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Structural studies of Escherichia coli K26 and K46-50 using chemical and microbiological methods Beynon, Linda M. 1985

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STRUCTURAL STUDIES OF ESCHERICHIA COLI K26 AND KkS-50 USING CHEMICAL AND MICROBIOLOGICAL METHODS by LINDA M. BEYNON B.A. (Hons.), The Open University, U.K., 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the reG^ir-ed--standard THE UNIVERSITY OF BRITISH COLUMBIA May 1985 ® Linda Margaretha Beynon In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis , for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of CHe.fV))ST^y  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e aiftv May |<?&5 D E - 6 rvsn ABSTRACT The capsular polysaccharides of Escherichia c o l i are immunogenic and antigenic. When conjugated to a c a r r i e r protein these polysaccharides can be used as vaccines. A knowledge of the structure of b a c t e r i a l capsular polysaccharides i s e ssential for understanding antibody-antigen i n t e r a c t i o n and also for understanding the chemical basis of se r o l o g i c a l d i f f e r e n t i a t i o n . For these reasons s t r u c t u r a l studies of some E. c o l i capsular polysaccharides are being undertaken i n th i s laboratory. In t h i s thesis a preliminary inves t i g a t i o n into the structures of capsular polysaccharides from E. c o l i serotypes K / f6, K / f7, K/+8, K 4 9 and K50 i s presented. The q u a l i t a t i v e composition of each polysaccharide was determined by varying the hydrolytic conditions used to cleave the glycosidic bonds between the monosaccharide u n i t s . Table I shows the sugars present i n each capsular polysaccharide. Capsular polysaccharide Rha Sugars Man GlcA Gal GlcNH 2 GalNH 2 K46 ;x X X X X. X X X X K48 X X X X X K 4 9 X X X K50 X X X X X X The r a t i o of the sugars i n each polysaccharide was I l l determined using methanolysis followed by reduction with sodium borohydride. The presence of amino sugars was confirmed by deamination of the hydrolyzed polysaccharide and detection of the product by g . l . c . G.l.c.-m.s., of the a l d i t o l acetates of the monosaccharides obtained by hydrolysis of the polysaccharides, was used to confirm the type of monsaccharide. present i n each polysaccharide, ^H-N.m.r. spectroscopy was u t i l i z e d to confirm the presence of deoxy sugars, amino sugars and non-carbohydrate substituents. E. c o l i Kl+7 and K50 capsular polysaccharides were both found to have pyruvate present as a substituent. A bacteriophage was i s o l a t e d from sewage for each of E. c o l i K47, K48 and Kk9 serotypes. Phage 47 also attacked E. c o l i K48 and K/+9 bacteria. The structure of E. c o l i K26 capsular polysaccharide was investigated using the techniques of acid hydrolysis, carbodiimide reduction and methanolysis followed by reduction with sodium borohydride. The polysaccharide was degraded using a bacteriophage-borne glycanase. The p o s i t i o n of cleavage found by methylation of the reduced oligosaccharide. Combination of the data obtained from the chemical analysis, n.m.r spectroscopy and the bacteriophage degradation gave the two following possible structures for the E. c o l i K26 capsular polysaccharide. -^GlcAp 1 ^Rhap1 ^Rhap1 ^Rhapl ^Galpl-6 Rhap t I V G l c A p ^ - ^ R h a p i - ^ R h a p ^ - ^ G a l p ^ ^Rhap-1 1Rhap p o s i t i o n of c l e a v a g e by the b a c t e r i o p h a g e - b o r n e g l y c a n a s e V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v i i LIST OF FIGURES • i x LIST OF SCHEMES x i ACKN OWLEDG EMENT S x i i PREFACE x i i i I INTRODUCTION 2 II ISOLATION AND PURIFICATION OF CAPSULAR POLYSACCHARIDES i 14 III METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES 1? 111.1 Separation Techniques 17 III.1.1 Paper chromatography . . • 17 I I I . 1.2 Gel chromatography 18 111.2 Instrumentation 19 111.2.1 Gas-liquid chromatography . 18 ' 111.2.2 Mass-spectrometry . • • • • 21 111.2.3 N.m.r 25 111.2.3.1 1H n.m.r. . . . 25 111.2.3.2 1 3 C n.m.r . . . 28 111.3 Characterization and quantitation of sugars • 50 v i 111.3.1 Total hydrolysis . . 30 111.3.2 Methanolysis 32 111.3.3 Carbodiimide reduction 32 III.3*4 Deamination 33 III.k Position of linkage . . . . . . . . . . . . 36 III.4*1 Methylation analysis 36 III.i+«2 Characterization and quantitation of methylated sugars 37 I I I . 5 Sugar sequence 39 111.5.1 P a r t i a l hydrolysis 39 111.5.2 Periodate oxidation and Smith hydrolysis kO 111.5.3 Uronic acid degradation (g-elimination 42 I I I . 5 » 4 Deamination kk IV MICROBIOLOGICAL ANALYSIS OF CAPSULAR POLYSACCHARIDES IV. 1 Introduction 46 IV.2 I s o l a t i o n of bacteriophage from sewage . . 51 IV.3 Propagation of bacteriophage 52 IV.k Bacteriophage cross-reactions 53 IV.5 Bacteriophage degradation of capsular polysaccharides' 54 IV.6 I s o l a t i o n of oligosaccharides 56 IV.7 Determination of the s i t e of cleavage by the phage-borne glycanase • • < 56 v i i V QUALITATIVE AND QUANTITATIVE SUGAR ANALYSIS OF E. COLI K26 CAPSULAR POLYSACCHARIDE ; . . . . 59 V.1 Introduction . . . 59 V.2 Results and discussion 60 V .3 Conclusion 65 V. 4 Experimental • • 6? VI BACTERIOPHAGE DEGRADATION OF E. COLI K26 CAPSULAR POLYSACCHARIDE 74 VI. 1 Introduction . . . . . 74 VI.2 Results and discussion . . . . . . . 74 VI.2.1 Bacteriophage degradation of K26 polysaccharide . • 74 VI.2.2 P u r i f i c a t i o n and analysis of depolymerization products of K26 polysaccharide • • • 76 VI.3. Conclusion 82 VI. 4 Experimental . . . . . . . . . . . 84 VII QUALITATIVE AND QUANTITATIVE SUGAR ANALYSIS OF E. COLI K46, K47, K48, K49 and K50 90 VII. 1 Introduction 90 VII.2 Results and discussion 90 VII. 3 Conclusion 101 VI 1.4 Experimental 107 VIII ISOLATION, PROPAGATION AND CROSS-REACTIONS OF E. COLI 0 47, 048 and 049 112 VIII. 1 Introduction 112 v i i i VIII.2 Results and discussion 112 VIII.2.1 Isolation and propagation of 0 4 7 , 0 4 8 and 0 4 9 • • • 112 VIII.2.2 Cross-reaction of 0 4 7 , 0 4 8 and 0 4 9 116 VIII.2.3 Conclusion 118 VIII.2.4 Experimental 119 IX BIBLIOGRAPHY 123 APPENDIX I: Known structures of E. c o l i K antigens 130 APPENDIX I I : N.m.r. spectra 138 APPENDIX I I I : Immunochemical cross-reactions of E. c o l i K26 149 i x LIST OF TABLES Table Page V.1 Sugar analysis of E. c o l i K26 polysaccharide.. 61 V.2 1H-n.ra.r. data for E . c o l i K26 capsular polysaccharide and derived oligosaccharide .. 63 V. 3 ^C-n.ro.r. data for E. c o l i K26 capsular polysaccharide 66 VI. 1 Propagation of bacteriophage 26 75 VI. 2 Methylation analysis of the reduced oligosaccharide from the bacteriophage degradation of E. c o l i K26 polysaccharide ... 79 VI.3 Methylation analysis of the product from the s e l e c t i v e hydrolysis of E. c o l i K26 polysaccharide 81 VII. 1 Sugar analysis of E. c o l i K 4 6 capsular polysaccharide 93 VII.2 Sugar analysis of E. c o l i KZ+7 capsular polysaccharide 93 X VII.3 Sugar analysis of E. c o l i K48 capsular polysaccharide ., 98 VII.4 Sugar analysis of E. c o l i K 4 9 capsular polysaccharide 98 VII.5 Sugar analysis of E. c o l i K50 capsular polysaccharide 100 VII.6 Results of the deamination analysis of E. c o l i capsular polysaccharides K46-5O . . . . . 102 VII.7 Results of the methanolysis and methanolysis plus reduction of E. c o l i K46-50 103 VII. 8 ^H-n.m.r. data for E. c o l i K46-50 capsular polysaccharides 104 VIII. 1 Cross-reactions of bacteriophage i s o l a t e d from sewage 114 VIII.2 Concentration of bacteriophage solutions a f t e r each test-tube l y s i s 114 VIII.3 Cross-reactions of E. c o l i 047, 048 and 049 • • 117 x i LIST OF FIGURES Figure Page 1 Diagrammatic representation of b a c t e r i a l c e l l showing thick murein layer of Gram-positive c e l l wall and two layers of Gram-negative c e l l wall 4 2 Section through capsulated E. c o l i treated with anti-capsule (K29) IgG 6 3 The three regions i n b a c t e r i a l l i p o -polysaccharides 7 4 Mass spectrum of hexa-O-acetyl-D-glucitol .... 23 3 Schematic representation of the di f f e r e n t regions i n the ^-n.m.r. spectrum of polysaccharides 27 6 Primary mass spectral fragment ions from 2,3-di -0_-methylpentitol triacetate-1-d and 3,^-di-jQ-methylpentitol triacetate-1-d ...... 38 7 Smith degradation - periodate oxidation followed by mild acid hydrolysis of the reduced product 41 x i i 8 Basic morphological types of bacteriophage with the types of nucleic acid .............. 4 6 9 Diagram of the coliphage T2 v i r i o n 4 7 10 E. c o l i capsule bacteriophage no.26 negatively stained with uranyl acetate 4 8 11 Diagrammatic representation of a paper chromatogram showing the res u l t s of the analysis of 026 degradation products ........ 7 6 12 Separation of E. c o l i K26 oligosaccharides using Bio-Gel P2 column 7 7 x i i i LIST OF SCHEMES Scheme Page 1 Reduction of carboxylic acid i n aqueous'solution using carbodiimide reagent. 34 2 Alternative ring contractions during the nitrous acid deamination of 3 - 0-substituted 2-amino-2-deoxy-D-glucopyranosides 35 3 N-Deacetylation-nitrous acid deamination of the Ll-specific polysaccharide chain from the S h i g e l l a dysenteriae type 1 lipopolysaccharide... 44 xiv ACKNOWLEDGEMENTS It i s d i f f i c u l t to thank i n d i v i d u a l l y a l l the people who have helped me during the course of t h i s work. But I would l i k e to express my sincere thanks to Professor G.G.S. Dutton for a l l his guidance, help and support. Thank you also to my friends and colleagues i n the laboratory, without whose advice t h i s work would have been far more d i f f i c u l t . Dr. G. Eigendorf and his s t a f f of the mass spectro-metry service have always been pleasant and very h e l p f u l . My thanks to them a l l , as. well as to Dr. S.O. Chan and members of the n.m.r. service. F i n a l l y my gra t e f u l thanks to my mother for making i t possible for me to undertake t h i s research. XV P R E F A C E * I n an e f f o r t t o f a m i l i a r i z e r e a d e r s who do n o t work 1n t h e p a r t i c u l a r a r e a o f o r g a n i c c h e m i s t r y t o w h i c h t h i s t h e s i s r e f e r s , t h e f o l l o w i n g e x p l a n a t i o n o f terms used i s o f f e r e d . F i s c h e r p r o j e c t i o n f o r m u l a e a r e used t o r e p r e s e n t t h e a c y c l i c m o d i f i c a t o n o f s u g a r s . Some examples a r e shown b e l o w . Numbering commences f r o m t h e c a r b o n y l group a t t h e t o p o f t h e c h a i n ( I ) . Note t h a t D - g l u c u r o n i c a c i d ( I I ) d i f f e r s from D - g l u c o s e ( I ) o n l y CHO 1 2 HOH h OH 3 4 1 - OH 5 - OH 6 C H 2 0 H CHO -OH HO—1 -OH —OH COOH' CHO —OH -OH HO —J HO CH. D - g l u c o s e D - g l u c u r o n i c a c i d L-rhamnose ( I ) ( I D ( I I I ) 1n t h a t C-6 1s o x i d i z e d t o a c a r b o x y l i c a c i d g r o u p . The C-6 o f . L-rhamnose ( I I I ) i s p a r t o f a m e t h y l group and 1s r e f e r r e d t o a l s o by a n o t h e r common n a m e , 6 - d e o x y - L - m a n n o s e . There a r e f o u r c h i r a l c e n t e r s 1n t h e s e s 1 x - c a r b o n c h a i n s (marked w i t h a s t e r i s k s 1n s t r u c t u r e I I I ) making 1t I m p o r t a n t t o a p p r e c i a t e t h e s p a t i a l arrangement o f atoms ( c o n f i g u r a t i o n ) t h a t 1s I m p l i e d by t h e s e F i s c h e r r e p r e s e n t a t i o n s . To s i m p l i f y t h e n o m e n c l a t u r e o f a l l t h e p o s s i b l e Isomers (16 f o r each o f I , I I , I I I ) , xvi a l l those having the hydroxy1 group a t the highest-numbered c h i r a l c e n t e r (C-5) p r o j e c t i n g to the r i g h t i n the F i s c h e r p r o j e c t i o n formulae belong to the D - s e r i e s , and the others t o the L - s e r i e s . — OH CH 20H D-series H 0 -CH 20H L - s e r i e s P h y s i c a l and chemical evidence i n d i c a t e s t h a t , i n f a c t , these six-carbon polyhydroxyaldehydes e x i s t i n a c y c l i c form. The r i n g c l o s u r e occurs by n u c l e o p h i l i c a t t a c k o f the oxygen atom a t C-5 on the a l d e h y d i c carbon atom, generating a new c h i r a l (anomeric) center a t C - l . T h i s r e s u l t s i n two anomers, represented below HO _ CH 20H a-D-glucose (IV) HO H \ / c . I — OH HO— — OH CHgOH B-D-glucose (V) x v i i i n the T o l l e n s formulae. I t should be noted t h a t C-l i s unique 1n having two attached oxygen atoms, f o r m a l l y making 1t a hemiacetal carbon. Since the T o l l e n s formulae have obvious l i m i t a t i o n s with t h e i r unequal bond lengths,Haworth developed a pe r s p e c t i v e method o f l o o k i n g a t the six-membered r i n g (VI and V I I ) . T h i s improvement recognizes t h a t the r i n g oxygen atom l i e s behind the carbon chain and th a t bond lengths are approximately equal. Often i n p r a c t i s e r e g u l a r hexagons are used i n Haworth projections-, OH OH a-D-glucopyranose e-D-glucopyranose pyran (VI) (VII) (VIII) which he r e l a t e d t o "such r i n g s a s the h e t e r o c y c l i c compound^pyran (VIII) and named them pyranoses. Note t h a t hydroxyl groups not i n v o l v e d i n r i n g formation on the r i g h t i n F i s c h e r and T o l l e n s formulae p o i n t down i n the Haworth p r o j e c t i o n s and those on the l e f t p o i n t up. S i m i l a r l y , f o r aldopyranoses, the group on C - 5 p o i n t s up f o r D (IX) and down f o r the L enantiomer ( X ) . I t f o l l o w s , then, that when sugar residues are attached there are two p o s s i b l e c o n f i g u r a t i o n s , an a - o r a e - p y r a n o s 1 d e , f o r each l i n k a g e . x v i i i HI OH HO OH HO OH a-D-rhamnopyranose (IX) The t r u e conformation o f pyranoid carbohydrates i s r e l a t e d to the c h a i r form of cydohexane. X-ray d i f f r a c t i o n a n a l y s i s has shown t h a t a hexose, such as a-D-glucose ( X I ) , c o n s i s t s of a puckered, six-membered, oxygen-containing carbon r i n g , with hydroxyl s u b s t i t u e n t s a t C-l through C-4, and a hydroxymethyl group at C-5. A l l s u b s t i t u e n t s on the r i n g , except f o r that a t C - l , are e q u a t o r i a l . Two isomers (anomers) are p o s s i b l e i n r e l a t i o n to the anomeric center ( C - l ) , depending on whether a s u b s t i t u e n t i s a x i a l (a-anomer; XII) OH (XI) xix o r e q u a t o r i a l (e-anomer; X I I I ) , where R • hydrogen, f o r monosaccharides, and R «= another sugar r e s i d u e , f o r d i - , o l l g o - , and p o l y s a c c h a r i d e s . Since H-l i s i n a d i f f e r e n t chemical environment f o r the two anomers, nuclear magnetic resonance spectroscopy can e a s i l y d i s t i n g u i s h between them and, thereby, provides i n v a l u a b l e a s s i s t a n c e i n a s s i g n i n g anomeric c o n f i g u r a t i o n s . OR H (XII) (XIII) Haworth p r o j e c t i o n s are most useful and w i l l be used i n t h i s t h e s i s , even though they give no i n d i c a t i o n o f three-dimensional molecular shape. There seems to be l i t t l e j u s t i f i c a t i o n f o r the use o f formulae which d e p i c t s t a t e s o f molecules as well as s t r u c t u r e s , when the true s t a t e s are o f t e n unknown o r v a r i a b l e . Reproduced with the kind permission of T.E. Folkman from h i s M.Sc. t h e s i s e n t i t l e d " S t r u c t u r a l Studies on K l e b s i e l l a Capsular P o l y s a c c h a r i d e s " , U n i v e r s i t y o f B r i t i s h Columbia, A p r i l 1979. 1 INTRODUCTION 2 1. INTRODUCTION Because o f t h e i r c a p a c i t y t o i n d u c e s y n t h e s i s of a n t i b o d i e s ( i . e . t h e y a r e iramunogens) and t h e i r r e a c t i v i t y w i t h a n t i b o d i e s ( i . e . t h e y a r e a n t i g e n s ) b a c t e r i a l p o l y -s a c c h a r i d e s have c o n t r i b u t e d t o the c l a s s i f i c a t i o n and i d e n t i f i c a t i o n o f b a c t e r i a , t o a g r e a t e r u n d e r s t a n d i n g o f the immune response and t o the d e f i n i t i o n o f the a c t i v e s i t e i n a n t i g e n - a n t i b o d y i n t e r a c t i o n . F i n a l l y t hey have been i n s t r u m e n t a l i n t h e d e t e c t i o n and p r e v e n t i o n o f human d i s e a s e caused by i n v a s i v e m i c r o - o r g a n i s m s . E s c h e r i c h i a c o l i was f i r s t i s o l a t e d by E s c h e r i c h i n 1855 from f a e c e s . I t i s p r e s e n t i n u r i n a r y t r a c t i n f e c t i o n s and has been a s s o c i a t e d w i t h i n f a n t i l e d i a r r h o e a . E. c o l i i s a Gram-negative, m o t i l e b a c t e r i u m , r o d shaped and p e r i t r i c h o u s -l y f l a g e l l a t e d . I t b e l o n g s t o the f a m i l y E n t e r o b a c t e r i a c e a e and the genera E s c h e r i c h i a . As t h e E s c h e r i c h i a genus i s heteroge n e o u s , a d d i t i o n a l c h a r a c t e r s have t o be i n t r o d u c e d f o r a c c u r a t e d e s c r i p t i o n s o f s i n g l e s t r a i n s . Each s p e c i e s may be d i v i d e d i n t o many d i f f e r e n t s e r o t y p e s . The s e r o t y p e scheme i s based on i d e n t i f i c a t i o n o f s u r f a c e 'K', so m a t i c •0' and f l a g e l l a 'H' a n t i g e n s . I n o r d e r t o make s e r o t y p i n g p r i n c i p l e s i n t e l l i g i b l e a s h o r t d e s c r i p t i o n o f the morphology and immunochemistry o f thos e s u r f a c e s t r u c t u r e s t h a t a r e i m p o r t a n t i n s e r o t y p i n g i s g i v e n . S u r f a c e S t r u c t u r e s ( i ) Morphology The c e l l w a l l o f t h e b a c t e r i a l c e l l i s i t s l i f e -s u p p o r t system i n a h o s t i l e and changeable environment. E l e c t r o n 3 m i c r o s c o p y of e n t e r o b a c t e r i a shows a r i g i d double l a y e r e d c e l l w a l l o u t s i d e t h e c y t o p l a s m i c membrane. The r i g i d i t y i s d e t e r m i n e d by t h e p e p t i d o g l y c a n l a y e r . There a r e two c e l l w a l l t y p e s (see F i g . 1 ) ; t h e s e can be r e l a t e d t o t h e Gram d e s i g n a t i o n o f b a c t e r i a . T h i s c l a s s i f i c a t i o n was i n t r o d u c e d by Gram 1 i n 1884 on the b a s i s of a c o l o u r s t a i n . Gram-negative b a c t e r i a have an outer-membrane s u r r o u n d i n g t h e c e l l w a l l . T h i s outer-membrane c o n t a i n s a n t i g e n s the main component b e i n g l i p o p o l y s a c c h a r i d e . When t h i s c o n t a i n s a h y d r o p h i l i c , h i g h m o l e c u l a r weight p o l y s a c c h a r i d e c h a i n (0 a n t i g e n ) , t h i s i s t h e p r i n c i p a l a n t i g e n of t h e b a c t e r i a and i s f u n c t i o n a l l y e q u i v a l e n t t o t h e c a p s u l a r p o l y s a c c h a r i d e (see b e l o w ) . I n G r a m - p o s i t i v e b a c t e r i a t h e r e i s no outermembrane, but the p e p t i d o g l y c a n l a y e r c o n t a i n s two t y p e s of c a r b o h y d r a t e a n t i g e n . One i s a r e l a t i v e l y s i m p l e p o l y s a c c h a r i d e , c o n t a i n i n g o n l y two o r t h r e e d i f f e r e n t m o n o s a c c h a r i d e s . T h i s p o l y s a c c h a r i d e i s the major group o r t y p e s p e c i f i c a n t i g e n of t h e b a c t e r i u m . The second c a r b o h y d r a t e i s a t e i c h o i c a c i d . I n some s p e c i e s , the t e i c h o i c 2 a c i d s have been shown t o be a n t i g e n i c d e t e r m i n a n t s . W h i l e t h e s e a r e b a s i c s t r u c t u r e s found i n a l l t y p i c a l E n t e r o b a c t e r i a c e a e , a d d i t i o n a l s t r u c t u r e s e x i s t which are i m p o r t a n t f o r s e r o t y p i n g . These s t r u c t u r e s a r e not n e c e s s a r y f o r t h e l i f e and r e p r o d u c t i o n of the b a c t e r i a , and t h e r e f o r e are not found i n a l l o r g a n i s m s . The f i r s t of t h e s e s t r u c t u r e s i s a p o l y s a c c h a r i d e c a p s u l e (K a n t i g e n ) which may e x i s t i n t h e form of a d i s c r e t e c a p s u l e s u r r o u n d i n g t h e b a c t e r i a l c e l l , o r i n the form of a l o o s e s l i m e u n a t t a c h e d t o t h e c e l l s u r f a c e . C a p s u l e s can be r e c o g n i z e d by t h e I n d i a i n k s t a i n i n g technique-^ © in 0) o % o o. © O to 83 © © . © © \ © © % ° %^ % t % © , T - , A $ ^ * D ^ ° ^ c6 © 9. »i r« 3 ° J * ° 03 © © ^ © © a V V* i> © ^ & ^ 9 v d a V* © w l A © o OO © © > .A 5 or by electron microscopy a f t e r reaction with s p e c i f i c antibody^" (see F i g . 2 ) . They usually have a high content of acidic constituents such as uronic acids, phosphate groups or acetals and are found most often i n pathogenic bacteria. The second structure occurs i n motile organisms. Long filamentous structures,used to propel the bacteria,are anchored i n the cytoplasmic membrane and project through the c e l l wall. These f l a g e l l a consist of f i b r e s of the protein flagellum. This protein i s the H antigen and d i f f e r s i n composition from one b a c t e r i a l species to another. Because of the enormous variety i n the chemical structure and the fundamental genetic s t a b i l i t y of the three structures, the lipopolysaccharide (0 antigen), the polysaccharide capsule (K antigen) and the protein f l a g e l l a (H antigen) make up the three fundamental serotyping antigens. Many enterobacteria, also carry fimbriae (or p i l i ) which have been l a b e l l e d the F-antigen.