2 3.4 2.5 6.4 7.5 a: Percentages d e r i v e d from f i g u r e s and t o t a l s i n Table XII 62. neoformans 365-11 bore more resemblance to the o t h e r nonpatho-g e n i c , s p e c i e s although, l i k e C r . neoformans 365-16 and 365-26, i t had a l a r g e amount o f a s p a r t i c a c i d . A comparison o f amino a c i d s i n e x t r a c e l l u l a r m a t e r i a l and c e l l w a l l s i s shown i n Tables XIV and XV. The e x t r a c e l l u l a r m a t e r i a l d i f f e r e d from c e l l w a l l preparations i n having l e s s b a s i c amino a c i d s and more s e r i n e and t h r e o n i n e . The amino a c i d composition o f somatic p r o t e i n s e x t r a c t e d from Cr. neoformans C-94 by Uzman, Rosen and F o l e y (1956) are i n c l u d e d i n Tables XIV and XV and compared w i t h the v a l u e s from c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l o b t a i n e d i n t h i s study. Ethanolamine was d e t e c t e d i n a l l the p r e p a r a t i o n s but i t was not p o s s i b l e to q u a n t i f y s i n c e three peaks were observed: two b e f o r e l y s i n e and one b e f o r e ammonia. A l l the s t r a i n s showed ethanolamine as a shoulder on the l y s i n e peak. Cr. a l b i d u s 367 and H1354 gave an e x t r a , separate peak w e l l b e f o r e l y s i n e and C r . l a u r e n t i i 371-1 a t h i r d peak b e f o r e ammonia. Both l i q u i d ethanolamine (BDH) and the s o l i d form (Calbiochem) gave fou r d i s t i n c t peaks (one j u s t b e f o r e l y s i n e and three around ammonia) but the p r o p o r t i o n s o f these peaks were d i f f e r e n t i n each case. (2) Amino Sugars The r e s u l t s i n Table XVI show t h a t glucosamine was r e -covered from a l l o f the e x t r a c e l l u l a r m a t e r i a l p r e p a r a t i o n s . No galactosamine was d e t e c t e d i n the s t r a i n s s t u d i e d here a l -though I found i t i n e x t r a c e l l u l a r m a t e r i a l from Cr. t e r r e u s 8157. The amounts o f glucosamine r e c o v e r e d were much s m a l l e r 63 TABLE XIV AMINO ACIDS IN THE EXTRACELLULAR MATERIAL AND CELL WALLS OF CR. NEOFORMANS 365-26 AND CR. LAURENTII 371-1 (Amino a c i d s as percent o f t o t a l ug anhydro amino a c i d s recovered) H y d r o l y s i s i n 6N HC1 Cr. l a u r . C r . l a u r C r . neof. Cr. neof. C r . neof. 371-1 371-1 365-26 365-26 C-94 C e l l w a l l a Ex. mat. 26 C e l l 26.Ext. w a l l mat. Lys 6.2 2.1 11.3 2.4 7.3 His 4.7 4.6 4.0. 0.6 2.2 Arg 5.9 2.7 7.3 1.1 3.9 Asx 11.0 11.8 9.2 14.7 9.4 Thr 5.7 9.0 5.4 8.9 6.0 Ser 6.0 10.4 6.8 13.8 6.6 Glx 13.5 13.8 12.0 7.5 9.9 Pro 5.3 7.7 5.5 13.6 4.2 Gly 4.5 5.8 5.7 2.7 7.5 A l a 7.4 10.8 6.9 11.9 9.6 Cys 0.7. 1.9 0.0 0.0 0.4 Va l 5.9 4.2 5.6 5.6 7.5 Met 1.2 0.6 1.8 0.1 0.0 H e 4.4 3.2 3.7 6.4 8.1 Leu 7.7 4.8 6.5 2.7 11.5 Tyr 4.5 3.3 4.3 0.4 1.8 Phe 5.5 3.4 4.2 7.5 4.6 100.1 99.7 100.2 99.9 100.5 a: Values from Table V I I I b: E x t r a c e l l u l a r i m a t e r i a l - v a l u e s from Table X I I I c: Amino a c i d composition o f somatic p r o t e i n s e x t r a c t e d from Cr. neoformans C-94 by treatment w i t h hot NaOH. P r o t e i n s (20 mg) h y d r o l y z e d w i t h 1 ml 6N HCl and 0.5 ml g l a c i a l a c e t i c a c i d f o r 24 hr a t HOC (Uzman, Rosen and F o l e y 1956) 64. TABLE XV AMINO ACID RATIOS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) AND THE CELL WALLS OF CR. NEOFORMANS (365-26) AND CR. LAURENTII (371-1) Ra t i o s o f per c e n t anhydro amino a c i d s 3 S t r a i n s G l y / A l a I l e / L e u Tyr/Phe Cr. a l b . HI354 #2 0.73 0.80 1.13 Cr . a l b . 367 0.51 0.97 1.18 Cr. l a u r . . b ex. mat. 0.54 0.67 0.97 Cr. l a u r . w a l l 0.61 0.57 0.83 Cr. neof. 365-11 0.54 0.69 1.56 Cr. neof. 365-16 0.32 2.16 0.20 Cr. neof. 365-26 ex. mat. 0.23 2.37 0.05 Cr. neof. 365-26 w a l l 0.83 0.57 1.02 Cr. neof. C-94 C 0.78 0.70 0.39 a: r a t i o s ; c a l c u l a t e d from percentage val u e s i n Table XIV b: e x t r a c e l l u l a r m a t e r i a l c: amino a c i d s o f somatic p r o t e i n s e x t r a c t e d from Cr. neoformans C-94 by treatment w i t h hot NaOH (Uzman, Rosen and F o l e y 1956) 65. TABLE XVI AMINO SUGARS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) (pg anhydro GlcNx/mg e x t r a c e l l u l a r m a t e r i a l ) H y d r o l y z i n g agent C r . a l b . H1354 #2 Cr . a l b . 367 C r . laur.371-1 C r . neof.365-11 Cr. neof.3fi5-i C r . neof.365-26 6N HCl + 1 mg/ml o x a l i c a c i d 2 HCl + 1 mg/ml o x a l i c a c i d 24 hr 1.50 0.66 1.63 1.51 0.41 0.49 Dur a t i o n o f H y d r o l y s i s Best 48 h r 0.57 0.41 1.41 1.26 0.37 0.33 72 h r 96 hr Estimate 0.90 0.65 1.89 1.04 0.37 0.34 1.26 0.68 2.11 1.35 0.39 0.50 1.50 0.68 2.11 1.51 0.41 0.50 a: maximum value 66. than those from the c e l l w a l l s (Table I X ) . (3) N e u t r a l Sugars The same four sugars (xylose, mannose, g a l a c t o s e and glucose) were d e t e c t e d i n the e x t r a c e l l u l a r m a t e r i a l as were found i n C r . l a u r e n t i i and C r . neoformans c e l l w a l l s , although the p r o p o r t i o n s d i f f e r e d . The r e s u l t s o f a n a l y s i s by two d i f -f e r e n t procedures are p r e s e n t e d i n Tables XVII and X V I I I . T o t a l r e c o v e r i e s were much h i g h e r by the r e s i n h y d r o l y s i s method o f Lehnhardt and W i n z l e r (1968) than a f t e r h y d r o l y s i s w i t h 2N TFA (Cameron 1973). Since the i n d i v i d u a l percentages o f t o t a l sugar were the same f o r both methods, the e r r o r may l i e i n the mannitol standard used i n the f i r s t procedure, or the myoino-s i t o l used i n the second. Maximum value s f o r mannose were o b t a i n e d a f t e r 72 h r h y d r o l y s i s w i t h 0.02N HCl and 4 hr w i t h 2N TFA. Xylose was r e l e a s e d and degraded more r a p i d l y , maxi-mum v a l u e s b e i n g 24 hr and 1 h r r e s p e c t i v e l y . G alactose and glucose gave b e s t r e c o v e r i e s between 48 and 72 hr and 2 - 4 h r . (4) U r o n i c A c i d s The r e s u l t s o f u r o n i c a c i d analyses are presented i n Table XIX. The pathogenic C r . neoformans s t r a i n s c o n t a i n e d much more u r o n i c a c i d than the nonpathogenic s p e c i e s . The r e l a t i v e p r o p o r t i o n s o f u r o n i c a c i d and n e u t r a l sugars are shown i n Table XXI. (5) O-Acetyl 0 - a c e t y l groups were d e t e c t e d o n l y i n the C r . neoformans s t r a i n s as shown i n Table XX. S t r a i n 365-11 had s u b s t a n t i a l l y TABLE XVII NEUTRAL SUGARS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11', 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) (ug anhydro sugar/mg e x t r a c e l l u l a r m a t e r i a l p r e p a r a t i o n ) A C r . A l b . 367 #3 C r . A l b . H1354 #3 Cr.Laur. 371-1 #3 Cr.Neof. 365-11 #2 Cr.Neof. 365-16 av. Cr.Neof. 365-26 av X y l 349.0 165.1 260.1 158.3 205.4 297.3 Man 591.1 392.9 404.0 707.7 414.6 538.0 Gal 90.8 359.2 195.9 119.3 270.2 92.4 Glc 56.2 231.3 36.0 29.6 99.8 26.4 T o t a l 1087.1 1148.5 896.0 1014.9 990.0 954.1 Recovery B X y l 215.3 89.7 200.4 103.1 138.7 187.8 a Man 355.7 214.2 287.5 368.5 251.4 319.8 Gal 46.7 177.4 138.5 67.0 159.5 59.2 Glc 35.1 117.7 26.8 15.8 58.3 17.0 T o t a l 652.8 597.0 653.2 554.4 607.9 583.8 Recovery A - n e u t r a l sugars determined by procedure [see Chapter I I , G ( 5 ) ] , a c c o r d i n g t o a m o d i f i c a t i o n o f Lehnhardt and W i n z l e r ' s method (1968). R e s u l t s are be s t e s t i m a t e s (maximum value) of d u p l i c a t e 24, 48 and 72 hr r e s i n h y d r o l y -ses w i t h 0.02N HC1. B - n e u t r a l sugars determined by procedure [see Chapter I I G(5)] a c c o r d i n g t o m o d i f i c a t i o n o f Cameron's method (1973). R e s u l t s are be s t e s t i m a t e s o f d u p l i c a t e 1, 2 and 4 hr h y d r o l y -ses w i t h 2N TFA. a: mean valu e s o f two-preparations taken f o r C r . neoformans 365-16 and 365-26. 68. TABLE XVIII NEUTRAL SUGARS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS ••(365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) ( n e u t r a l sugars as per c e n t t o t a l pg anhydro n e u t r a l sugar recovered) A C r . A l b . 367 #3 C r . A l b . H1354 #3 Cr.Laur. 371-1 #3 Cr. Neof. 365-11 #2 Cr.Neof. 365-16 av Cr.Neof. . 365-26 av X y l 32.1 14.4 29.0 15.6 20.8 31.2 Man 54.4 34.2 45.1 69.7 41.9 56.4 Gal 8.4 31.3 21.9 11.8 27.3 9.7 Gl c 5.2 20.1 4.0 2.9 10.1 2.8 T o t a l 100.1 Recovery 100.0 100.0 100.0 100.1 100.1 B . X y l 32.9 15.0 30.7 18.6 22.8 32.2 Man 54.5 35.8 44.0 66.5 41.4 54.8 Gal 7.2 29.6 21.2 12.1 26.2 10.2 G l c 5.4 19.7 4.1 2.9 9.6 2.9 T o t a l 100.0 100.1 100.0 100.1 100.0 100.1 Recovery A - n e u t r a l sugars determined by procedure [Chapter I I G(5)] a c c o r d i n g t o a m o d i f i c a t i o n o f Lehnhardt and W i n z l e r ' s method (1968) B - n e u t r a l sugars determined by procedure [Chapter I I G(5)] ac c o r d i n g to a m o d i f i c a t i o n o f Cameron's method (1973) a: percentages c a l c u l a t e d from v a l u e s i n Table XVII 69. TABLE XIX URONIC ACIDS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) (ug u r o n i c acid/mg e x t r a c e l l u l a r m a t e r i a l ) 3 C r . A l b . C r . A l b . Cr.Laur. Cr.Neof. Cr. Neof. Cr.Neof. 367 H1354 371-1 365-11 365-16 365-26 b 73.1 56.0 32.2 158.0 112.2 151.2 a: Determined s p e c t r o p h o t o m e t r i c a l l y (Blumenkrantz and Asboe-Hansen 1973) b: Mean o f d u p l i c a t e o f two p r e p a r a t i o n s o f each s t r a i n TABLE XX O-ACETYL GROUPS IN THE EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) (pg O-acetyl/mg e x t r a c e l l u l a r m a t e r i a l ) 3 C r . A l b . C r . A l b . Cr.Laur. Cr.Neof. C r . Neof. Cr.Neof. 367 H1354 371-1 365-11 365-16 365-26 0.0 0.0 0.0 67.1 14.2 18.1 a: Detected as methyl a c e t a t e s by gas chromatography. For d e t a i l s o f procedure see Chapter I I , G (b). 70. TABLE XXI NEUTRAL SUGARS AND URONIC ACIDS OF CRYPTOCOCCAL EXTRACELLULAR MATERIAL EXPRESSED AS MOLE PERCENT 3 OF TOTAL NM SUGAR C r . A l b . C r . A l b . Cr.Laur. Cr.Neof. Cr.Neof. Cr.Neof. 367 H1354 371-1 365-11 365-16 365-26 X y l 34.4 16.4 33.8 17.5 22.9 30.1 Man 46.3 31.9 39.5 51.0 33.9 41.8 Gal 6.1 26.4 19.0 9.3 21.5 7.7 Glc 4.6 17.5 3.7 2.2 7.9 2.2 Uronic ! 8.8 7.7 4.1 20.2 13.9 18.2 A c i d 100.2 99.9 100.1 100.2 100.1 100.0 a: Mole percentages were c a l c u l a t e d from the f i g u r e s i n Tables XVIII and XIX d i v i d e d by the anhydro molecular weight f o r each sugar. TABLE XXII NEUTRAL SUGARS, URONIC ACIDS AND O-ACETYL GROUPS OF EXTRACELLULAR MATERIAL FROM CR. NEOFORMANS (365-11, 365-16 AND 365-26) EXPRESSED AS MOLE PERCENT 3 OF TOTAL NM SUGAR Cr. Neof. Cr. Neof. Cr. Neof. 365-11 365-16 365-26 X y l 13.0 21.4 27.7 Man 37.7 31.6 38.4 Gal 6.9 20.0 7.1 Gl c 1.6 7.3 2.0 Ur o n i c a c i d 14.9 13.0 16.7 O r - a c e t y l 25.9 6.7 8.1 100.0 100.0 100.0 a: Mole percentages were c a l c u l a t e d from the f i g u r e s i n Tables XVIII, XIX and XX d i v i d e d by the anhydro molecular weight f o r each sugar. 71. l a r g e r amounts than e i t h e r 365-16 or 365-26. Table XXII shows the r e l a t i v e p r o p o r t i o n s o f u r o n i c a c i d , n e u t r a l sugar and O - a c e t y l i n these three s t r a i n s . From these r e s u l t s I est i m a t e d t h a t 35% of the t o t a l p o l y s a c c h a r i d e was O - a c e t y l a t e d i n the e x t r a c e l l u l a r m a t e r i a l from C r . neoformans 365-11. The value s f o r 365-16 and 365-26 were 7% and 9% r e s p e c t i v e l y . I t was not p o s s i b l e to determine from these f i g u r e s where the O - a c e t y l groups were s u b s t i t u t e d or i n which sugars they o c c u r r e d . (6) Elemental and ash a n a l y s i s Two d i f f e r e n t p r e p a r a t i o n s o f e x t r a c e l l u l a r m a t e r i a l from each s t r a i n were analyzed; the r e s u l t s are shown i n Table XXIII. In most o f the p r e p a r a t i o n s there was a s u b s t a n t i a l d i f f e r e n c e between the n i t r o g e n value determined by Organic M i c r o a n a l y s i s and the t o t a l n i t r o g e n c a l c u l a t e d from amino a c i d n i t r o g e n p l u s ammonia n i t r o g e n as d e t e c t e d on the amino a c i d a n a l y z e r . The n i t r o g e n values balanced i n o n l y one o f the p r e p a r a t i o n s , Cr. a l b i d u s HI354 #3. T h i s p r e p a r a t i o n gave a much h i g h e r r e c o v e r y o f t o t a l amino a c i d s and amino sugars than any o f the other p r e p a r a t i o n s . Table XXIV com-pares the r e c o v e r i e s o f amino a c i d s and amino sugars i n Cr. a l b i d u s HI354 p r e p a r a t i o n s #2 and #3. (7) Complete a n a l y s i s o f e x t r a c e l l u l a r m a t e r i a l The t o t a l r e c o v e r y o f e x t r a c e l l u l a r m a t e r i a l compon-ents f o r the s i x Cryptococcus s t r a i n s i s summarized i n Table XXV. Approximately 80% o f the weight o f each e x t r a c e l l u l a r m a t e r i a l p r e p a r a t i o n was recovered, l e a v i n g 20% unaccounted. 