Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Characterization of the fibrinogen and fibrin adhesin of Staphylococcus aureus Martin, Alexis Wilma 1982

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1982_A6_7 M38.pdf [ 5.49MB ]
Metadata
JSON: 831-1.0095711.json
JSON-LD: 831-1.0095711-ld.json
RDF/XML (Pretty): 831-1.0095711-rdf.xml
RDF/JSON: 831-1.0095711-rdf.json
Turtle: 831-1.0095711-turtle.txt
N-Triples: 831-1.0095711-rdf-ntriples.txt
Original Record: 831-1.0095711-source.json
Full Text
831-1.0095711-fulltext.txt
Citation
831-1.0095711.ris

Full Text

CHARACTERIZATION OF THE FIBRINOGEN AND FIBRIN ADHESIN OF STAPHYLOCOCCUS AUREUS by ALEXIS WILMA MARTIN B . S c , U n i v e r s i t y of V i c t o r i a , 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1982 (c) A l e x i s Wilma M a r t i n , 1982 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 his 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 The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT The i n t e r a c t i o n between Staphylococcus aureus and c r o s s - l i n k e d and non-cross-linked f i b r i n has been c h a r a c t e r i z e d and the c e l l w a l l compo-nent mediating f i b r i n o g e n induced clumping has been i d e n t i f i e d . Binding of S. aureus to f i b r i n and clumping of the c e l l s i n f i b r i n o g e n , appear to be mediated by the same c e l l w a l l component, c a l l e d clumping f a c t o r ( C l f ) . The b i n d i n g of S. aureus to f i b r i n was maximal at 37°C w i t h a pH optima of 6-8 and was independent of d i v a l e n t c a t i o n s . The i n t e r a c t i o n between the two surfaces occurred w i t h i n minutes and once bound, c e l l s were d i f f i c u l t to remove. The f i b r i n - b i n d i n g r e a c t i o n was i n h i b i t e d by 1M NaCl and 4M urea but not by any p r o t e i n , g l y c o p r o t e i n , g l y c o l i p i d , or any s p e c i f i c sugar t e s t e d . Heating the c e l l s , t r e a t i n g them w i t h protease, or a c e t y l a t i n g amino groups abrogated t h e i r b i n d i n g a c t i v i t i e s . A g l y c o p r o t e i n , b e l i e v e d to be the C l f , has been p a r t i a l l y p u r i f i e d from l y s o s t a p h i n s o l u b i l i z e d c e l l w a l l s of S. aureus, C l f + s t r a i n s , by a f f i n i t y chromatography on immobilized f i b r i n o g e n . The C l f a c t i v e f r a c t i o n bound to f i b r i n o g e n and e l u t e d w i t h 2M NaCl. A n a l y s i s of the f r a c t i o n by g e l e l e c t r o p h o r e s i s revealed a s i n g l e band, molecular weight approximately 90,000 d a l t o n s , which stained p o s i t i v e l y f o r p r o t e i n and carbohydrate. This g l y c o p r o t e i n could not be i d e n t i f i e d i n c e l l w a l l s of a C l f negative s t r a i n of jS. aureus. Immunoelectrophoretic a n a l y s i s of the clumping f a c t o r f r a c t i o n , showed that i t formed a s i n g l e p r e c i p i -t i n l i n e w i t h a n t i - C l f + S_. aureus antiserum ( a n t i - S . aureus-1) but not I l l w i t h a n t i s e r u m r a i s e d a g a i n s t t h e C l f - m u t a n t . A n a s s a y was d e v e l o p e d t o q u a n t i t a t e t h e s o l u b l e C l f , a n d s h o w e d t h a t t h e C l f a b s o r b e d o r n e u t r a l i z e d s p e c i f i c c l u m p i n g i n h i b i t i n g a n t i b o d i e s p r e s e n t i n a n t i -_S. a u r e u s - 1 , t h a t p r e v e n t e d f i b r i n o g e n i n d u c e d c l u m p i n g o f S. a u r e u s . O t h e r C l f - m u t a n t s o f S. a u r e u s a n d n o n - s t a p h y l o c o c c a l b a c t e r i a w e r e e x a m i n e d f o r f i b r i n a n d f i b r i n o g e n b i n d i n g a n d f i b r i n o g e n c l u m p i n g a c t i v i t e s . T h e m a j o r i t y o f b a c t e r i a o b s e r v e d t h a t c o u l d b i n d t o f i b r i n a n d f i b r