^ P i l i are thread-l i k e protein structures protruding from the surface; they probably a r i s e i n or close to the c e l l membrane, ( i i ) Chemistry Enterobacterial lipopolysaccharides are composed of three s t r u c t u r a l regions (see F i g . 3)» Region III (Lipid'A) i s attached at the outermembrane l i p o p r o t e i n layer of the b a c t e r i a l c e l l wall. It consists of glucosamine phosphate and f a t t y acids. L i p i d A i s responsible for the : b i o l o g i c a l (endotoxic) properties of lipopolysaccharides^. Region II (core) i s linked to L i p i d A v i a a carbohydrate component 2-keto-^deoxymannulosoctonic acid (KD0).:which i s also found 6 Figure 2. U l t r a t h i n section through capsulated E . c o l i ' treated with high concentration (0.5 mg/mL) of anti-capsule(K29) IgG before dehydration and embedding. (From Reference 4) O - s p e c i f i c Chain- J L - ' P o l y s a c c h a r i d e L •Core •J >-Lipid A-L i p i d -(3' Monosaccharide #:Phosphate Ethanolamine : Long C h a i n (Hydroxy) F a t t y A c i d F i g u r e 3« The t h r e e r e g i o n s i n b a c t e r i a l l i p o p o l y s a c c h a r i d e s . 8 i n some K a n t i g e n s of Gram-negative b a c t e r i a . Region I c o n s i s t s of o l i g o s a c c h a r i d e r e p e a t i n g u n i t s , and t h e c o m p o s i t i o n and s t r u c t u r e of r e g i o n I a r e the c h e m i c a l b a s i s o f t h e 0 a n t i g e n i c s p e c i f i c i t y o f Gram-negative b a c t e r i a . The c a p s u l a r m a t e r i a l o f a l l E. c o l i c e l l s i s of a p o l y s a c c h a r i d e n a t u r e , but the c a p s u l a r p o l y s a c c h a r i d e from s t r a i n s w i t h t h i c k c a p s u l e s and t h o s e from b a c t e r i a w i t h t h i n c a p s u l e s are c h e m i c a l l y d i f f e r e n t . Most, however, are a c i d i c p o l y s a c c h a r i d e s and t h e r e f o r e n e g a t i v e l y c h a r g e d . I t was d i s c o v e r e d i n e a r l y s e r o l o g i c a l s t u d i e s o f E. c o l i t h a t c a p s u l e s which c o v e r t h e c e l l w a l l (0) a n t i g e n s i n t e r f e r e d w i t h t h e i r s e r b l o g i c a l d e t e c t i o n • S i n c e t h a t time the non-a g g l u t i n a b i l i t y of an E . c o l i s t r a i n by homologous 0 a n t i s e r a ( a n t i s e r a r a i s e d a g a i n s t the 0 a n t i g e n o f t h a t p a r t i c u l a r s t r a i n of E . c o l i ) h a s been seen as an i n d i c a t i o n t h a t t h e b a c t e r i u m has a c a p s u l e . Kauffmann^ f i r s t c l a s s i f i e d K a n t i g e n s (from the German word f o r c a p s u l e , K a p s e l ) i n t o t h r e e d i f f e r e n t t y p e s termed the L,A, and B a n t i g e n s . E . c o l i s t r a i n s c o n t a i n i n g A a n t i g e n s are n o n a g g l u t i n a b l e i n homologous 0 a n t i s e r a b e f o r e and a f t e r h e a t i n g However s t r a i n s w i t h e i t h e r L or B a n t i g e n s are n o n a g g l u t i n a b l e b e f o r e h e a t i n g , but a g g l u t i n a b l e a f t e r h e a t i n g i n homologous 0 a n t i s e r a . A f t e r h e a t i n g , the t h e r m o l a b i l e K a n t i g e n s expose the u n d e r l y i n g c e l l w a l l (0 a n t i g e n ) , t h u s a l l o w i n g t h e c e l l t o a g g l u t i n a t e i n i t s homologous a n t i s e r a . S t r a i n s which c o n t a i n t h e t h e r m o l a b i l e L o r B a n t i g e n s do not u s u a l l y p o s s e s s m o r p h o l o g i c a l c a p s u l e s . S t r a i n s w i t h t h e r m o s t a b l e A a n t i g e n s are e n c a p s u l a t e d . 9 This c l a s s i f i c a t i o n was l a t e r revised by 0rskov and o coworkers 0. These authors found that the K antigens of E. coli, s t r a i n s can be divided into two groups, according to th e i r electrophoretic mobility. The f i r s t group consists of those with rather high electrophoretic mobility, formerly c a l l e d L antigens, the second i s made :up of those with low electro-phoretic mobility i . e . a l l former A antigens and some former B antigens. K antigens with high electrophoretic mobility are acidic polysaccharides with molecular weights below 50,000 daltons and high charge density. Those with low electro-phoretic mobility have a lower charge density but the i r molecular weights are above 150,000-200,000 daltons. Acidic polysaccharides with high molecular weights and low charge density form thick capsules, while those with low molecular weights form thin patchy capsules. I t i s noteworthy that, although capsular polysaccharides with high electrophoretic mobility belong to many 0 groups, those with low electro-phoretic mobility belong predominantly to groups 8 and 9> with only a few exceptions i n 0 groups 20 and 101. Immunology of polysaccharides When certain foreign substances or bacteria (covered with antigenic surface components) enter the human body, an immune response i s i n i t i a t e d leading to the production of a n t i -bodies or immunoglobulins. These are proteins which can combine non-covalently with th e i r antigen molecules i n a s p e c i f i c way. The a n t i g e n i c i t y and immunogenicity of polysaccharides have been known for some time beginning with the studies of Avery and Heidelberger 9» 1 0 , . Subsequently the concept of 10 the d e t e r m i n a n t group was proposed when i t was shown t h a t o n l y a s m a l l p a r t o f a p o l y s a c c h a r i d e i s the s i t e of a n t i b o d y s p e c i f i c i t y . The presence of the same d e t e r m i n a n t group was a l s o shown t o be r e s p o n s i b l e f o r t h e i r s e r o l o g i c a l c r o s s -r e a c t i v i t y , i . e . the c a p a c i t y of a p o l y s a c c h a r i d e from one s p e c i e s of b a c t e r i u m t o p r e c i p i t a t e a n t i b o d i e s r a i s e d a g a i n s t a p o l y s a c c h a r i d e from a n o t h e r s p e c i e s . The i m m u n o l o g i c a l s p e c i f i c i t y of p o l y s a c c h a r i d e a n t i g e n s r e s i d e s i n t h e i r s t r u c t u r e . P o l y s a c c h a r i d e s can be l i n e a r o r branched, the branches may be s i n g l e - u n i t s i d e c h a i n s o r may be of i n t e r m e d i a t e l e n g t h (3-7 u n i t s ) . They can c o n s i s t o f a s i n g l e type of monosaccharide r e s i d u e s , i . e . they a r e homopolymers e.g. d e x t r a n , or they can c o n s i s t o f s e v e r a l d i f f e r e n t monosaccharides i . e . t h e y are h e t e r o p o l y m e r s . The m a j o r i t y of b a c t e r i a l p o l y s a c c h a r i d e s are composed of r e l a t i v e l y s m a l l r e p e a t i n g u n i t s ( o l i g o s a c c h a r i d e s ) . Sugars which are most o f t e n found i n p o l y s a c c h a r i d e a n t i g e n s i n c l u d e D-glucose, D - g a l a c t o s e , D-mannose, D - g l u c u r o n i c a c i d , D - g a l a c t u r o n i c a c i d , D-mannuronic a c i d , L-rhamnose, L - f u c o s e , D^-glucosamine and D-g a l a c t o s a m i n e . V a r i a t i o n s i n s t r u c t u r e are a l s o i n t r o d u c e d by the p o s i t i o n and c o n f i g u r a t i o n o f the l i n k a g e s between a d j a c e n t s u g a r s and by t h e p r e s ence of h y d r o x y l s u b s t i t u e n t s such as a c e t a t e , phosphate and p y r u v a t e . The c o m b i n a t i o n o f a l l t h e s e v a r i a t i o n s i n p o l y s a c c h a r i d e s t r u c t u r e p r o v i d e s f o r an e x t r e m e l y l a r g e range of i m m u n o l o g i c a l s p e c i f i c i t i e s . I m m unological s t u d i e s o f p o l y s a c c h a r i d e s a r e aimed a t f i n d i n g out which p a r t of the m o l e c u l e i s r e s p o n s i b l e f o r i m m u n o l o g i c a l s p e c i f i c i t y , i n o t h e r words,' which p a r t of the 11 p o l y s a c c h a r i d e r e p e a t i n g u n i t o r o l i g o s a c c h a r i d e i s the de t e r m i n a n t group. Because i t i s r e s p o n s i b l e f o r immuno-s p e c i f i c i t y a d e t e r m i n a n t group i s the p a r t o f the p o l y -s a c c h a r i d e which e n t e r s the a n t i b o d y combining s i t e . S t u d i e s by Kabat i n d i c a t e d t h a t i n a l i n e a r p o l y s a c c h a r i d e the i m m u n o l o g i c a l s p e c i f i c i t y r e s i d e s p r i m a r i l y i n the t e r m i n a l s u g a r r e s i d u e and extends a l o n g the p o l y s a c c h a r i d e c h a i n . The a n t i s e r u m produced i n response t o such a p o l y s a c c h a r i d e c o n t a i n s many a n t i b o d y m o l e c u l e s w i t h d i f f e r e n t s i z e d combining s i t e s , e a c h d i r e c t e d towards the same d e t e r m i n a n t r e g i o n . I n branched p o l y s a c c h a r i d e s the immunodominant s u g a r s a r e u s u a l l y i p t h o s e t h a t form the branches . C e r t a i n n o n c a r b o h y d r a t e groups e.g. p y r u v a t e may f u n c t i o n as a n t i g e n i c d e t e r m i n a n t s when p r e s e n t as s u b s t i t u e n t s i n p o l y s a c c h a r i d e s . The Role o f B a c t e r i a l P o l y s a c c h a r i d e Whether a b a c t e r i a l i n f e c t i o n d e v e l o p s defends on a . complex i n t e r a c t i o n between h o s t and organism. B a c t e r i a l v i r u l e n c e i s t h e a b i l i t y of t h e organism t o r e s i s t t he h o s t ' s defense system, so t h e organism m u l t i p l i e s , damages and maybe f i n a l l y k i l l s t h e h o s t . The p r i m a r y r o l e o f 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 t o p r o t e c t t h e organism from d e s t r u c t i o n by the p h a g o c y t i c c e l l s o f t h e body. The O-antigen i s not a n t i -p h a g o c y t i c , u n l i k e t h e K a n t i g e n , but i t does c o n f e r some p r o t e c t i o n a g a i n s t t h e r a p i d b a c t e r i c i d a l a c t i v i t y o f serum c o m p l e m e n t ^ . N o n - c a p s u l a t e d organisms a r e u s u a l l y phago-c y t i z e d r e a d i l y because of t h e a b i l i t y of o t h e r exposed s u r f a c e a n t i g e n s e.g. t e i c h o i c a c i d s t o i n i t i a t e t he f a s t e r a c t i n g 12 a l t e r n a t e pathway o f complement mediated c e l l l y s e s . C a p s u l a r P o l y s a c c h a r i d e s as V a c c i n e s Pneumococcal and men i n g o c c a l p o l y s a c c h a r i d e v a c c i n e s based on c a p s u l a r p o l y s a c c h a r i d e s a re now a v a i l a b l e . However t h e r e i s a major problem a s s o c i a t e d w i t h c a p s u l a r p o l y s a c c h a r i d e v a c c i n e s . Young c h i l d r e n (under two y e a r s ) do not d e v e l o p p r o t e c t i v e l e v e l s of serum a n t i b o d i e s , i . e . t h e r e i s no anamnestic (memory) response t o subsequent b o o s t e r i n j e c t i o n s . T h i s problem can be overcome by c o n j u g a t i n g p o l y s a c c h a r i d e s t o an a n t i g e n i c c a r r i e r p r o t e i n . The most p r o m i s i n g approach seems t o be c o u p l i n g t h r o u g h a s i n g l e t e r m i n a l f r e e aldehyde group g e n e r a t e d i n . t h e p o l y s a c c h a r i d e t o an a c c e p t a b l e p r o t e i n , t e t a n u s t o x o i d 1 ^ . The l i n k a g e does not a f f e c t a n t i g e n i c s t r u c t u r e and i s s a f e f o r human v a c c i n a t i o n . I n t h i s t h e s i s a p r e l i m i n a r y i n v e s t i g a t i o n i n t o t h e q u a l i t a t i v e and q u a n t i t a t i v e c o m p o s i t i o n of t h e p o l y s a c c h a r i d e c a p s u l e s o f s i x d i f f e r e n t s t r a i n s of E. c o l i b a c t e r i a i s p r e s e n t e d . E. c o l i K26 c a p s u l a r p o l y s a c c h a r i d e has been s u b j e c t e d t o d e g r a d a t i o n i n t o i t s component o l i g o s a c c h a r i d e s by a b a c t e r i p p h a g e a s s o c i a t e d g l y c a n a s e . T h i s p r o j e c t i s t h e r e f o r e an i n t e r d i s c i p l i n a r y one i n v o l v i n g b o t h c h e m i c a l and m i c r o b i o l o g i c a l methods o f s t r u c t u r e d e t e r m i n a t i o n . These w i d e l y d i f f e r e n t methods complement each o t h e r , i n f o r m a t i o n o b t a i n e d from t h e b a c t e r i o p h a g e degraded p r o d u c t not b e i n g o b t a i n a b l e u s i n g c h e m i c a l t e c h n i q u e s o f s t r u c t u r e d e t e r m i n a t i o n (see S e c t i o n I V ) . 13 11. ISOLATION AND PURIFICATION OF CAPSULAR POLYSACCHARIDES 14 1 1 . ISOLATION AND PURIFICATION OF CAPSULAR POLYSACCHARIDES As was pointed out previously, a knowledge of the chemical structure of b a c t e r i a l capsular polysaccharides i s important for understanding not only the chemical basis of s e r o l o g i c a l d i f f e r e n t i a t i o n but also the a n t i g e n i c i t y of the polysaccharide. Before the structure can be determined however, the capsular polysaccharide must be obtained i n a pure form and i n reasonable amounts. E . c o l i bacterium serotype K 2 6 was received as a stab culture from Dr. Ida 0rskov (Copenhagen). The bacteria were streaked on Mueller Hinton agar plates and incubated at 37°» The i n d i v i d u a l colonies of E. c o l i thus propagated were replated sucessively u n t i l large, a c t i v e l y growing colonies of bacteria were obtained. A b a c t e r i a l culture was obtained by the inoculation of Mueller Hinton broth(5mL) with a single b a c t e r i a l colony. The culture was shaken overnight at 37°» and then added to 50mL Mueller Hinton broth. This culture was shaken for 4h at 37° u n t i l b a c t e r i a l growth was evident (the culture became cloudy). This a c t i v e l y growing (log phase) b a c t e r i a l culture was subsequently incubated on a small tray of Mueller Hinton agar for 6d. Bacteria were grown on a t o t a l of s i x trays i n a l l . The lawn of bacteria was harvested by scraping from the agar surface, and the bacteria k i l l e d by the addition of 1$ phenol s o l u t i o n . The b a c t e r i a l suspension was dialysed to remove the phenol and then ultracentrifuged to separate the capsular polysaccharide.,^rom the b a c t e r i a l c e l l . The viscous golden supernatant was freeze-dried and 15 weighed. This crude capsular polysaccharide was p u r i f i e d by the addition of Cetavlon (cetyltrirnethylammonium bromide) solution to a solution of the polysaccharide. The a c i d i c polysaccharide was precipitated and any neutral polysaccharide was.left behind i n solution. Centrifugation was used to separate the Cetavlon-polysaccharide complex from the neutral polysaccharide s o l u t i o n . F i n a l l y the polysaccharide complex was dissolved i n 3M sodium chloride solution, precipitated i n e.thanol, redissolved i n water and dialysed against running tap water for 2d. After l y o p h i l i z a t i o n the polysaccharide obtained had a styrofoam-like appearance, and was shown to be pure by nuclear magnetic resonance spectroscopy. 16 M E T H O D O L O G Y O F S T R U C T U R A L A N A L Y S I S O F P O L Y S A C C H A R I D E S 17 U J . METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES 111.1 S e p a r a t i o n t e c h n i q u e s 1 1 1 . 1 . 1 Paper chromatography The modern concept o f paper chromatography was f i r s t d e s c r i b e d i n 1944 by Consden, Gorden and M a r t i n 1 ^ f o r the s e p a r a t i o n o f complex m i x t u r e s of amino a c i d s . Today the method i s used w i d e l y f o r sugar a n a l y s i s . M i x t u r e s of monosaccharides and o l i g o s a c c h a r i d e s can be s e p a r a t e d c l e a n l y , , w i t h o u t the n e c e s s i t y of p r e p a r i n g d e r i v a t i v e s . Paper chromatography i s t h e r e f o r e employed t o o b t a i n p r e l i m i n a r y i n f o r m a t i o n on t h e c o n s t i t u e n t s u g a r s , e i t h e r as t h e f r e e s u g a r s o r a l d i t o l s } from h y d r o l y s i s o f the n a t i v e p o l y s a c c h a r i d e s . P r e p a r a t i v e paper chromatography i s used as a method of s e p a r a t i n g o l i g o m e r s r e s u l t i n g from a b a c t e r i o p h a g e d e g r a d a t i o n . S e p a r a t i o n by paper chromatography i s dependent upon d i f f e r e n c e s , o f t e n v e r y s m a l l , i n the p a r t i t i o n c o e f f i c i e n t s of t h e s u g a r s . I t i n v o l v e s c o u n t e r c u r r e n t p a r t i t i o n between a s t a t i o n a r y c e l l u l o s e - w a t e r complex ( f i l t e r paper c o n t a i n i n g 20% w a t e r ) , and a m o b i l e phase composed of an o r g a n i c s o l v e n t , o r a m i x t u r e o f s o l v e n t s c o n t a i n i n g some w a t e r . The c h o i c e of s o l v e n t i s v e r y i m p o r t a n t . The water c o n t e n t i s a c r i t i c a l f a c t o r , s i n c e the s o l u b i l i t y o f a suga r i n the m o b i l e phase w i l l g overn i t s r a t e o f movement on the paper chromatogram, and t h u s a f f e c t i t s s e p a r a t i o n from o t h e r s u g a r s i n the m i x t u r e . A f t e r chromatography s u g a r s a re d e t e c t e d w i t h e i t h e r 18 1) alkaline s i l v e r n i t r a t e , 2 ) p-anisidine hydrochloride i n aqueous 1-butanol followed by heating at 1 1 0 ° for 5 rain, or 3 ) ninhydrin i n acetone followed by heating at 1 2 0 ° for 1 rain. 111.1 . 2 Gel chromatography Gel chromatography (also c a l l e d gel f i l t r a t i o n or molecular sieve chromatography), i s a l i q u i d chromatographic technique which separates molecules primarily according to differences i n molecular s i z e . This f i e l d has been reviewed 17 by Churms . I t i s p a r t i c u l a r l y suitable for separating oligosaccharides obtained by bacteriophage degradation, as i t i s a gentle separation method which ra r e l y removes any l a b i l e substituents. Gel chromatography i s based on the decreasing permeability of a three dimensional network of a swollen gel to molecules of increasing s i z e . The smaller the molecule the further i t penetrates into the gel's pores and,therefore, the more i t i s retarded i n i t ' s migration through the g e l . The smallest molecules are thus the l a s t to emerge from the column. One of the Bio-Gel P series of neutral hydrophilic polyacrylamide gels was used i n t h i s study. Bio-Gels are of a completely synthetic nature and have two main advantages over polysaccharide,' derived gels.. F i r s t l y they are resi s t a n t to microbial growth. Secondly, i f degradation of the polyacrylamide 19 m a t r i x o c c u r s then c o n f u s i o n between r e a c t i o n p r o d u c t s and sample b i o m o l e c u l e s i s u n l i k e l y . Because p o l y a c r y l a m i d e i s a r e l a t i v e l y i n e r t s u b s t a n c e , few r e s t r i c t i o n s a r e p l a c e d upon the c h e m i c a l c o m p o s i t i o n of of e l u a n t s . A w a t e r - p y r i d i n e - a c e t i c a c i d b u f f e r (1000:10:4) was the e l u a n t chosen i n t h i s s t u d y as i t i s e a s i l y removed from t h e e f f l u e n t f r a c t i o n s by l y o p h i l i z a t i o n . The M o l i s c h t e s t was used t o l o c a t e t h e i n d i v i d u a l c a r b o h y d r a t e c o n t a i n -i n g f r a c t i o n s and t h e s e a re th e n l y o p h i l i z e d and weighed. 111.2 I n s t r u m e n t a t i o n 111.2.1 G a s - l i q u i d chromatography ( g . l . c . ) The f i r s t r e p o r t on g a s - l i q u i d chromatography of c a r b o h y d r a t e s was p u b l i s h e d i n 1958 1 ^. S i n c e then t h e r e have been many developments i n t h i s f i e l d . D u t t o n has p u b l i s h e d an e x t e n s i v e r e v i e w of t h e a p p l i c a t i o n s of g . l . c . t o c a r b o h y d r a t e s The c h r o m a t o g r a p h i c p r o c e s s i n gas chromatography c o n s i s t s o f s o l u t e p a r t i t i o n i n g between two phases, t h e s t a t i o n a r y l i q u i d phase and the m o b i l e gas phase. The e q u i l i b r i u m c o n s t a n t i s d e t e r m i n e d o n l y by the compound ( s o l u t e ) , the l i q u i d phase, and t h e t e m p e r a t u r e . I t does not depend on column t y p e . The d i s t r i b u t i o n c o n s t a n t , Kp, i s d e f i n e d b y ; c o n c e n t r a t i o n i n the l i q u i d phase c o n c e n t r a t i o n i n t h e gas phase 20 wt. of s o l u t e i n l i q u i d p hase/vol. of l i q u i d phase wt. of s o l u t e i n gas phase/vol. of gas _ wt. i n l i q u i d phase v o l . of gas wt. i n gas phase v o l . of l i q u i d phase = k g where k i s c a l l e d the p a r t i t i o n r a t i o or c a p a c i t y r a t i o and B i s the phase r a t i o . The t o t a l amount of time a s o l u t e spends i n the column i s i t s r e t e n t i o n time, t R • A l l s o l u t e s must spend the same amount of time i n the gas phase s i n c e whenever a s o l u t e molecule l e a v e s the l i q u i d ' phase and enters the gas phase i t must move along with the c a r r i e r gas flow. Thus the a c t u a l s e p a r a t i n g a b i l i t y of a column i s r e l a t e d to the amount of time a s o l u t e spends i n the l i q u i d phase. In g i l . c . the degree of r e s o l u t i o n and the time necessary f o r a s e p a r a t i o n i s a f u n c t i o n of s i z e i n t e r r e l a t e d parameters:-column l e n g t h , column i n t e r n a l diameter, f i l m t h i c k n e s s , type of c a r r i e r gas, c a r r i e r gas v e l o c i t y and column temperature. The f i r s t three parameters are f i x e d , and are c h a r a c t e r i s t i c of a p a r t i c u l a r column, but the remaining t h r e e are o p e r a t i o n a l parameters which can be changed to e f f e c t a maximum s e p a r a t i o n . Column temperature e s p e c i a l l y , has a very marked e f f e c t on a n a l y s i s . The lower the temperature the h i g h e r the c a p a c i t y r a t i o ( k ) . High c a p a c i t y r a t i o s i n d i c a t e t h a t a s o l u t e i s spending more time i n the l i q u i d phase and i t s r e t e n t i o n time w i l l t h e r e f o r e be g r e a t e r . 