72. TABLE XXIII ASH, PHOSPHORUS,AND NITROGEN ANALYSIS OF EXTRACELLULAR MATERIAL FROM CR. NEOFORMANS (365-11, 365-16 AND 365-26), CR. ALBIDUS (367 AND HI354) AND CR. LAURENTII (371-1) (ug/mg e x t r a c e l l u l a r m a t e r i a l p r e p a r a t i o n ) A s h 3 P b N C T o t a l C r . a l b . 371 #2 4.6 1.1 ND d ND Cr. a l b . 371 #3 6.9 1.3 1.4 0.5 Cr. a l b . HI354 #2 8.5 1.5 ND 1.0 Cr. a l b . H1354 #3 . 3.6 1.6 1.8 1.8 Cr. l a u r . 371-1 #1 43.0 0.1 1.2 1.0 Cr . l a u r . 371-1 #3 5.7 1.3 1.4 1.0 Cr . neof. 365-11 #1 9.6 1.9 ND ND Cr . neof. 365-11 #2 4.5 0.4 1.8 0.6 Cr . neof. 365-16 av.^ 2.2 0.2 1.1 0.5 Cr. neof. 365-26 av. 2.2 0.2 1.1 0.5 a: determined g r a v i m e t r i c a l l y . See Chapter I I G ( l ) f o r de-t a i l s o f procedure. b: determined s p e c t r o p h o t o m e t r i c a l l y (Ames 1966) c: a n a l y s i s performed by Organic M i c r o a n a l y s i s , M o n t r e a l , Quebec. d: ND = not determined. e: n i t r o g e n c a l c u l a t e d from amino n i t r o g e n p l u s ammonia n i t r o g e n as d e t e c t e d on the amino a c i d a n a l y z e r . f : l i t t l e d i f f e r e n c e between d u p l i c a t e p r e p a r a t i o n s o f C r . neoformans 365-16 and 365-26, t h e r e f o r e average v a l u e p r e s e n t e d . TABLE XXIV DIFFERENCES IN RECOVERIES OF AMINO ACIDS AND AMINO SUGARS IN TWO SEPARATE PREPARATIONS OF EXTRACELLULAR MATERIAL FROM CR. ALBIDUS HI354 pg anhydro amino mg e x t r a c e l l u l a r a c i d r e c g v e r e d / m a t e r i a l Amino a c i d s as perc e n t o f t o t a l ug anhydro amino a c i d s r e c o v e r e d Cr . a l b i d u s HI354 #2 Cr. a l b i d u s H1354 #3 Cr. a l b i d u s HI354 #2 Cr. a l b i d u s H1354 #3 Lys 0.84 2.04 1.8 1.9 His' 0.78 1.14 1.6 1.1 Arg 1.28 2.53 2.7 2.4 Asx 4.12 9.08 8.7 8.5 Thr 4.72 10.63 9.9 9.9 Ser 5.12 13.11 10.8 12.2 Glx 7.44 15.41 15.7 14.4 Pro 2.60 7.34 5.5 6.8 Gly 3.55 8.30 7.5 7.7 A l a 4.81 9.93 10.1 9.3 Cys 2.11 4.16 4.5 3.9 V a l 2.86 5.41 6.0 5.0 Met 0.20 0.39 0.4 0.4 H e 1.42 3.18 3.0 3.0 Leu 1.77 5.19 3.7 4.8 Tyr 1.23 3.08 2.6 2.9 Phe 1.12 2.52 2.4 2.4 G l c N x b 1.50 3.84 3.2 3.6 T o t a l jcovery 47.47 107.28 100.1 100.2 a: h y d r o l y z e d i n 6N HC1 w i t h 1 mg/ml o x a l i c a c i d f o r 24, 48 and 72 h r . Values r e p r e s e n t b e s t estimates (maximum v a l u e ) . b: h y d r o l y z e d i n 6N HC1 w i t h 1 mg/ml o x a l i c a c i d f o r 24 and 48 hr and i n 2N HC1 f o r 72 and 96 h r . Values r e p r e s e n t b e s t estimates (maximum v a l u e ) . TABLE XXV COMPLETE ANALYSIS OF EXTRACELLULAR MATERIAL OF CR. NEOFORMANS (365-11, 365-16, 3 6 5 - 2 6 ) , CR. ALBIDUS (367, H1354) AND CR. LAURENTII (371-1) (pg'/mg e x t r a c e l l u l a r m a t e r i a l p r e p a r a t i o n ) C r . a l b . C r . a l b . C r . l a u r . C r . n e o f . C r . n e o f . C r . n e o f 367 #3 H1354 #3 371-1 #2 365-11 #2 365-16 a v . 365-26 P o l y s a c c h a r i d e anhydro n e u t r a l i s u g a r 652.8 599.0 653.2 554.4 607.9 583.8 anhydro amino s u g a r 0.7 3.8 2.1 1.5 0.4 0.5 anhydro u r o n i c a c i d 73.1 56.0 32.2 158.0 112.2 151.2 O - a c e t y l 0.0 0.0 0.0 67.1 14.2 18.1 P r o t e i n anhydro amino a c i d 24.2 103.4 49.0 28.0 20.9 33.1 A s h 6.9 3.6 11.6 4.5 2.2 2.2 Phosphorus 1.3 1.6 1.3 0.4 0.2 0.2 T o t a l R e c o v e r y 759.0 767.4 749.4 813.9 757.7 788.7' a : a v e r a g e o f c o m p l e t e a n a l y s i s o f two p r e p a r a t i o n s o f 365-16 and 365-26 b: t o t a l n e u t r a l s u g a r v a l u e s t a k e n f r o m T a b l e X V I I (Cameron's method 1973) c: t o t a l s o f C r . neoformans 365-11, 365-16 and 365-26 c o r r e c t e d f o r t h o s e H atoms on t h e s u g a r m o l e c u l e w h i c h were s u b s t i t u t e d by O-a c e t y l groups ( c a l c u l a t e d f r o m number o f moles o f O - a c e t y l ) . 75. L i p i d d e t e r m i n a t i o n s were not i n c l u d e d s i n c e g r a v i m e t r i c methods ( B a r t n i c k i - G a r c i a and N i c k e r s o n 1962; Weete, p e r s o n a l communi-c a t i o n ) were found to be u n r e l i a b l e on the small q u a n t i t i e s a v a i l a b l e . However, the values o b t a i n e d were never l e s s than 3% f o r the three s t r a i n s t e s t e d (Cr. neoformans 365-26, Cr. a l b i d u s 367 and Cr. l a u r e n t i i 371-1). 9. Gel e l e c t r o p h o r e s i s and S t a i n i n g A. P olyacrylamide g e l s The e l e c t r o p h o r e t i c p a t t e r n s o f Cr. a l b i d u s e x t r a c e l l u -l a r m a t e r i a l , as d e t e c t e d by amido b l a c k ( f o r p r o t e i n ) and p e r -i o d i c a c i d - S c h i f f s t a i n ( f o r carbohydrate) are shown i n F i g u r e 7. I n i t i a l runs w i t h the PAS s t a i n i n d i c a t e d 5 t h a t f a r l e s s m a t e r i a l was needed per g e l to g i v e good r e s o l u t i o n of carbohydrate than was needed to d e t e c t a t h i n p r o t e i n band w i t h amido b l a c k . A l l the Cr. neoformans s t r a i n s were too v i s c o u s i n s o l u t i o n f o r s u f -f i c i e n t q u a n t i t i e s to be a p p l i e d to d e t e c t p r o t e i n . Congruence o f the PAS and amido b l a c k bands suggest the presence o f one or more g l y c o p r o t e i n m o i e t i e s , o r the presence o f two s p e c i e s w i t h the same m o b i l i t y . P r o t e i n band C was very f a i n t , p r e s e n t on o n l y two out o f f i v e g e l s . B. C e l l u l o s e a c e t a t e s t r i p s The e l e c t r o p h o r e t i c p a t t e r n s of C r y p t o c o c c a l e x t r a -c e l l u l a r m a t e r i a l from f o u r d i f f e r e n t s t r a i n s are shown i n F i g u r e s 8 and 9. S t r i p s were s t a i n e d f o r p r o t e i n , carbohydrate and c a r b o x y l m o i e t i e s a f t e r e l e c t r o p h o r e s i s . The Coomassie blue s t a i n d i d not d e t e c t p r o t e i n presumably because i t proved i m p o s s i b l e to apply s u f f i c i e n t amounts to the s t r i p s to v i s u a l -i z e p r o t e i n because the m a t e r i a l was too v i s c o u s and o f l i m i t e d L L A C+ ) B FIGURE 7. E l e c t r o p h o r e t i c p a t t e r n o f Cr. a l b i d u s 367 #1 e x t r a c e l l u l a r m a t e r i a l a p p l i e d to p o l y a c r y l a -mide g e l s . A: 3 mg m a t e r i a l ( i n 0.2 ml) ap-p l i e d t o g e l , and p r o t e i n d e t e c t e d w i t h 1% w/v amido b l a c k . B: 1 mg m a t e r i a l ( i n 0.2 ml) ap-p l i e d t o g e l , and carbohydrate d e t e c t e d by modi-f i e d PAS s t a i n (Page and Stock 1974). F: e l e c -t r o p h o r e t i c f r o n t . C: p r e s e n t on two g e l s only, Band p o s i t i o n s drawn t o s c a l e (1 cm r e p r e s e n t s 0.5 cm l e n g t h ) and taken from mean o f 5 g e l s per s t a i n . See Chapter I I H ( l ) f o r c o n d i t i o n s o f e l e c t r o p h o r e s i s . 77.a FIGURE 8 E l e c t r o p h o r e t i c p a t t e r n o f c r y p t o c o c c a l e x t r a c e l l u l a r m a t e r i a l on c e l l u l o s e a c e t a t e s t r i p s . A: s t a i n e d w i t h A l c i a n blue alone to d e t e c t c a r b o x y l groups B: s t a i n e d f o r g l y c o p r o t e i n by A l c i a n blue method o f Wardi and A l l a n (1972). Band p o s i t i o n s taken as mean of d u p l i c a t e samples and drawn to s c a l e . P: p o i n t o f a p p l i c a t i o n o f sample (5 See Chapter I I H(2) f o r c o n d i t i o n s of e l e c t r o p h o r e s i s . 77 b A B Cr. neoformans 365-11 1 Cr. neoformans 365-26 Hep a r i n standard FIG. 8 78. a FIGURE 9 E l e c t r o p h o r e t i c p a t t e r n o f c r y p t o c o c c a l e x t r a c e l l u l a r m a t e r i a l a p p l i e d t o c e l l u l o s e a c e t a t e s t r i p s . A: s t a i n e d w i t h A l c i a n blue alone to d e t e c t c a r b o x y l groups B: s t a i n e d f o r g l y c o p r o t e i n by A l c i a n blue method o f Wardi and A l l a n (1972) Band p o s i t i o n s taken as mean o f d u p l i c a t e samples and drawn to s c a l e . P: p o i n t o f a p p l i c a t i o n o f sample (5 u l ) F"*": very f a i n t band 2 F : f a i n t band See Chapter I I H(2) f o r c o n d i t i o n s o f e l e c t r o p h o r e s i s 7S _ C r . albidus 367' I — , 1 V V v v\ : x\\ W • M l \\v : \v l i p C r . 'laurentii 371-11 1 ; + H e p a r i n standard FIG.9 79. s o l u b i l i t y i n water or t r i s - b a r b i t a l - s o d i u m - b a r b i t a l b u f f e r . P r e p a r a t i o n s from the d i f f e r e n t s p e c i e s s t a i n e d e q u a l l y w i t h the PAS technique but both C r . neoformans p r e p a r a t i o n s s t a i n e d much more i n t e n s e l y w i t h A l c i a n b l u e alone than d i d C r . l a u r e n t i i o r C r . a l b i d u s . A comparison o f Rp value s shows the congruence of p r o t e i n and carbohydrate bands (Table XXVI) and o f p o s s i b l e g l y c o p r o t e i n and a c i d i c p o l y s a c c h a r i d e (Table XXVII) i n p r e p a r a -t i o n s o f e x t r a c e l l u l a r m a t e r i a l from Cryptococcus s p e c i e s . T h i s again i n f e r s the p o s s i b i l i t y o f the presence o f a g l y c o p r o t e i n complex c o n t a i n i n g a c i d i c groupings. 10. Gel Chromatography The f r a c t i o n a t i o n p a t t e r n s o f p u r i f i e d C r . neoformans 365-26 e x t r a c e l l u l a r m a t e r i a l on a DEAE BioGel-A column are shown i n F i g u r e 10. The m a t e r i a l e l u t e d as a s i n g l e s h a r p l y d e f i n e d band j u s t a t the s t a r t o f the NaCl g r a d i e n t . P r o t e i n and carbohydrate m o i e t i e s were c o i n c i d e n t w i t h i n t h i s band sug g e s t i n g t h a t the small p r o t e i n f r a c t i o n may be bound t o the l a r g e carbohydrate moiety. The m a t e r i a l d i d not appear t o be contaminated w i t h any oth e r m olecular s p e c i e s . 11• I n f r a r e d Spectroscopy C r y p t o c o c c a l e x t r a c e l l u l a r m a t e r i a l from the s i x d i f f e r -ent s t r a i n s and C r . l a u r e n t i i c e l l w a l l s showed i d e n t i c a l spec-t r a l p a t t e r n s f o r wavenumbers between 2000 and 4000 cm-"'" (Figure 11). A u t h e n t i c mannan showed the same a b s o r p t i o n maxima at 2900 and 3250 - 3550 c m - 1 ( I i d a and F i n n e r t y 1973). The ' p r e p a r a t i o n s a l s o had s i m i l a r p a t t e r n s f o r wavenumbers between 80. TABLE XXVI MEAN Rp VALUES OF GLYCOPROTEIN AND PROTEIN BANDS OF EXTRACELLU-LAR MATERIAL!?FROM CRYPTOCOCCUS ALBIDUS 367 #1 ON POLYACRYLAMIDE GELS P e r i o d i c A c i d 3 Amido Black''3 S c h i f f S t a i n S t a i n Band No. 1 0.18 ND C 2 0.28 d 0.27 d 3 ND 0.72 e a: PAS s t a i n s f o r carbohydrate b: amido b l a c k s t a i n s f o r p r o t e i n c: not d e t e c t e d d: darkest band e: f a i n t band Rp = band m o b i l i t y TABLE XXVII MEAN Rp VALUES OF GLYCOPROTEIN AND CARBOXYLIC ACID BANDS OF EXTRACELLULAR MATERIAL FROM CRYPTOCOCCUS SPECIES ON CELLULOSE ACETATE STRIPS 1 A l c i a n B l u e 3 S t a i n _ i • b A l c i a n Blue S t a i n U r o n i c A c i d ug/mg Cr. a l b . 367 #1 0.28° 0.27 41.3 C r . l a u r . 371-1 #1 0.48 0.46 21.5 C r . neof. 365-11 #1 0.31 0.31 100.1 C r . neof. 365-26 #1 0.34 ' 0.34 151.2, a: s t a i n s f o r c a r b o x y l groups b: s t a i n s f o r carbohydrate (Wardi and A l l a n 1972) c: f a i n t bands 81.a FIGURE 10 Chromatography o f e x t r a c e l l u l a r p o l y s a c c h a r i d e from C r . neoformans 365-26 on a DEAE BioGel-A column (37 x 2.5 cm). A convex g r a d i e n t from 0 - 3M NaCl i n 0.02 M p y r i dine-HCl b u f f e r (pH 5.5) was a p p l i e d a f t e r e l u t i o n w i t h 400 ml p y r i d i n e - H C l b u f f e r . F r a c t i o n s o f 10 ml were co l e c t e d . • = carbohydrate O = p r o t e i n A = NaCl g r a d i e n t 83. 650 and 1800 cm-"'" (Figure 12) but there were some d i f f e r e n c e s i n i n t e n s i t y p a r t i c u l a r l y a t 1730, 1650, 1550, 1375, 1250 and 800 cm ^. F i g u r e 13 shows the s p e c t r a o f Cr. l a u r e n t i i c e l l w a l l , Cr. l a u r e n t i i e x t r a c e l l u l a r m a t e r i a l and standard f o r GlcN NH1. These s p e c t r a a l l l a c k e d the i n t e n s e peak at 1730 cm which i s due to c a r b o n y l s t r e t c h i n g v i b r a t i o n s . T h i s was c h a r a c t e r i s t i c o f the C r . neoformans s t r a i n s . The v i b r a t i o n s at 1550 and 1620 - 35 cm - 1 were a t t r i b u t e d to C• = 0 and NH groups (Beran e t a l . 