i n o g e n , c o u l d n o t c l u m p i n t h e p r e s e n c e o f f i b r i n o g e n . ' A m o d e l i s p r e s e n t e d t o d e s c r i b e a d h e s i n s t h a t , b i n d t o f i b r i n o g e n b u t a r e u n a b l e t o f o r m c e l l - a g g r e g a t e s . i v INTRODUCTION TABLE OF CONTENTS Page 1 I I . MATERIALS AND METHODS 17 A. B a c t e r i a and c u l t u r a l c o n d i t i o n s 17 B. Mutagenic procedures 1^ C. Pr e p a r a t i o n of f i b r i n o g e n 19 D. I s o l a t i o n of f i b r i n o g e n chains 19 E. P r e p a r t i o n of f i b r i n v i a l s 20 F. Clumping t i t r e of b a c t e r i a 21 1. Tube d i l u t i o n method 21 2. M i c r o t i t r e method 21 G. Adherence of b a c t e r i a to f i b r i n 22 H. C h a r a c t e r i z a t i o n of the b i n d i n g receptors of jS. aureus 1. M o d i f i c a t i o n of whole c e l l s 23 a. A u t o c l a v i n g 23 b. S o n i c a t i o n 23 c. A c e t y l a t i o n 23 d. SDS 23 e. NaOH, HC1 24 f • Proteases • 24 2. M o d i f i c a t i o n of c e l l w a l l s 24 a. T r i c h l o r a c e t i c a c i d e x t r a c t i o n 24 b. Phenol e x t r a c t i o n 24 c. Sodium periodate treatment 25 d. Formamide 25 I. I n h i b i t o r s of the j>. aureus f i b r i n b i n d i n g r e a c t i o n 25 J . I s o l a t i o n and s o l u b i l i z a t i o n of c e l l w a l l s 25 1. I s o l a t i o n of w a l l s 25 2. S o l u b i l i z a t i o n of w a l l s 26 K. A n a l y s i s of s o l u b i l i z e d c e l l w a l l f r a c t i o n s 26 1. Phosphorus, p r o t e i n and hexose content 26 2. A f f i n i t y chromatography 26 3. Gel e l e c t r o p h o r e s i s 27 4. Immunological procedures 28 5. Soluble clumping f a c t o r assay 29 a. F i b r i n o g e n - c e l l clumping i n h i b i t i o n t e s t . . . . . . . 29 b. Soluble clumping f a c t o r assay 30 L. Hemagglutination assay 30 M. "Interactions of other b a c t e r i a w i t h f i b r i n and f i b r i n o g e n 31 N. Absorption of f i b r i n o g e n 31 0. E l e c t r o n microscopy 32 P. Chemicals 32 V Page I I I . RESULTS 33 A. C h a r a c t e r i z a t i o n of the jS. a u r e u s - f i b r i n b i n d i n g r e a c t i o n . . . 33 1. Adherence of EL aureus to f i b r i n 33 2. Parameters of the S_. a u r e u s - f i b r i n b i n d i n g r e a c t i o n .... 34 a. C e l l numbers versus b i n d i n g to f i b r i n 34 b. R e l a t i o n s h i p of time of i n c u b a t i o n to adherence .... 38 c. E f f e c t of pH 38 d. Temperature 41 3. P h y s i c a l and chemical treatment of S_. aureus 41 a. P h y s i c a l treatment 41 b. Chemical treatment 46 4. Protease treatment 48 5. I n h i b i t o r s of the b i n d i n g r e a c t i o n 48 6. E f f e c t of a n t i s e r a on the J5. aureus-f i b r i n b i n d i n g r e a c t i o n 51 a. A n t i s e r a prepared against f i b r i n o g e n and a , g, and Y chains • • • > - • • • 51 b. A n t i s e r a r a i s e d against S_. aureus-1 and j>. aureus-5 55 B. I s o l a t i o n and c h a r a c t e r i z a t i o n of C l f 56 1. Pr e p a r a t i o n of c e l l w a l l s 56 2. Adherence p r o p e r t i e s of c e l l w a l l s 58 3. M o d i f i c a t i o n ' 58 4. S o l u b i l i z a t i o n of c e l l w a l l s 60 5. C h a r a c t e r i z a t i o n of s o l u b i l i z e d c e l l w a l l s 60 a. P r o t e i n , phosphorus and hexose 60 b. Immunoelectrophoresis 64 6. I s o l a t i o n of C l f 64 7. C h a r a c t e r i z a t i o n of C l f 73 a. Gel e l e c t r o p h o r e s i s 73 b. Immunoelectrophoresis 74 c. Measurement of the a c t i v i t y of s o l u b l e C l f . , . . , . . . 74 C. Reactions of other b a c t e r i a w i t h f i b r i n and f i b r i n o g e n .... 84 1. Clumping i n f i b r i n o g e n 84 2. Binding to f i b r i n 84 3. Absorption of f i b r i n o g e n 84 D. Summary 86 1. Comparison of the f i b r i n and f i b r i n o g e n b i n d i n g adhesins 86 2. R e l a t i o n s h i p of C l f to f i b r i n and f i b r i n o g e n b i n d i n g , and to fibrinogen-induced clumping 87 IV. DISCUSSION 92 V. LITERATURE CITED. 