21 Most p o l y s a c c h a r i d e s are not s u f f i c i e n t l y v o l a t i l e t o be used f o r g . l . c . so t h e y must f i r s t be c o n v e r t e d t o v o l a t i l e compounds. The most u s u a l d e r i v a t i v e s f o r t h i s purpose are t r i m e t h y l s i l y l (Me^Si) e t h e r s . However the t r i m e t h y l s i l y l a t i o n o f a r e d u c i n g s u g a r w i l l , g i v e r i s e t o a t l e a s t f o u r i s o m e r s , i . e . the a and g anomers of the f u r a n o s i d e s and p y r a n o s i d e s which are p r e s e n t a t e q u i l i b r i u m . T h i s problem has been overcome by the r e d u c t i o n a t t h e anomeric c e n t r e t o the a l d i t o l , which i s r e a d i l y a c e t y l a t e d t o y i e l d a v o l a t i l e d e r i v a t i v e . To i d e n t i f y t h e s u g a r s p r e s e n t i n an o l i g o s a c c h a r i d e o r p o l y s a c c h a r i d e , t h e sample i s f i r s t h y d r o l y s e d t o c o n v e r t t h e p o l y s a c c h a r i d e t o m o nosaccharides. These are t h e n reduced, a c e t y l a t e d and s u b j e c t e d t o a n a l y s i s by g . l . c . DB 1 7 , a c a p i l l a r y column of i n t e r m e d i a t e p o l a r i t y , was the column of c h o i c e f o r s e p a r a t i n g a l d i t o l a c e t a t e s and a l s o f o r a n a l y z i n g m i x t u r e s from m e t h y l a t i o n a n a l y s i s (see S e c t i o n 111.4.1). I d e n t i f i c a t i o n o f the unknown su g a r s i s a c h i e v e d by comparison of the r e t e n t i o n t i m e s w i t h those of a u t h e n t i c samples, and by g.l.c.-m.s. (see S e c t i o n 111.2.2). For q u a n t i f i c a t i o n of 22 peaks, molar response f a c t o r s are t a k e n i n t o c o n s i d e r a t i o n ; 111.2.2 Mass s p e c t r o m e t r y (m.s.) Mass s p e c t r o m e t r y has now become a v e r s a t i l e and i m p o r t a n t method i n p o l y s a c c h a r i d e s t r u c t u r e a n a l y s i s . T h i s f i e l d was r e v i e w e d by Lonngrenand Svensson i n 1 9 7 4 ^ . The i n t r o d u c t i o n of combined g a s - l i q u i d chromatography-mass 2 2 spectrometry (g.l.c.-m.s.), i n which the components from the chromatographic column are introduced d i r e c t l y into the i o n i z a t i o n chamber of the mass spectrometer, has s i m p l i f i e d the inves t i g a t i o n of complex mixtures. For m.s., as for g . l . c , the sugars must be converted into v o l a t i l e derivatives, again i t i s advantageous to convert the aldoses to the non-cyclic a l d i t o l s and then acetylate to give a single v o l a t i l e deriva-t i v e . Carbohydrate derivatives give either weak molecular ions, or none at a l l , on electron impact mass spectrometry. A l d i t o l acetates do not give a molecular ion, but (M-CH^CC^-)* i s found i n low abundance. As the mass spectra of stereo-isomers are almost i d e n t i c a l , the mass spectrum of D-glucitol hexaacetate (see F i g . 4) represents a l l peracetylated h e x i t o l s . The base peak i n the spectra of a l l a l d i t o l acetates i s the acetylium ion, m/e 43 (CH^C—0). Primary ions are formed from alditol._acetatesby cleavage of the a l d i t o l chain , as shown i n F i g . 4. The primary fragments are degraded by elimination(s) of acetic acid (60), ketene (42), or acetic anhydride(102). The simple and well established f i s s i o n patterns obtained from a l d i t o l acetates upon electron impact, makes these derivatives very suitable for the i d e n t i f i c a t i o n of sugars. Methylation analysis i s an important method i n s t r u c t u r a l polysaccharide chemistry. Lindberg and co-workers systematically investigated the mass spectra of p a r t i a l l y methylated a l d i t o l acetates 2 i f. Their work led to the following generalizations:-2 3 43 -p •H C D 8 6 -4 -2 _ x 0.5 139 217 VJ4!> 73 10 200 289 361 300 CH?0Ac J3. I t I 361 HCOAc L45_ I I 289 AcOCH 2 1 7 I I 217 HCOAc 2 S 9 I T 145 HCOAc 36.1 I I 73 CH OAc 2 Figure 4» Mass spectrum of Hexa-O-acetyl-D-g l u c i t o l (above) and cleavage pattern of same ( l e f t ) . 24 1) Similar mass spectra are obtained from derivatives with the same substitution pattern. 2) Cleavage between a methoxylated and an acetoxylated carbon i s preferred over cleavage between two acetoxylated carbons. 3) Cleavage between two adjacent methoxylated carbons i s preferred over cleavage between one of these and an adjacent acetoxylated carbon. 4) Secondary fragments are generated by the the single or consecutive loss of acetic acid (m/e 60), ketene (m/e 42), methanol (m/e 32) or formaldehyde (m/e 30) from the primary fragments. This information can be used to i d e n t i f y sugars for which no standard spectra are available. When an acetamidodeoxy sugar i s present retention times are higher than the corresponding neutral sugar. In unmethylated compounds fragmentation i s governed by the acetamido group. Secondary fragments are obtained by the loss of acetic acid (m/e 60), acetamide (m/e 59)» and ketene (m/e 42). In a p a r t i a l l y methylated acetamidodeoxy sugar, cleavage between a methoxyl and acetamido group i s preferred. The development of fast-atom-bombardment mass spectro-metry (f.a.b. m.s.) i s becoming increasingly important i n 25 carbohydrate structure elucidation . F.a.b. spectra are r e l a t i v e l y easy to acquire and only small amounts of unmodified carbohydrates are needed. 25 111 .2 .3 N u c l e a r magnetic resonance (n.m.r.) s p e c t r o s c o p y I n r e c e n t y e a r s n.m.r. s p e c t r o s c o p y has become the most w i d e l y used t e c h n i q u e f o r s t r u c t u r a l , c o n f i g u r a t i o n a l and c o n f o r m a t i o n a l a n a l y s i s of p o l y s a c c h a r i d e s . ^C-N.m.r., i n p a r t i c u l a r , i s now e x t e n s i v e l y used i n p o l y s a c c h a r i d e s t u d i e s as i t g i v e s b e t t e r s i g n a l s e p a r a t i o n t h a n ^H-n.m.r. owing t o the w i d e r range of c h e m i c a l s h i f t s i n v o l v e d . 111.2.3*1 P r o t o n magnetic resonance (p.m.r.) s p e c t r o s c o p y Two problems have t o be overcome i n o r d e r t o o b t a i n a w e l l r e s o l v e d 1H-n.m.r. spectrum of a p o l y s a c c h a r i d e ; 1) i n t e r f e r e n c e by exchangeable p r o t o n s (0-H and N-H) and 2) l i n e b r o a d e n i n g o f s i g n a l s . The f i r s t problem i s p a r t i a l l y overcome by p r i o r t r e a t m e n t of the p o l y s a c c h a r i d e w i t h .. de u t e r i u m o x i d e . N e v e r t h e l e s s , a s t r o n g peak due t o r e s i d u a l w ater (HOD s i g n a l ) , as w e l l as s i d e bands o f the peak a r e o f t e n o b t a i n e d . I n c r e a s i n g the temp e r a t u r e t o a p p r o x i m a t e l y 95° w i l l however s h i f t t h e HOD s i g n a l u p f i e l d , t h u s e x p o s i n g more o f the spectrum. The problem of s i g n a l b r o a d e n i n g i s a t t r i b u t e d l a r g e l y t o the f a c t t h a t p o l y s a c c h a r i d e p r o t o n s have s h o r t r e l a x a t i o n t i m e s . The s p i n - s p i n r e l a x a t i o n time (T2) i s r e l a t e d t o the n a t u r a l l i n e w i d t h ( v ) a c c o r d i n g t o t h e e x p r e s s i o n v 2 = — — • Decreased l i n e w i d t h can a l s o TV T^ be o b t a i n e d by r a i s i n g the temp e r a t u r e o f t h e s o l u t i o n , t h u s d e c r e a s i n g the m o l e c u l a r r o t a t i o n a l c o r r e l a t i o n time. The t h r e e f o l l o w i n g n.m.r. parameters g i v e u s e f u l 26 information on polysaccharide structure. (a) Chemical s h i f t . A .large portion of the information available i n a spectrum i s obtained by the measurement of the chemical s h i f t of a proton, which i s i n d i c a t i v e of both the electronic and geometric environment of the proton. The ^H-n.m.r. spectrum can be divided into three regions (see F i g . 5). In the anomeric region signals which appear downfield of 6 5*0 are a r b i t r a r i l y assigned to a-linkages (equatorial protons) and those appearing u p f i e l d are assigned to g-linkages ( a x i a l protons). Anomeric resonances are well separated from signals produced by most of the other n u c l e i . This fact helps greatly i n determining the number of d i f f e r e n t residues per repeating unit of a polysaccharide, and also i n estimating t h e i r r e l a t i v e proportions. The fact that polysaccharides,which have molecular weights greater than 10 , give good interpretable spectra i s evidence for a structure of regular repeating units. (b) Relative i n t e n s i t y of the signals. The heights of the i n t e g r a l s are proportional to the areas under each peak. The areas are i n the same r a t i o as the number of hydrogen atoms that give r i s e to each s i g n a l . Consequently the r e l a t i v e amounts of each sugar present i n the polysaccharide can be determined from the r e l a t i v e heights of each i n t e g r a l . It i s also possible to ascertain whether or not a non-carbohydrate substituent such as pyruvate i s present on every repeating unit. An oligosaccharide w i l l give two signals i n the anomeric region. The reducing end exhibits mutarotation and so has a signal for the a- and 3- anomers. C H 3 of 6-deoxy po 6 ( p . p . a . ) Figure 5» Schematic representation of different regions i n the H-n.m.r. spectrum of polysaccharides. 2 8 (c) Coupling constants. This parameter i s useful for determining the configuration and/or conformation of carbo-hydrates as i± bears a relationship to the torsion angle 26 (dihedral angle 0) between v i c i n a l protons. Karplus demonstrated th i s r e l a t i o n s h i p and gave an empirical equation which enables us to calculate coupling constants. The coupling constant depends on a variety of parameters besides the torsion angle; these include the electronegativity of substituents and angle s t r a i n . In spite of t h i s , i t i s s t i l l a useful way of assigning t r a n s - d i a x i a l protons (0 = 180°, 3-linked), and equatorial-axial or equatorial-equatorial protons (gauche conformation, 0 = 6 0 ° a - l i n k e d ) . The former display a large coupling constant ( J 1 2 7 "9 H z ) whereas a smaller coupling constant (J-j H Z ) ^ s obtained for the l a t t e r . Although there i s generally a f a i r l y close correspon-dence between chemical s h i f t s of sugar residues i n a poly-saccharide and those of t h e i r monomeric counterparts, i t i s not possible to i d e n t i f y unambiguously, without supporting chemical evidence, monosaccharide constituents of a poly-saccharide from a spectrum of the l a t t e r . 111.2.3.2 vC-n.m.r. spectroscopy '» Carbon-13 nuclear magnetic resonance spectroscopy complements 1H-n.m.r. spectroscopy. With the advent of the pulse-Fourier transform method spectra of polysaccharides 1 z can be obtained using only t h e i r natural abundance ^C atoms. 29 Broad band d e c o u p l i n g c o l l a p s e s s p i n m u l t i p l e t s i n t o s i n g l e t s . These two advanced t e c h n i q u e s g i v e w e l l d e f i n e d ^C-n.m.r. s p e c t r a which p r o v i d e i n f o r m a t i o n on c h e m i c a l and p h y s i c o c h e m i c a l p r o p e r t i e s . The main parameter used f o r assignment of s p e c t r a i s the c h e m i c a l s h i f t . ^C-N.m.r. s p e c t r a can be d i v i d e d i n t o f o u r main r e g i o n s , 1) c a r b o n y l and c a r b o x y l groups ( a p p r o x i -m a t e l y 170 p.p.m.), 2) anomeric carbons ( a p p r o x i m a t e l y 93 -110 p.p.m.), 3) r i n g carbons and p r i m a r y a l c o h o l s ( a p p r o x i -m a t e l y 60 - 85 p.p.m.) and 4) m e t h y l groups o f 6-deoxysugars ( a p p r o x i m a t e l y 17 p.p.m.), a c e t a t e s (21 p.p.m.) and p y r u v a t e s ( a p p r o x i m a t e l y 23 p.p.m.). The most u s e f u l r e g i o n i s the anomeric r e g i o n . An a r b i t r a r y d i v i s i o n a t 101 p.p.m. has been a c c e p t e d . However, u n l i k e p.m.r., a-anomeric carbons u s u a l l y r e s o n a t e u p f i e l d o f t h i s , i . e . the C - l resonance o f the a x i a l i s o m e r i s s h i e l d e d r e l a t i v e t o t h a t of the e q u a t o r i a l i s o m e r . The anomeric carbon o f f r e e s u g a r s ( t h e r e d u c i n g end) appears u p f i e l d i n the approximate r e g i o n of 93 - 97 p.p.m. I n t h e r i n g carbon r e g i o n s i g n a l s due t o the carbons of p r i m a r y a l c o h o l s can be d i f f e r e n t i a t e d i n t o those due t o n o n - l i n k e d s u g a r s (60 - 62 p.p.m.) and th o s e due t o l i n k e d s u g a r s which have been s h i f t e d 7 - 1 0 p.p.m. d o w n f i e l d . The methyl groups which appear i n the h i g h f i e l d r e g i o n a re e a s i l y d i s t i n g u i s h e d . I t has a l s o been shown t h a t i t i s p o s s i b l e t o a s s i g n the a b s o l u t e c o n f i g u r a t i o n o f an a c e t a l l i n k e d p y r u v a t e group by the c h e m i c a l s h i f t o f the m e t h y l group 2 9 , V a l u e s o f J r„ can i n d i c a t e g l y c o s i d i c c o n f i g u r a t i o n , 30 and, sometimes, the mac r o m o l e c u l a r c o n f o r m a t i o n , as e x p r e s s e d by g l y c o s i d i c t o r s i o n - a n g l e s . I n f o r m a t i o n a t t h e macromolecular l e v e l can be g a i n e d through v a l u e s o f and measurements o f s p i n - l a t t i c e r e l a x a t i o n time may a l s o g i v e i n f o r m a t i o n on m o l e c u l a r motion and c h e m i c a l s t r u c t u r e - ^ . The most u s e f u l method of a n a l y s i s of ^C-n.m.r. s p e c t r a i s based on the c o r r e l a t i o n of the c h e m i c a l s h i f t s of the carbon atoms o f p o l y s a c c h a r i d e s w i t h t h o s e o f t h e i r p r e v i o u s l y a s s i g n e d monosaccharide and o l i g o s a c c h a r i d e u n i t s . When i n t e r p r e t i n g the 1^C-n.m.r. s p e c t r a of p o l y s a c c h a r i d e s i t i s assumed t h a t the c h e m i c a l s h i f t s of the p o l y s a c c h a r i d e s i g n a l s a re the same as t h o s e of the component monosaccharides h a v i n g the a p p r o p r i a t e c o n f i g u r a t i o n , and t h a t a s t r o n g , down-f i e l d d i s p l a c e m e n t of t h e O - g l y c o s y l a t e d resonance ( a - e f f e c t ) t a k e s p l a c e , w i t h s m a l l e r u p f i e l d d i s p l a c e m e n t s of the s i g n a l ( s ) o f a d j a c e n t carbon atoms ( s - e f f e c t ) . 111 .3 C h a r a c t e r i z a t i o n and q u a n t i t a t i o n of s u g a r s 111.3.1 T o t a l H y d r o l y s i s To i d e n t i f y the c o n s t i t u e n t s u g a r s o f E. c o l i h e t e r o -p o l y s a c c h a r i d e s , the i n i t i a l s t e p i s an a c i d h y d r o l y s i s , t o r e l e a s e the monosaccharides. A l l monosaccharides a r e degraded t o some e x t e n t by a c i d , t h e r e f o r e i t i s e s s e n t i a l t h a t the c o n d i t i o n s of h y d r o l y s i s a re c a r e f u l l y chosen and c o n t r o l l e d . D i f f e r e n t g l y c o s i d i c l i n k a g e s have d i f f e r e n t s t a b i l i t i e s t o a c i d h y d r o l y s i s . T h i s f a c t t o g e t h e r w i t h t h e d i f f e r e n t s t a b i l i t i e s 31 of monosaccharides to acid degradation means that no one method of hydrolysis w i l l necessarily be s u f f i c i e n t on i t s own to cleave every linkage i n a polysaccharide. For example, poly-saccharides containing uronic acid residues are f a i r l y resistant to acid hydrolysis, a furanosyl linkage being hydrolysed 300x faster than a uronosyl linkage. Glycosaminoglycans are also very stable to acid hydrolysis. Acids commonly used for hydrolysis are sulphuric, 20 hydrochloric, formic and t r i f l u o r o a c e t i c . Dutton has reviewed the advantages and disadvantages of these acids and the hydrolytic conditions used. T r i f l u o r o a c e t i c acid (TFA) i s v o l a t i l e and thus e a s i l y removed and also results i n l e s s sugar degradation than sulphuric or hydrochloric acid. For example, hydrolysis of E. c o l i K 50 polysaccharide with 2M HCl for h, resulted i n complete degradation of 2-amino-2-deoxy-glucose and 2-amino-2-deoxygalactose, whereas both sugars were released from the polysaccharide when hydrolysed under the same conditions with TFA. After hydrolysis the most informative f i r s t analysis i s made with paper chromatography. Using d i f f e r e n t solvent systems, hexoses, 6-deoxyhexoses, ac i d i c sugars and aminosugars are i d e n t i f i e d . G.l.c. i s used i n analysis of the correspond-ing a l d i t o l acetates and to determine the quantitative composi-t i o n . It i s essential therefore to hydrolyse a l l the glycosidic linkages with the minimum of sugar degradation. 32 111.3«2 M e t h a n o l y s i s M e t h a n o l y s i s i s an a l t e r n a t i v e t o h y d r o l y s i s and o f t e n g i v e s b e t t e r r e s u l t s . Treatment o f the p o l y s a c c h a r i d e w i t h m e t h a n o l i c hydrogen c h l o r i d e c l e a v e s most g l y c o s i d i c bonds t o form t h e met h y l g l y c o s i d e s and a t t h e same t i m e , the methyl e s t e r s of any u r o n i c a c i d s p r e s e n t . I n the case o f p o l y -s a c c h a r i d e s c o n t a i n i n g amino s u g a r s , t r a n s g l y c o s y l a t e d amino sug a r m e t h y l g l y c o s i d e s and ami n o u r o n i c a c i d methyl g l y c o s i d e methyl e s t e r s a re o b t a i n e d - ^ . Some u r o n o s y l l i n k a g e s c o u l d s t i l l remain i n t a c t , so r e d u c t i o n o f the u r o n i c e s t e r t o i t s c o r r e s p o n d i n g a l c o h o l and subsequent a c i d h y d r o l s i s e nsures complete r e l e a s e o f the sugar r e s i d u e s . By comparing the r a t i o of n e u t r a l s u g a r s r e l e a s e d by m e t h a n o l y s i s w i t h o u t t h e r e d u c t i o n s t e p , w i t h t h o s e which are r e l e a s e d . a f t e r m e t h a n o l y s i s f o l l o w e d by r e d u c t i o n , i t i s p o s s i b l e not o n l y t o i d e n t i f y the u r o n i c a c i d and the a l d o b i o u r o n i c a c i d ( u r o n i c a c i d l i n k e d t o a n e u t r a l s u g a r ) , but a l s o t o determine the molar p r o p o r t i o n s of the component s u g a r s . The r e d u c t i o n s t e p a l s o a l l o w s a c l e a r d i f f e r e n t i a t i o n between amino s u g a r s and a m i n o u r o n i c a c i d s . 111.3*3 C a r b o d i i m i d e r e d u c t i o n . 32 T a y l o r and Conrad^ have developed an a l t e r n a t i v e method f o r d e t e r m i n i n g the t o t a l s ugar r a t i o of a p o l y s a c c h a r i d e c o n t a i n i n g one o r more u r o n i c a c i d s u g a r s . T h e i r method i n v o l v e s the t r e a t m e n t of the a c i d i c p o l y s a c c h a r i d e ( i n 33 aqueous s o l u t i o n ) w i t h a water s o l u b l e c a r b o d i i m i d e t o g i v e an O - a c y l i s o u r e a , which i s then reduced w i t h sodium b o r o h y d r i d e (see Scheme 1 ) . R e d u c t i o n o f the c a r b o d i i m i d e r e a c t i o n p r o d u c t s w i t h sodium b o r o h y d r i d e i s v e r y s e n s i t i v e t o a l k a l i n e pH, so a d d i t i o n of the.aqueous sodium b o r o h y d r i d e t o the c a r b o -d i i m i d e r e a c t i o n m i x t u r e i s b e s t c a r r i e d out at a p p r o x i m a t e l y pH 4 . 7 5 * The p r o d u c t i s r e c o v e r e d by d i a l y s i s and l y p p h i l i z a -t i o n . Two c y c l e s of t r e a t m e n t may be n e c e s s a r y t o a c h i e v e complete r e d u c t i o n . A f t e r r e d u c t i o n the p o l y s a c c h a r i d e can r e a d i l y be h y d r o l y s e d w i t h d i l u t e a c i d which i s then a b l e t o c l e a v e a l l the g l y c o s i d i c bonds. 1 1 1 . 3 . 4 Deamination '*)'*> Over a hundred y e a r s ago L e d d e r h o s e ^ i n v e s t i g a t e d the d e a m i n a t i o n o f 2-amino-2-deoxy-D-glucose w i t h n i t r o u s a c i d , a l t h o u g h i t was not u n t i l l a t e r t h a t the p r o d u c t was i d e n t i f i e d as 2,5-anhydro-D-mannose. Amino s u g a r s n o r m a l l y o c c u r as t h e i r N - a c e t y l d e r i v a t i v e s , and N - d e a c e t y l a t i o n has t o be c a r r i e d out b e f o r e d e a m i n a t i o n . T h i s i s u s u a l l y a c c o m p l i s h e d by t r e a t m e n t w i t h sodium h y d r o x i d e i n d i m e t h y l sulphoxide-^ . 