1972). Only Cr. l a u r e n t i i ( c e l l w a l l and e x t r a c e l l u l a r m a t e r i a l ) and Cr. a l b i d u s HI354 had peaks at 1550 cm which c o r r e l a t e d w i t h the l a r g e r amounts o f amino a c i d and glucosamine recovered from these s t r a i n s compared w i t h Cr. neoformans s t r a i n s and C r . a l b i d u s 367. The peaks at 800 and 900 cm are i n d i c a t i v e o f e q u a t o r i a l pi -H-C^ and a l s o occur i n y e a s t mannan. Cr. l a u r e n t i i c e l l w a l l , which was l a r g e l y glucan and glucosamine had very weak v i b r a t i o n s at these wavenumbers but a s l i g h t peak at 890 cm~^~ i n d i c a t i v e of /} - l i n k a g e s and a l s o c h a r a c t e r i s t i c of y e a s t glucan (Beran e_t aJL.1972). The l a r g e peak at 1620 - 1650 cm p r e s e n t i n a l l the s t r a i n s , was a c h a r a c t e r i s t i c peak o f carbohydrates such as mannan and glucan (Beran e t a l . 1972). The i n c r e a s i n g i n t e n s i t y of v i b r a t i o n s a t 1250 cm - 1 (CO o f a c e t y l ) and 1730 cm - 1 (C = 0) from spectrum b - f c o r r e l a t e d w i t h the i n c r e a s e i n both a c e t y l a t i o n and u r o n i c a c i d content i n the s i x s t r a i n s (Table XXV). A s i m i l a r s l i g h t i n c r e a s e i n methyl H a t 1375 cm ^ and i n c a r b o x y l a t e anion a t 1425 cm ^ a l s o confirmed the i n c r e a s e i n O - a c e t y l and c a r b o x y l i c a c i d content. Cr. neoformans 365-11 had the most i n t e n s e bands at a l l these wavenumbers. 84. a FIGURE 12 I n f r a r e d a b s o r p t i o n s p e c t r a o f e x t r a c e l l u l a r m a t e r i a l from Cryptococcus s p e c i e s . a: Cr. a l b i d u s 367 b: Cr. l a u r e n t i i 371-1 c: Cr. a l b i d u s 371 -1 d: Cr. neoformans 365- 16 e : Cr. neoformans 365- 26 f : Cr. neoformans 365- 11 84 b FIG. 12 85. a FIGURE 13 I n f r a r e d a b s o r p t i o n s p e c t r a o f c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l from Cr. l a u r e n t i i 371-1 compared w i t h spectrum from glucosamine HCl a: Cr. l a u r e n t i i c e l l w a l l b: C r . l a u r e n t i i e x t r a c e l l u l a r m a t e r i a l c: Glucosamine HCl 85 b 86. 12. V i r u l e n c e T e s t i n g w i t h Mice The r e s u l t s o f v i r u l e n c e t e s t i n g w i t h mice are. expressed i n Table XXVIII. C r . a l b i d u s s t r a i n s 367 and H1354 and Cr. l a u r e n t i i 371-1 a l l f a i l e d t o k i l l any o f the mice. Cr. neo-formans 365-11 k i l l e d 50% o f the mice a f t e r f i v e days, w h i l e the m o r t a l i t y r a t e f o r 365-16 was 50% on day 18. The mice showed c h a r a c t e r i s t i c symptoms o f domed s k u l l , s t a g g e r i n g , t w i t c h i n g , d u l l f u r and no i n c r e a s e i n s i z e . 80% o f the mice were s t i l l a l i v e 28 days a f t e r i n o c u l a t i o n w i t h 365-26. Mice were k i l l e d and smears and c u l t u r e s made from the b r a i n t i s s u e . C e l l s from b r a i n t i s s u e o f a mouse k i l l e d by t h i s s t r a i n were s u b c u l t u r e d and r e i n o c u l a t e d i n t o a f u r t h e r 12 mice. A f t e r t h i s passaging, 50% m o r t a l i t y o c c u r r e d a f t e r 19 days. F i g . 14 shows c e l l s from mouse b r a i n b e f o r e and a f t e r passaging, demon-s t r a t i n g the i n c r e a s e i n c e l l s i z e i n b r a i n s o f mice k i l l e d by the y e a s t . T h e r m a j o r i t y o f c e l l s a l s o had l a r g e r capsules i n the more v i r u l e n t forrru „ 87. TABLE XXVIII INOCULATION 3 OF MICE INTRACEREBRALLY WITH VIABLE CELLS OF CR. NEOFORMANS (365-11, 365-26 AND 365-16), CR. ALBIDUS (367 AND H1354) AND CR. LAURENTII (371-1) No. o f deaths/day , From 12 mice 4 5 6 8 14 18 21 25 28 % s u r v i v a l Cr. a l b i d u s 367 0 0 0 0 0 0 0 0 0 100 Cr. a l b i d u s H1354 0 0 0 0 0 0 0 0 0 100 Cr. l a u r e n t i i 371-1 0 0 0 0 0 0 0 0 0 100 Cr. neoformans 365-11 2 4 4 2 0 r Cr. neoformans 365-16 0 0 0 0 1 6 5 _ _ 0 Cr. neoformans 365-26 0 0 0 0 0 0 0 1 1 83 Cr. neoformans 0 365-26 0 0 0 0 0 1 1 0 2 66 Cr. neoformans^ 365-26 0 0 0 0 1 a: mice i n o c u l a t e d i n t r a c e r e b r a l l y w i t h 0.02 - 0.04 ml o f a 10 suspension o f c e l l s o f each s t r a i n i n s t e r i l e s a l i n e , b: experiment terminated on day 28. c: i n o c u l a t i o n s repeated a f t e r s u b c u l t u r i n g s t r a i n twice on Sabouraud agar a t 36C. d: i n o c u l a t i o n s repeated w i t h c e l l s removed from b r a i n t i s s u e o f mouse k i l l e d by t h i s s t r a i n and s u b c u l t u r e d a t 36C. 88 FIGURE 14. CR. NEOFORMANS 365-26 IN BRAIN SMEARS OF SWISS WHITE MICE. Smears were s t a i n e d w i t h f u n g i c i d a l I n d i a i n k to show the cap -s u l e . Photographs x 200. a. C r . neoformans 365-26 i n b r a i n t i s s u e o f mouse not k i l l e d by the organism. b. C r . neoformans 365-26 i n b r a i n t i s s u e o f mouse k i l l e d by the organism a f t e r p a s s a g i n g through mice and s u b c u l t u r i n g . 89. CHAPTER IV DISCUSSION P r e l i m i n a r y S t u d i e s and Growth C o n d i t i o n s o f Cryptococcus Species P r e l i m i n a r y s t u d i e s were undertaken to f i n d a s u i t a b l e method f o r k i l l i n g Cr. neoformans c e l l s t o a v o i d h e a l t h hazards d u r i n g h a r v e s t and e x t r a c t i o n . . Previous workers used 1% phenol (Evans and K e s s e l 1951), 0.5% f o r m a l i n (Gadebusch and Johnson 1968, Kobayashi e t a l . 1974), 3% c h l o r o f o r m ( F a r h i , Bulmer and Tacker 1970) or a u t o c l a v i n g a t 82C f o r 35 min (Goren and M i d d l e -brook 1967). Phenol was shown to reduce amino a c i d content o f C r . l a u r e n t i i c e l l w a l l s (Table I I I ) and f o r m a l i n caused f l o c c u -l a t i o n o f C r . neoformans c e l l w a l l s w i t h whole c e l l s d u r i n g w a l l e x t r a c t i o n . A u t o c l a v i n g under the a c i d c o n d i t i o n s o f the medium (Goren and Middlebrook 1967) probably degraded r e a d i l y r e l e a s e d sugars such as x y l o s e . I found t h a t m e t a b o l i c poisons such as potassium cyanide d i d not k i l l the c e l l s . F a r h i (1969) r e p o r t e d t h a t n e i t h e r sodium f l u o r i d e nor potassium i o d o a c e t a t e i n h i b i t e d glucose u t i l i z a t i o n by a s t r a i n o f C r . neoformans. Perhaps the capsule prevented these chemicals from e n t e r i n g the c e l l . As m i l d heat treatment d i d not a f f e c t chemical composition o f w a l l s or capsules (Table I V ) , t h i s method was chosen to k i l l c e l l s p r i o r t o h a r v e s t , f o r a l l p r e p a r a t i o n s made i n t h i s study. S l i g h t d i f f e r e n c e s i n temperature and h o l d i n g time were needed f o r c e l l s grown i n d i f f e r e n t media f o r d i f f e r e n t i n c u b a t i o n 90. p e r i o d s . In a d d i t i o n , f o r e x t r a c e l l u l a r p r e p a r a t i o n s , the pH o f the medium was a d j u s t e d to about 6.0 before heat treatment to a v o i d h e a t i n g under deg r a d a t i v e a c i d c o n d i t i o n s . Bulmer and Sans (1968) and Golubev e t a l . (1971) ex-t r a c t e d adhered c r y p t o c o c c a l c a p s u l a r m a t e r i a l and r e p o r t e d q u a l i t a t i v e a n a l y s i s . I was unable to o b t a i n such p r e p a r a t i o n s by any o f the p u b l i s h e d methods (Bulmer and Sans 1968, Goren and Middlebrook 1967, Vogel 1966) without c e l l w a l l or c y t o p l a s -mic contaminants. Standard c o n d i t i o n s o f temperature, pH, i n c u b a t i o n time and v i t a m i n requirements f o r maximum capsule p r o d u c t i o n i n LCM were determined f o r each s t r a i n (Figures 2 - 5 ) . A temperature o f 25C was chosen f o r a l l l a r g e s c a l e p r e p a r a t i o n s s i n c e n e i t h e r o f the Cr. a l b i d u s s t r a i n s grew at 37C but both showed optimum growth a t 25C. The l a c k o f growth o f C r . a l b i d u s HI354 above 33C i s o f i n t e r e s t i n view o f the supposed p a t h o g e n i c i t y o f t h i s s t r a i n , which was i s o l a t e d from human c e r e b r o s p i n a l f l u i d (Wieser 1973). C a s t e l l a n i (1963, c i t e d by Phaff and F e l l 1970), i s o l a -t e d two Cryptococcus s p e c i e s from l e s i o n s on male g e n i t a l organs. These c a p s u l a t e d i s o l a t e s showed s i m i l a r a s s i m i l a t i o n p a t t e r n s to Cr. a l b i d u s but d i d not a s s i m i l a t e n i t r a t e or grow at 37C. Tang and Howard (1973) showed t h a t whereas Cr. neoformans c o u l d u t i l i z e glutamate a t 37C, a t t h i s temperature uptake o f L g l u t a -mic a c i d by C r . a l b i d u s was s e v e r e l y i n h i b i t e d . I t may be t h a t although Cr. a l b i d u s c e l l s c o u l d not grow i n v i t r o a t 37C, i n v i v o some Cr. a l b i d u s s t r a i n s c o u l d i n c o r p o r a t e and u t i l i z e L glutamic a c i d . There are a l s o s e v e r a l examples o f f u n g i being 9 1 . unable to grow above c e r t a i n temperatures u n l e s s s u p p l i e d w i t h e s s e n t i a l v i t a m i n s or amino a c i d s ( D e v e r a l l i 9 6 5 ) . Perhaps con-d i t i o n s i n v i v o p r o v i d e these substances which Cr. a l b i d u s H1354, f o r example, i s able to i n c o r p o r a t e . However, Tang and Howard (1973) r e p o r t e d t h a t both adenine i n c o r p o r a t i o n i n t o DNA and n u c l e a r m i g r a t i o n p r i o r t o n u c l e a r d i v i s i o n by C r . a l b i d u s were a l s o temperature s e n s i t i v e . I f t h i s were t r u e o f a l l C r . a l b i d u s s t r a i n s , i t i s s t i l l u n c l e a r why C r . a l b i d u s H1354 was a b l e to grow i n v i v o , but not i n v i t r o , a t 37C. I t i s a l s o p o s s i b l e t h a t the organism may have mutated to a lower maximum tempera-t u r e as Wieser (1973)suggested. A l l the s t r a i n s grew p o o r l y above pH 7.0, consequently media f o r e x t r a c t i o n s were a d j u s t e d t o the optimum pH, 6.9 -7.0, b e f o r e a u t o c l a v i n g . T h i s c o n t r a s t s w i t h Bulmer and Sans' work (1968) where maximum capsule s i z e was o b t a i n e d on s o l i d LCM a t pH 7.5. The c e l l s must a l s o be a b l e to grow at pH 7.34 ( s p i n a l f l u i d ) and 7.39 (blood plasma) i n v i v o , thus i t i s un-c l e a r why the s t r a i n s grew p o o r l y above pH 7.0 h e r e . A l -though the optimum pH f o r c e l l growth and subsequent capsule p r o d u c t i o n was 6.5 - 7.0 a t i n o c u l a t i o n , c a p s u l a r m a t e r i a l and v i s i b l e c a p s u l e s d i d not appear i n the medium or on the c e l l s , u n t i l the pH had dropped to 5.0 or lower. A l l the s t r a i n s r e q u i r e d thiamine f o r growth, and a l l except Cr . a l b i d u s 367 c o u l d u t i l i z e i t i n the heat separated form (F i g u r e s 2 - 5 ) . C r . a l b i d u s 367 c o u l d not u t i l i z e t h i a -z o l e and p y r i m i d i n e as w e l l as i t c o u l d use the i n t a c t thiamine molecule a l t h o u g h capsule s i z e was l i t t l e reduced i n a u t o c l a v e d thiamine. F a r h i (1969) r e p o r t e d no e f f e c t o f thiamine w i t h i n 24 hr i n c u b a t i o n e i t h e r on growing o r r e s t i n g c e l l s , but w i t h the c e l l s t e s t e d here ( i n c l u d i n g v i t a m i n d e p l e t e d c e l l c o n t r o l s ) growth enhancement was observed a f t e r 16 h r . As 10 ug/ml thiamine gave maximum capsule p r o d u c t i o n i n these s t u d i e s , t h i s was added to LCM i n s t e a d o f 1 ug/ml as recommended by Littman (1958). The r e s u l t s from these growth s t u d i e s , which showed d i f f e r e n c e s among the s t r a i n s , emphasized the need t o determine and s t a n d a r d i z e the optimum growth c o n d i t i o n s f o r each s t r a i n b e f o r e any f u r t h e r s t u d i e s or e x t r a c t i o n s were c a r r i e d out. T h i s should a l s o be co n s i d e r e d i n f u t u r e work. C e l l Morphology o f Yeast and Hyphal Forms o f Cryptococcus There were d i f f e r e n c e s i n both c e l l shape and capsule s i z e among the s t r a i n s (Figure 5 ) . C r . a l b i d u s c e l l s were t y p i -c a l l y o v a l w i t h p o l a r budding whereas C r . neoformans and C r . l a u r e n t i i c e l l s were-'always s p h e r i c a l . Cr. neoformans 365-26 had l a r g e c a p s u l e s o f up to 4.5 u i n width whereas 365-11 and 365-16 capsules were s l i g h t l y s m a l l e r . The c e l l s o f C r . neo-formans 365-11 were a l s o s m a l l e r than the other two s t r a i n s . Both Cr. a l b i d u s HI354 and C r . l a u r e n t i i 371-1 had smal l cap-s u l e s . I t i s d i f f i c u l t to compare the capsule s i z e s observed here w i t h those o f other workers s i n c e many authors do not s t a t e how capsule width was c a l c u l a t e d . L i t t m a n (1958) f o r example, r e f e r r e d merely to capsule diameter, whereas Bulmer and Sans (1968) d e f i n e d capsule width as 1/2 (diameter o f c e l l and cap-s u l e - diameter o f c e l l ) . 