104 v i LIST OF TABLES Page I. Comparison of S. aureus - 1 and S. aureus - 5 35 I I . E f f e c t of p h y s i c a l and chemical teatment on the S. a u r e u s - f i b r i n b i n d i n g r e a c t i o n . .. 47 I I I . E f f e c t of protease treatment on the S[. aureus-f i b r i n b i n d i n g r e a c t i o n 7. .777777 49 IV. E f f e c t of i n h i b i t o r s on the J^. a u r e u s - f i b r i n b i n d i n g r e a c t i o n . 52 V. E f f e c t of a n t i s e r a on the j3. a u r e u s - f i b r i n b i n d i n g r e a c t i o n . . . 57 VI. E f f e c t of chemical, p h y s i c a l and enyzmatic treatment on the fibrinogen-induced clumping a c t i v i t y of S^. aureus c e l l w a l l s 59 V I I . P r o t e i n , phosphorus, and hexose content of l y s o s t a p h i n -s o l u b i l i z e d c e l l w a l l s and c e l l w a l l s 63 V I I I . Influence of clumping i n h i b i t i n g a n t i b o d i e s on f i b r i n o g e n -induced clumping of S. aureus 82 IX. Assay f o r s o l u b l e clumping f a c t o r 83 X. Reaction of other b a c t e r i a w i t h f i b r i n and f i b r i n o g e n 85 v i i LIST OF FIGURES Page 1. Influence of c e l l numbers on the j ^ . a u r e u s ~ f i b r i n b i n d i n g r e a c t i o n 37 2. E f f e c t of time on the S^. aureus-f i b r i n , b i n d i n g r e a c t i o n 40 3. E f f e c t of pH on the S. a u r e u s - f i b r i n b i n d i n g r e a c t i o n 43 4. E f f e c t of temperature on the S. a u r e u s - f i b r i n b i n d i n g r e a c t i o n .777777 45 5. The e f f e c t of NaCl on the S[. aureus-f i b r i n b i n d i n g r e a c t i o n ... 54 6. S o l u b i l i z a t i o n of S. aureus c e l l w a l l s w i t h l y s o s t a p h i n 62 7. Immunoelectrophoretic a n a l y s i s of s o l u b i l i z e d c e l l w a l l s of j>. aureus-1 and j>. aureus-5 against anti-S_. aureus-1 and a n t i - S . aureus-1 absorbed w i t h j>. aureus-5 c e l l s 66 8. F i b r i n o g e n - a f f i n i t y chromatography of S^. aureus-1 s o l u b i l i z e d c e l l w a l l s 7. .777777 . 6 8 9. F i b r i n o g e n - a f f i n i t y chromatography of JS. aureus-5 s o l u b i l i z e d c e l l w a l l s 7. .777777 7 0 10. P r o t o c o l f o r the i s o l a t i o n of s o l u b l e clumping f a c t o r from jS. aureus 72 11. (a,b) SDS 10% PAGE a n a l y s i s of s o l u b i l i z e d c e l l w a l l f r a c t i o n s of S. aureus-1 and S. aureus-5 76,77 12. Immunoelectrophoretic a n a l y s i s of l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l f r a c t i o n s of j ^ . aureus-1 and JS. aureus-5 79 13. Models of complete, incomplete and masked clumping f a c t o r of S. aureus 91 v i i i ACKNOWLEDGEMENT I wish to acknowledge my indebtedness to Dr. Barry McBride f o r h i s constant encouragement, optimism and p a t i e n t guidance. Working w i t h him i n research has been a h i g h l y i n s t r u c t i v e and rewarding e f f o r t . ' I thank the members of my committee f o r t h e i r h e l p f u l advice and c o n s t r u c t i v e c r i t i c i s m . Thanks are a l s o due to Meja Shim f o r d u p l i -c a t i n g some experiments and ensuring that my r e s u l t s were not b i a s e d . I wish to thank my husband David f o r h i s help and understanding under t r y i n g times. L a s t l y , I dedicate my t h e s i s to my son Drew who, above a l l has taught me patience. INTRODUCTION In many environments, the a b i l i t y of a microorganism to s e l e c t i v e l y adhere to a surface i s an e s s e n t i a l step i n the c o l o n i z a t i o n of that area. Adherence to a s o l i d surface may prevent the organism's removal by hydro-dynamic or other p h y s i c a l forces and may a s s i s t i n i t s a c q u i s i t i o n of food p a r t i c l e