2-Amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-g a l a c t o s e are the most w i d e l y o c c u r r i n g amino sugar c o n s t i t u e n t s of p o l y s a c c h a r i d e s and have been the most e x t e n s i v e l y s t u d i e d . D e a mination o f 2-amino-2-deoxy-D-glucopyranosides f o l l o w s m a i n l y pathway (a) i n Scheme 2, w i t h g l y c o s i d i c c l e a v a g e and f o r m a t i o n of 2,5-anhydro-D-mannose. However, the same s t e r e o -e l e c t r o n i c r e q u i r e m e n t s f o r the l o s s o f n i t r o g e n from the 3k RCOOH + RCOCTH H pH475 RCH20H N0BH4 RCH NHR' N0BH4 pH 5-7 NHR + 0=6 + H+ NHR' E.D.C. = 1-ethyl -3-(-dimethylaminopropyl)carbodiimide C.M.C. = 1-cyclbhexyl -3 - (2-morpholinoethyl)carbodiimide metho-p_-toluene sulphonate Scheme 1. Reduction of carboxylic acid i n aqueous solution using carbodiimide reagent. Scheme 2. A l t e r n a t i v e r i n g c o n t r a c t i o n s d u r i n g t h e n i t r o u s a c i d d e a m i n a t i o n of 3 - 0 - s u b s t i t u t e d 2-amino , - 2-deoxy-D-glucopyranosides r e s u l t i n g i n (a) s e l e c t i v e g l y c o s i d e c l e a v a g e w i t h f o r m a t i o n of 2 ,5-anhydro-D-mannose d e r i v a t i v e s w i t h l i b e r a t i o n of ag l y c o n e and (b) f o r m a t i o n of 2-C-formyl p e n t o f u r a n o s i d e s ( t h i s r e a c t i o n o c c u r s w i t h e p i m e r i z a t i o n at C-2) w i t h l i b e r a t i o n o f 3 - 0 - g l y c o s y l s u b s t i t u e n t s . (From R e f e r e n c e 58) 36 intermediate diazonium ion with concomitant ring contraction i s met not only by p a r t i c i p a t i o n by the electrons of the 0-C-1 bond, but also by p a r t i c i p a t i o n of the C-3—C-4 bond (path-way b). In the l a t t e r case, 2-C-formyl pentofuranoside i s produced, with the l i b e r a t i o n of any 3-0 substituents which may be present. Thus deamination of amino sugars usually gives a variety of products. 111.4 Position of linkage 111.4.1 Methylation analysis Methylation analysis was developed by Haworth and his co-35 workers , and i s s t i l l the most important method i n s t r u c t u r a l carbohydrate chemistry. It involves the methylation of a l l free hydroxy-groups i n the polysaccharide and hydrolysis of the methylated polysaccharide to a mixture of p a r t i a l l y methylated monosaccharides. The free hydroxy-groups i n the hydrolysate, then indicate the positions at which the sugar residues were substituted i n the o r i g i n a l polysaccharide. Thus methylation analysis can give information on : 1) the number and type of sugars per repeating unit, 2) ring s i z e , 3) linkage positions, 4) i d e n t i t y of terminal unit(s) and branching units, 5) the position of base-stable substituents e.g. pyruvate. Methylation does not, however, give information on sequences or on anomeric configuration. 37 The most e f f e c t i v e method of methylation was devised 36 by Hakomori-^ . The polysaccharide , dissolved i n dimethyl-., sulphoxide, i s treated with sodium methylsulphinylmethanide, and subsequently with methyl iodide. As indicated by i n f r a -red spectroscopy, complete methylation i s usually achieved i n one step. In cases of incomplete methylation a subsequent 37 Purdie-Irving methylation ' (Ag-fi i n refluxing Mel) i s conducted. Any methyl esters formed by methylation of an uronic acid must be reduced before complete depolymerization can be achieved. Lithium aluminium hydride, i n tetrahydro-furan i s the reducing agent used. The methylated reduced poly-saccharide i s then remethylated and hydrolysed. The p a r t i a l l y methylated monosaccharides are converted to t h e i r a l d i t o l acetates to y i e l d v o l a t i l e derivatives for g.l.c.-m.s. analysis. Comparison of methylation" data from native, reduced and depyruvylated polysaccharides gives valuble information on presence of uronic acids and the position of pyruvate sub- . sti t u e n t s . 111.4.2 Characterization and quantitation of methylated sugars Paper chromatography i s used, i n the f i r s t instance, to characterize the p a r t i a l l y methylated monosaccharides released upon t o t a l hydrolysis of the permethylated poly-saccharide (or oligosaccharide). The'papers, when sprayed with p-anisidine and subsequently heated, show d i f f e r e n t coloured spots according to the methylated sugars. Preliminary i d e n t i f i c a t i o n of the methylated sugars can be made from t h e i r 38 m o b i l i t i e s (Rf values) and colours. For confirmation of this C H D O A c C H D O A c H - - O M e 118 H - -OAc H - - O M e 189 H - -OMe 190 H - OAc H - - O M e 117 C H 2 O A c CHjOAc ( D (2) Figure 6. Primary mass spectral fragment ions from 2,3-di-JD-methylpentitol triacetate-1-d^ (1) and 3,4-di-(>-methylpentitol t r i a c e t a t e - ! - ^ (2) preliminary q u a l i t a t i v e analysis, and for the quantitative analysis, g.l.c.-m.s.can be performed. An extensive review 2^-1-of this f i e l d has been done by Lindberg and coworkers . The methylation pattern of a component i s evident from i t s mass spectrum. Usually a l l components i n a mixture can be i d e n t i f i e d provided that the sugar composition i s known. Consideration of the r e l a t i v e retention times and co- " chromatography with authentic samples allows unambiguous i d e n t i -f i c a t i o n of the methylated sugars present. The main l i m i t a t i o n of the use of p a r t i a l l y methylated sugars l i e s i n the s t r u c t u r a l symmetry that may exist when the primary hydroxyl group i s not e s t e r i f i e d . Introduction of deuterium at C-1, by reduction of the sugar with sodium borodeuteride can overcome th i s problem. For example i n F i g . 6. the p a r t i a l l y methylated a l d i t o l acetates from 2,3- ( 1 ) and 3,4- (2) di-O-methylpenitols can be d i f f e r e n t i a t e d by observation of the relevant i s o t o p i c s h i f t s i n primary fragment ions. 39 111.5 Sugar sequence Iso l a t i o n and i d e n t i f i c a t i o n of oligosaccharide fragments i s a common method used for sequence analysis of polysaccharides. It i s not only a useful method for elucidating the sequence of the monosaccharides i n a polysaccharide, but i t also aids i n making assignments i n the n.m.r. spectra. There i s no standard technique for obtaining oligosaccharide fragments. Each polysaccharide presents i t s own problems. The oldest method i s p a r t i a l acid-hydrolysis, but the most commonly used method i s the Smith degradation, devised by J O F. Smith ? , a former co-worker of Haworth. 3-elimination (uronic acid degradation) i s another method used as well as the deamination of amino-sugar residues. Although these techniques have not been used i n t h i s work, a b r i e f summary of each i s being included i n order to give the reader a clearer understanding of the t o t a l process of polysaccharide s t r u c t u r a l analysis. 111.5.1 P a r t i a l hydrolysis This method i s p a r t i c u l a r l y valuble when a polysaccharide contains a li m i t e d number of linkages which are e s p e c i a l l y sensitive to acid hydrolysis. For example, furanosides are hydrolyzed faster than the corresponding pyranosides by factors of 10-10^. When furanosides occupy terminal or near terminal positions i n polysaccharide structures, they can be s e l e c t i v e l y hydrolyzed under mild 40 c o n d i t i o n s w i t h l i m i t e d d i s r u p t i o n of the r e s t of the p o l y s a c c h a r i d e c h a i n . On t h e o t h e r hand, u r o n o s y l l i n k a g e s i n p o l y s a c c h a r i d e s a re more r e s i s t a n t t o a c i d h y d r o l y s i s t h a n o t h e r g l y c o s i d i c l i n k a g e s . T h e r e f o r e i t i s r e l a t i v e l y easy t o i s o l a t e a c i d i c d i s a c c h a r i d e s ( a l d o b i o u r o n i c a c i d s ) and h i g h e r o l i g o s a c c h a r i d e s . 2-Amino-2-deoxy g l y c o s i d e s a r e a l s o v e r y r e s i s t a n t t o a c i d h y d r o l y s i s due t o an i n d u c t i v e e f f e c t of the "protonated amino group. By v a r y i n g a c i d t y p e and c o n c e n t r a t i o n , a l o n g w i t h t e m p e r a t u r e and r e a c t i o n t i m e , i t i s p o s s i b l e t o o b t a i n m i x t u r e s of mono-, d i - and h i g h e r o l i g o s a c c h a r i d e s . These may be i s o l a t e d from the r e s i d u a l p o l y s a c c h a r i d e by d i a l y s i s . The o l i g o m e r s a r e then s e p a r a t e d by e i t h e r g e l or p a p e r chromatography. A major drawback o f t h i s t e c h n i q u e i s t h a t any a c i d -l a b i l e n o n - c a r b o h y d r a t e s u b s t i t u e n t s , such as a c e t a t e s and p y r u v a t e s , a re c l e a v e d d u r i n g the p r o c e s s . D e p o l y m e r i z a t i o n by a b a c t e r i o p h a g e - b o r n e g l y c a n a s e i s an a l t e r n a t i v e method which may the n be employed (see S e c t i o n 1V.4)» 111.5.2 P e r i o d a t e o x i d a t i o n and Smi t h h y d r o l y s i s Sugars which c o n t a i n v i c i n a l h ydroxy-groups a r e o x i d i z e d by p e r i o d a t e . Cleavage of t h e car b o n c h a i n t a k e s p l a c e w i t h the f o r m a t i o n o f two a l d e h y d i c groups, one mole of p e r i o d a t e b e i n g consumed'(see F i g . 7)« I n t h e case of a & - y - t r i o l s , a double c l e a v a g e of the car b o n c h a i n o c c u r s w i t h the f o r m a t i o n of two a l d e h y d i c groups and the l i b e r a t i o n 41 R HCOH HCOH CH,OH F i g u r e 7. S m i t h D e g r a d a t i o n - p e r i o d a t e o x i d a t i o n f o l l o w e d by m i l d a c i d h y d r o l y s i s o f the reduced product. of 1 mole of f o r m i c a c i d . The p e r i o d a t e consumption can be m o n i t o r e d s p e c t r o p h o t o m e t r i c a l l y and t h e amount of f o r m i c a c i d r e l e a s e d can be measured by a c i d - b a s e t i t r a t i o n . I n a d d i t i o n , the presence o f s u g a r s t h a t a r e s u b s t i t u t e d i n such a manner t h a t l e a v e s no d i o l group s u s c e p t i b l e t o o x i d a t i o n may be a s c e r t a i n e d by l i b e r a t i o n o f the suga r a f t e r h y d r o l y s i s . R CHO HCOjH + HCHO 42 I n t h e Smith d e g r a d a t i o n - ^ , the p e r i o d a t e - o x i d i z e d p o l y s a c c h a r i d e i s reduced w i t h sodium b o r o h y d r i d e t o a ' p o l y a l c o h o l 1 • The a c e t a l l i n k a g e s i n t h e m o d i f i e d a c y c l i c r e s i d u e s are much more s e n s i t i v e t o a c i d h y d r o l y s i s than th e g l y c o s i d i c l i n k a g e s of the i n t a c t sugar r e s i d u e s , and can be s e l e c t i v e l y c l e a v e d under m i l d c o n d i t i o n s . C h a r a c t e r i z a t i o n o f t h e p r o d u c t , which may be p o l y m e r i c o r c o n s i s t o f s i n g l e u n i t g l y c o s i d e s , o f t e n g i v e s c o n s i d e r a b l e i n f o r m a t i o n on the n a t u r e and p r o p o r t i o n o f the g l y c o s i d i c l i n k a g e s p r e s e n t i n t h e p o l y s a c c h a r i d e . However, r e s u l t s o b t a i n e d by p e r i o d a t e o x i d a t i o n must be c o n f i r m e d by r e s u l t s from m e t h y l a t i o n a n a l y s i s . 39 111•5•3 U r o n i c a c i d d e g r a d a t i o n ( 3 - e l i m i n a t i o n ) • D u r i n g a Hakomori m e t h y l a t i o n the c a r b o x y l group of any uronic" a c i d r e s i d u e s i n the p o l y s a c c h a r i d e w i l l be e s t e r i f i e d . The e s t e r , b e i n g e l e c t r o n w i t h d r a w i n g , i n c r e a s e s the a c i d i t y o f the r i n g p r o t o n a t C - 5 . On t r e a t m e n t of the m e t h y l a t e d p r o d u c t w i t h base, 3 - e l i m i n a t i o n o c c u r s . The sub-s t i t u e n t a t C-k on the u r o n i c a c i d ( e i t h e r a methoxyl group o r a s u g a r r e s i d u e ) i s e l i m i n a t e d as P^OH, and an u n s a t u r a t e d u r o n i c a c i d r e s i d u e ( I I ) i s formed. I n c o n t r a s t t o the p a r e n t a c i d - r e s i s t a n t g l y c o s i d u r o n i c a c i d l i n k a g e , the hex-4-eno-p y r a n o s i d u r o n i c a c i d l i n k a g e i s e x t r e m e l y l a b i l e and can be c l e a v e d under m i l d a c i d i c c o n d i t i o n s , w i t h accompanying h y d r o l y s i s of normal g l y c o s i d i c l i n k a g e s , t o expose th e h y d r o x y l group t o which the u r o n i c a c i d was a t t a c h e d . T h i s 43 free hydroxyl group can then be i d e n t i f i e d by further a l k y l a t i o n with methyl iodide, ethyl iodide or trideutero-methyl iodide. When R^ OH i s a single residue or chain of sugar residues, a second g-elimination reaction occurs, and the next sugar may be released on the subsequent mild acid treatment, g-Elimination i n conduction with methylation analysis i s thus an excellent method for obtaining oligo-saccharide fragments and for i d e n t i f y i n g the sugar attached to the uronic acid residue. 111 .5 .4 Deamination The deamination reaction can also be used for the controlled degradation of amino sugar containing polysaccharides to y i e l d oligosaccharides. One example i s the application of the N-deacetylation-nitrous acid deamination sequence to the O-specific polysaccharide chain=of S h i g e l l a dysenteriae type I. lipopolysaccharide to give two oligosaccharides. One i s from the normal rin g contraction and the other from the 40 alternative ring contraction- . -3i-a-L-Rha/)-(1 - > 3 H » L - R h a p - ( 1 - . 2 M - D - G a ! / > - U - 3 ) - a - D - G l c p N A c - l f-( D (2) Scheme 3. N-Deacetylation-nitrous acid deamination of the O-specific polysaccharide chain from the Sh i g e l l a  dysenteriae type 1 lipopolysaccharide leads to the i s o l a t i o n of two oligosaccharides, (1) from the alternative ring contraction as the 3-0-substituent of the N-acetyl-D-glucosamine and (2)from the normal ring contraction. 45 MICROBIOLOGICAL ANALYSIS CAPSULAR POLYSACCHARIDES 46 IV. MICROBIOLOGICAL ANALYSIS OF CAPSULAR POLYSACCHARIDES IV.1 Introduction The word 'bacteriophage' was coined by d'Herelle i n 1 9 1 7 ^ The f i r s t account however of a bacteriophage was fio published by Twort i n 1915 • He attributed the l y s i s of some colonies of bacterium to a virus s i m i l a r to those that i n f e c t plants and animals. Bacteriophage, usually abbreviated to 'phage' (designated by the Greek l e t t e r 0 ) , have b a s i c l y the same structure as other viruses, i n that they have a protein coat (caps'id) which surrounds a nucleic acid core. The protein coat i s an assembly of i d e n t i c a l morphological sub-units, and the nucleic acid core may be either DNA or RNA. The nucleic acid i s i n the form of a long filamentous molecule and may be double or single stranded. 2-DNA 2-DNA 2-DNA 1-DNA 1-ENA 1-DNA Figure 8. Basic morphological types of bacteriophage with the types of nucleic acid (from Reference 41). 47 F i g u r e 9. Diagram o f the C o l i p h a g e T2 V i r i o n showing the p r i n c i p a l p a r t s i n the normal r e l a t i o n t o one a n o t h e r . 48 Although the basic structure i s the same for a l l viruses, phages exhibit a larger d i v e r s i t y of form than any other group. Bradley*^ has c l a s s i f i e d phages into s i x basic morphological types (see F i g . 8). Type A i s the most complex, having a polyhedral head and a t a i l with a c o n t r a c t i l e sheath attached to i t . The t a i l i s usually r i g i d and may have various appendages e.g. end plate, t a i l pins and fibres (see Fig 9). The E . c o l i capsule bacteriophage (K phages) belong mainly to Bradley group C. K phages carry 'spikes' only, which i n the majority of cases are linked d i r e c t l y to the head (see Fig 10). Figure 10. E. c o l i capsule bacteriophage no. 26 negatively stained with uranyl acetate, x 384»000 (from Reference 42). 49 These K phages adsorb to and lyse some capsular strains of bacteria but not acapsular mutant strains of t h e i r host organisms. Neither do they usually adsorb to s i m i l a r strains with serological and chemically d i f f e r e n t capsules. After studying 15 capsule phages, Stirm and his c o l l e a g u e s ^ concluded that because no K phage was seen to carry t a i l f i b r e s but a l l had spikes, these spikes were the most l i k e l y candidates for bearing the " s p e c i f i c s i t e s " responsible for capsular polysaccharide recognition. The phage l y t i c cycle or i n f e c t i v e cycle can be divided into four phases: 1) Absorption of the phage p a r t i c l e to the susceptible host; 2) Injection of v i r a l DNA or RNA into the host; 3) Replication of phage nucleic acid and synthesis of phage protein; 4) Phage maturation and release.. Thus, the absorption of a phage to i t s host i s a multistep process, i n which the v i r i o n attaches to, and penetrates, the polysaccharide. After reaching the outer membrane, the virus p a r t i c l e attaches to i t s surface, comes into contact with the adhesion s i t e , and then releases i t s DNA. A l l phage p a r t i c l e s capable of capsule penetration carry a receptor degrading enzyme which i s associated with the t a i l t i p of the v i r i o n or i s retained i n the v i r a l s p i k e s ^ . These receptor degrading enzymes have high substrate s p e c i f i c i t y . When the E. c o l i ^29 was tested against 82 heterologous b a c t e r i a l glycans i t cleaved only one other 50 polysaccharide, that of K l e b s i e l l a K 3 1 . Phage 29 depoly-merizes the p-linkage between glucose and glucuronic acid. This cleavage s i t e i s present i n both E. c o l i K29 and Kleb- s i e l l a K 3 1 4 4 . The action of the receptor degrading enzymes i s responsible for the occurrence of haloes around the clear plaques i n lawns of capsulated bacteria. The plaque i s caused by a single phage p a r t i c l e . It f i r s t i n f e c t s a single c e l l . I t then m u l t i p l i e s inside the c e l l causing i t to lyse and release many progeny phages. These diffuse away, i n f e c t i n g other c e l l s and l y s i n g them also. The process i s repeated, so that the area of l y s i s , (known as a "plaque") becomes v i s i b l e to the naked eye. The haloes surrounding the plaque are the res u l t of overproduction of the depolymerizing enzyme. Within the halos, the bacteria have l o s t t h e i r capsules. Plaque c h a r a c t e r i s t i c s are often useful i n i d e n t i f y i n g phages. Phage-bound receptor-depolymerizing enzymes can be either endoglycanases (hydrolyzing glyco s i d i c linkages), esterases (hydrolyzing J)-acetyl substituents) or lyases. For most capsular polysaccharides there i s a s p e c i f i c phage which w i l l cleave the polysaccharide chain into oligosaccharides which may correspond to one or more repeating units. The a b i l i t y of the phage to depolymerize i t s corresponding capsular polysaccharide makes i t an excellent tool for obtaining an oligosaccharide repeating unit with i t s acid- or base-l a b i l e non-carbohydrate substituent (e.g. acetate or a k e t a l -linked pyruvate) i n t a c t . Such oligosaccharides are i d e a l for the production of synthetic antigens by l i n k i n g them to protein 51 c a r r i e r s . They are also useful as substrates for n.m.r. and mass spectral studies. IV.2 Isolation of bacteriophage from sewage Standard methods (see Stirm and Freund-Molbert 1961) are used for i s o l a t i o n of bacteriophage from sewage. Crude sewage i s combined with 10-fold concentrated Mueller Hinton broth. A log-phase culture of the prospective capsular host i s added immediately and the mixture incubated at-37°« An aliquot of the mixture i s removed, s t e r i l i z e d with a few drops of chloroform, and centrifuged. The cl e a r supernatant i s subsequently assayed for phage. Ten-fold d i l u t i o n s are prepared. Using a Pasteur pipette one drop of each d i l u t i o n i s spotted onto a b a c t e r i a l lawn of_the phage's host bacterium. A clear plaque on the ba c t e r i a l lawn indicates the possible presence of a phage. To confirm that a phage i s responsible for the plaque formation, a p u r i f i c a t i o n step i s carried out. A single plaque i s removed from the plate and added to s t e r i l e broth. A s e r i a l d i l u t i o n i s made of t h i s solution and again each of the d i l u t i o n s i s spotted onto a lawn of the phage's homologous bacterium. This p u r i f i c a t i o n step i s repeated at least once more. Propagation of the phage on th e i r host bacterium by t e s t - l y s i s i s used to p o s i t i v e l y confirm that a phage has been i s o l a t e d for the p a r t i c u l a r bacterium under study. 