93. The photographs (Figure 5) show two d i s t i n c t zones i n the c a p s u l e . Stoetzner and Kemmer (1971) and Al-Doory (1971) have a l s o d e s c r i b e d two capsule r e g i o n s i n a few s t r a i n s o f C r . neoformans, although many oth e r s t r a i n s have no i n d i c a t i o n o f a mucous h a l o . A number o f workers have p u b l i s h e d e l e c t r o n m i c r o s c o p i c evidence ( L u r i e e t a l . 1971) and f l u o r e s c e n c e s t u d i e s (Vogel 1966) to show t h a t the outer w a l l and i n n e r cap-su l e are s i m i l a r i n s t r u c t u r e . Takeo et. a l . (1973) d e s c r i b e d a two l a y e r e d w a l l w i t h dense, 20 nm diameter p a r t i c l e s most p r e v a l e n t i n the outer l a y e r and a l s o p r e s e n t i n the i n n e r cap-s u l e . Because c a p s u l a r m i c r o f i b r i l s were r e c o g n i z e d by f r e e z e e t c h i n g but not f r e e z e f r a c t u r e , the authors c o n s i d e r e d the capsule to be h i g h l y hydrated. Capsule width i n c r e a s e d c o n s i d -e r a b l y when the C r . neoformans s t r a i n s were' t r a n s f e r r e d i n t o mouse b r a i n s ( c f . F i g u r e s 5 and 14) where v i r u l e n c e appeared to be r e l a t e d to capsule s i z e . When Cr. neoformans 365-26 produced predominantly small capsules i t d i d not k i l l the mice. The c e l l s o f v i r u l e n t . s t r a i n s were s l i g h t l y l a r g e r i n v i v o than i n v i t r o . I t was p o s s i b l e t h a t once the y e a s t reached the b r a i n , c e l l d i v i s i o n slowed down and m e t a b o l i c a c t i v i t y was d i r e c t e d towards s y n t h e s i s o f w a l l , capsule and c y t o p l a s m i c m a t e r i a l . The f r e e z e e t c h i n g s t u d i e s o f Takeo e_t a l . (1973) support t h i s view s i n c e the l a r g e vacuoles and accumulation o f storage o r g a n e l l e s observed i n the cytoplasm of in. v i v o c e l l s suggested a lower r a t e o f m u l t i p l i c a t i o n , and the l a r g e number of v e s i c l e s suggested a h i g h s e c r e t i o n a c t i v i t y . The p r o d u c t i o n o f branching, septate hyphae w i t h clamp 94. connections (Figure 6 a, b) i n the Coward s t r a i n o f Cr. neoformans supports the view of s e v e r a l authors (L u r i e and Shadomy 1971, Kurtzman 1973, Gordon and Devine 1970) t h a t some s p e c i e s o f Cryptococcus may be the i m p e r f e c t forms o f b a s i d i o m y c e t e s . I was not able t o c o n f i r m a p e r f e c t basidiomycete s t a t e s i n c e d i k a r y o t i c hyphae were not d e t e c t e d and no s e x u a l l y r e a c t i v e c u l -t u r e s were found i n any o f the s t r a i n s on the s p o r u l a t i o n media t e s t e d . Conjugation tubes and hyphal outgrowths were observed i n C r . neoformans 365-26 a f t e r heat treatment (Figure 6 c , d ) . These resembled the s t r u c t u r e s d e s c r i b e d by Gordon and Devine from sodium deoxycholate induced mutants. U n l i k e these authors, I d i d not observe endogenous s p o r u l a t i o n . Cr. neoformans 365-11 and 365-26 are probably type A s t r a i n s (as judged by O - a c e t y l peak i n i n f r a r e d s p e c t r a , F i g u r e 12), and s i n c e GordQn and Devine d e t e c t e d s p o r u l a t i o n i n type C o n l y , t h i s may be one reason why endogenous s p o r u l a t i o n was not observed i n Cr. neoformans 365-11 and 365-26. O l d c u l t u r e s o f both Cr. a l b i d u s s t r a i n s and Cr. t e r r e u s 8157 developed many hyphal bodies and c i g a r - s h a p e d c e l l s . These were s i m i l a r i n appearance to c e l l s o f C r . a l b i d u s which had been incubated at 37C f o r 18 h r (Tang and Howard 1973). There-f o r e i t appeared t h a t i n these cases, hyphal p r o d u c t i o n was i n response to the adverse c o n d i t i o n s of h i g h temperature and nu-t r i e n t d e f i c i e n c y as was suggested by Evans (1969). The Coward s t r a i n , however, d i d produce t r u e m y c e l i a under normal c u l t u r a l c o n d i t i o n s , and on the b a s i s o f the h i g h degree o f DNA homology between t h i s s t r a i n and o t h e r non hyphal C r . neoformans s t r a i n s (Erke and Schneidau 1973), can be c l a s s i f i e d as C r . neoformans. T h i s s t r a i n d i d not produce hyphae i n the l i q u i d media SAB and LCM, and t h e r e f o r e I was unable to o b t a i n hyphal c e l l w a l l s . I s o l a t i o n o f the C r y p t o c o c c a l C e l l Envelope E x t r a c e l l u l a r m a t e r i a l was i s o l a t e d from three s t r a i n s o f C r . neoformans, two o f C r . a l b i d u s and one o f Cr. l a u r e n t i i . In agreement w i t h work by Abercrombie et. a l . (1960) and Foda e t a l . (1973) I found t h a t v r a l l the s t r a i n s here produced l a r g e amounts o f h e t e r o p o l y s a c c h a r i d e i n LCM a t an i n i t i a l pH o f 7.0 (Table V I ) . Recoveries o f e x t r a c e l l u l a r m a t e r i a l were poor when c e l l s were h a r v e s t e d above pH 4.5. No amylose was d e t e c -t e d i n any o f the s t r a i n s although t h i s polymere has been r e -p o r t e d f o r o t h e r Cryptococcus s t r a i n s (Foda and Phaff 1969), Kooiman 1963, G o r i n e t a_l. 1966). T h i s may have been because the pH d i d not drop below 3.0 i n any i n s t a n c e . I d i d i s o l a t e a w a t e r - i n s o l u b l e f r a c t i o n ( a f t e r e t h a n o l p r e c i p i t a t i o n ) from the c u l t u r e medium o f the nonpathogenic s p e c i e s . T h i s , l i k e the n e u t r a l p o l y s a c c h a r i d e i s o l a t e d from Cr. l a u r e n t i i by Abercrombie e t a_l. (1960), d i d not s t a i n blue w i t h i o d i n e . A f t e r f i v e days i n c u b a t i o n i n LCM the Cr. neoformans c u l t u r e s were much more mucilaginous and s l i m y than the non pathogens and t h e i r e t h a n o l p r e c i p i t a t e s i n c u l t u r e f l u i d were more f i b r o u s (Table V I ) . The i n c r e a s e i n v i s c o s i t y o f these p r e -c i p i t a t e s seemed to be accompanied by an i n c r e a s e i n O - a c e t y l and g l u c u r o n i c a c i d content (Tables VI, XIX and XX). C e l l w a l l s were e x t r a c t e d from Cr. neoformans 365-26 and Cr. l a u r e n t i i 371-1. I have a l r e a d y mentioned the d i f f i -c u l t i e s i n v o l v e d i n o b t a i n i n g w a l l p r e p a r a t i o n s from Cr . neoformans 96. s t r a i n s . Erke and Schneidau (1973), i n t h e i r DNA e x t r a c t i o n s from Cryptococcus s p e c i e s , a l s o found C r . neoformans c e l l s much more d i f f i c u l t t o d i s r u p t than those o f Cr. l a u r e n t i i due to the presence o f c a p s u l a r m a t e r i a l . They were unable to r e c o v e r s p o o l a b l e DNA from the C r . neoformans s t r a i n s even a f t e r f r e e z e -thaw procedures and d i s r u p t i o n i n a French Pressure c e l l . They c o n s i d e r e d the French p r e s s u r e c e l l more e f f e c t i v e than the Braun homogenizer whereas Phaff (personal communication) found the l a t t e r t o be more e f f e c t i v e w i t h C r . l a u r e n t i i . D e v l i n (1969) r e p o r t e d o n l y 20% d i s r u p t i o n o f Cr. neoformans w i t h the Braun homogenizer but o b t a i n e d 80% by s o n i c o s c i l l a t i o n and an even h i g h e r value by the z e o l i t e e x t r a c t i o n procedure (Zipper and Person 1966). I d i d not o b t a i n more than 60% breakage w i t h z e o l i t e , s o n i c o s c i l l a t i o n o r w i t h an Edebo p r e s s (AB B i o t e c Sweden X25 491). I found these methods u n s u i t a b l e s i n c e there must be a t l e a s t 90% breakage to a v o i d aggregation o f the w a l l s w i t h remaining whole c e l l s and c y t o p l a s m i c d e b r i s . T h i s aggre-g a t i o n may be due to b i n d i n g c a p a c i t i e s o f the h i g h l y charged c a p s u l a r m a t e r i a l ( a c i d groups) w i t h s i t e s on the w a l l s or r e -maining whole c e l l s . A s u c c e s s f u l procedure f o r breakage and subsequent i s o l a t i o n o f pure c e l l w a l l s remains to be developed f o r C r . neoformans s t r a i n s . Chemical Composition o f C e l l W a l l s The c e l l w a l l s i s o l a t e d from C r . l a u r e n t i i and C r . neo-formans showed some s t r i k i n g s i m i l a r i t i e s i n chemical composition to the w a l l s o f other basidiomycetes. Previous s t u d i e s on 97. basidiomycete c e l l w a l l s (Crook and Johnston 1962, O'Brien and Ralph 1966, Angyal et. a l . 1974) showed glucose and g l u c o -samine to be the major monosaccharides w i t h mannose and x y l o s e i n l e s s e r amounts and t r a c e s o f g a l a c t o s e or f u c o s e . Galac-tose found i n both Cryptococcus s p e c i e s (Tables X and X I ) , was p r e s e n t o n l y i n U s t i l a q o maydis (Crook and Johnston 1962), Coniophora c e r e b e l l a (O'Brien and Ralph 1966) and T r e m e l l a  mesenterica (Cameron 1973). The Cryptococcus s p e c i e s c o n t a i n e d no fucose and much s m a l l e r amounts o f glucosamine than most basidiomycetous y e a s t s , except T r e m e l l a . Kobayashi e t a l . (1974) claim e d to have d e t e c t e d r i b o s e or rhamnose i n an endo-t o x i c substance (hot phenol-water e x t r a c t ) from C r . neoformans. The peak, which they observed by g a s - l i q u i d chromatography as a TMS d e r i v a t i v e , was a l s o p r e s e n t i n small amounts i n my w a l l and e x t r a c e l l u l a r m a t e r i a l samples. However, I d i d not c o n s i d e r i t had the same r e t e n t i o n time as e i t h e r r i b o s e , rhamnose or a r a b i n o s e . Arabinose has been claimed to be a minor component of C r . l a u r e n t i i c e l l w a l l s (Ankel e_t a l . 1969). However, t h i s peak seemed most l i k e l y to be one o f the f i r s t minor x y l o s e peaks. I n f r a r e d spectroscopy (Figure 13) showed t h a t the c e l l w a l l o f Cr. l a u r e n t i i had both o< - and yS-linked glycans (840 and 890 cm-"*" r e s p e c t i v e l y ) . The spectrum was very s i m i l a r to t h a t o f a C r . a l b i d u s c e l l w a l l p r e p a r a t i o n examined by Jones e t a l , (1969). These authors found t h a t the eH. -glucan composition was c o n s i d e r -a b l y reduced by growing the y e a s t under unfavourable c o n d i t i o n s . Kanetsuna and C a r b o n e l l (1970, 1971) a l s o found t h a t oC-glucan decreased and/J-glucan s y n t h e s i s i n c r e a s e d , d u r i n g the y e a s t to m y c e l i a l c o n v e r s i o n which was a s s o c i a t e d w i t h the l o w e r i n g o f temperature i n P a r a c o c c i d i o i d e s b r a s i l i e n s i s and Blastomyces  d e r m a t i t i d i s P o l y s a c c h a r i d e s from the c e l l w a l l o f Polyporus  tumulosus i n c l u d e /3-1/ 3-glucan, •<-glucan and c h i t i n as w e l l as a xylomannan which has s i m i l a r molar p r o p o r t i o n s o f mannose and x y l o s e (1.2:1) to those i n Cr. l a u r e n t i i c e l l w a l l (Angyal e t a l . 