s that have adsorbed to the same surface (11,22,23,31,39,41, 72,74). E a r l y s t u d i e s of microorganisms i n n a t u r a l , aqueous environments showed them capable of a t t a c h i n g to a v a r i e t y of s o l i d m a t e r i a l s ; c l a y , sand, g l a s s , c e l l u l o s e , p l a s t i c , or other b a c t e r i a l and euk a r y o t i c c e l l s (21,22,23,72). Many of the adherent organisms were found to possess a v a r i e t y of s p e c i a l i z e d surface s t r u c t u r e s , i m p l i c a t e d as the f a c t o r s mediating b i n d i n g (9,11,12,42,72). Recently, a t t e n t i o n has focused on the adherence of b a c t e r i a to mammalian t i s s u e s . The reader i s r e f e r r e d to s e v e r a l e x c e l l e n t reviews on the subject (12,39,41,82,91,92). Organisms that populate the mammalian s k i n or mucosa of the r e s p i r a t o r y , alimentary, or u r o g e n i t a l t r a c t s , are co n s t a n t l y being challenged by defence mechanisms of the host. These mechanisms, be they a mucosal blanket and c i l i a r y or p e r i s t a l t i c movements, can be l a r g e l y surmounted by a microbe i f i t has the means f o r attachment. Most i n v e s t i g a t o r s i n the f i e l d of adherence concur w i t h the statement made by Gibbons, 1977, (39), "The attachment of an organism to a host t i s s u e therefore appears to be the f i r s t e s s e n t i a l step f o r p e r s i s t e n t c o l o n i z a t i o n . " S e l e c t i v e nature of b a c t e r i a l attachment to mammalian t i s s u e I n c r e a s i n g evidence i n d i c a t e s that microorganisms do not adhere to host t i s s u e i n d i s c r i m i n a t e l y , but show a s p e c i f i c i t y f o r c e r t a i n c e l l s urfaces. Much of our present knowledge on the s p e c i f i c i t y of b a c t e r i a l adherence i n the mammalian host has r e s u l t e d from s t u d i e s of the o r a l ecosystem. This area i s e a s i l y a c c e s s i b l e and has r e c e i v e d much a t t e n t i o n because of the u n i v e r s a l incidence of m i c r o b i a l l y induced d e n t a l d i s e a s e s . Gibbons and c o l l a b o r a t o r s , (32, 39,'40,41,42,43,62), pioneered work i n t h i s area and have reached s e v e r a l c o n c l u s i o n s : ( i ) B a c t e r i a indigenous.to the o r a l c a v i t y adhere s e l e c t i v e l y to e p i t h e l i u m , t o the tooth s u r f a c e , or to other b a c t e r i a , ( i i ) The a f f i n i t y of each b a c t e r i a l species f o r a p a r t i c u l a r o r a l surface c o r r e l a t e s p o s i t i v e l y w i t h t h e i r n a t u r a l occurrence at that s i t e . Streptococcus s a l i v a r i u s and Streptococcus m i t i s show a p r e d i l e c t i o n f o r s p e c i f i c e p i t h e l i a l s urfaces. S^. s a l i v a r i u s p r e f e r s the dorsum of the tongue whereas S^. m i t i s adheres t o , and c o l o n i z e s n o n - k e r a t i n i z e d b u c c a l mucosa (41,62). Other o r a l b a c t e r i a l species adhere to the tooth p e l l i c l e or to b a c t e r i a l c e l l s , and form den t a l plaque (43). The acquired p e l l i c l e on the tooth surface i s an organic f i l m , absorbed to the enamel and c o n s i s t s of- s a l i v a r y p r o t e i n s , g l y c o p r o t e i n s and immunoglobulins. Streptococcus mutans and Streptococcus sanguis are examples of o r a l micro-organisms that s e l e c t i v e l y adhere to the p e l l i c l e (40). The accumulation of b a c t e r i a i n plaque a l s o i n v o l v e s i n t e r a c t i o n s of b a c t e r i a w i t h c e l l s of the same or d i s s i m i l a r species. These adherent i n t e r a c t i o n s appear to be mediated by substances that are e i t h e r produced by the b a c t e r i a or are adsorbed to t h e i r c e l l surfaces from the surrounding m i l i e u . Some examples of these i n t e r b a c