52 1V. 3 P r o p a g a t i o n o f b a c t e r i o p h a g e F o r a t e s t - t u b e l y s i s , an a c t i v e l y growing b a c t e r i a l c u l t u r e i s added t o M u e l l e r H i n t o n b r o t h . The i n o c u l a t e d b r o t h i s i n c u b a t e d u n t i l the c u l t u r e becomes c l o u d y . . An a l i q u o t o f b a c t e r i o p h a g e - c o n t a i n i n g s o l u t i o n ( p r e p a r e d by a d d i n g one b a c t e r i o p h a g e plaque t o lmL M u e l l e r H i n t o n b r o t h ) i s added t o the t e s t - t u b e . The c u l t u r e i s a g a i n i n c u b a t e d u n t i l t h e c l o u d y b a c t e r i a l c u l t u r e c l e a r s (due t o c e l l l y s i s by the b a c t e r i o p h a g e ) . A few drops o f c h l o r o f o r m a r e added to the c o n t e n t s o f the t e s t - t u b e t o p r e v e n t b a c t e r i a l growth. C e n t r i f u g a t i o n i s used t o remove dead b a c t e r i a l c e l l s and the phage s o l u t i o n i s then assayed u s i n g the "plaque a s s a y t e c h n i q u e " . S e r i a l d i l u t i o n s o f the phage s o l u t i o n a r e p r e p a r e d . U s i n g a c a l i b r a t e d p i p e t t e , one drop from each d i l u t i o n i s t h e n s p o t t e d onto a b a c t e r i a l lawn of the phages's h o s t b a c t e r i u m . The p l a t e i s l e f t a t room tem p e r a t u r e o v e r -n i g h t . At h i g h phage c o n c e n t r a t i o n s (10~ 2,10""^) i n d i v i d u a l p l a q u e s cannot be seen. However, a t s u i t a b l e d i l u t i o n s («>•» 1 0 ) i n d i v i d u a l p l a q u e s can be d i s t i n g u i s h e d . Sometimes t h e s e a r e s u r r o u n d e d by h a l o e s . The t i t r e of the b a c t e r i o p h a g e . s o l u t i o n i s t h e n c a l c u l a t e d . B a c t e r i o p h a g e t i t r e = no. o f p l a q u e s x d i l u t i o n x drop s i z e ( p . f . u . p e r mL)* * p l a q u e f o r m i n g u n i t s p e r mL An i n c r e a s e i n the t i t r e o f the b a c t e r i o p h a g e s o l u t i o n a f t e r l y s i s , t o g e t h e r w i t h the c l e a r i n g o f the b a c t e r i a l c u l t u r e 53 a f t e r the addition of the bacteriophage, i s good evidence for the presence of bacteriophage s p e c i f i c for the host bacterium. IV. hf Bacteriophage cross reactions The host surface carbohydrate degrading enzymes carried by phage p a r t i c l e s are s p e c i f i c for only one, or at most, a few substrates. Stirm and Rieger-Hug y carried out an extensive survey using 7h d i f f e r e n t K l e b s i e l l a capsular polysaccharides and a t o t a l of 55 bacteriophage, the p a r t i c l e s of which catalyzed host capsular glycan depolymerization. Twenty-eight cross reactions were detected, i n which a phage carrying a glycanase s p e c i f i c for the degradation of the capsular polysaccharide of a p a r t i c u l a r K l e b s i e l l a serotype, was also able to degrade that of another serotype. The v i r a l depolymerases thus proved to be very s p e c i f i c (33 cross-reacting with none, 18 with one, 2 with two and 1 each with three or four heterologous polysacharides). From t h e i r r e s u l t s , Stirm and Rieger-Hug were able to reach the following general conculsions about the chemical c h a r a c t e r i s t i c s of glycosidic linkages i n acidic heteropolysaccharides suceptible to phage glycanase. 1) In most cases, where one polysaccharide i s acted upon by several phage enzymes, the same bonds are s p l i t ; 2) Hydrolysis occurs on either side of the sugar unit carrying the negative charge but reducing glucuronic acids are not produced; 3) The reducing end sugar formed i s often substituted at 54 position 3. Determination of the reducing end (see Section 1V . 7 ) leads d i r e c t l y to the i d e n t i f i c a t i o n of the glycosidic bonds s p l i t by the v i r a l enzyme. Thus, cross-reactions can be used to make predictions about the p o s s i b i l i t y of a certain bond and chemical structures (see Section 1 V . 5 ) being present i n a polysaccharide of unknown structure. In order to ascertain whether a phage w i l l degrade heterologous polysaccharides, test tube lyses are carried out as for a bacteriophage propagation. However the phage i s added to a b a c t e r i a l culture other than that of i t ' s host bacterium. The phage solution i s then assayed i n the usual manner except that the phage solution i s spotted onto a b a c t e r i a l lawn of the same s t r a i n as the culture to which the phage was added. The a b i l i t y of a phage to form plaques on a b a c t e r i a l lawn other than that of i t ' s host bacterium, along with an increase i n the number of phage p a r t i c l e s over that added to the b a c t e r i a l culture, i s taken as evidence that a cross-reaction has occurred. 1V. 5 Bacteriophage degradation of capsular polysaccharides Although a number of highly s p e c i f i c "K" bacteriophag have been found for E. c o l i capsular s t r a i n s ^ 2 , t h e i r enzymatic action had not been thoroughly investigated u n t i l 47 recently. Stirm and colleagues studied the capsule-degrading enzymic a c t i v i t y of two E. c o l i bacteriophage (092 and 0 1.2). Their re s u l t s demonstrated not only that 5 5 phage-borne enzymes are endoglycanases but also that the r e l a t i v e distances of the substrate functional groups in t e r a c t i n g with the enzyme.protein are decisive. Elsasser-48 Beile and Stirm had previously shown, while working on the cross-reactions of 06 ( K l e b s i e l l a ) glycanases, that the enzyme proteins recognize disaccharide portions of t h e i r substrate. Within these "recognition s i t e s " one carboxyl and one to three hydroxyl groups at the correct distance from the suceptible g l y c o s i d i c oxygen are necessary for recognition. In order for a bacteriophage to be able to depolymerize the capsular polysaccharide of i t ' s host bacterium i t must be present i n the correct concentration. Therefore, before a phage degradation i s undertaken, the bacteriophage i s propagated on i t ' s host bacterium by means of successive flask lyses u n t i l the crude lysate contains approximately 1 0 ^ p.f.u. This amount i s s u f f i c i e n t to depolymerize one gram of the polysaccharide. The polysaccharide i s added to the crude phage solution and depolymerization i s allowed to proceed for approximately two days. After the addition of chloroform to prevent b a c t e r i a l growth, the mixture i s con-centrated, and dialysed against tap water, u n t i l the Molisch . test shows that no more oligosaccharides are. idialysing out. The dialysates are then combined, concentrated, treated with ion-exchange resin and subsequently freeze-dried. 56 IV. 6 I s o l a t i o n o f o l i g o s a c c h a r i d e s The m i x t u r e o f o l i g o s a c c h a r i d e s o b t a i n e d by phage d e p o l y m e r i z a t i o n may c o n s i s t o f c h a i n s o f one, two o r more r e p e a t i n g u n i t s . These may be s e p a r a t e d e i t h e r by p r e p a r a t i i v e paper chromatography o r g e l chromatography. O f t e n b o t h t e c h n i q u e s a r e a p p l i e d w i t h paper chromatography b e i n g used t o p u r i f y the o l i g o m e r s s e p a r a t e d by g e l chromatography. The l y o p h i l i z e d p r o d u c t from t h e phage d e g r a d a t i o n i s d i s s o l v e d i n a minimum amount of wa t e r , a p p l i e d t o a B i o - G e l column, and c o l l e c t e d by a m i c r o f r a c t i o n a t o r i n weighed t e s t - t u b e s . The c a r b o h y d r a t e c o n t a i n i n g f r a c t i o n s are l o c a t e d u s i n g the M o l i s c h t e s t . These f r a c t i o n s a re then f r e e z e - d r i e d and weighed. A graph i s p l o t t e d o f t e s t -tube number v e r s u s w e i g ht o f p r o d u c t t o i d e n t i f y t he p o s i t i o n o f the s i n g l e ( i f any) r e p e a t i n g u n i t . ^H-N.m.r. i s employed t o determine the degree o f p o l y m e r i z a t i o n and p u r i t y of the p r o d u c t . 1V. 7 D e t e r m i n a t i o n o f the phage-borne g l y c a n a s e ' s c l e a v a g e p o s i t i o n Once an o l i g o s a c c h a r i d e has been o b t a i n e d by means o f m i c r o b i o l o g i c a l methods, t h e n c h e m i c a l methods o f s t r u c t u r e d e t e r m i n a t i o n a r e a p p l i e d t o t h e p r o d u c t . When d e t e r m i n i n g t h e s t r u c t u r e of an o l i g o s a c c h a r i d e the same p o i n t s must be e s t a b l i s h e d as f o r a p o l y s a c c h a r i d e , i . e . t he i d e n t i t y and sequence o f c o n s t i t u e n t monosaccharides, 57 the position and configuration of the glycosidic linkages, and the size of the monosaccharide rings. Two additional points need to established however for oligosaccharides; these are the degree of polymerization and the nature of the reducing end. I d e n t i f i c a t i o n of the reducing end w i l l be conclusive evidence for the point of cleavage by the phage-borne enzyme. To i d e n t i f y the reducing end the reduced ol i g o -saccharide i s methylated using the Hakomori method. After hydrolysis the p a r t i a l l y methylated monosaccharides are converted to t h e i r a l d i t o l acetates, and analyzed by g . l . c -m.s. The reducing end sugar i s now methylated at CI and C5 as well as at the other o r i g i n a l l y unsubstituted positions. The non-reducing end sugar becomes a terminal residue which must have been linked to the reducing end sugar i n the native polysaccharide. Proton n.m.r. i s used not only for i d e n t i f i c a t i o n of the reducing end, but also for determining the degree of poly-merization of the oligosaccharide. A spectrum of P 1 ( o l i g o -saccharide consisting of one repeating unit) w i l l show the absence of a signal as compared to the native polysaccharide. P 1 , w i l l have instead, two anomeric signals, one for the a-anomer of the reducing end residue and one for the $- anomer. 1 ^ The -^C-n.m.r. spectrum w i l l show the anomeric carbons of free sugars (reducing end) appearing u p f i e l d , in''the region 93-97 p.p.m. 58 V. QUALITATIVE AND QUANTITATIVE SUGAR ANALYSIS OF E. COLI K26 CAPSULAR POLYSACCHARIDE 59 V. QUALITATIVE AND QUANTITATIVE SUGAR ANALYSIS OF E. COLI K26 CAPSULAR POLYSACCHARIDE .V.I Introduction E. c o l i K26 (09a K26 H~) capsular polysaccharide has a high molecular weight and low electrophoretic mobility. I t , thus, belongs to the group of K antigens formerly c a l l e d A antigens. Like other A antigens i t forms a thick and copious capsule around the b a c t e r i a l c e l l . K antigens with low electrophoretic mobility can be divided into two groups; one group which i s acidic due to the presence of uronic acids and possibly pyruvate, and another group which consists of lipopolysaccharides. E. c o l l with these K antigens, in..fact, contain two c e l l wall lipopolysaccharides: an acidic one which i s termed a K antigen i n addition to a neutral one (08, 09 or010,antigen).The acidic l i p o p oly-saccharides are not capsular (K) antigens i n the true sense. In the nomenclature of Kauffmann they were termed B antigens^. E. c o l i K26 capsular polysaccharide i s classed as a true capsular polysaccharide, i t s negative charge being due to glucuronic acid and pyruvate. The purpose of t h i s study was to investigate the sugar composition and sugar r a t i o of E. c o l i K26 capsular polysaccharide. U n t i l recently, i t was thought that true capsular polysaccharides do :not contain amino sugars*^. This has been v e r i f i e d for a l l K l e b s i e l l a K antigens, which are true capsular polysaccharides and has also been found to be 60 correct for E. c o l i K26 polysaccharide. However, work now being done i n this laboratory shows that some true capsular polysaccharides do contain amino sugars (see Section VI1). V.2 Results and discussion Composition and n.m.r. spectra E. c o l i K26 bacteria were grown on Mueller Hinton agar, and the capsular polysaccharide was p u r i f i e d by one pr e c i p i t a t i o n with Cetavlon (as described i n Section 11). Paper chromatography of an acid hydrolyzate. showed the presence of rhamnose, glucose and/or galactose and glucuronic a c i d . Also found was a f a i n t spot which could be attributed to an aldobiouronic acid. Conversion of the monosaccharides to t h e i r a l d i t o l acetates, followed by g . l . c . analysis gave rhamnose, galactose and glucose i n the r a t i o 1:1:0.2. Further sugar analysis by methanolysis, followed by reduction, hydrolysis and conversion of the released sugars to t h e i r a l d i t o l acetates gave rhamnose, galactose and glucose i n the r a t i o 2.5:1:0.5. However the reduction was only 50% successful. Hydrolysis of the carbodiimide reduced polysaccharide gave rhamnose, galactose and glucose i n the r a t i o 3.5:1:0.6.(see Table V.1). These re s u l t s indicated that the polysaccharide consisted of a hexasaccharide repeating unit, i n which an acidic sugar, glucuronic acid, was present. As the reduction of the polysaccharide also increased the amount of rhamnose released on hydrolysis, the hexasaccharide repeating unit contained an aldobiouronic acid 61 TABLE V.I SUGAR ANALYSIS OF K26 POLYSACCHARIDE Sugars Mole r a t i o (as a l d i t o l acetates) I s- IT> II T> Rhamnose 1.1 3.5 2.5 Galactose 1.0 1.0 1 .0 Glucose 0.2 0.6 0.5 I, o r i g i n a l a c i d i c polysaccharide; I I , carbodiiraide-reduced polysaccharide; III, carboxyl-reduced polysaccharide a. Using SP 2340 column programmed for 195° for 2 min 2°/ min to 260°. b_ Using DB 1? column programmed for 180° for 2 min 5 ° / min to 220°. 62 composed of glucuronic acid linked to rhamnose. The s l i g h t l y low amounts of rhamnose and glucose can be explained by i n -complete reduction of the glucuronic acid. Auto hydrolysis of the K26 polysaccharide reduced the v i s c o s i t y by removing the pyruvate substituent. The and ^C-n.m.r. spectra substantiated the hexasaccharide repeating unit. The 1H-n.m.r. spectrum indicated that three of the sugars are a-linked and three are g-linked. The presence of deoxy-sugars and pyruvate (before autohydrolysis) were also shown by the 1H-n.m.r. r e s u l t s . The downfield . signal at Sk»k was attributed to the H-5 of a g-glucuronic acid. It was not possible to assign peaks to a d e f i n i t e sugar without further evidence e.g. from oligosaccharides obtained by p a r t i a l hydrolysis or 6 degradation. However, by reference to the known chemical s h i f t s of the corresponding mono-saccharides, and to the chemical s h i f t s of the same sugar, with the same anomeric configuration i n other polysaccharides, i t was possible to make tentative assignments -(see Table V.2). The ^C-n.m.r. spectrum showed five resonance peaks i n the anomeric region, along with a signal for the CH-^  of rhamnose (17.61 p.p.m.) and one for the CH-^  of pyruvate (23»61 p.p.m.). The signal at 102.99 p»p«rn. had twice the in t e g r a l of the other peaks. In 1-^C-n.m.r. spectra, the chemical s h i f t s of the anomeric carbon atoms of sugars having the manno-configuration are very s i m i l a r regardless of whether they are a- or f$- l i n k e d , ^ 0 . The 1^C-n.m.r. spectrum of E. c o l i K26 polysaccharide showed no signals u p f i e l d from 101 p.p.m. which could be assigned to an a-rhamnosyl residue. TABLE V.2 H^-N.M.R. DATA FOR E. COLI K26 CAPSULAR POLYSACCHARIDE AND DERIVED OLIGOSACCHARIDES a b Compound 6 J1 2 Integral Assignment Chemical s h i f t ' of aldohexo-(Hz) pyranose (6) K26 5.10 3 a _ R h a 5.1. 4.91 2 J g-Rha 4.86 i e - G a l c 4 . 7 4 2 g-GlcA 4.7 1.59 CH^ of pyruvate 1 .35 CH^ of rhamnose K26 (depyruvylated) 5*08 4 . 9 0 4 . 7 3 1 .35 3 2 8 ct-Rha /B-Rha \ g-Gal B-GlcA CH^ Of rhamnose K26 o l i g o -s a c c h a r i d e 5.36 5.11 5.08 4.97 4.88 4.76 4.67 8 8 0.67 2 0.33 1 1 i m p u r i t y a-Rha-OH a-Rha i m p u r i t y 3-Rha-OH a Chemical s h i f t r e l a t i v e t o i n t e r n a l acetone; 2.23 d o w n f i e l d from sodium d i m e t h y l - 4 - s i l a p e n t a n e - 1 - s u l p h o n a t e (D.S.S.). b Chemical s h i f t s are r e c o r d e d o n l y f o r anomeric p r o t o n s , c Y. M. Choy and G.G.S. Dut t o n , Can. J . Chem., V o l . 51, 198-209, (1973). 65 During the course of a comparative n.m.r. study of rhamno-51 bioses, Dutton and co-workdrs-^ showed that an a-rhamnose sugar linked 1 - 5 to another a-rhamnose resonates at 1 0 3 * 1 1 p.p.m. As can be seen from Table V . 3 , a signal was present at 1 0 3 . 1 1 p.p.m. which can therefore be assigned to an a-rhamnosyl residue linked to another a-rhamnosyl unit. After depyruvylation, the signal at 1 0 2 . 0 4 p.p.m. sh i f t e d u p f i e l d to 99«75 p.p.m., demonstrating that t h i s peak may be assigned to a sugar residue, which i n the native poly-saccharide had a pyruvate substutuent. More precise assignments could not be made u n t i l the n.m.r. spectra (both ^ C and ^ H) of an oligosaccharide was a v a i l a b l e . V . 3 Conclusion These preliminary results therefore indicate that the structure of E. c o l i K26 capsular polysaccharide i s based on a hexasaccharide repeating u n i t . The hexasaccharide contains a g-glucuronic acid linked to a rhamnose residue. Possibly t h i s rhamnose sugar has an a-configuration and i s linked 1 - 3 to another a-rhamnose. Immunological cross-reactions of E. c o l i K26 with the anti-sera to a number of Pneumococcal K antigens were studied by Dr. M. Heidelberger. He attributed some of the cross-reactions to the possible presence of 3 - l i n k e d L-rhamnosyl residues (see Appendix XLT for his r e s u l t s ) . He also con-cluded that the glucuronic acid was hindered either con-TABLE V.3 13C-N.M.R. DATA FOR E. COLI K26 CAPSULAR POLYSACCHARIDE Compound p.p.m.— Assignment K26 polysaccharide 10/+. 16 103.39 103.11 a-Rha-(1-3)-a-Rha 102.99 102.04 23.61 CH^ of pyruvate 17.61 CH^ of rhamnose a Chemical s h i f t in p.p.m. downfield from Me^Si r e l a t i v e to in t e r n a l acetone; 31.07 p.p.m. downfield from D.S.S. 67 f o r r a a t i o n a l l y o r by a s u b s t i t u e n t . T h i s may be the e x p l a n a t i o n f o r t h e d i f f i c u l t y e n c o u n t e r e d i n the p r o c e s s of the p r e s e n t work i n a c h i e v i n g a good r e d u c t i o n of the g l u c u r o n i c a c i d r e s i d u e . The f o l l o w i n g p a r t i a l s t r u c t u r e i s c o n s i s t e n t w i t h the i n f o r m a t i o n p r e s e n t e d i n t h i s s e c t i o n . — ' G l c A p 1 — Rhap 1 2 Rhap 1 — g a a V.4 E x p e r i m e n t a l G e n e r a l methods. S o l u t i o n s were c o n c e n t r a t e d under d i m i n i s h e d p r e s s u r e a t b a t h t e m p e r a t u r e s not above 40°. The c o n c e n t r a t e d s o l u t i o n was f r o z e n u s i n g a d r y i c e - a c e t o n e m i x t u r e , and then f r e e z e - d r i e d on a U n i t r a p 11 f r e e z e -d r y e r t o g i v e the p o l y s a c c h a r i d e i n a s t y r o f o a m - l i k e form. D e i o n i z a t i o n s were performed on a column of A m b e r l i t e IR-120 ( H + ) r e s i n . The i n f r a r e d ( i . r . ) s p e c t r a of m e t h y l a t e d d e r i v a t i v e s were r e c o r d e d on a P e r k i n - E l m e r model 457 s p e c t r o p h o t o m e t e r . The s o l v e n t used was s p e c t r o s c o p i c grade carbon t e t r a c h l o r i d e . A n a l y t i c a l paper chromatography was performed by the d e s c e n d i n g method u s i n g Whatman No. 1 paper. Two s o l v e n t systems were used: 1) 18:3:1 ;4 e t h y l a c e t a t e - a c e t i c a c i d - p y r i d i n e - w a t e r and 2) 8:2:1 e t h y l a c e t a t e - p y r i d i n e - w a t e r . Sugars were d e t e c t e d a f t e r paper chromatography u s i n g a l k a l i n e 68 s i l v e r n i t r a t e . Unless otherwise s p e c i f i e d , analytic g . l . c . separations were performed with a Hewlett-Packard 5890A instrument f i t t e d with dual flame-ionization detectors. A Hewlett-Packard 3392A Integrator was used to quantify the peak areas. A c a p i l l a r y column, DB-17» was used with a helium c a r r i e r gas flow-rate of 25mL/min, programmed for 180° for 2min, followed by 5°/min to 220°. The re s u l t s of analysis by g . l . c . were confirmed by g.l.c,-ra.s. using a Carloerba 4160- Kratos M58ORFA instrument. A helium carrier-gas was used with a flow-rate of 30 cm/sec. Spectra were recorded at 70 eV with an i o n i z a t i o n current of 100/*.A and an ion source at 200°. Proton magnetic resonance spectra were recorded on a Bruker WH-400 instrument. Spectra were recorded at a temperature of 90^ ± 5° and acetone was used as an i n t e r n a l standard. A l l values are given r e l a t i v e l y to that of external sodium-4,4-dimethyl-4-silapentanesulphonate taken as 0. Samples (10-20 mg) were prepared by dissolving i n D 20 and freeze-drying 2-3 times from D^ O solutions. Tubes 5 ram i n diameter were used. Carbon-13 magnetic resonance spectra were recorded on a Bruker WH-400 spectrometer at ambient temperature. Acetone was used as the i n t e r n a l standard (3^07 p.p.m.). Samples (30-50 mg) were dissolved i n D^O and placed i n n.m.r. tubes of diameter sizes 5 ^ or 10 mm. 69 Preparation and properties of E. c o l i K26 capsular polysaccharide A stab culture of Escherichia c o l i bacteria K26 was obtained from Dr. I 0rskov (Copenhagen). The following media were used to grow the bacteria. 1) Mueller Hinton Broth, dehydrated, 2) Mueller Hinton agar, dehydrated. 0.5$ (w/v) NaCl was added to both the broth and agar media. Agar plates were made by pouring the s t e r i l e , l i q u i d nutrient agar into disposable P e t r i dishes (8.5 cm diameter). Six s t e r i l e , small trays (30 x 50 cm) each containing 1.5 L of s t e r i l e Mueller Hinton agar were used for the propagation of the bacteria. S t e r i l i z a t i o n of glassware and nutrient media was performed i n an American S t e r i l i z e r model 57-CR for 15 min at 121° and 15-20 p . s . i . The bacteria were grown and the polysaccharide i s o l a t e d as described i n Section 11. 