1974). Other basidiomycete c e l l w a l l s (O'Brien and Ralph 1966) have s i m i l a r mannose:xylose p r o p o r t i o n s except T r e m e l l a which has much l a r g e r amounts o f x y l o s e than mannose. T h i s a g a i n i n f e r s a c l o s e r e l a t i o n s h i p between Cryptococcus and the b a s i d i o m y c e t e s . The main d i f f e r e n c e i n sugar content between the w a l l s o f the two Cryptococcus s p e c i e s was the much l a r g e r amount o f glucan i n Cr. neoformans; 93%, i n c o n t r a s t to 74% i n C r . l a u r e n t i i (Tables X and X I ) . The l a t t e r f i g u r e was s i m i l a r to the 75% glucose 1.recovered from Cr . a l b i d u s w a l l s (Bacon e_t aJL. 1968). The other sugars, i n c l u d i n g glucosamine, were p r e s e n t i n s m a l l e r amounts i n Cr. neoformans (Tables IX, X and X I ) , although the p r o p o r t i o n o f glucosamine was s t i l l h i g h e r than i n T r e m e l l a c e l l w a l l s . Cr. neoformans 365-26 a l s o c o n t a i n e d a small amount o f galactosamine which has not p r e v i o u s l y been d e t e c t e d i n y e a s t c e l l w a l l s . In the o n l y p u b l i s h e d study o f the c e l l w a l l s of Cr. neoformans Cook e_t a l . (1970) r e p o r t e d a very low r e c o v e r y o f hexosamine (0.3%) i n w a l l s o f both p a t i e n t and s o i l i s o l a t e s . However, they d i d not use c r i t i c a l c s e r i a l h y d r o l y s e s and t h e r e -f o r e p r o b a b l y underestimated the glucosamine. D e v l i n (1969) a l s o examined c r y p t o c o c c a l c e l l w a l l s and " z e o l i t e ghosts" by 99. q u a l i t a t i v e chemical methods. He f a i l e d t o d e t e c t glucosamine, but the m i l d h y d r o l y t i c c o n d i t i o n s employed(1.5N HC1 f o r 3 hr at 97C) were probably i n s u f f i c i e n t t o r e l e a s e glucosamine. He was a l s o unable to r e l e a s e N - a c e t y l glucosamine by h y d r o l y s i s w i t h p u r i f i e d c h i t i n a s e and concluded t h a t the w a l l s c o n t a i n e d no c h i t i n . In view o f h i s poor chemical techniques, i t i s l i k e l y t h a t he d i d not d e t e c t the r e l a t i v e l y s m a l l amount o f N - a c e t y l glucosamine t h a t I c o n s i d e r to be p r e s e n t . V i b r a t i o n s a t 1380, 1550, 1640 and 2940 cm" 1 i n the i n f r a r e d s p e c t r a o f C r . l a u r e n t i i c e l l w a l l s (Figure 13), are t y p i c a l o f c h i t i n , which suggests t h a t the glucosamine p r e s e n t i s N - a c e t y l a t e d and -1 may be o c c u r r i n g as c h i t i n . The shoulder a t 1725 cm i s i n -d i c a t i v e o f c a r b o n y l v i b r a t i o n s and may be due to N - a c e t y l a -t i o n (perhaps o f glucosamine) r a t h e r than to c a r b o x y l a t i o n , i n C r . l a u r e n t i i c e l l w a l l s . However, c h i t i n does.not have a peak at t h i s wavenumber. Bowden and Hodges (1970) p o s t u l a t e d t h a t the s m a l l amounts o f glucosamine p r e s e n t i n the y e a s t w a l l as N - a c e t y l glucosamine, need not n e c e s s a r i l y occur as a c h i t i n -l i k e polymer but r a t h e r a c t as attachment p o i n t s f o r p e p t i d e m o i e t i e s to mannan and glucan components. Nakajima and B a l l o u (1974) have r e c e n t l y used enzymic techniques to i s o l a t e an o l i g o s a c c h a r i d e c o r r e s p o n d i n g to the l i n k a g e r e g i o n between p o l y s a c c h a r i d e and p r o t e i n p a r t s o f Saccharomyces c e r e v i s i a e mannan. A s i n g l e N - a c e t y l glucosamine was a t t a c h e d to the r e -ducing end o f 12 mannose u n i t s by ay3-l, 4 l i n k a g e . I f the glucan l a y e r was combined w i t h or c o v e r i n g the c e l l w a l l c h i t i n (Domanski and M i l l e r 1968) then c h i t i n a s e (as used by D e v l i n 100. 1969) would.not r e l e a s e N - a c e t y l glucosamine^without p r i o r ap-p l i c a t i o n o f a y j l - 3 glucanase. Very few f u n g a l c e l l w a l l s have been examined q u a n t i -t a t i v e l y f o r amino a c i d s and o n l y one study employs c o r r e c t i v e s e r i a l a c i d h y d r o l y s e s (Cameron 1973). The amino a c i d p a t t e r n s o f C r . l a u r e n t i i and C r . neoformans c e l l w a l l s were very s i m i -l a r t o those o f T r e m e l l a mesenterica (Table V I I I ) . A l l were h i g h i n Asx and Glx, the 1amino a c i d s a s s o c i a t e d w i t h A l k a l i s t a b l e g l y c o p e p t i d e bond for m a t i o n . Nakajima and B a l l o u (1974) proposed t h a t y e a s t mannan i s l i n k e d t o p r o t e i n through a d i -N - a c e t y l c h i t o b i o s e to asparagine: t h i s may be the case i n Cryptococcus c e l l w a l l s . C r . l a u r e n t i i c e l l w a l l s , i n f a c t , not o n l y had more glucosamine but a l s o had a s l i g h t l y h i g h e r percentage o f Asx than C r . neoformans (Tables VI, IX) suggest-i n g more o f these g l y c o p r o t e i n l i n k a g e s . Most workers have f a i l e d t o d e t e c t c y s t e i n e / c y s t i n e i n the f u n g i examined (Roy and Landau 1972) but Pine (1972) d e t e c t e d very small amounts i n Histoplasma capsulatum and H. d u b i o s i i c e l l w a l l s . I de-t e c t e d c y s t e i n e / c y s t i n e o n l y i n C r . l a u r e n t i i . H y d r o x y p r o l i n e has not been p r e v i o u s l y r e p o r t e d i n f u n g i w i t h c h i t i n o u s c e l l w a l l s ( B a r t n i c k i - G a r c i a 1968) and i t was not d e t e c t e d i n the Cryptococcus s p e c i e s t e s t e d h e r e . I t i s p o s s i b l e t h a t the p r e -sence o f h y d r o x y p r o l i n e i n T r e m e l l a mesenterica c e l l w a l l s (Cameron 1973) was r e l a t e d t o the absence o f c h i t i n . These c e l l w a l l s have y e t to be t e s t e d f o r the presence o f N - a c e t y l a t e d glucosamine. C r y p t o c o c c a l c e l l w a l l s were a l s o s i m i l a r u l t r a s t r u c t u r a l l y 101. -to those o f other basidiomycetous y e a s t s (Kreger-van R i j and Veenhuis 1971) i n having a l a m e l l a r w a l l s t r u c t u r e and a s i m i l a r method o f bud formation and s e p t a t i o n . These f e a t u r e s d i f f e r e d from those r e c o r d e d f o r ascomycetous y e a s t s . Cryptococcus and T r e m e l l a d i f f e r e d mainly from o t h e r basidiomycetous y e a s t s i n having l e s s glucosamine. T h i s seemed to be r e p l a c e d by l a r g e r amounts o f glucan but i t i s u n c l e a r a t p r e s e n t e x a c t l y how the polymer i s arranged to form the two l a y e r e d l a m e l l a r w a l l observed by e l e c t r o n microscopy. R e l a t i o n o f C e l l W a l l Chemistry to P a t h o g e n i c i t y I t i s d i f f i c u l t t o r e l a t e d i f f e r e n c e s i n c e l l w a l l chemi-s t r y t o p a t h o g e n i c i t y . Cox and Best (1972) r e l a t e d h i g h e r w a l l p h o s p h o l i p i d , c h i t i n and p r o t e i n content to v i r u l e n c e i n B l a s t o -myces d e r m a t i t i d i s : l e s s v i r u l e n t s t r a i n s had more g l u c a n . On the other hand, Cook e_t a l . (1970) found more hexose and l e s s l i p i d and hexosamine i n the c e l l w a l l o f the p a t i e n t i s o l a t e o f Cr. neoformans than i n the s o i l i s o l a t e . However, they d i d not determine the degree o f v i r u l e n c e o f the two s t r a i n s f o r mice. Although I d i d not perform l i p i d and phosphorus a n a l y s e s , my r e s u l t s agree w i t h Cook e_t a l ' s i n t h a t the pathogen, C r . neo-formans 365-26, had much l a r g e r amounts o f glucan ( e q u i v a l e n t to hexose, Cook e t a l . ) and a s m a l l e r amount o f glucosamine and probably p r o t e i n i n the c e l l w a l l than the nonpathogenic C r . l a u r e n t i i . C r . neoformans 365-26 c e l l w a l l a l s o had a substan-t i a l l y l a r g e r percentage o f l y s i n e than C r . l a u r e n t i i . I t i s premature to conclude t h a t l a r g e r amounts o f glucose and s m a l l e r amounts o f glucosamine and p o s s i b l y g l y c o p r o t e i n i n 102. the c e l l w a l l s o f Cryptococcus s p e c i e s , are r e l a t e d t o patho-g e n i c i t y . C e l l w a l l s from more s p e c i e s and s t r a i n s must be examined both c h e m i c a l l y and s t r u c t u r a l l y , as w e l l as t e s t e d f o r a n t i g e n i c i t y . Chemical Composition o f E x t r a c e l l u l a r M a t e r i a l The e x t r a c e l l u l a r m a t e r i a l i s o l a t e d from the s i x Cryptococcus s t r a i n s c o n t a i n e d the same monosaccharide con-s t i t u e n t s : mannose, x y l o s e , g a l a c t o s e , small amounts of glucose and g l u c u r o n i c a c i d as w e l l as O - a c e t y l s u b s t i t u t i o n s (Tables XVII t o XX). The mannose, x y l o s e and g l u c u r o n i c a c i d o c c u r r e d i n approximately the same molar p r o p o r t i o n s as those determined by other workers ( F a r h i e t al.1970, Blandamer and Danishefsky 1966, S l o d k i e t a l . 1966, Helms e t a l . 1969). The presence o f g a l a c t o s e i s a p o i n t o f d i s p u t e s i n c e some workers c o n s i d e r the c a p s u l a r m a t e r i a l t o be a mixture o f two p o l y s a c c h a r -i d e s , one c o n t a i n i n g g a l a c t o s e and the other not (Evans and T h e r i a u l t 1953, Rebers e t a l . 1958). C r . neoformans 365-16 and Cr. a l b i d u s HI354 p r e p a r a t i o n s c o n t a i n e d c o n s i d e r a b l y more g a l a c t o s e than the other s t r a i n s (Tables XVII, X V I I I ) . Judging from the'-'diffuse carbohydrate band i n g e l e l e c t r o p h o r e s i s o f Cr. a l b i d u s e x t r a c e l l u l a r m a t e r i a l i ( F i g u r e 7) i t i s q u i t e poss-i b l e t h a t the p o l y s a c c h a r i d e p o r t i o n i s heterogeneous. However, u r o n i c a c i d and carbohydrate m o i e t i e s d i d migrate t o g e t h e r i n the c e l l u l o s e a c e t a t e s t r i p s ( Figures 8 and 9) sugges t i n g t h a t the molecule may be homogeneous. Golubev e t a l . (1971) c o n s i d e r e d the capsule and e x t r a c e l l u l a r m a t e r i a l t o be d i f f e r e n t on the b a s i s o f l a c k o f g a l a c t o s e i n the former. However, F a r h i e t a l . (1970) found no d i f f e r e n c e i n percentage g a l a c t o s e between 103. adhered and s o l u b l e p o l y s a c c h a r i d e s . Perhaps Golubev e t a l . l o s t the g a l a c t o s e d u r i n g the a u t o c l a v i n g procedure they used to remove adhered c a p s u l e . Glucose was not d e t e c t e d i n any p r e v i o u s s t u d i e s . The major d i f f e r e n c e s between the s t r a i n s were i n u r o n i c a c i d content and the degree o f O - a c e t y l s u b s t i t u t i o n . ( 0 - a c e t y l has not been estimated q u a n t i t a t i v e l y p r e v i o u s l y and some authors ( F a r h i e t a l . 1970, Blandamer and Danishefsky 1966) d i d not t e s t f o r i t at a l l ) . These d i f f e r e n c e s were c l e a r l y demonstrated i n the i n f r a -r e d s p e c t r a (Figure 12). Although no methyl a c e t a t e s were de-t e c t e d i n t r a n s e s t e r i f i e d samples o f C r . a l b i d u s (H1354 and 367) and C r . l a u r e n t i i 371-1, Cr. a l b i d u s 367 does have a peak at 1725 cm - 1 ( i n d i c a t i v e o f C = 0) and Cr. a l b i d u s H I 3 5 4 a s l i g h t s h o u l d e r . D i f f i c u l t i e s i n i n t e r p r e t a t i o n o f c e l l w a l l and p o l y -s a c c h a r i d e s p e c t r a o f t e n a r i s e because o f the a d d i t i v e e f f e c t s and i n f l u e n c e s o f the a b s o r p t i o n s o f the d i f f e r e n t chemical groups p r e s e n t . Thus the more heterogeneous the compound, the more complicated becomes the i n t e r p r e t a t i o n . For example,the peaks at 1730 and 1250 cm"''' are regarded by some authors (Bland-amer and Danishefsky 1966, Maschessault 1962) to be i n d i c a t i v e o f c a r b o x y l groups whereas Levine e t a l . 1959, Goren and M i d d l e -brook 1967 and Kobayashi e t a l . 1974 c o n s i d e r these v i b r a t i o n s to be due to a c e t y l f u n c t i o n s . The peak at 1250 cm ^ i s un-doubtedly due to C-0 s t r e t c h i n g v i b r a t i o n s , but the l i n e b e t -ween e s t e r f u n c t i o n at 1725 - 1750 cm ^ and c a r b o x y l a t 1650 -1725 i s h a r d to d i s t i n g u i s h e s p e c i a l l y i f both f u n c t i o n s are 104. known t o be p r e s e n t . Ukai e t a l . (1974) working w i t h T r e m e l l a p o l y s a c c h a r i d e and Goren and Middlebrook w i t h C r . neoformans, found t h a t the e s t e r f u n c t i o n , presumed to be O - a c e t y l , was h y d r o l y t i c a l l y c l e a v e d under m i l d a l k a l i n e c o n d i t i o n s w i t h r e -i s u i t i n g l o s s o f a b s o r p t i o n a t 1725 and 1250 cm "*". Levine e_t a l . (1959) c o n s i d e r e d the peaks at 1650 and 1425 cm""'" to r e -present c a r b o x y l a t e but both glucan and mannan have l a r g e peaks at 1650 as does glucosamine. Because none of the nonpathogenic s t r a i n s c o n t a i n e d O - a c e t y l f u n c t i o n s , but a l l had u r o n i c a c i d , i t i s p o s s i b l e t h a t the v i b r a t i o n s a t 1730 cm""'" were due to both a c e t y l and c a r b o x y l f u n c t i o n s . T h i s would c e r t a i n l y c o r -r e l a t e w i t h the i n c r e a s e i n peak s i z e at t h i s wavenumber from the t r a c e i n C r . l a u r e n t i i t o the l a r g e peak i n C r . neoformans 365-11 (Figure 12). I f the e f f e c t were a d d i t i v e i t would ex-p l a i n why the d i f f e r e n c e i n peak s i z e between C r . neoformans 365-11 and the oth e r two C r . neoformans s t r a i n s i s not as marked as would be expected from the d i f f e r e n c e s i n t h e i r O - a c e t y l content. None o f the s t r a i n s showed a c a r b o x y l s a l t peak at 1600 cm - 1. The h i g h e r glucosamine content o f Cr. a l b i d u s HI354 #3 and C r . l a u r e n t i i 371-1 (Table XVI) can a l s o be observed i n the s p e c t r a from the v i b r a t i o n at 1550 cm-"'" (Figure 12). Thus the s l i g h t - shoulder at 1730 cm""'" on the H1354 spectrum may a l s o be due to N - a c e t y l a t i o n . Both glucose and glucosamine have not p r e v i o u s l y been d e t e c t e d i n c r y p t o c o c c a l e x t r a c e l l u l a r p o l y -s a c c h a r i d e s or capsules although o t h e r y e a s t s and both y e a s t and m y c e l i a l phases o f S p o r o t h r i x s c h e n c k i i produced e x t r a c e l l u l a r 105. p o l y s a c c h a r i d e s which c o n t a i n e d glucosamine and N - a c e t y l g l u c o -samine (Jeanes e t a l . 