t e r i a l aggregations are: (i) the "corncob "formations" of Bacteroides matruchotii and o r a l streptococci, or Actinomyces naeslundii and sanguis (43). ( i i ) j>. s a l i v a r i u s and V e i l l o n e l l a alcalescens (99). ( i i i ) S^. sanguis or S^  inutans bound to Actinomyces viscosus (15,68) . Selective adherence was also observed with the indigenous microflora i the g a s t r o i n t e s t i n a l t r a c t of rodents and chickens; these microorganisms showed a preference for e i t h e r secreting, or nonsecreting, k e r a t i n i z e d c e l l s (85,86). Aly et a l (3), found a high degree of s p e c i f i c i t y i n the adherence of b a c t e r i a to nasal mucosal c e l l s . Organisms most commonly found i n the nose included species of Staphylococcus aureus and Staphylococcus epidermidis. These b a c t e r i a were found to adhere to nasal c e l l s to a higher degree than organisms not normally present i n the nose. Adherence and pathogenicity Although the pathogenicity of a microbe i s not determined s o l e l y by i t s a b i l i t y to bind to host ti s s u e , i t has been suggested that adherence i s a necessary prerequisite to a number of microbial i n f e c t i o n s . Some examples are: o (i) streptococcal pharyngitis by Streptococcus pyogenes (32) ( i i ) dental c a r i e s (40,43). ( i i i ) gonorrhea (80,93) (iv) cholera (36,52,53). Strains of V i b r i o cholera that could attach s p e c i f i c a l l y to the brush borders of rabb i t i n t e s t i n a l c e l l s appeared to have an advantage 4 i n c o l o n i z i n g that area over nonadherent s t r a i n s (v). D i a r r h e a , caused by e n t e r o t o x i g e n i c s t r a i n s of E s c h e r i c h i a c o l i t hat could bind to porcine brush border c e l l s . S t r a i n s of E_. c o l i that f a i l e d to adhere to these c e l l s d i d not cause the dise a s e . (6,54). ( v i ) b a c t e r i a l e n d o c a r d i t i s (46,81). Organisms most f r e q u e n t l y i s o l a t e d from p a t i e n t s w i t h b a c t e r i a l e n d o c a r d i t i s , are e n t e r o c o c c i , v i r i d a n s s t r e p t o c o c c i , s t a p h y l o c o c c i and Pseudomonas aeruginosa (46). These b a c t e r i a were a l s o found to adhere more r e a d i l y to damaged.heart valves i n v i t r o , than could other organisms not g e n e r a l l y a s s o c i a t e d w i t h the disease. The f i r s t step i n the i n i t i a t i o n of these diseases appears to be the adherence of the microbe to the host t i s s u e . A f t e r adherence has occurred, i n d u c t i o n of an inflammatory response, r e l e a s e of b a c t e r i a l t o x i n s , or i n v a s i o n of host t i s s u e can f o l l o w . Competition between indigenous m i c r o f l o r a and new invaders Where an indigenous m i c r o f l o r a has e s t a b l i s h e d i t s e l f i n the host, a competition would e x i s t f o r the a v a i l a b l e adherence s i t e s between these microorganisms and any newcomers to the area. A greater a v i d i t y f o r these s i t e s by the indigenous m i c r o f l o r a would be advantageous to the host to help prevent the establishment of a pathogen. I f the pathogen i s not su c c e s s f u l i n competing f o r a space i n the host, i t s c o l o n i z a t i o n would be impaired. Savage, i n h i s review (86), c i t e s s e v e r a l examples where the indigenous m i c r o f l o r a of the mammalian mucosal e p i t h e l i u m i n t e r f e r e s w i t h invading 5 pathogens. One example i n c l u d e s the indigenous m i c r o f l o r a , l a c t o b a c i l l i and yeast, of the mouse stomach. As long as the l a c t o b a c i l l i remain and are not d i s p l a c e d w i t h agents such as p e n i c i l l i n , other microbes seem unable to e s t a b l i s h themselves i n t h i s area (85,86). I f the invading organism possesses a strong adherence mechanism, i t