1H-N.m.r. spectroscopy was performed on the o r i g i n a l polysaccharide. However, the v i s c o s i t y of the solution was high. A better spectrum was obtained a f t e r the v i s c o s i t y was reduced by the removal of pyruvate, by autohydrolysis for 1 h, on a steam-bath. See Appendix 11 for the reproduction of a l l n.m.r. spectra. Sugar analysis A-sample (7.2 mg) of K26 polysaccharide was hydrolysed with 2M t r i f l u o r o a c e t i c acid (TFA) overnight on a steam-bath, 70 followed by removal of the excess acid by c o d i s t i l l a t i o n with water. Paper chromatography, using solvent 2, showed four spots, rhamnose, galactose and/or glucose, glucuronic acid and an aldobiouronic aci d . The sugars released were converted to th e i r a l d i t o l s by reduction with sodium borohydride (~100 mg) i n water for 1 h. The mixture was neutralized with I.R. 120 (H +) resi n , f i l t e r e d and evaporated to dryness. The borate, produced by the decomposition of the excess sodium borohydride was removed by successive addition of methanol (5 mL), followed by evaporation to dryness. The product was then acetylated (1:1 acetic anhydride pyridine) f o r 1 h on a steam-bath, under anhydrous conditions, and analyzed by g . l . c . using a SP 2340 column programmed for 193° 2 min 2 % i i n 260° 32 min and a Hewlett-Packard 5700 instrument. The resu l t s showed the a l d i t o l acetates of rhamnose, galactose and glucose i n the r a t i o s 1:1:0.2 (see Table V . l ) . A sample of K26 polysaccharide (5 mg) was dried i n vacuo under an i . r . lamp (2 h), and hydrolysed overnight with methanolic hydrogen chloride(3%) on a steam-bath, under anhydrous conditions. The excess acid was neutralized with lead carbonate,.and the resultant lead chloride p r e c i p i t a t e was removed by centrifugation. The supernatant was evaporated to dryness, and the uronic ester reduced with sodium boro^ hydride i n anhydrous methanol overnight at room temperature. The mixture was neutralized with I.R. 120 (H +) resin, f i l t e r e d , evaporated to dryness and the borate ion removed by c o d i s t i l l a t i o n with methanol (3 times). The residue was 71 hydrolyzed overnight with 2M TFA. The sugars released, were converted to t h e i r a l d i t o l acetates by reduction with sodium borohydride, followed by acetylation. Analysis by g . l . c . gave rhamnitol hexaacetate, g a l a c t i t o l hexaacetate and g l u c i t o l hexaacetate i n the r a t i o 2.5:1:0.5 (see Table V . I ) . Carbodiimide reduction of K26 polysaccharide A sample of K26 polysaccharide was converted to the H + form by passage through an I.R. 120 (H +) resin column, and dissolved i n water (100 mL),' the resultant pH was if.5. 1-Cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulphonate (CMC-0.4 g) was added and as the reaction proceeded (with the consumption of hydrogen ions), the pH was maintained at 4.75 by the dropwise addition of 0.1M hydrochloric acid. After about 1 h, when the uptake of hydrogen ions had ceased, aqueous sodium borohydride solution (3M) was added dropwise (15 mL). Foaming was controlled by the addition of a few drops of 1-octanol. The pH of the solution was maintained at approximately 7 by the dropwise addition of hydrochloric acid (4M). After the addition of a few drops of hydrochloric acid to destroy any excess sodium borohydride, the polysaccharide solution was dialysed against tap water for three days, followed by l y o p h i l i z a t i o n ( y i e l d 49.6 g). A small sample of the reduced polysaccharide (5 nig) was hydrolysed with 2M TFA on a steam-bath overnight, followed by reduction with sodium borohydride and acetylation to i t s a l d i t o l acetates. The g. l . c . analysis indicated that the 72 reduction of the glucuronic acid was 66% complete (see Table V . I ) . However a second carbodiimide reduction did not improve the percentage reduction. 73 VI. BACTERIOPHAGE DEGRADATION OF E. COLI K26 CAPSULAR POLYSACCHARIDE 74 VI. BACTERIOPHAGE DEGRADATION OF E . c o l i K26 CAPSULAR POLYSACCHARIDE V1.1 Introduction The presence of pyruvate as a substituent makes the E. c o l i K26 capsular polysaccharide an i d e a l candidate for depolymerization by a phage-borne glycanase. The l a b i l e i pyruvate i s e a s i l y removed by any chemical techniques designed to produce oligosaccharides. In the present work, depoly-merization of K26 polysaccharide was carried out using phage 26 i s o l a t e d from sewage (courtesy of Dr. S. Stirm, Freiburg Germany). Phage 26 belongs to Bradley group C. It has an isometric head, both the length and diameter of which are greater than 600 nm. The t a i l consists of a base plate about 3.0 nm i n width and 41»0 nm i n length, carrying nine spikes of 12.0 to 14.0 nm i n length and 4.0 nm diameter (see F i g . 10). When plated out on a b a c t e r i a l lawn of i t s host bacterium, the plaques produced by 0 26 exhibit large haloes, demonstrating that the phage produces more than s u f f i c i e n t glycanase for depolymerization of the b a c t e r i a l capsule. V1.2 Results and discussion VI.2.1. Bacteriophage depolymerization of K26 polysaccharide Bacteriophage 26 was propagated on i t s host s t r a i n 75 by test-tube l y s i s under various conditions. When the experimental parameters had been established for obtaining optimum phage concentration, larger scale flask l y s i s was conducted. The res u l t s of the phage assays from test-tube l y s i s and flask l y s i s are tabulated i n Table VI. 1. TABLE V1.1 PROPAGATION OF BACTERIOPHAGE 26 I - II T i t r e (p.f.u./mL)- 109 9 x 109 Volume (mL) 15 2400 Total 16 (p.f.u.) 1.5 x 10 1 0 2.1 x 10 1 3 a. I, test-tube l y s i s ; I I, flask l y s i s ; b p.f.u. = plaque forming units The phage solution was dialysed for two days, concentrated, and added to a solution of p u r i f i e d K26 capsular polysaccharide. The mixture was incubated for two days at 37°. After 24 h a sample of the mixture was removed, exchanged with I.R. 120 (H +) r e s i n , and concentrated. Analysis by paper chromatography against a reference standard indicated that depolymerization had occurred. The reaction mixture was incubated for a further 24 h, after which the progress of the 76 reaction was again checked by paper chromatography.- The result of the second analysis was i d e n t i c a l to that of the f i r s t , i n d i c a t i n g that there had been no further depolymerization (see F i g . 11). The depolymerized products were separated from the polysaccharide-phage mixture by d i a l y s i s , p u r i f i e d by cation exchange using I.R. 120 (H +) r e s i n , and f i n a l l y l y o p h i l i z e d . K 1 7 * P2 K26 o 1 s K 1 7 PI u 0 0 0 \J 0 0 0 V 0 Figure 11. Paper chromatogram showing the r e s u l t s of the analysis of 26 degradation products. • K l e b s i e l l a K 1 7 i s a pentasaccharide containing rhamnose, glucose and glucuronic acid. V1.2.2 P u r i f i c a t i o n and analysis of products of depolymerization  of E. c o l i K26 capsular polysaccharide After freeze-drying, the depolymerization products were dissolved i n a minimum amount of d i s t i l l e d water and added to a column of Bio-Gel P2. The elution pattern i s shown i n F i g . 12. The contents of test-tubes 7 to 17 i n c l u s i v e were pooled to give a y i e l d of 120 mg. Analysis of the sugars obtained a f t e r acid hydrolysis 77 78 of a small sample of the oligosaccharide showed the presence of a large amount of impurity. Reduction of the oligosaccharide and removal of the impurity by preparative paper chromatography gave a reasonably pure product (15 .7 mg)« Methylation of the reduced oligosaccharide, followed by hydrolysis, d e r i v a t i z a t i o n as a l d i t o l acetates, and g . l . c -m.s. analysis gave the values shown i n Table V I . 2 . The oligosaccharide was examined by 1H-n.m.r. spectroscopy. The spectrum was very s i m i l a r to that of the native polysaccharide a f t e r depolymerization with d i l u t e acid. Examination of the spectrum permitted the assignment of the reducing end. The signal at 6 5.11 was assigned to ct- rhamnose and the signal at 6 4*88 was assigned to B-rhamnose The s p l i t t i n g of the a-rhamnose signal at 6 5.08 was attributed to an a-rhamnose being linked to either an a-rhamnose or a 8-rhamnose residue. The 1 H -n.m.r. data are summarized i n Table V . 2 . The presence of some remaining impurities made any further assignments d i f f i -cult . The data demonstrated that the bacteriophage enzyme was an a-rhamnosidase and that rhamnose was present at the reducing end of the oligosaccharide. This rhamnose was linked to galactose which became the non-reducing terminal a f t e r enzymatic cleavage of the polysaccharide. The oligo-saccharide was not a single repeating unit, since 1,3,5-tri-O-acetyl-2,4,6-tri-0-methyl g a l a c t i t o l was seen a f t e r methylation of the reduced oligosaccharide. This residue became the terminal 2,3,4,6-tetra-0-methyl g a l a c t i t o l a f t e r phage degradation. 79 TABLE VI . 2 METHYLATION ANALYSIS OF THE REDUCED OLIGOSACCHARIDE FROM THE BACTERIOPHAGE DEGRADATION OF E. COLI K26 POLYSACCHARIDE Methylated sugars— (as a l d i t o l acetates) Mole# 1 ,2,4,5-Rha O.O-2,3,4-Rha 4.8 2,4-Rha 22.2 2,3,4,6-Gal 21 .6 2,4,6-Gal 15.3 a 2,4,6-Gal = 1,3,5-0-acetyl -2,4,6-tri-0-methyl_galactitol etc. b Values are corrected by the use of the ef f e c t i v e carbon-. response factors given by Albersheim and. coworkers . c Certain sugars were absent or present i n only low concentra-tions due to the high v o l a t i l i t y of the derivative (1,2,4>5-Rha) or incomplete hydrolysis of the glucosyl-uronic linkage (2,4-Rha). 80 The r e s u l t s of the m e t h y l a t i o n a n a l y s i s of the o l i g o s a c c h a r i d e i n d i c a t e d the presence o f a t e r m i n a l rhamnose su g a r . A m i l d h y d r o l y s i s o f t h e E. c o l i . K26 p o l y s a c c h a r i d e was t h e r e f o r e c a r r i e d out t o t r y t o s e l e c t i v e l y remove the t e r m i n a l rhamnose s u g a r . Removal of t h i s s u g a r would show th e p o s i t i o n of the b r a n c h p o i n t i n the p o l y s a c c h a r i d e c h a i n . The h y d r o l y z e d p r o d u c t was m e t h y l a t e d , reduced, h y d r o l y z e d and c o n v e r t e d t o i t s a l d i t o l a c e t a t e s . The r e s u l t s o f the g . l . c . a n a l y s i s a r e g i v e n i n T a b l e V1 . 3 . The presence of 2 , 4-di-C)-methyl g l u c i t o l i n d i c a t e d t h a t g l u c u r o n i c a c i d was the b r a n c h p o i n t and t h a t the rhamnose r e s i d u e was l i n k e d t o p o s i t i o n Zf. The presence o f 2 - 0-methyl g l u c i t o l i n d i c a t e d t h a t not a l l the t e r m i n a l rhamnose had been removed. A l t h o u g h the a c i d h y d r o l y s i s c o n d i t i o n s were m i l d some c l e a v a g e of the p o l y s a c c h a r i d e c h a i n had a l s o o c c u r r e d , as i n d i c a t e d by the presence of 2 , 3 » 4 , 6-tetra - 0 rmethyl g a l a c t i t o l . No new t e r m i n a l s u g a r s were produced t h e r e f o r e the t e r m i n a l rhamnose u n i t i s l i n k e d d i r e c t l y t o the g l u c u r o n i c a c i d . 81 TABLE VI . 3 METHYLATION ANALYSIS OF THE PRODUCT FROM THE SELECTIVE HYDROLYSIS OF E. COLI K26 POLYSACCHARIDE Methylated sugars 3-(as a l d i t o l acetates) Mole# 2 , 3 , 4 - R h a 2 , 4 - R h a 2 3 . 3 2 , 3 , 4 , 6 - G a l 3 . 7 2 , 4 , 6 - G a l 8 . 5 2 , 4 - G l c 3 . 9 2-Glc 4 . 5 a 2 , 4 , 6 - G a l = 1 , 3 > 5 - 0 - a c e t y l - 2 , 4 , 6 - t r i - O - m e t h y l g a l a c t i t o l etc. b Values are corrected by the use of the ef f e c t i v e carbon-response factors given by Albersheim and pp coworkers . 82 VI.3 Conclusion E. c o l i K26 polysaccharide was previously under investi g a t i o n by another member of thi s laboratory 52 1 .The number and type of sugars contained by t h i s polysaccharide were established, and the aldobiouronic acid, g-GlcA-C1-3)-Rha, was i s o l a t e d . E. c o l i K26 polysaccharide has proved d i f f i c u l t to work with, due to the large number of rhamnose sugars i t contains. Rhamnose derivatives tend to be v o l a t i l e and e a s i l y l o s t during methylation analyses. However, combination of the data from Sections V and VI resulted i n a number of st r u c t u r a l features of the polysaccharide becoming clear: 1) The polysaccharide has a side chain and g-glucuronic acid i s the branch point. 2) Rhamnose i s the terminal sugar of the side chain, and i s d i r e c t l y linked to the 4 position of glucuronic acid. 3) The main chain contains the disaccharide a-Rha-(1-3)-ct- Rha. 4) Pyruvate i s present as a substituent, either on the terminal rhamnose or the 3-linked galactose. 5) The 3-linked galactose i s present i n the main chain and i s linked to rhamnose. The position of the pyruvate substituent could not be determined using the oligosaccharide obtained by phage degradation because the 1H-n.m.r. spectrum showed that the E. c o l i K26 polysaccharide had been produced without pyruvate. 8 3 Two alternative structures may be written for the E. c o l i K26 polysaccharide: 1) -^GlcAp} ^Rhap1 ^Rhap1 -^ Rhap- ^Galp 1— a a a 6 6 Rhap 2) -^GlcAp 1 ^Rha1 ^Rha- -^Galp- ^Rhap— a k 6 1 B R h a p Absolute configuration of the sugars may be ascertained by 55 measurement of c i r c u l a r dichroism of the a l d i t o l acetates-^ . Anomeric configuration' i s obtained by chromic acid oxidation of the f u l l y a c e t y l a t e d aldopyranosides with equatorially attached aglycons i n the most stable chair form ( i . e g-anoaers)^, Further experiments are underway to establish which of the two possible structures i s correct for E. c o l i K26 polysaccharide. However, t h i s work was temporarily halted when fi v e new polysaccharides, E. c o l i K<+6-50, became avai l a b l e . Nimmich has reported the qu a l i t a t i v e composition of most K l e b s i e l l a K serotypes^, but no such comprehensive screening has been undertaken for E. c o l i K serotypes. Thus, i t was necessary for a preliminary s t r u c t u r a l i n v e s t i g a t i o n to be carried out on these new E. c o l i polysaccharides. 8 4 VI.k Experimental General methods The equipment for n.m.r. spectroscopy, g . l . c . and g.l.c.-m.s. was the same as used i n the investi g a t i o n of E. c o l i K26 polysaccharide (see Section V . 4 ) . Descending paper chromatography and preparative paper chromatography were carried out using as solvent a mixture of ethyl acetate, acetic acid, formic acid and water (18 : 3".1 : 4 ) and Whatman No. 1 chromatography paper. Unless otherwise s p e c i f i e d , g . l . c . separations were carried out using a DB 17 c a p i l l a r y column (programme - 180° for 2 min, 5°/min to 220°). Q Using an o r i g i n a l phage solution of 4 x 1 0 p.f.u./mL concentration, a t r i a l t e s t - l y s i s was undertaken to determine the most favourable conditions for phage propagation. An active E. c o l i K26 colony was added to 5 mL s t e r i l e Mueller Hinton broth and incubated overnight at 3 7 ° . Six test-tubes of s t e r i l e Mueller Hinton broth ( 5 mLO were each inoculated with 0.2 mL of the a c t i v e l y growing b a c t e r i a l culture and further incubated for 1 h. At 1 5 min i n t e r v a l s , consecutive tubes were inoculated with phage solution (0.2 mL). Incubation was continued for a further 5 h. The f i r s t test-tube cleared a f t e r 120 min but the succeeding f i v e did not cle a r . Chloroform (1 mL) was added to a l l the tubes. The contents of the tubes were then centrifuged, and both the solution which cleared and those that did not were assayed to determine the phage concentration. The b a c t e r i a l lawn required for the phage assay was 85 prepared as follows. A single b a c t e r i a l colony was incubated i n 5 mL Mueller Hinton broth u n t i l the culture just began to turn cloudy (approximately 3-4 h). 3 mL of the culture was then pipetted onto an agar plate which had been dried i n an incubator for 1 h at 37°» After 15 min the excess l i q u i d was removed and the plate allowed to dry i n the incubator with the l i d p a r t i a l l y removed. The phage assay was performed as follows. A Pasteur pipette was drawn to a fine t i p and calibra t e d for drops per mL. The phage suspension (0.1 mL) was dil u t e d hundred-fold by adding to s t e r i l e Mueller Hinton broth (9»9 mL). This process was repeated successively u n t i l a d i l u t i o n range of 10""1 to 10*"10 was obtained. One drop of each phage d i l u t i o n was applied (with the calibrated pipette) to the dried agar plate. The plate was l e f t at room temperature overnight and the p.f.u. per mL were calculated from the number of plaques formed. Four propagations by flask l y s i s were necessary to obtain s u f f i c i e n t quantities of 0 26. Six Erlenmeyer flasks (250 mL) each containing 100 mL s t e r i l e Mueller Hinton broth were inoculated with an a c t i v e l y growing b a c t e r i a l culture (5 mL) and incubated at 37° for 1 h. If mL phage solut i o n (concentra-ti o n 3 x 10 9 p.f.u./mL) was added to each flask and incubation continued f o r k h u n t i l the f l a s k contents had cleared. The procedure was then the .same as for test-tube l y s i s . Bacteriophage depolymerization of K26 capsular polysaccharide Successive 0 26 propagations using test-tube l y s i s 55 and f l a s k l y s i s were carr i e d out (see Table VI. 1), Stirirr-' 86 had shown that 1 0 ^ bacteriophage are needed to degrade 1 g of the corresponding b a c t e r i a l capsular polysaccharide. The technique used to depolymerize E. c o l i K26 polysaccharide was developed i n t h i s l a b o r a t o r y ^ . The crude phage solution (2.1 x l O 1 ^ p.f.u.) i n broth was concentrated by evaporation i n vacuo and dialysed against tap water for 2 d. After further concentration, the phage solution was added to an aqueous solution of the p u r i f i e d polysaccharide (1 g dissolved i n 250 mL water). Depolymeriza-tion was allowed to proceed for 2 d i n a water-bath at 3 7 ° , chloroform being added to prevent b a c t e r i a l growth. The progress of the reaction was monitored by paper chromatography. The mixture was concentrated and dialyzed (cut off 3500 daltons) against d i s t i l l e d water u n t i l the Molisch test was no longer positive (6X). The dialyzates were combined and treated with I.R. 120 (H +) cation-exchange r e s i n . After each-exchange, the dialyzate was freeze-dried and weighed. This process was repeated u n t i l the weight of the l y o p h i l i z e d product was constant. The product was dissolved i n 1 mL d i s t i l l e d water and applied to a Bio-Gel P2 column (exclusion l i m i t 1800 daltons). The column was eluted with buffer (water—pyridine-acetic acid, 1000:10:4) at a flow-rate of 10 . 5 mL/h. The eluant was collected i n weighed test-tubes ( 2 . 5 mL per test-tube). The re s u l t indicates c l e a r l y that there was only a single component of which 120 mg were obtained to give a y i e l d of 12$. 87 Total hydrolysis A 5 rag sample was hydrolyzed overnight with 2M TFA on a steam-bath. The monosaccharides obtained were reduced with sodium borohydride (100 mg), i n water, to t h e i r a l d i t o l and then acetylated with a 1:1 mixture of acetic anhydride and pyridine. G.l.c. analysis showed an extremely large impurity peak with a retention time s i m i l a r to that of glucose/galactose. Reducing end determination The oligosaccharide (16 mg) was reduced with sodium borohydride overnight and dried under the in f r a - r e d lamp for 1 h. The sample was methylated by the Hakomori procedur and extracted with dichloromethane. The f i n a l traces of dimethyl sulphoxide were removed by passage through a column of LH 20 r e s i n . The p a r t i a l l y methylated oligosaccharide was hydrolyzed overnight, with 2M TFA on a steam-bath. The p a r t i a l l y methylated monosaccharides were converted to t h e i r a l d i t o l acetates and analyzed by g.l.c.-m.s. (column DB 225 programmed for 1 7 0 ° for 1 min then 3°Aiin to 220°). P a r t i a l hydrolysis A sample (10 mg) of E. c o l i K26 polysaccharide was hydrolyzed with 0.1M TFA for 15 min. The hydrolyzate was evaporated to dryness and c o d i s t i l l e d with water (3X) to remove the l a s t traces of acid. The residue was dissolved i n water and dialyzed against tap water for 1 d. The retentate was concentrated and freeze-dried. The sample 8 8 was then methylated using the Hakomori method, reduced with lithium aluminium hydride and hydrolyzed with 2M TFA for k h. The p a r t i a l l y methylated monosaccharides were converted to t h e i r a l d i t o l acetates and analyzed by g.l.c.-tn.s. (column DB 1? programmed for 180° f o r i min then 2%>in to 220°). 89 QUALITATIVE AND QUANTITATIVE SUGAR ANALYSIS OF E. COLI K 4 6 , KV7, K48, Kk9 and K50 CAPSULAR POLYSACCHARIDES 90 VII. QUALITATIVE AND QUANTITATIVE SUGAR ANALYSIS OF E. COLI Ki+6, KZ+7, K48, K/+9 AND K50 CAPSULAR POLYSACCHARIDES V11.1 Introduction E. c o l i K46, K 4 7 , K48, KZ+9 and K50 serotypes belong toO group 8 and were formerly c l a s s i f i e d as belonging to the A group of antigens, i . e . they are true capsular polysaccharides and have high molecular weights and low electrophoretic m o b i l i t i e s . As previously mentioned i n Section V.1, K antigens with low electrophoretic mobility can be divided into two groups: 1) The true capsular polysaccharides which do not contain amino sugars, and 2) the lipopolysaccharides, the polysaccharide moiety of which contains one or more amino sugars^ 9. This has been true for a l l K l e b s i e l l a K antigens and also, u n t i l recently, for the E. c o l i K antigens whose compositions are known. However, work at present being undertaken i n this laboratory now indicates that true capsular polysaccharides (A antigens) may contain amino sugars. The results of a preliminary q u a l i t a t i v e and quantitative sugar analysis of E. c o l i capsular polysaccharides K46-50 are presented here. V11.2 Results and Discussion A l l E. c o l i K/+6-50 bacteria were grown on Mueller Hinton agar and p u r i f i e d by one p r e c i p i t a t i o n with Cetavlon. 