1971, T o r i e l l o and M a r i a t 1974). A l l the s t r a i n s showed peaks at 800 and 900 cm "*" r e p r e s e n t a t i v e o f o(-l i n k e d g l y c a n . Beran e t a l . (1972) observed t h a t these v i b r a -t i o n s , as w e l l as v i b r a t i o n s a t 980 cm""'", were p r e s e n t i n the spectrum o f y e a s t mannan but absent from t h a t o f y e a s t g l u c a n . Since Cr.neoformans 365-11, which c o n t a i n e d more mannose than the other s t r a i n s , a l s o had the most i n t e n s e peak at 800 cm - 1, i t i s p o s s i b l e t h a t the mannose pr e s e n t i s oC-linked. The p o l y s a c c h a r i d e s were s i m i l a r i n composition to those i s o l a t e d from T r e m e l l a s p e c i e s ( S l o d k i et. a l . 1966, F r a s e r et. a l . 1973a), i n t h a t they c o n t a i n e d mannose, x y l o s e , g l u c u r o n i c a c i d and O - a c e t y l groups. S l o d k i e t a l . (1966) and Helms e t a l . (1969) a l s o found three C r . l a u r e n t i i s t r a i n s which c o n t a i n e d O - a c e t y l groups although many do not. However, S l o d k i e_t a l . (1970) showed t h a t the phosphorus content o f mannans may be r e p l a c e d by O - a c e t y l groups i n phosphate l i m i t i n g medium. The medium he used c o n t a i n e d 0.01% phosphorus whereas the LCM used here c o n t a i n e d 0.2%. Cadmus e t a l . (1962) found t h a t tncre^ase i n c o n c e n t r a t i o n s o f phosphorus i n h i b i t e d capsule p r o d u c t i o n i n a C r . l a u r e n t i i s t r a i n . T h i s may e x p l a i n why I d e t e c t e d l e s s cap-s u l e and c a p s u l a r m a t e r i a l from Cr. l a u r e n t i i than from the o t h e r s t r a i n s , and why no 0 - a c e t y l groups were d e t e c t e d . Ukai t e t a l . (1974) i s o l a t e d a homogeneous p o l y s a c c h a r i d e from hot water e x t r a c t s o f T r e m e l l a f u c i f o r m i s and o b t a i n e d s i m i l a r molar r a t i o s o f sugars to those o b t a i n e d from o t h e r T r e m e l l a s p e c i e s ( S l o d k i e_t a l . 1966). The main d i f f e r e n c e between 106 T r e m e l l a and Cryptococcus i s t h a t T r e m e l l a has more x y l o s e than mannose i n the e x t r a c e l l u l a r p o l y s a c c h a r i d e s and i n the c e l l w a l l The C r . neoformans s t r a i n s - I examined, a l s o had more u r o n i c a c i d and O - a c e t y l groups; than T r e m e l l a s p e c i e s . F r a s e r e t a l , (1973b) demonstrated t h a t the O - a c e t y l groups o c c u r r e d on the C-3 p o s i t i o n s o f the g l u c u r o n i c a c i d r e s i d u e s . T h i s study i s the f i r s t r e p o r t o f amino a c i d a n a l y s e s from C r y p t o c o c c a l p o l y s a c c h a r i d e . The o n l y o t h e r amino a c i d a n a l y s i s r e l a t e s to somatic p r o t e i n s e x t r a c t e d from C r . neo-formans w i t h hot a l k a l i (Uzman 1956). From 20 t o 100 p.g amino a c i d s were r e c o v e r e d per mg e x t r a c e l l u l a r m a t e r i a l f o r a l l the s t r a i n s and a l l c o n t a i n e d a t l e a s t 1% n i t r o g e n (Tables XII and X X I I I ) . Most authors have regarded the n i t r o g e n component as a contaminant and have used the d e p r o t e i n i z a t i o n procedures o f Kabat and Mayer (1967) d u r i n g p u r i f i c a t i o n (Kozel and C a z i n 1970, F a r h i e t a l . 1970, Murphy and Cozad 1972). These p r o -cedures, i n v o l v i n g c h l o r o f o r m - b u t a n o l e x t r a c t i o n s , would not have removed c o v a l e n t l y bound p r o t e i n . A f t e r d e p r o t e i n i z a t i o n , about 1.7% p r o t e i n was d e t e c t e d i n the c r y p t o c o c c a l p o l y s a c -c h a r i d e by the F o l i n Lowry procedure. However, the F o l i n Lowry method i s o f t e n i n a c c u r a t e s i n c e c e r t a i n amino a c i d sequences are f a r more chromogenic than o t h e r s and hence the value ob-t a i n e d depends on the s t a n d a r d used and on the composition o f the p r o t e i n (Chou and G o l d s t e i n 1960). Thus th e r e was p r o b a b l y more p r o t e i n p r e s e n t i n the o r i g i n a l c r y p t o c o c c a l e x t r a c e l l u l a r m a t e r i a l than these authors d e t e c t e d . A l s o the c e l l s had been k i l l e d w i t h 3% c h l o r o f o r m or 1% phenol (which reduces amino 107. a c i d r e c o v e r i e s i n c e l l w a l l s , Table II) bef o r e p o l y s a c c h a r i d e e x t r a c t i o n and t h i s may have caused a d d i t i o n a l l o s s e s i n p r o -t e i n r e c o v e r y . Both g e l e l e c t r o p h o r e s i s (Figure 7, Tables XXVI and XXVII) and column chromatography (Figure 10) showed t h a t the e x t r a c e l l u l a r m a t e r i a l (at l e a s t o f C r . neoformans 365-26 and j Cr. a l b i d u s 367) migrated as a s i n g l e s p e c i e s , thus I c o n s i d e r the p r o t e i n p o r t i o n to be a t r u e component r a t h e r than contam-i n a t i n g m a t e r i a l . The use o f c h e m i c a l l y d e f i n e d media [with (NH^^SO^ a s the s o l e n i t r o g e n source] and e x t e n s i v e d i a l y s i s and r e p r e c i p i t a t i o n d u r i n g p u r i f i c a t i o n , r u l e out the p o s s i b i l -i t y o f t h i s p r o t e i n b e i n g e i t h e r a medium contaminant or from f r e e amino a c i d s produced by the c e l l s ' metabolism or breakdown o f the c e l l w a l l s . There were some i n t e r e s t i n g s i m i l a r i t i e s and d i s t i n c t d i f f e r e n c e s i n the amino a c i d p a t t e r n s o f e x t r a c e l l u l a r m a t e r i a l from the s i x Cryptococcus s t r a i n s . Threonine, s e r i n e and g l u t a -mate were the predominant amino a c i d s i n both C r . a l b i d u s s t r a i n s whereas C r . neoformans 365-11 had l a r g e amounts o f as-p a r t a t e , s e r i n e and glutamate (Tables XII and X I I I ) . In the other two C r . neoformans s t r a i n s a s p a r t a t e and s e r i n e were the predominant amino a c i d s . Cr. l a u r e n t i i 371-1, l i k e the c e l l w a l l p r e p a r a t i o n s , had l a r g e amounts o f a s p a r t a t e and glutamate. The f a c t t h a t the percentages o f thr e o n i n e and s e r i n e were g e n e r a l l y h i g h e r i n the e x t r a c e l l u l a r m a t e r i a l than i n the c e l l w a l l p r e p a r a t i o n s , and t h a t there was l e s s glucosamine, might suggest the presence o f a d i f f e r e n t r . t y p e o f g l y c o p e p t i d e l i n k a g e . I t i s i n t e r e s t i n g t h a t both C r . l a u r e n t i i and C r . neoformans 108. 365-11 e x t r a c e l l u l a r m a t e r i a l , which, were h i g h i n a s p a r t a t e , a l s o had l a r g e r amounts of glucosamine than the o t h e r two C r n e o f o r m a n s s t r a i n s . However, t h i s was not the case f o r the' Cr. a l b i d u s s t r a i n s . Both 'CrV neoformans 365—16 and 365-25 were u n u s u a l l y h i g h i n p r o l i n e and p h e n y l a l a n i n e compared to the o t h e r s t r a i n s . As the percentage of glutamate i s a l s o n o t i c e a b l y lower i n these two s t r a i n s , i t may be t h a t they are d e f i c i e n t i n p r o l i n e oxidase which converts p r o l i n e to A ' - p y r r o l i n e - 5 - ^ c a r b o x y l i c a c i d and then to g l u tamic a c i d (Meister 1965). Andersoh e t a l . (1971) found a very high percentage o f p r o l i n e i n the amino a c i d content of a c u l t u r e f i l t r a t e from the pathogen C o c c i d i o i d e s i m m i t i s . They suggested t h a t p r o l i n e along w i t h C—methyl mannose and mannose c o n s t i t u t e d the major components of s k i n t e s t a c t i v i t y . Bradley e t a l . (19 74) a l s o found a high p r o l i n e l e v e l i n H and M r e a c t i v e components of h i s t o p l a s m i n , a d i a g n o s t i c agent f o r h i s t o p l a s m o s i s . Perhaps the h i g h percentage o f p r o l i n e i n these two Cryptococcus s t r a i n s i s connected w i t h a n t i g e n i c a c t i v i t y . I found t h a t comparison of the r a t i o s of G l y : A l a , l i e : L e u and Tyr:Phe (Table XV) r e v e a l e d some c o n s i s t e n t p a t t e r n s i n the p r o t e i n s . The r a t i o s were very s i m i l a r i n the c e l l w a l l p r e p a r a -t i o n s . That o f Cr. l a u r e n t i i was almost i d e n t i c a l t o T r e m e l l a mesen-t e r i c a . However, Cr. neoformans 365-16 and 365-26 showed some s t r i k -i n g d i f f e r e n c e s . The G l y : A l a and Tyr:Phe r a t i o s were much lower than the other s t r a i n s and the I l e : L e u r a t i o was much, h i g h e r . The Tyr:Phe r a t i o f o r Cr. neoformans 365-11 was the h i g h e s t of a l l the s t r a i n s . The somatic p r o t e i n e x t r a c t o f Cr. neoformans (Uzman e t a l . 1956) had a s i m i l a r G l y : A l a r a t i o to 'Cr'.' neoformans 109. 365-26, the I l e : L e u value was s l i g h t l y h i g h e r but the Tyr:Phe value resembled the e x t r a c e l l u l a r m a t e r i a l . I t i s p o s s i b l e t h a t Cr. neoformans 365-16 and 365-26 have developed a d e f i -c i e n c y i n p h e n y l a l a n i n e h y d r o l a s e and hence are unable to con-v e r t p h e n y l a l a n i n e to t y r o s i n e (Chandra and V i n i n g 1968). These two s t r a i n s a l s o l a c k e d c y s t e i n e which was p r e s e n t i n t r a c e amounts i n 365-11 and Uzman's p r e p a r a t i o n and was 2 - 5% o f the t o t a l amino a c i d s i n the other s t r a i n s . The c l o s e r e -l a t i o n s h i p o f 365-16 and 365-26 i s a l s o borne out by t h e i r s i -m i l a r glucosamine, u r o n i c a c i d and O - a c e t y l content (Tables XVI, XIX and XX). T h e i r i n f r a r e d s p e c t r a were very a l i k e (Figure 12)„although Cr. neoformans 365-26 had l a r g e r peaks at 1730 and 1250 cm 1 i n d i c a t i v e o f s l i g h t l y h i g h e r O - a c e t y l and u r o n i c a c i d content. Cr. neoformans 365-16 and 365-26 appeared more c l o s e l y r e l a t e d to each ot h e r than to 365-11 which seemed more s i m i l a r i n G l y : A l a , Tyr:Phe and I l e : L e u r a t i o s t o the ndhpathogens and had s l i g h t l y more b a s i c amino a c i d s . Ethanolamine, d e t e c t e d i n a l l the s t r a i n s , has o n l y r e c e n t l y been r e p o r t e d i n f u n g a l e x o c e l l u l a r g l y c o p e p t i d e s (Rick e_t a l . 1974). Although I d i d not q u a n t i f y the e t h a n o l a -mine i t i s s i g n i f i c a n t t h a t i t o c c u r r e d i n l a r g e amounts i n those s t r a i n s t h a t a l s o c o n t a i n e d more phosphorus (Table X X I I I ) . C r . neoformans 365-16 and 365-26 had small ethanolamine peaks and o n l y 0.2%.phosphorus. Perhaps the ethanolamine was a t t a -ched to t h e , g l y c o p r o t e i n through a phosphodiester l i n k a g e . However, i t i s d i f f i c u l t to make any c o n c l u s i o n s s i n c e although 110. I e s t a b l i s h e d the presence o f l i p i d s i n the e x t r a c e l l u l a r mater-i a l , I o b t a i n e d no q u a n t i t a t i v e d a t a . Phosphorus val u e s Were l a r g e r f o r the nonpathogenic s t r a i n s (Table X X I I I ) , which seems to r u l e out the p o s s i b i l i t y t h a t a h i g h p h o s p h o l i p i d content i s r e l a t e d t o v i r u l e n c e i n Cr. neoformans as was p o s t u l a t e d by Cox and Best (1972) f o r Blastomyces d e r m a t i t i d e s . Ash content ranged from 2 - 11% ex-cept f o r one p r e p a r a t i o n of Cr. l a u r e n t i i which had 43% ash (Tables VI and X X I I I ) . T h i s p r e p a r a t i o n was not used f o r t o t a l a n a l y s e s . Although I d i d not analyze any o f the m a t e r i a l f o r s u l p h a t e , i t has r e c e n t l y been d e t e c t e d i n the capsule by auto-r a d i o g r a p h i c and h i s t o c h e m i c a l techniques (Mahvi _^.__1. 