91 E. c o l i K46 capsular polysaccharide .Paper chromatography of the acid hydrolyzate (18 h, 2M TFA) of the K46 polysaccharide showed the presence of galactose, rhamnose, glucuronic acid and possibly fucose. Conversion of the sugars to t h e i r a l d i t o l acetates, followed by g . l . c . analysis gave only galactose and glucose i n the r a t i o 1:0.05« The hydrolytic conditions were gradually increased from 18 h to 48 h, s t i l l using 2M TFA. After 24 h hydrolysis, g . l . c . analysis of the a l d i t o l acetates gave galactose, glucose and rhamnose i n the r a t i o 1.0:0.2:0.1; these ra t i o s remained s u b s t a n t i a l l y the same even a f t e r 48 h hydrolysis (see Table V l l .1). However, when the polysaccharide was hydrolyzed , for 4 h with 4M HCl, 2-amino-2-deoxy glucose (.glucosamine) and a small amount of 2-amino-2-deoxy galactose (galactosamine) were released. These results indicated that E. c o l i K46 polysaccharide contained rhamnose, glucose, galactose and glucosamine (and possibly galactosamine). Confimation of the presence of glucosamine was obtained when deamination of the hydrolysed polysaccharide, followed by g . l . c . analysis , gave 2,5-anhydromannose. No 2,5-anhydro-talose was observed which was unexpected since t h i s sugar should have been produced from the deamination of galactos-amine (see Table V11.6). However, the amount of the 2,5-anhydromannose was less than half the amount of glucosamine observed i n a control sample of the polysaccharide. Thus, the absence of 2,5-anhydrotalose may have been due to the low yi e l d s of the deamination products obtained. Methanolysis followed by reduction gave an increase 92 i n the amount of glucose, thereby i n d i c a t i n g that the polysaccharide contained a glucuronic acid residue (see Table VI1 .7). The r a t i o for glucose to galactose increased to 0 . 3 :1 .0 . The 1H-n.m.r. spectrum of the E. c o l i K46 poly-saccharide exhibited three main peaks i n the anomeric region at 6 4.58, 64.98 and 6 5-16. The peak at 64.58 was assigned to g-galactose, since a signal i s observed i n this area i n a l l E. c o l i K46-50 polysaccharide ^H-n.m.r. spectra. Each polysaccharide contains more than one glactose residue, and the i r respective spectra a l l show a large signal at approximately 64*58. The peak at 64*98 was not assigned , but the 65.16 signal may be assigned t e n t a t i v e l y to an a-rhamnose residue. The two signals observed at 6 2 .02 and 6 2 .0 were assigned to two amino sugars and the signal at 6 1.32 was attributed to the CH^ of rhamnose. The poor quality of the H-n.m.r. spectrum (not only for t h i s polysaccharide, but also for K47-K49 polysaccharides) made i t d i f f i c u l t to draw any conculsion about the number of sugars i n the repeating unit. Unfortunately a l l attempts to improve the spectrum for example, by mild hydrolysis with 0.1M NaOH, or autohydrolysis at pH 3-4,or by cation exchange with I.R. 120 (H +) res i n , were unsuccessful. 93 TABLE VII.1 SUGAR ANALYSIS OF E. COLI K 4 6 CAPSULAR POLYSACCHARIDE Sugars^ (as a l d i t o l Mole r a t i o acetates) I II I I I IV Rhamnose 0.12 0.14 0.14 0.1 1 Glucose 0.05 0 . 2 3 0.24 0.23 0 . 2 3 Galactose 1.0 1.0 1.0 1 .0 1.0 Glucosamine 0.16 Galactosamine 0.04 TABLE V11.2 SUGAR ANALYSIS OF E. COLI K47 CAPSULAR POLYSACCHARIDE Sugars- Mole r a t i o (as a l d i t o l acetates) I II I I I IV V ^ Fucose 0.08 0 . 0 5 Glucose 0.04 0 . 0 5 0.07 0.07 0.07 Mannose 0.07 0.07 0.08 0.08 0.08 Galactose 1 .0 1.0 1.0 1 .0 1.0 Glucosamine 0.19 Galactosamine trace a Using DB 1 7 column programmed f o r 180° fo r 2 min 5 /mm to 220°. b H y d r o l y t i c conditions - I, 18 h 2M TFA; I I , 2 4 h 2MTFA; I I I , 3 6 h 2M TFA: IV, 48h 2M TFA; V, 4 h HCl. 94 E. c o l i K47 capsular polysaccharide After hydrolysis of a sample of E. c o l i K47 polysaccha-saccharide (18 h 2M TFA), paper chromatography showed the presence of galactose, glucose, mannose, glucuronic acid and fucose. Conversion of the sugars to th e i r a l d i t o l acetates, followed by g . l . c . analysis gave glucose, mannose and galactose i n the r a t i o 0.04:0.07:1.0. Increasing the hydrolysis time to 24 h showed the presence of fucose,(see Table VI1.2). However,when the hydrolysis time was increased to 48h, no fucose was observed on g . l . c . analysis of the a l d i t o l acetates. Hydrolysis of E. c o l i K47 polysaccharide using 4M HCl for 4 h and conversion of the hydrolyzate to the a l d i t o l acetates, gave glucose, mannose, galactose, glucosamine and a trace of galactosamine i n the r a t i o 0.07:0.08:1.0:0.19. Deamination of a sample of hydrolyzed polysaccharide followed by g . l . c . analysis of the a l d i t o l acetates, showed the presence of 2,5-anhydromannose. S i g n i f i c a n t l y 2,5-anhydro-talose was not observed. Methanolysis, followed by reduction, increased the amount of glucose released upon hydrolysis (see Table VI1.7) . There was also a concomitant increase i n the amount of glucos-amine released, thus i n d i c a t i n g that the glucuronic acid i s linked to glucosamine. The 1H-n.m.r. spectrum of E. c o l i K47 polysaccharide exhibited s i x peaks i n the anomeric region (see Table V11.8). The signal at 6 4.58 was assigned to e-galactose. The signal at 6 5.16 was te n t a t i v e l y assigned to an a-mannose residue. The rest of the anomeric signals were unassigned. 9 5 The signal at 6 2.09 was assigned to the N-acetyl group of glucosamine. The signal at 61.6 had an i n t e g r a l that correlated to 3 hydrogens. Although i t occurred at lower f i e l d than was usual for the CH^ of pyruvate (-^1.5) i t was s t i l l i n the correct region of the spectrum and was therefore assigned to a pyruvate substituent. The most noticeable feature of the E. c o l i KZ+7 capsular polysaccharide was the large amount of galactose present, as shown by the ^H-n.m.r. spectrum and acid hydrolysis. Reprecipitation with Cetavlon did not remove any of t h i s galactose. Before any further studies are carried out on th i s polysaccharide, i t must be regrown to confirm that the galactose i s part of the polysaccharide structure and not an impurity. E. c o l i K48 capsular polysaccharide After hydrolysis for 18 h with 2M TFA, paper chromatography of the acid hydrolyzate of E. c o l i KZ+8 polysaccharide gave three d e f i n i t e spots, i n d i c a t i n g the presence of galactose, glucose and glucuronic acid. A fa i n t spot was also observed possibly due to the presence of fucose. Conversion of the sugars to t h e i r a l d i t o l acetates, followed by g . l . c . analysis gave glucose and galactose i n the r a t i o 0.2:1.0 (see Table V11.3). However analysis by g.l.c.-m.s. showed that a small amount of rhamnose was present. The presence of rhamnose was. confirmed when g. l . c . analysis of the acid hydrolyzate ( 3 6 h 2M TFA) a l d i t o l acetates gave rhamnose, glucose and galactose i n the r a t i o 0.09:0.3:1«0. 96 Hydrolysis with 4M HCl for 4 h, followed by conversion of the sample to i t s a l d i t o l acetates gave rhamnose, glucose, galactose, glucosamine and galactosamine i n the r a t i o 0.04: 0 . 3 : 1 . 0 : 0 . 6 : 0 . 0 9 (see Table V11 . 3 ) . . Deamination of a sample of E. c o l i K48 polysaccharide resulted i n the production of 2,5-anhydromannose, the amount of which agreed with the amount of glucosamine present i n the o r i g i n a l polysaccharide ( 0 . 5 7 and 0 . 6 mole% respectively, see Table V 1 1 . 6 ) No 2,5-anhydrotalose was observed. Methanolysis of E. c o l i K48 gave rhamnose, glucose, galactose, glucosamine and galactosamine i n the r a t i o 0 . 0 3 : 0./+: 1 • 0 : 0 . 4 5 : 0 . 7 . Methanolysis followed by reduction gave an increase i n the amount of glucose. However, the r a t i o of galactose to glucosamine showed the most s i g n i f i c a n t change going from the r a t i o 1 . 0 : 0 . 4 5 before reduction to the r a t i o 1 . 0 : 0 . 9 5 a f t e r reduction. These results indicated that the glucosamine was present as an.acidic amino sugar. It i s probable that the polysaccharide also contained glucuronic acid, and that the low amounts of galactosamine and rhamnose were due to these sugars being linked to the ac i d i c sugars. The ^H-n.m.r. spectrum of E. c o l i K48 polysaccharide showed a signal at 61 . 3 6 which confirmed the presence of rhamnose. Two signals at 61 . 9 7 and 6 2.04 (with an i n t e g r a l of 2) were attributed to the CH^ of the acetyl groups of three amino sugars (see Table V I 1 . 8 ) . The anomeric region of the spectrum was not well resolved and the only assignment made was that of the signal at 6 5*05 which was attributed to rhamnose. 97 E. c o l i K49 capsular polysaccharide A small sample of E, c o l i K49 polysaccharide was hydrolyzed for 18h with 2M TFA. Paper chromatography of the hydrolyzate showed the presence of galactose, glucose, gluc-uronic acid and a trace of fucose. After conversion of the hydrolyzate to i t s a l d i t o l acetates, g . l . c . analysis gave glucose, galactose i n the r a t i o 0.2:1.0. G.l.c.-m.s. showed that a very small amount of fucose was also present. After hydrolysis for 24 h with 2M TFA no glucose was observed on g. l . c . analysis of the hydrolyzate. However, when hydrolysis was extended to 3 6 h, glucose was again released, i n the same r a t i o as previously. Anomalies of t h i s type were attributed to unintentional variations i n the hydrolytic conditions. Hydrolysis of the polysaccharide with i+MHCl for if h gave the a l d i t o l acetates of glucose, galactose, glucosamine and galactosamine i n the r a t i o 0.2:1.0:0.04:0.1 4. Deamination of the polysaccharide followed by g . l . c . analysis of the a l d i t o l acetates showed the presence of 2,5-anhydrotalose. No 2.5-anhydromannose was observed. Methanolysis of E. c o l i K49 capsular polysaccharide gave the a l d i t o l acetates of glucose and galactose i n the r a t i o of 0.2:1.0. After methanolysis followed by reduction, the r a t i o of glucose to galactose increasedto 0.3:1.0. This result indicated that E. c o l i KZ+9 polysaccharide contained a glucuronic acid residue. The 1H-n.m.r. spectrum of E. c o l i K49 polysaccharide was poor. A large signal at 64.58 was again observed, but resolution of the rest of the spectrum was inadequate and was 9 8 TABLE V I I . 3 SUGAR ANALYSIS OF E. COLI K48 CAPSULAR POLYSACCHARIDE <- a Sugars— (as a l d i t o l Mole r a t i o acetates) I II I I I IV Rhamnose 0.09 0.04 Glucose 0.2 0.22 0 .3 0.26 Galactose 1.0 1.0 1.0 1.0 Glucosamine 0.6 Galactosamine 0.09 TABLE VII.4 SUGAR ANALYSIS OF E. COLI Kif9 CAPSULAR POLYSACCHARIDE Sugars— (as a l d i t o l Mole r a t i o acetates) I II I I I IV Glucose 0.2 0.2 0.21 Galactose 1.0 1.0 1.0 1.0 Glucosamine 0.04 Galactosamine 0.14 a Using DB 17 column programmed f o r 180° f o r 2min, 5°/ min to 220°. b H y d r o l y t i c conditions - I, 18 h 2M TFA: I I , 24 h 2M TFA; I I I , 36 h 2M TFA; IV, 48 h 2M TFA; V, 4 h 4M HCl. 99 not enhanced by the usual methods employed to improve 1H-n.m.r. spectra of polysaccharides (see discussion of KZ+6 capsular polysaccharide). E. c o l i KSO capsular polysaccharide Paper chromatography of the acid hydrolyzate of E.  c o l i K50 polysaccharide (18 h 2M TFA) indicated the presence of rhamnose, galactose, glucose, mannose, glucuronic acid, and a trace of fucose. G.l.c. analysis, a f t e r conversion of the sample to i t s a l d i t o l acetates, gave rhamnose, mannose, glucose and galactose i n the r a t i o 0.25 :0.15 :0.1:1 .0 . These r a t i o s remained f a i r l y constant even though the hydrolysis times were gradually increased to 48 h (see Table V11.5). No amino sugars were observed for E. c o l i K50 a f t e r hydrolysis with 4M HCl for 4h. However, hydrolysis with 2M TFA for 44 h, followed by g . l . c . analysis of the a l d i t o l acetates revealed glucosamine and galactosamine. G . l . c -m.s. analysis confirmed the presence of the two amino sugars. Deamination of E. c o l i K5Q polysaccharide gave 2,5-anhydromannose and thus confirmed that the polysaccharide contained glucosamine. . 2,5-Anhydrotalose was not observed -even though the amount of galactosamine obtained by the hydrolysis of the o r i g i n a l polysaccharide was s u f f i c i e n t for the deaminated product to have been detected. Methanolysis of the native polysaccharide gave rhamnose, mannose, glucose, galactose, glucosamine and galactosamine i n the r a t i o 0.19:0.5:0.15:1.0:0.34:0.24. After reduction of the methanolysate, the amounts of glucose, rhamnose and galactos-100 TABLE VI 1 . 5 SUGAR ANALYSIS OF E. COLI K 5 0 CAPSULAR POLYSACCHARIDE Sugars-3 (as a l d i t o l . Mole r a t i o acetates I II III IV V V1*> Rhamnose 0 . 2 5 0.26 0 . 3 6 0 . 3 0 . 2 5 0.21 Mannose 0 . 1 5 0.15 0 . 1 5 0.16 0 . 3 1 0.12 Glucose 0.1 0.11 0.11 0 . 1 3 0.11 0 . 1 3 Galactose 1.0 1.0 1.0 1.0 1.0 1.0 Glucosamine 0.2 Galactosamine 0.16 a Using DB 17 column programmed for 180° for 2 min, 5°/min to 220°. b Hydrolytic conditions - I, 18 h 2M TFA ; II , 24 h 2M TFA; I I I , 36 h 2M TFA; IV, 48 h 2M TFA; V, 4 h 4M HCl; V1, 44 h 2M TFA. 101 amine increased (see Table V11.7), i n d i c a t i n g that the poly-saccharide contained glucuronic acid possibly linked to rhamnose* The increase i n galactosamine suggested either that i t was an acidic amino- sugar or, a l t e r n a t i v e l y , that i t , and not rhamnose, was linked to the glucuronic acid residue. The 1H-n.m.r. spectrum of E. c o l i K50 polysaccharide showed seven signals i n the anomeric region (see Table VI1 .8); the signal at 6 4 . 5 8 had an i n t e g r a l of three and was assigned .to a g-galactose residue. The signal at 6 5.06 was assigned to a-rhamnose, the remaining f i v e signals being unassigned. The t o t a l number of signals observed i n the anomeric region corresponded to nine sugars per repeating unit, while the t o t a l sugar r a t i o obtained from methanolysis plus reduction was only eight. Therefore i t i s probable that one of the signals was due to an impurity. The peak at 6 5 * 1 5 had a low i n t e g r a l and was thus a l i k e l y candidate. Two signals were observed at 62.12 and 62.08; these were assigned to the CH^ of the acetyl.groups of two amino sugars. The two signals at 61.49 and 61.3 were assigned to the CH^ groups of pyruvate and rhamnose respectively. V11.3 .Conclusion A l l the capsular polysaccharides i n this study (E. c o l i K46-50) have proved to be acid i c polysaccharides containing glucuronic acid. In the case of E. c o l i K 5 0 (and possibly Ki+7) polysaccharide, pyruvate was a substituent. In thi s they conform to the previously accepted s t r u c t u r a l pattern TABLE VI1.6 RESULTS OF THE DEAMINATION ANALYSIS OF E. COLI CAPSULAR POLYSACCHARIDES KZ+6-50 E. c o l i capsular polysaccharide Amino sugar composition before deamination— (mole ratio) I II Amino sugar composition af t e r deamination— (mole ratio) III 1V K46 K47 K/+8 K49 K50 0.17 0.19 0.60 0.04 0.20 0.04 0.09 0.14 0.16 0.07 0.06 0.57 0.08 0.19 a After hydrolysis with 2M HCl for 4 h. I = glucosamine; II = galactosamine; III = 2,5-anhydromannose; 1V = 2,5-anhydrotalose 103 TABLE VI1.7 RESULTS OF THE KETHANOLYSIS AND METHANOLYSIS PLUS REDUCTION OF E. COLI K 46 - 5 0 E. c o l i Mole r a t i o polysaccharide Rha Man Glc Gal GlcKH 2 GalNHg K 4 6 I-I M t-i 0 . 1 7 0.10 0.28 0 . 3 3 1.0 1.0 K47 I II 0 . 0 9 0.08 0.08 0 . 1 4 1.0 1.0 0.06 K 4 8 I II 0.03 0 . 1 3 0 . 4 0 . 5 2 1.0 1.0 0 . 4 5 0 . 9 5 0.07 0.11 K 4 9 I II 0.20 0 . 3 1 1.0 1.0 K50 I 0 . 1 9 0 . 3 0 0 . 1 5 1.0 0 . 3 4 0 . 2 4 II 0.27 0 . 3 8 0 . 2 6 1.0 0 . 3 5 0 . 3 3 I - methanolysis without reduction. II - methanolysis followed by reduction. 104 TABLE V 1 1 . 8 H-N.M.R. DATA FOR K46-50 CAPSULAR POLYSACCHARIDE E. c o l i polysaccharide 6 a 1 H-n.m.r. data Integral (H) J 1 , 2 (Hz) Assignment K46 5 . 1 6 4 . 9 8 4 . 5 8 2 . 0 6 2 . 0 1 . 9 3 1 . 3 2 1 .0 1 .0 5.0 1 .0 1 . 0 0.5 ct-Rha 6-Gal CH^ of acet-amido sugars Impurity CH^ of rhamnose 5 . 4 2 1.0 5 . 1 6 1 . 0 ct-Rha 4 . 9 3 8 1.0 K47 4 . 8 6 1 . 0 4 . 6 8 2.0 4 . 5 8 e-Gal 2.09 CH^ of acet-amido sugar 1 .6 CH-j of rhamnose 5 . 0 5 1 . 0 a-Rha K48 4 . 5 4 2.04 2.0 CH,of acet-amido sugar 1 0 5 TABLE VI1.8 (continued) K48 (-continued) 1.97 1.36 K50 3.15 5.09 5.06 4.89 4.85 4.68 4.58 2.12 2.08 1.49 1.35 1.0 CH,-.of acet-amido sugar CH^ of rhamnose 0.75 1.0 1.0 1 .0 1 .0 1 .0 3.0 1 .0 1 .0 a-Rha g - G a l CH-, of acet-. amido sugar CH^ of pyruvate CH^ of rhamnose a Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; 6 2.23 down-f i e l d from sodium Z+, 4-dimethyl-4-silapentane-1-sulphonate (D.S.S). 106 for the true capsular polysaccharides (A antigens). However, E. c o l i K46-50 capsular polysacharides also contain amino sugars. True capsular polysaccharides were previously thought not to contain amino sugars. K./+6-50 polysaccharides are not the only true capsular polysaccharides which have been shown to contain amino sugars. E. c o l i and K45 capsular polysaccharides, also being investigated i n t h i s laboratory, 56 have been found to contain amino sugars- . To summarize, the st r u c t u r a l information obtained i n th i s preliminary study of E. c o l i K46-50 capsular poly-saccharides has shown the following:-1) E. c o l i K46 capsular polysaccharide - heptasaccharide containing rhamnose, glucuronic acid, galactose, glucosamine and galactosamine i n the r a t i o 1:1:3:1:1« 2) E. c o l i K47 capsular polysaccharide - i t was not possible to determine the number of sugars i n the repeating unit due to the large amount of galactose. However, the polysaccharide contains glucuronic acid, mannose, galactose and glucosamine. 3) E. c o l i K48 capsular polysaccharide - heptasaccharide containing rhamnose, glucuronic acid, galactose, glucosamine and galactosamine i n the r a t i o 1:1:2:2:1. 4) E. c o l i K49 capsular polysaccharide - as with E. c o l i KV7, the information i s not conclusive enough to determine the number of sugars per repeating unit, but the polysaccharide contains glucuronic acid, galactose, galactosamine and possibly glucosamine. 5) E. c o l i K50 capsular polysaccharide - octasaccharide containing rhamnose, mannose, glucuronic acid, galactose, 107 glucosamine and galactosamine i n the r a t i o 1:1:1:3:1:1. V11.4 Experimental General methods. The equipment for n.m.r. spectroscopy, g . l . c . and g.l.c.-m.s. was the same as used i n the investigation of E. c o l i K26 polysaccharide (see Section V.4). For descending paper chromatography, the solvent systems used for E. c o l i K26 polysaccharide were employed. A n a l y t i c a l g . l . c . separations were carried out with a DB 17 c a p i l l a r y column, which was shown to give good separation of the a l d i t o l acetates of amino sugars (programme - 180° for 2 min, 5° / min to 220°). Preparation and Properties The cultures of E. c o l i K46-50 were obtained from Dr. I. 0rskov (Copenhagen). The polysaccharides were is o l a t e d as previously described (see Section 111). Each s t r a i n was grown on one small tray (30 x 50 cm) containing 1.5 L Mueller Hinton agar, for 4 d at 37° and harvested on the 5th day. On harvesting E. c o l i K47 and K 4 9 gave a watery slime. In contrast, E. c o l i K48 and K50 both produced more viscous slimes, with that of E. c o l i K 4 8 being p a r t i c u l a r l y glutinous. E. c o l i K 4 6 did not give a slime; the b a c t e r i a l growth on the agar was very dry. After i s o l a t i o n and p u r i f i c a t i o n of the polysaccharides, the solutions were ultracentrifuged a second time to remove any remaining l i p o -polysaccharide. The best y i e l d of polysaccharide was obtained 108 for E. c o l i K48 (263*2 mg) which produced the most viscous slime when growing. The worst y i e l d was obtained for E. c o l i K/+6 (99.8 mg) which produced no slime at a l l . Analysis of constituent sugars A small sample of each polysaccharide (3-k mg) was hydrolyzed for varying lengths of time (18 h, 24 h, 36 h, 48 h) with 2M T F A on a steam-bath. The monosaccharides released on hydrolysis were i d e n t i f i e d i n the f i r s t instance by descending paper chromatography. The sugars were detected with alkaline s i l v e r n i t r a t e (see Section V11.2 for the results of the paper chromatography). The sugars released were converted to t h e i r a l d i t o l s by reduction with sodium borohydride, followed by acetylation (1:1 pyridine - acetic anhydride) for \ h on a steam-bath. G.l.c. analysis of the a l d i t o l acetates gave the results shown i n Tables VI1 1-5 • Only E. c o l i K50 polysaccharide, on hydrolysis for kk h with 2M T F A , released amino sugars. However, hydrolysis of the other polysaccharides with 4M HCl for 4 h, followed by conversion of the released sugars to t h e i r a l d i t o l acetates, and analysis by g.l.c.-m.s., showed each polysaccharide contained amino sugars (see Tables V11. 1-5). Samples of 4-5 mg of each polysaccharide were dried for 2 h i n vacuo under the I.R. lamp. Methanolysis was performed with 3% methanolic hydrogen chloride (1 mL) for 8 h on a steam-bath, under anhydrous conditions. The acid was then neutralized with s o l i d s i l v e r carbonate and the methanolyzate was reacetylated overnight i n the same flask, 109 with acetic anhydride ( 0 . 1 mL) at room temperature^ • S i l v e r s a l t s were removed by f i l t r a t i o n and washed with anhydrous methanol ( 2 x 1 mL). The f i l t r a t e was evaporated to dryness and traces of acetic acid removed from the residue by repeated d i s s o l u t i o n i n methanol and evaporation to dryness. The residue was dissolved i n anhydrous methanol (1 mL) and treated with sodium borohydride overnight at room temperature. Sodium ions were removed using Amberlite I . R . 1 2 0 (H +) cation exchange r e s i n . Samples were evaporated to dryness and boric acid d i s t i l l e d off as methyl borate by repeated additions of methanol (3 X). The residues were hydrolyzed with 4M hydrochloric acid for 2 h at 95°. The solutions were then evaporated to dryness. The sugars released were reduced with aqueous sodium borohydride solution for 4 5 min at room temperature. After the usual workup, the a l d i t o l s were acetylated ( 1 : 1 acetic anhydride-pyridine mixture) for h on a steam-bath, under anhydrous conditions. The a l d i t o l acetates were extracted with chloroform and water, evaporated to dryness and analyzed by g . l . c . using column DB 17 (see Table VI1 . 7 ) . The results were confirmed by g.l.c.-m.s. analysis. Deamination of E. c o l i K46-50. A sample of each polysaccharide (5 -6 mg) was hydrolyzed with 2M H C l for 4 h (apart from E. c o l i K 5 0 which was hydrolyzed for 44 h with 2M TFA). The samples were evaporated to dryness and c o d i s t i l l e d with water (3X). One half of each hydrolyzed polysaccharide was dissolved i n water ( 0 . 5 mL) and 1 10 treated with a mixture of 33$ acetic acid (1 mL) and 5$ aqueous sodium n i t r i t e solution (1 mL). The solution was s t i r r e d for 1 h at room temperature, diluted with water (3 mL) and subsequently freeze-dried. The deaminated products were dissolved i n water (2 ml.) and reduced with sodium borohydride for 2 h at room temperature. The mixture was a c i d i f i e d with 50$ acetic acid, evaporated to dryness and washed with methanol ( 3 x 5 mL). A white precipitate remained due to sodium ions. The samples were dried under the i . r . . lamp for 1 h and acetylated (1:1 pyridine and acetic anhydride) for 1 h on a steam-bath, under anhydrous conditions. Excess pyridine was removed by co-d i s t i l l a t i o n i n vacuo with- water. The deaminated samples were analyzed by g . l . c . For each polysaccharide, the presence of an amino sugar was confirmed by the appearance of the deaminated product of the amino sugar. The second half of the hydrolyzed polysaccharides was converted to i t s a l d i t o l acetates and analyzed by g . l . c . for comparison with the deaminated samples. 111 V I 1 1 . ISOLATION,PROPAGATION AND CROSS-REACTIONS OF E.COLI 047, 048 and 049 112 VI 11. ISOLATION, PROPAGATION AND CROSS-REACTIONS OF E.COLI. 047, 048 and 049 V111.1 I n t r o d u c t i o n As y e t , l i t t l e i s known about the c h e m i c a l p r i n c i p l e s o f s u b s t r a t e s p e c i f i c i t y of the b a c t e r i a l g l y c a n a s e s . R i e g e r -45 Hug and S t i r m ^ u n d e r t o o k the i s o l a t i o n o f a l a r g e number of K l e b s i e l l a b a c t e r i o p h a g e s and c h a r a c t e r i z e d them w i t h r e s p e c t t o t h e h e t e r o p o l y s a c c h a r i d e s t h e y degraded. The purpose o f t h i s work was t o determine whether the h y d r o l a s e s u s c e p t i b l e g l y c o s i d i c l i n k a g e s i n the h e t e r o p o l y s a c c h a r i d e s u b s t r a t e s e x h i b i t e d any common c a r b o h y d r a t e c h a r a c t e r i s t i c s , and hence, which h e t e r o g l y c a n s of known s t r u c t u r e can be expected to be a c t e d upon by a g i v e n phage p a r t i c l e . From t h e i r r e s u l t s t h e y deduced what c h e m i c a l f e a t u r e s can be i d e n t i f i e d from the enzymic s u s c e p t i b i l i t y of a p o l y s a c c h a r i d e of unknown s t r u c t u r e ( see S e c t i o n V .4 and V . 5 ) . Because t h e r e has been no comparable work u s i n g E.  c o l i b a c t e r i o p h a g e , t h e p r e s e n t s t u d y was u n d e r t a k e n u s i n g the E. c o l i #47, #48 and #49 i s o l a t e d from sewage. The r e s u l t s of the c r o s s - r e a c t i o n s of E. c o l i #47» #48 and #49 w i t h h e t e r o p o l y s a c c h a r i d e s from E. c o l i K46, K47 , K48, K49 and K50 s e r o t y p e s a r e p r e s e n t e d h e r e . VI11.2 R e s u l t s and d i s c u s s i o n V111.2.1 I s o l a t i o n and p r o p a g a t i o n of E . c o l i #47, #48 and #49 An attempt t o i s o l a t e , from sewage, b a c t e r i o p h a g e f o r 113 E. c o l i K46-50 serotypes, was undertaken as previously described (see Section 1V.2). After assaying the clear supernatant obtained, i n d i v i d u a l clear plaques were observed on b a c t e r i a l lawns of E. c o l i K 4 6 , K 4 7 , K 4 8 , K49 and K50 (at di l u t i o n s of 10 and 10"^ of # sol u t i o n ) . Phage 49 gave plaques surrounded by haloes, while the other phage formed plaques of the pinhole type. To puri f y the bacteriophage, an i n d i v i d u a l plaque was removed from each b a c t e r i a l lawn and added to s t e r i l e broth. Ten-fold d i l u t i o n s (3X) of each were prepared and a drop of each d i l u t i o n was spotted on a lawn of i t s host bacterium. A drop of concentrated phage solution was also spotted on a lawn of each of the phage's heterologous b a c t e r i a l s t r a i n s . The results are tabulated i n Table VI11.1. The results indicated that a phage had been iso l a t e d for each of the b a c t e r i a l serotypes. Cross-reactions occurred between phages 4 6 , 4 7 and 48 and heterologous polysaccharides from E. c o l i K 4 6 - 4 9 sero-types. Phage 50, however, did not cross-react with any heterologous bacteria. While phage 49 did cross-react with heterologous b a c t e r i a l s t r a i n s , i t did not produce the large haloes which were evident when i t was plated on i t s host bacterium. However such cross-reactions are only meaning-f u l i f they continue to occur at high d i l u t i o n s . The bacteriophage were each propagated by means of three successive test-tube lyses. The phage solutions were o r i g i n a l l y prepared by adding one phage plaque from the p u r i f i e d phage agar plate, to Mueller Hinton broth. The concentrations of the phage solutions a f t e r each propagation are given i n 114 TABLE VI11.1 CROSS-REACTIONS OF BACTERIOPHAGE ISOLATED FROM SEWAGE E. coli Bacteriophage serotype 046 047 048 049 050 K46 + + + _ + K47 + + + - + K48 + + + - + . K49 + + + + + + K50 - ••- + + area of bacterial lawn where phage solution was applied became clear; ++ area of bacterial lawn where phage solution was applied became clear and surrounded by a halo; - no clearing of the bacterial lawn occurred where the phage solution was applied. TABLE V111.2 CONCENTRATION OF BACTERIOPHAGE SOLUTIONS AFTER EACH TEST-TUBE LYSIS Bacteriophage I ConcentrationCp.f.u II III ./mL) IV 046 9 .5x10 6 1,1x106 047 -.?.6x10 6 I.OxlO7 3*6x1O8 9 .5x10 9 048 3.0x10 5 1.2x105 2.3x1O8 1.2x109 049 1.5x105 7 .6x10 5 2.0x10 8 1.3x109 0 5 0 - - - — I - original phage solution; II - test-tube lysis 1 ; III - test-tube lysis 11; IV - test-tube lysis 111. 115 Table VI11.2. Although phages 46 and 50 had each produced plaques on a lawn of i t s homologous bacteria a f t e r the p u r i f i c a t i o n step, no plaques were observed for either phage after the f i r s t test-tube l y s i s was assayed. The test-tube l y s i s was repeated for each of phages 46 and 50 using a freshly prepared phage solution. Again the bacteriophage assay showed no results for phage 50'but phage 46 showed an increase i n the t o t a l number of phage p a r t i c l e s . An in t e r e s t i n g result was obtained for phage 49 • The o r i g i n a l phage solution exhibited large haloes when plated out on i t s host bacterium but afte r the f i r s t test-tube l y s i s the plaques were no longer surrounded by haloes. The test-tube l y s i s was repeated using fresh phage solution and th i s time the plaques exhibited haloes a f t e r the assay. It was concluded that E . c o l i K 4 9 serotype was attacked by two phage, one which produced an excess of the glycanase and one which did not. When selecting a phage plaque for the phage solution for the f i r s t test-tube l y s i s , one without a halo had been selected. After the second test-tube l y s i s , phage 46 again gave no result on assaying the phage solution obtained. It was decided that the phage i s o l a t e d must be weak, and afte r achieving suitable concentrations of phage 4 7 , 4 8 and 49 i t would be more pr o f i t a b l e to see i f one of these would cross-react with E . c o l i K 4 6 . 116 VI 1 1.2.2 Cross-reactions of #47, #48 and #49 Phages 47> 48, and 49 were propagated by test-tube l y s i s u n t i l concentrations of approximately 10^  p.f.u./mL were reached. Each of the bacteriophage was then propagated on each of E . c o l i K 4 6 -50 b a c t e r i a l s t r a i n s . The results of the cross-reactions are tabulated i n Table VI11.3• In only three cases was there an actual increase i n the number of phage plaque forming units i . e . when #47 was added to a b a c t e r i a l culture of E . c o l i K48 or K 4 9 , and when #48 was added to E. c o l i K47. In these cases i t seemed f a i r l y conculsive that #47 cross-reacts with E. c o l i K48 and K49, and #48 cross-reacts with E. c o l i K 4 7 . Equally i t was obvious that none of the phages cross-reacted with E. c o l i K 5 0 . An i n t e r e s t i n g cross-reaction was that of #48 with E. c o l i K 4 6 . Although there had been no increase i n the number of phage p a r t i c l e s , the b a c t e r i a l culture of K 4 6 did clear on addition of #48. It should be noted that the optimum conditions for the propagation of each i n d i v i d u a l phage were not established. General conditions,which experience with #26 had shown to give the best phage y i e l d , were used for the propagation of the phage. In only one case was the test-tube l y s i s assay negative where the preliminary screening (see Section V111.2.1) had indicated that a cross-reaction might occur, i . e . the cross-reaction of #48 with E . c o l i K49. 117 TABLE VI11.3 CROSS-REACTIONS OF E. COLI 047, 048 AND 049 Bacteriophage K46 E. c o l i K47 serotype K48 K49 K 5 0 I 047 II 2.8x109 2.5x108 2.8x109 5.5x109 2.8x109 4.8x1O9 2.8X109 I 048 T 1 3.6x108 4.5x10° 3.6x108 1.4x10* 3.6x108 3.6x108 I 049 II 3.9X10 8 ** 6.0x105 3.9x108 ** 1.1x107 3.9x108 ** 2.2x107 3.9X 1 0 8 I - t o t a l number of phage added (p.f.u.); I I - t o t a l number of phage a f t e r cross-reaction (p.f.u.); * - t'est-tube contents had cleared; **- phage plaques were pin-hole sized and did not exhibit haloes. 1 18 VI11.2.3 Conclusion These preliminary results indicated that E. c o l i K 4 7 , Ki+8 and K49 capsular polysaccharides have some str u c t u r a l features i n common as they are a l l degraded by E. c o l i # 4 7 . Stirm and coworkers^ showed that the enzyme proteins of K l e b s i e l l a glycanases recognise disaccharide portions of t h e i r substrates. Within these disaccharides, the correct distance of a carboxyl group from the suceptible glycosidic oxygen i s important for recognition. Further studies on the substrate s p e c i f i c i t i e s of K l e b s i e l l a #6 and #13 glycanases and other enzymes of t h i s type were carried out by Stirm and his 5 7 associates. . They found that, i f the carboxylate groups of the glycans were reduced to primary hydroxyls, the enzymes no longer degraded them. These results indicated that K l e b s i e l l a #6 and #13 glycanases both recognize, not complete sugars, but single functional groups distributed around the susceptible glycosidic oxygens within the glycan repeating units. It would be a valuable study to see i f Stirm- and his coworker's conclusions are v a l i d for E. c o l i K46-50 b a c t e r i a l glycanases since a l l t h e i r substrates contain a glucuronic acid residue. Since the cross-reactions between #48 and E. c o l i K 4 7 and #47 and E. c o l i K48 are r e c i p r o c a l , t h i s i s a strong . i n d i c a t i o n that the two polysaccharides contain a disaccharide unit i n commom. K l e b s i e l l a #2 w i l l attack K l e b s i e l l a K2 and and K 1 3 , and #13 w i l l attack K l e s i e l l a K2 and K 1 3 . The two polysaccharides both contain the disaccharide -3)-B-D-Glcp-(l-4)-g-D-Manp-( l-» , the (1-4) linkage between glucose and 119 and mannose being attacked by both 02 and 013. It w i l l be i n t e r e s t i n g to see i f this conclusion i s substantiated once the structures of E. c o l i K47 and K48 have been determined. VI11.2.4 Experimental General methods. The following media were used to grow the bacteria; 1) Mueller Hinton broth - dehydrated, 2) Mueller Hinton agar - dehydrated. 0.5$ (w/v) NaCl was added to both broth and agar medium. Agar plates were prepared and s t e r i l i z a t i o n carried out as described previously (see Section V. 4). The b a c t e r i a l lawns were prepared and phage assays were carried out as described i n Section V1.2.4« Isolation of Bacteriophage from sewage Ten times concentrate Mueller Hinton broth(100 mL) was prepared and combined with an a c t i v e l y growing b a c t e r i a l culture (30 mL). This culture was prepared by inoculating 30 mL s t e r i l e Mueller Hinton broth with three b a c t e r i a l colonies of the prospective capsular host, and shaking for 3-4 h u n t i l the culture was cloudy. The concentrated Mueller Hinton broth and the log-phase b a c t e r i a l culture were added to the raw sewage effluent (900 mL) and the mixture was incubated at 37°. After 18 h, 10 mL of the mixture was removed and chloroform (10 mL) was added to the sample. The mixture was then centrifuged to remove any s o l i d debris, and 120 the clear supernatant assayed. The o r i g i n a l sewage mixture was further incubated for another 2 4 h. Again a sample ( 1 0 mL) was removed and worked up as for the f i r s t sample. Assays of both samples showed no increase i n the phage concentration a f t e r the further 24 h incubation. At t h i s point, the res u l t s of the assays indicated that a phage s p e c i f i c for each of E. c o l i K46-50 had been i s o l a t e d . The phage were p u r i f i e d by taking a single plaque from the agar plate with a s t e r i l e wire loop and adding i t to 1 mL Mueller Hinton broth. S e r i a l d i l u t i o n s of the r e s u l t i n g solutions were prepared ( 1 0 ~ , 1 0 " , 10" 4 ") and one drop was spotted onto a b a c t e r i a l lawn of the phage's host bacterium. A l l the b a c t e r i a l lawns again showed clear plaques, which could be attributed to the presence of a bacteriophage s p e c i f i c for the bacteria. The plaques on the b a c t e r i a l lawn of E. c o l i K 4 9 were surrounded by haloes which spread i f the plates were l e f t at room temperature for a few days. Propagation of bacteriophage The bacteriophage i s o l a t e d from sewage were each propagated by three successive test-tube lyses. An a c t i v e l y growing b a c t e r i a l culture ( 0 . 2 mL) was added to s t e r i l e Mueller Hinton broth (5 mL), and incubated for 1 h u n t i l the b a c t e r i a l culture was s l i g h t l y cloudy. 0.3 mL of phage solution was added to the culture of i t s host bacterium. The mixture was then shaken for 3-4 h u n t i l the contents of the test-tube became clear, or u n t i l i t was apparent that no 121 clearing was going to occur. Chloroform (1 mL) was added to the test-tube and the contents shaken. After centrifugation, the clear phage solution was assayed to determine the phage concentration. After the t h i r d successive propagation the concentration of 0 4 7 , 0 4 8 and 0 4 9 had reached approximately 10 9 p.f.u./mL (see Table V I 1 1.2). Cross-reactions of bacteriophage 4 7 , 4 8 and 4 9 To investigate the cross-reactions of 0 4 7 , 0 4 8 and 0 4 9 each phage was propagated by means of a test-tube l y s i s on each of the heterologous bacteria. The test-tube l y s i s was carried out using the same conditions as those for the bacteriophage propagation. However, whereas i n the bacterio-phage propagation the phage solution was added to i t s homologous bacterium, i n these experiments the phage solutions (from the t h i r d bacteriophage propagation) were added to cultures of heterologous bacteria. The results of the cross-reactions are given i n Table V111 . 3 * 122 I X . BIBLIOGRAPHY 123 IX. BIBLIOGRAPHY 1. C . Gram, F o r t s c h r . Med., 2, 185-189, (1884). 2. M.M. B u r g e r , P r o c . N a t l . Acad. S c i . U.S.A., ^6, 910- 917, (1966). 3. E . M . B o l t , C.W. Bonynge and R.L. J o y c e , J . I n f e c t D i s . , 58, 5-9, ( 1 9 3 6 ) . 4. M.E. Bayer, and H. Thurow, J . 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S t i r m , V i r o l o g y , 56, 134-151, (1973). 56. D.N. K a r u n a r a t n e , p e r s o n a l communication. 57. F. Fehmel. H. B e i l h a r z , H. Niemann and S. S t i r m , J . V i r o l o g y , J_6, 591-601, (1975). 58. G.O. A s p i n a l l , 'The P o l y s a c c h a r i d e s ' , I_ (Academic P r e s s I n c . U.S.A. 1983). 59. F. W. Twort, L a n c e t , 2, 189, 1241, (1915) 60. F. d ' H e r e l l e , C.R. Acad. S c i . P a r i s , 165, 373, (1917) 128 APPENDICES 129 APPENDIX I: Known structures of E. c o l i K antigens* 130 K. c o l l K. antigens 8 „ 2 — NANA5AC — a E. c o l l K l «• 1 2 1(3) , 5 1 2 1(3) x _ P _ G a l _ G l y _L_2 ~ ( - P - G a l f — Gly - A - i ) n E. c o l i K2 h i v l — GlcA s - GlcNAc — P o E. c o l i K3 2 1 2 1 7 2 3 1 7 2 - R i b f — R i b f — KDO — or — ^ I i b j — KDO — Rib, E. c o l i 6a E. c o l l K6 131 — ManNAcA Glc olc E. t o l l K7 and K56 — Rha •*-=*• Rha ^ KDO -Sr o a 7 / o p 1 2 n L . J _ 5 778 I OAc E. c o l i K12 and K82 R l b f -y- KDO -2p I OAc E. c o l i K13 — GalNAc KDO T OAc E. c o l i K14 — GlcNAc —• KDO p^-E. c o l i K15 — | l b f ^-r- KDO OAc E. c o l l K20 132 Rlb f - y KDO -2pf E . c o l i K23 — Glc GlcA Fuc 31 ot a a Gal E . c o l i K27 — Glc GlcA Fuc 0 ? o r 3 1 Gal 1 •Ac E . c o l i K28 — Man Cal ±v GlcA Gal E . c o l i K30 133 — Gal Glc — GlcA -ijp Rha ^ Rha E. coll K31 OAc j G l c L v C A L J _ GlcA E. coli K32 -I Glc ^  GlcA Y Fuc V ll« + 0 A c Pyf Gal ~ E. coli K33 — Gal y GalA ± ^ Fuc E. coli K42 — Gal Fru E. coli K52 + OAc + OPr 2/U GlcA Man Man JLA or | 1 2 Rha J _ 3 GlcNAc IcA 1_1 Man i - i GlcNAc ? Man •*—4 Man Rha E. coli K85 134 GlcA FucNAc — GlcNAc 1 P Glc-E. coli K87 — NANA5AC NANA5Ac E. coli K92 Rib f ribitol — 0 — E. coli K100 135 References Kl E.J. McGulre and S.B. Binkley, Biochemistry, 3 (1964) 247-251. K2 K. Jann, B. Jann, and A.M. Schmidt,J. Bact., 143 (1980) 1108-1115. K5 W.F. Vann, M.A. Schmidt, B. Jann, and Ki Jann, Eur. J. Biochem., 116 (1981) 359-364. K6 P. Messner and F.M. Unger, Biochem. Biophys. Res. Commun., 96, (1980) 1003-1010. K6a H.J. Jennings, K.-G. Rosell, and K.G. Johnson, Carbohydr. Res., 105 (1982) 45-56. K7-K56 F.-P. Tsui, R.A. Boykins, and W. Egan, Carbohydr. Res., 102 (1982) 263-271. K12-K82 M.A. Schmidt, B. Jann, and K. Jann, FEMS Microbiol. Lett., 14 (1982) 69-74. K13 W.F. Vann and K. Jann, Infect. Immun., 25 (1979) 85-92. W.F. Vann, T. Soderstrom, W. Egan, F.-P. Tsui, R. Schneerson, I. 0 r s k o v , and F. 0 r s k o v , Infect. Immun., 39 (1983) 623-929. K14 B. Jann, P. Hofmann, and K. Jann, (from K. Jann and B. Jann, Prog. Allergy, 33 (1983) 53-79. K15 W. Vann, unpublished results. (From K. Jann and B. Jann, Prog. Allergy, 33 (1983) 53-79). K20.K23 W.F. Vann, T. Soderstrom, W. Egan, F.-P. Tsui, R. Schneerson, I. 0 r s k o v , and F. 0 r s k o v , Infect. Immun., 39 (1983) 623-629. K27 K. Jann, B. Jann, K.F. Schneider, F. fhrskov, and I. frskov, Eur. J. Biochem., £ (1968) 456-465. A. K. Chakraborty, Macromol. Chem., 183 (1982) 2881-2887. K28 E. Altman and G.G.S. Dutton, Carbohydr. Res., (in press). K29 Y.-M. Choy, F. Fehmel, N. Frank, and S. Stirm, J. Virol., 16 (1975) 581-590. K30 A.K. Chakraborty, H. Frielbolin, and S. Stirm, J. Bacterid., 141 (1980) 971-972. K31 K. Jann, unpublished results. (From I. 0 r s k o v , F. 0 r s k o v , B. Jann, and K. Jann, Bacterlol. Rev., 41 (1977) 667-710. 136 K32 E. Altman, unpublished r e s u l t s . K33 B.A. Lewi8, unpublished r e s u l t s . K42 H. Niemann, A.K. Chakraborty, H. F r i e b o l i n , and S. Stirm, J .  B a c t e r i d . , 133 (1978) 390-391. K52 P. Hofmann, B. Jann, and K. Jann, Int. Symp. Carbohydr. Chem., 12th, 1984 Abstracts, p. 367. K85 K. Jann, B. Jann, F. ^rskov, and I. 0 r s k o v , Biochem. Z., 346 (1966) 368-385. K87 L. Tarcsay, B. Jann, and K. Jann, Eur. J . Biochem., 23, (1971) 505-514. K92 W. Egan, T.-Y. L u i , D. Dorow, J.S. Cohen, J.D. Robbins, E.C. Gotschlich, and J.B. Robbins, Biochemistry, 16 (1977) 3687-3692. K100 W. Egan, F.P. T s u i , R. Schneerson, and J.B. Robbins, J . B i o l . Chem., (In press). * Reproduced with the kind permission of Dr. E. Altman from her Ph.D. thesis e n t i t l e d 'Capsular Antigens of Gram-negative Bacteria', University of B r i t i s h Columbia, Sept 1984. 137 APPENDIX I I : N.m.r. s p e c t r a H-n.m.r. - c h e m i c a l s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e ; 2.23 d o w n f i e l d from sodium k,4-dimethyl-4-silapentane-1-sulphonate c h e m i c a l s h i f t i n p.p.m. d o w n f i e l d from Me, S i . r e l a t i v e t o i n t e r n a l a c e tone; 31.07 p.p.m. d o w n f i e l d from D.S.S. K26 polysaccharide Spectrum No.1 Co K26 o l i g o s a c c h a r i d e ^-n.m.r. 400 MHz, 95° Spectrum No.3 K26 p o l y s a c c h a r i d e ( h y d r o l y z e d 1 h 0.1M TFA) 400 MHz 95° Spectrum No.5 5.0? 5 - A J 4.86 4.81 \ | ^ / 4 . 7 6 1.35 K26 p o l y s a c c h a r i d e ^C-n.m.r. 100,6 MHz, amb. temp. 180.67 C,=0 from acetone 103.11 102.99 103.39^ J 102.04 104.16 , "—I— T 1 ' f • r t r u r n N o . 6 ac etone 31.07 K26 depyruvylated (autohydrolysis) •^ C-n.m. r. 1 0 0 . 6 MHz amb. temp. 3.000 1.500 1 0 2 . 9 2 1 0 2 . 9 5 1 0 3 . 1 5 10 i | . 30 9 9 . 7 5 1.000 soo Spectrum No.7 1 7 . 5 0 acetone 3 1 . 0 7 KZf8 p o l y s a c c h a r i d e In H-n.m.r. 4 0 0 MHz 95° 5 Spectrum No.10 K50 polysaccharide H-n.m.r. 400 MHz 9 5 ° 5 

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