1974). Perhaps sulphate comprises a small;.percentage o f the m a t e r i a l t h a t was unaccounted f o r i n my t o t a l r e c o v e r i e s . C r . neoformans 365-16 and 365-26 were c o n s i s t e n t i n a l l r e s p e c t s i n the d u p l i -cate batches, whereas other s t r a i n s showed s l i g h t v a r i a t i o n s e s p e c i a l l y i n ash content. C r . a l b i d u s HI354 p r e p a r a t i o n s #2 and #3 showed l a r g e d i f f e r e n c e s i n p r o t e i n c o n t e n t . Although I suspected t h a t Cr. a l b i d u s HI354 #3 r e p r e s e n t e d the t r u e amino a c i d and amino sugar content and t h a t the o t h e r s t r a i n s were underestimated f o r p r o t e i n , the i n f r a r e d s p e c t r a seem to i n d i c a t e t h a t t h i s s t r a i n d i d have more amino groups. However, i t i s obvious from the d i s c r e p a n c i e s i n n i t r o g e n r e c o v e r i e s (Table XXIII) t h a t l o s s e s i n amino a c i d s and amino sugars o c c u r r e d . The e x t r a c e l l u l a r m a t e r i a l c o n t a i n e d l a r g e amounts o f p o l y s a c c h a r i d e which caused humin p r o d u c t i o n d u r i n g a c i d h y d r o l y s i s (James 1972). 111. T h i s may have been r e s p o n s i b l e f o r d e g r a d a t i v e l o s s e s i n amino a c i d , amino sugar and n e u t r a l sugar components. Robel (1973) and A l t o s a a r (1974) showed t h a t l o s s e s o f c e r t a i n amino a c i d s were due to a d s o r p t i o n to the g l a s s tubes d u r i n g d e s i c c a t i o n . . Robel's procedure f o r c o r r e c t i n g such l o s s e s was t r i e d i n t h i s study, but was found i m p r a c t i c a l s i n c e p r o t e i n content o f the e x t r a c e l l u l a r m a t e r i a l was so s m a l l . The t o t a l n e u t r a l sugar val u e s i n c l u d e d i n Table XXV are from Cameron's method (1973). Values from Lehnhardt and W i n z l e r ' s method (Table XVII) appear to be o v e r e s t i m a t i o n s , and i t may be t h a t the t r u e sugar value l i e s between the two s e t s o f data p r e s e n t e d . These d i s c r e p a n c i e s i n t o t a l n e u t r a l sugar emphasize the need f o r an accurate and r e l i a b l e i n t e r n a l standard. More s t u d i e s must be a l s o done on r e l e a s e and sub-sequent d e g r a d a t i o n o f glucosamine, e s p e c i a l l y , from complex p o l y s a c c h a r i d e s such as these. I t i s w e l l known t h a t the ex-t e n t o f d e g r a d a t i o n v a r i e s f o r i n d i v i d u a l monomers, depending on the type o f l i n k a g e between them and on the c o n d i t i o n s o f h y d r o l y s i s . Although p r o t e i n , hexose and a c i d f u n c t i o n s migrated together i n e l e c t r o p h o r e s i s and g e l chromatography, su g g e s t i n g p r o t e i n and p o l y s a c c h a r i d e are t i g h t l y bound, i t i s q u i t e l i k e l y t h a t chemical f r a c t i o n a t i o n would r e v e a l s e v e r a l components. Is the p r o t e i n s t r u c t u r a l , a by-product o f c e l l metabolism, or i s i t an enzyme c l o s e l y bound to the p o l y s a c c h a r i d e ? Yeasts have been shown to produce i n v e r t a s e and acid^phosphatases which are l o c a t e d a t the c e l l s u r f a c e (Odds and T r u j i l l o - G o n z a l e s 112. 1974, Odds and H i e r h o l z e r 1973). These authors showed t h a t C r . neoformans had a h i g h l e v e l o f a c i d phosphatase a c t i v i t y at pH 3.9 and t h a t a s i m i l a r enzyme from Cr. a l b i d u s was manno-p r o t e i n i n nature w i t h a h e x o s e : p r o t e i n r a t i o o f 7:1. They a l s o suggested t h a t the hexose p r o t e i n was a n t i g e n i c . Mahvi e t a l . (1974) r e c e n t l y demonstrated a c i d phos-phatase a c t i v i t y i n the capsule and c e l l w a l l o f C r . neoformans. They r e p o r t e d g r e a t e r a c t i v i t y a t 25 than a t 37C. Perhaps g e n e t i c r e p r e s s i o n o f enzyme s y n t h e s i s a t the h i g h e r tempera-t u r e causes slow growth o f some s t r a i n s at 37C. They proposed t h a t the v i s c o u s p o l y s a c c h a r i d e together w i t h a c i d phosphatase w i t h i n the capsule serve i n h o l d i n g and degrading n u t r i e n t s at the c e l l s u r f a c e . However, L i u (1959) found a l k a l i n e phospha-tase i n the capsule under i n v i v o c o n d i t i o n s o n l y . He concluded t h a t the enzyme was induced and s e c r e t e d i n the, p a r a s i t i c s t a t e . P r o t e o l y t i c a c t i v i t y has a l s o been demonstrated i n C r . enoformans (Muller and S e t h i 1972). The fungus was able to degrade human «C zHS,-glycoprotein and to d i g e s t human f i b r i n o g e n i n v i t r o . As there are d i f f e r e n c e s i n amino a c i d composition among the s t r a i n s i t i s i n t e r e s t i n g to s p e c u l a t e whether these may have any r e l a t i o n to enzymic f u n c t i o n and perhaps v i r u l e n c e . In view o f the d i f f e r e n c e s i n w a l l and e x t r a c e l l u l a r m a t e r i a l chemistry, i t seems u n l i k e l y to me t h a t e x t r a c e l l u l a r m a t e r i a l i s e n t i r e l y a r e s u l t o f over s y n t h e s i s or a u t o l y s i s o f m a t e r i a l as suggested by K i k u c h i et. aJL. (1973) and Carmo-Sousa and Barroso-Lopes (1970) working w i t h Candida s p e c i e s . Phaff (1971) has suggested t h a t some components o f the capsule 113. and e x t r a c e l l u l a r slime may be r e s u l t s o f over s y n t h e s i s , but t h a t o t h e r components are r e t a i n e d i n the w a l l . However, the Cryptococcus c e l l w a l l i s l a r g e l y glucose (with s m a l l e r amounts o f x y l o s e , mannose, glucosamine and g a l a c t o s e ) and the capsule i s predominantly mannose, u r o n i c a c i d and x y l o s e . The amino a c i d s o f c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l a l s o showed d i f -f e r e n c e s : the c e l l w a l l s c o n t a i n e d more b a s i c amino a c i d s (Table XIV). The Tyr:Phe and G l y : A l a r a t i o s were much l a r g e r i n the c e l l w a l l o f C r . neoformans 365-26, than i n the e x t r a -c e l l u l a r m a t e r i a l , whereas the I l e : L e u r a t i o was much s m a l l e r (Table XV). T h e r e f o r e , I c o n s i d e r the c e l l w a l l and e x t r a c e l l u -l a r polymers to be s y n t h e s i z e d independently, to a l a r g e e x t e n t . Ankel e t a l . (1970) i s o l a t e d an enzyme from C r . l a u r e n t i i which 1 J.4 14 c a t a l y z e d the i n c o r p o r a t i o n o f Cmannose from GDP- C mannose i n t o an endogenous p o l y s a c c h a r i d e a c c e p t o r . T h e i r r e s u l t s i n d i -14 c a t e d t h a t C mannose was t r a n s f e r r e d o n l y to the n e u t r a l w a l l h e t e r o p o l y s a c c h a r i d e s u g g e s t i n g t h a t the a c i d i c heteropolymer was assembled by a d i f f e r e n t mannosyl t r a n s f e r system. A l s o , x y l o s y l t r a n s f e r to a c i d i c e x t r a c e l l u l a r p o l y s a c c h a r i d e , un-l i k e mannosyl t r a n s f e r , was dependent on the presence of added primer o b t a i n e d from the a c i d i c p o l y s a c c h a r i d e . The a c t u a l mode of capsule p r o d u c t i o n has y e t to be e l u c i d a t e d . Takeo e t a l . (1973) proposed t h a t capsule m a t e r i a l p r e c u r s o r s were s y n t h e s i -zed i n v e s i c l e s or paramural bodies and s e c r e t e d to the c e l l w a l l . At the o u t e r l a y e r o f the w a l l , p o l y m e r i z a t i o n occurs from the m i c r o f i b r i l s o f the c a p s u l e . The p a r t i c l e s they ob-served i n the o u t e r w a l l and i n n e r capsule appeared to be i n -v o l v e d i n t h i s p o l y m e r i z a t i o n . 114. R e l a t i o n o f S t r a i n V i r u l e n c e to Chemistry o f the C r y p t o c o c c a l  C e l l Envelope A c c o r d i n g to t h e r v i r u l e n c e t e s t s t h a t I made w i t h Swiss white mice, Cr. neoformans 365-11 was the most v i r u l e n t , C r . neoformans 365-16 and 365-26 ( a f t e r passaging) were l e s s v i r u -l e n t , and the o t h e r three s t r a i n s were a v i r u l e n t . T h i s does not agree w i t h p r e l i m i n a r y t e s t s made by Dr. L. Kapica w i t h the same Cr. neoformans s t r a i n s f i v e y e a rs ago. A l l the s t r a i n s k i l l e d 100% o f Ajax mice when i n j e c t e d i n t r a p e r i t o n e a l l y . 365-16 k i l l e d 80% o f C-57 b l a c k mice, 365-26, 30% and 365-11 k i l l e d none. Townley-Price and Bulmer (1974) working w i t h Cr. neoformans, and Chick and Roberts (1974) u s i n g Histoplasma  capsulatum, a l s o found v a r i a t i o n s i n v i r u l e n c e of the patho-gens when i n j e c t e d i n t r a p e r i t o n e a l l y i n t o d i f f e r e n t s t r a i n s of mice. However, Goren and Middlebrook (1967) r e p o r t e d t h a t v i r -ulence d e c l i n e d over a p e r i o d o f two years s u b c u l t u r i n g , even though morphology and c a p s u l a r appearance were the same. There-f o r I assume the r e s u l t s I"Obtained to be v a l i d f o r the three Cr. neoformans s t r a i n s at the time they were t e s t e d , and w i l l attempt to r e l a t e these d i f f e r e n c e s i n v i r u l e n c e to the chemi-s t r y o f c e l l w a l l s and e x t r a c e l l u l a r m a t e r i a l a l s o e x t r a c t e d a t t h i s time. There are s e v e r a l i n t e r e s t i n g p o s s i b i l i t i e s . The i n f r a -r e d s p e c t r a , u r o n i c a c i d and 0 - a c e t y l data, and c e l l u l o s e ace-t a t e s t r i p e l e c t r o p h o r e s i s a l l i n d i c a t e the h i g h e r c a r b o x y l and e s t e r content o f the C r . neoformans s t r a i n s : 365-11 (the most pathogenic) b e i n g the h i g h e s t and 365-26 and 365-16 115. somewhat l e s s . 365-11, the most v i r u l e n t , a l s o has more man-nose and glucosamine (and phosphate) than the o t h e r two patho-gens. However, i t i s d i f f i c u l t to r e l a t e these f a c t o r s alone to v i r u l e n c e s i n c e nonpathogenic s p e c i e s do have some u r o n i c a c i d and O - a c e t y l components, but perhaps i t i s the p o l y m e r i c s t r u c t u r e which i s important. I t i s s i m i l a r l y hard to conclude t h a t the d i f f e r e n c e s i n amount o f w a l l glucan between Cr. neo-formans and C r . l a u r e n t i i might a f f e c t p a t h o g e n i c i t y , without doing f u r t h e r s t u d i e s on more s p e c i e s . Bulmer e t al.(1967) r e l a t e d a c t u a l presence o f capsule or c a p s u l a r m a t e r i a l to v i r u l e n c e i n unencapsulated Cr . neoformans mutants which were a v i r u l e n t f o r -..mice. They based t h e i r evidence f o r l a c k o f capsule on a c i d h y d r o l y s i s (3N HC1 at 100C f o r 4 hr) o f whole c e l l s : o n l y glucose was recovered from unencapsulated mutants but x y l o s e , mannose, g a l a c t o s e and g l u c u r o n i c a c i d were rec o v e r e d from e n c a p s u l a t e d c e l l s . E i t h e r the mutants' c e l l w a l l s were d e f i c i e n t i n x y l o s e , mannose and g a l a c t o s e or the s t r o n g con-d i t i o n s o f h y d r o l y s i s employed had d e s t r o y e d the s m a l l amounts of these sugars t h a t I have demonstrated i n Cr. neoformans c e l l w a l l s . Thus c e l l w a l l d e f i c i e n c i e s as w e l l as d e f i c i e n c i e s i n capsule s y n t h e s i s may have a f f e c t e d p a t h o g e n i c i t y i n these mutants. I have a l r e a d y s t a t e d t h a t the r e a c t i v i t y of the d i f -f e r e n t serotypes o f c r y p t o c o c c a l p o l y s a c c h a r i d e s depends to a c e r t a i n e x t e n t on the degree o f a c e t y l a t i o n (Levine e t a l . 1959, Goren and Middlebrook 1966). These serotypes have not been assessed f o r d i f f e r e n c e s i n v i r u l e n c e which might be a t t r i -b u t a b l e to O - a c e t y l . Vogel (1966) suggested t h a t B and C 116. antigens (having l e s s O - a c e t y l ) were b u i l t on b a s i c A s t r u c t u r e but w i t h B or C a n t i g e n i c components having a p e r i p h e r a l o r i e n -t a t i o n on the c a p s u l e . Removing the c a p s u l e by a c i d treatment (0.5N HCl at 60C f o r 2 hr) transformed B a n t i g e n i c c e l l s i n t o A a n t i g e n s . Assuming t h a t e x t r a c e l l u l a r m a t e r i a l and capsule are of s i m i l a r composition, I propose t h a t A a n t i g e n types, such as 365-11, have more mannose, O - a c e t y l and u r o n i c a c i d but l e s s x y l o s e (13% see Table XXII) s i d e c h a i n s . I f the B types had more x y l o s e (as do 365-16 and 365-26 - 27% Table XXII) then a c i d treatment and h e a t i n g would remove the l a b i l e x y l o s e c h a i n s r e v e a l i n g the s t a b l e mannose and a c i d i c groupings, which, i n now a l t e r e d p r o p o r t i o n s , would g i v e A r e a c t i o n s . A c c o r d i n g to B e M i l l e r (1967) any r e s t r i c t i o n of the f l e x i b i l i t y o f a p o l y -s a c c h a r i d e c h a i n decreases the r a t e o f h y d r o l y s i s . Consequently the l a r g e r amounts o f glucosamine and p r o t e i n i n C r . neoformans 365-11 might reduce f l e x i b i l i t y o f the p o l y s a c c h a r i d e and p r o -v i d e r e s i s t a n c e to h y d r o l y s i s . In t h i s way the p a r t i c u l a r s t r u c t u r e o f the c a p s u l a r m a t e r i a l might a l s o c o n f e r r e s i s t a n c e to p h a g o c y t i c enzymes s i n c e i t i s known ( M i t c h e l l and Friedman 1972) t h a t the a b i l i t y o f macrophages to k i l l e n g u l f e d c e l l s i s l a r g e l y s t r a i n dependent and u n r e l a t e d to capsule t h i c k n e s s . I f the capsule i s a s o l u b l e e x t e n s i o n o f the a n t i g e n i c component found i n the c e l l w a l l ( D e v l i n 1969) then i t i s pos-s i b l e t h a t the a n t i g e n may be mannan-protein. I d i d not t e s t the C r . neoformans c e l l w a l l f r a c t i o n f o r u r o n i c a c i d s or 0-a c e t y l : these groupings, i f p r e s e n t , might a l s o have a n t i g e n i c p r o p e r t i e s . K o z e l and C a z i n (1971) and Bulmer (personal 117. communication) found t h a t i n j e c t i o n o f c a p s u l a r p o l y s a c c h a r i d e from a v i r u l e n t s t r a i n o f C r . neoformans d i d not i n h i b i t phago-c y t o s i s , i n . v i t r o , o f a non e n c a p s u l a t e d s t r a i n o r o f Cr. l a u r e n t i i . F a r h i (1969) proposed t h a t f o r i n h i b i t i o n o f phag o c y t o s i s t o o c c u r , c a p s u l a r m a t e r i a l must be able t o adhere to s p e c i f i c s i t e s on the c e l l w a l l . I f t h i s i s t r u e , these s i t e s a g a i n may i n v o l v e p r o t e i n o r h i g h l y charged a c e t y l and a c i d i c groupings. Vogel (1966) a l s o mentioned t h a t c u l t u r a l c o n d i t i o n s a f f e c t e d the a n t i g e n i c p r o p e r t i e s o f the c e l l s : B types might gi v e A r e a c t i v i t y w i t h c e r t a i n media, and i f they were p o o r l y e n c a p s u l a t e d . In t h i s study I found t h a t e x t r a c e l l u l a r m a t e r i a l p r e p a r a t i o n s from batches o f c e l l s t h a t grew p o o r l y o r were ha r v e s t e d b e f o r e the pH had dropped t o about 3.5 were l e s s v i s c o u s and had a lower u r o n i c a c i d c o n t e n t . D i f f e r e n t growth c o n d i t i o n s s t i m u l a t e the p r o d u c t i o n o f d i f f e r e n t polymers. Perhaps d i f f e r e n t mouse s t r a i n s p r o v i d e d i f f e r e n t environments f o r any one s t r a i n which might induce d i f f e r e n c e s i n the e x t r a -c e l l u l a r o r w a l l polymer produced and hence l e a d t o d i f f e r e n c e s i n v i r u l e n c e . The p o s s i b i l i t y t h a t C r . neoformans s e c r e t e s e x t r a c e l l u -l a r enzymes, perhaps p r o t e o l y t i c , may be important t o patho-g e n i c i t y . I do not know whether the p r o t e i n i s o l a t e d here i s enzymic, but a t t h i s stage a l t h o u g h amino a c i d c o m p o s i t i o n may a f f e c t a n t i g e n i c i t y , i t does not seem r e l a t e d t o v i r u l e n c e . I b e l i e v e t h a t the amino a c i d c o m p o s i t i o n o f c r y p t o c o c c a l e x t r a -c e l l u l a r m a t e r i a l and c e l l w a l l s i s under t i g h t e r g e n e t i c c o n t r o l 118 than the h e t e r o g l y c a n p o r t i o n o f the polymer. The l a t t e r i s e a s i l y a f f e c t e d by c u l t u r a l c o n d i t i o n s . Amino a c i d p a t t e r n s seem constant and d i s t i n c t i v e f o r each s t r a i n and show the r e -l a t i o n s h i p s between s t r a i n s and s p e c i e s . Uzman e_t a l . (1956) a l s o found t h a t the somatic p r o t e i n s o f two s t r a i n s which d i f -f e r e d i n degree o f e n c a p s u l a t i o n , had s i m i l a r amino a c i d compo-s i t i o n s . C r . a l b i d u s H1354, the c e r e b r o s p i n a l f l u i d i s o l a t e , w a s a v i r u l e n t f o r mice, had a lower u r o n i c a c i d content than Cr. a l b i d u s 367, no O - a c e t y l a t i o n and d i d not grow at 37C. However, l i k e C r . neoformans 365-16, Cr. a l b i d u s H1354 had a l a r g e amount o f g a l a c t o s e and l i k e C r . neoformans 365-11, a r e l a t i v e l y l a r g e percentage o f glucosamine and the same n i t r o -gen c o n t e n t . I f the p r o t e i n p o r t i o n was enzymic (or a n t i g e n i c ) then these s i m i l a r i t i e s t o C r . neoformans 365-11 may be the e x p l a n a t i o n f o r H1354's p a t h o g e n i c i t y . I t would seem t h a t o t h e r enzymic systems have become heat s e n s i t i v e on the t r a n s -f e r from i n v i v o t o i n v i t r o , or t h a t the s p i n a l f l u i d was p r o v i d i n g unknown f a c t o r s to account f o r d e f i c i e n c i e s a t 37C. Otherwise t h i s s t r a i n was v e r y s i m i l a r to. the o t h e r C r . a l b i d u s s t r a i n except i n n e u t r a l sugar p r o p o r t i o n s . 119. CONCLUSIONS Heat treatment was found to be a. s u i t a b l e method f o r k i l l i n g Cr. neoformans c e l l s without a l t e r i n g w a l l o r capsule chemistry, p r o v i d i n g the pH o f the medium was a d j u s t e d p r e -v i o u s l y t o about 6.0. K i l l i n g w i t h both phenol and f o r m a l i n a l t e r e d c e l l w a l l chemistry. Thiamine was shown to be growth s t i m u l a t o r y and to cause i n c r e a s e i n capsule t h i c k n e s s i n c o n c e n t r a t i o n s of up to 10 ug/ml. Cr. a l b i d u s 367 alone r e q u i r e d unautoclaved t h i a -mine. None o f the s t r a i n s grew w e l l i n v i t r o above pH 7.0 a l -though the pathogen i s known to grow w e l l i n v i v o i n s p i n a l f l u i d , pH = 7.4,and i n b l o o d plasma pH=7.39. N e i t h e r o f the C r . a l b i d u s s t r a i n s grew at 37C. These r e s u l t s emphasized the need f o r c a r e f u l l y c o n t r o l l e d and s t a n d a r d i z e d growth c o n d i t i o n s i n f u t u r e work: optimum c o n d i t i o n s must be determined f o r each s t r a i n . I observed s e p t a t e hyphae and clamp connections w i t h the Coward s t r a i n o f Cr. neoformans and conclude t h a t t h i s i s a member o f the h e t e r o b a s i d i o m y c e t e s . I was not able to demon-s t r a t e d i k a r y o t i c hyphae or e s t a b l i s h mating s t r a i n s . C e l l w a l l s were i s o l a t e d o n l y from two s p e c i e s of Cryptococcus: Cr. neoformans and Cr. l a u r e n t i i . Previous methods i n the l i t e r a t u r e were of l i m i t e d success i n b r e a k i n g the c e l l w a l l s . A s u c c e s s f u l method must be developed f o r f u t u r e s t u d i e s . 120. The pathogenic Cryptococcus s t r a i n had more glucose and l e s s glucosamine, g l y c o p r o t e i n , mannose and x y l o s e i n the c e l l w a l l s than d i d the nonpathogen, Cr . l a u r e n t i i . Cr. neo-formans a l s o had a t r a c e o f galactosamine. Only the c e l l w a l l s of C r . l a u r e n t i i c o n t a i n e d c y s t e i n e / c y s t i n e . More s t r a i n s must be examined before these d i f f e r e n c e s can be r e l a t e d to patho-g e n i c i t y . I n f r a r e d 1 s p e c t r a demonstrated the presence o f and ft-linked glycans i n the w a l l s o f Cr. l a u r e n t i i . High q u a n t i -t i e s o f Asx and Glx i n amino a c i d composition o f both s p e c i e s suggested the presence o f a l k a l i s t a b l e g l y c o p e p t i d e bonds. Per-haps mannan i s l i n k e d t o p r o t e i n through d i - N - a c e t y l c h i t o b i o s e to a sparagine. The amino a c i d composition of Cryptococcus c e l l w a l l s was ve s i m i 1 RT ir> fha f - cf T r e m e l l a c e l l w a l l s . The mannose :xylose r a t i o was s i m i l a r t o those of other basidiomycete w a l l s although d i f -f e r e n t from T r e m e l l a . Again a r e l a t i o n s h i p to Tremella and the lower basidiomycetes i s suggested. A l l the s t r a i n s produced an e x t r a c e l l u l a r p o l y s a c c h a r i d e containing«C-linked mannan, x y l o s e , g a l a c t o s e , g l u c u r o n i c a c i d and small amounts o f g l u c o s e . Only the three C r . neoformans s t r a i n s had O - a c e t y l s u b s t i t u e n t s . These p o l y s a c c h a r i d e s were s i m i l a r i n composition to those produced by T r e m e l l a mesenterica and Candida humicola except t h a t T r e m e l l a had more x y l o s e than mannose w h i l e Cr. neoformans s t r a i n s had more 0 - a c e t y l and g l u -c u r o n i c a c i d . T h i s f u r t h e r supports the r e l a t i o n s h i p to lower basidiomycetes. 121. ' On the b a s i s o f congruent bands i n e l e c t r o p h o r e s i s and g e l chromatography, I c o n s i d e r e d the p r o t e i n p o r t i o n o f the e x t r a -c e l l u l a r m a t e r i a l might be l i n k e d to the p o l y s a c c h a r i d e moiety and thus the m a t e r i a l may c o n t a i n a g l y c o p r o t e i n component. T h i s " g l y c o p r o t e i n " c o n t a i n e d the same amino a c i d s as the c e l l w a l l g l y c o p r o t e i n although p r o p o r t i o n s were d i f f e r e n t . Percent-ages o f s e r i n e and threonine were h i g h e r than i n the w a l l p r e -p a r a t i o n s : they may a l s o have been i n v o l v e d i n g l y c o p e p t i d e l i n k a g e . Ethanolamine was p r e s e n t i n a l l the p r e p a r a t i o n s . Comparison of the G l y / A l a , Tyr/Phe and I l e / L e u r a t i o s showed s t r i k i n g s i m i l a r i t i e s between Cr. neoformans s t r a i n s 365-16 and 365-26. These were d i f f e r e n t from the o t h e r 4 s t r a i n s . These two s t r a i n s had l a r g e amounts o f p r o l i n e and p h e n y l a l a -nine ( p o s s i b l y due to d e f i c i e n c i e s i n p r o l i n e oxidase and p h e n y l a l a n i n e h y d r o l a s e ) and both l a c k e d c y s t e i n e / c y s t i n e . T h e i r i n f r a r e d s p e c t r a were much a l i k e and they had s i m i l a r glucosamine, O - a c e t y l and u r o n i c a c i d content. 365-16 had more g a l a c t o s e than 365-26. On the b a s i s o f t h i s work these two s t r a i n s seem to be c l o s e l y r e l a t e d . Amino a c i d composition was l e s s a f f e c t e d by growth c o n d i t i o n s than were carbohydrate c o n s t i t u e n t s . A c c o r d i n g to mouse v i r u l e n c e t e s t s , Cr. neoformans 365-11 was the most pathogenic. The p o l y s a c c h a r i d e produced by t h i s s t r a i n i n v i t r o c o n t a i n e d more mannose, g l u c u r o n i c a c i d , O - a c e t y l and p r o t e i n than the other C r . neoformans s t r a i n s . The combination of these f a c t o r s and t h e i r s p a t i a l arrangement may be r e l a t e d to v i r u l e n c e , or f u n c t i o n i n the a n t i g e n i c i t y 122. o f the organism. E x t r a c e l l u l a r m a t e r i a l and c e l l w a l l composition were s u f f i c i e n t l y d i f f e r e n t to suggest independent s y n t h e s i s . The degree o f capsule s y n t h e s i s and the composition of the polymers were e a s i l y a f f e c t e d by growth c o n d i t i o n s such as pH, tempera-t u r e and n u t r i e n t supply. The y e a s t produced l a r g e r c a p s u l e s i n s u s c e p t i b l e mice than i t d i d e i t h e r i n r e s i s t a n t mice or i n  v i t r o . Perhaps both the composition and r a t e o f s y n t h e s i s o f the capsule are a f f e c t e d by v a r i a t i o n s i n the environments p r o -v i d e d by s u s c e p t i b l e h o s t s as opposed to normal h o s t s . T h i s i n t u r n c o u l d a f f e c t p r o d u c t i o n o f antibody to the p a r t i c u l a r a n t i -gen s y n t h e s i z e d by the y e a s t and a b i l i t y o f complement to a t t a c h to the a n t i b o d y - a n t i g e n complex or cause o p s o n i z a t i o n . Once i n s i d e the p h a g o c y t i c l e u k o c y t e s death of the y e a s t c e l l may be r e l a t e d to r e s i s t a n c e o f d i f f e r e n t capsule s t r u c -t u r e s t o p h a g o c y t i c enzymes. S t r a i n s w i t h l e s s x y l o s e and more mannose and p r o t e i n may be more r e s i s t a n t . C r . a l b i d u s H1354, the pathogenic i s o l a t e , showed few s i m i l a r i t i e s to the C r . neoformans s t r a i n s 365-16 and 365-26. I t d i d not grow a t 37C and was very s i m i l a r to the other C r . a l b i d u s s t r a i n / s u g g e s t i n g a c l o s e r e l a t i o n s h i p . However, Cr. a l b i d u s H1354 had the same n i t r o g e n content as the v i r u l e n t s t r a i n 365-11. I f the p r o t e i n p o r t i o n were enzymic or a n t i -g e nic t h i s might e x p l a i n the p a t h o g e n i c i t y i n v i v o o f t h i s temperature s e n s i t i v e s t r a i n . BIBLIOGRAPHY Abercrombie, M.J., J.K.N. Jones, M.V. 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