can become e s t a b l i s h e d i n the host. For example, the pathogen K[. gonorrhoea, can adhere more s t r o n g l y to human v a g i n a l c e l l s than can L a c t o b a c i l l u s a c i d o p h i l u s and other indigenous microorganisms (71,80). Pathogenic Candida a l b i c a n s , i s shown to adhere t o , and c o l o n i z e v a g i n a l c e l l s to a much greater degree than nonpathogenic species (57). Forces involved i n b a c t e r i a l - c e l l surface i n t e r a c t i o n s P r e s e n t l y there are two schools of thought regarding the fo r c e s that mediate attachment of b a c t e r i a to a s o l i d s u r f ace. One school suggests that the m a j o r i t y of ''.the:- b i n d i n g i n t e r a c t i o n s i n v o l v e e l e c t r o s t a t i c f o r c e s such as those observed when b a c t e r i a bind to g l a s s (72), or to hydroxyapatite c r y s t a l s (43). However, i n c r e a s i n g evidence tends to support the second school, which f e e l s that a more s p e c i f i c bonding occurs between b a c t e r i a and host t i s s u e . These bonds have been l i k e n e d to those involved i n antigen-antibody or l e c t i n - s u g a r i n t e r a c t i o n s (11,12,22,42,43). Before any bonds can be formed between a b a c t e r i a l , and another c e l l s u r f ace, the a t t r a c t i v e f o r c e s between them must be s u f f i c i e n t l y strong to overcome the r e s p u l s i v e e l e c t r i c a l f o r c e s created by the j u x t a p o s i t i o n of the two n e g a t i v e l y charged surfaces (9,11,100). A t t r a c t i v e f o r c e s may include van der Waals' forces where a f l u c t u a t i o n i n charge or dipolemove-ment could a t t r a c t and draw the two surfaces together. A l t e r n a t i v e l y , s u i t a b l e alignment of the two surfaces could r e s u l t i n the formation of 6 hydrogen or i o n i c bonds. Hydrophobic i n t e r a c t i o n s between b a c t e r i a l and euka r y o t i c c e l l s have been suggested as a major a t t r a c t i v e force (10,11, 12,100). Experimental r e s u l t s i n d i c a t e that the l i p i d p o r t i o n of l i p o -t e i c h o i c acids found on s t r e p t o c o c c a l c e l l s , i n t e r a c t s w i t h hydrophobic areas of euk a r y o t i c c e l l membranes (10,12); t h i s b i n d i n g r e a c t i o n i s i n h i b i t e d by g a n g l i o s i d e s or albumen (10,12,100). I n h i b i t i o n may r e s u l t when the l i p o t e i c h o i c acids form an i n t e r a c t i o n w i t h the l i p i d moiety of ga n g l i o s i d e s or w i t h areas of the albumen molecule known to have a high a f f i n i t y f o r f a t t y a c i d molecules (10,100). C e l l surface components involved i n adherence Adhering b a c t e r i a are thought to possess areas on t h e i r c e l l surface which are a c c e s s i b l e and i n an arrangement that f a c i l i t a t e s the formation of bonds w i t h complementary molecules on eukaryotic c e l l s u r f a c e s . I n v e s t i -gators studying m i c r o b i a l adherence are attempting to i d e n t i f y these c e l l surface components. The adhesive molecules on the b a c t e r i a l c e l l have been termed ligands (26), or adhesins (12), and those on animal c e l l s have been c a l l e d receptors (11,12). Experimental procedures used to determine the character of adhesins and receptors i n c l u d e : ( i ) I n h i b i t i n g the b i n d i n g r e a c t i o n w i t h "haptens" or analogues of the adhesin, or w i t h a n t i b o d i e s d i r e c t e d against antigens present i n the adhesin. ( i i ) Modifying or d e s t r o y i n g the adhesin w i t h s p e c i f i c p h y s i c a l , enzymatic, or chemical treatments, ( i i i ) I s o l a t i n g and p u r i f y i n g the adhesin or r e c e p t o r , ( i v ) C h a r a c t e r i z i n g mutants which l a c k the a b i l i t y to mediate the b i n d i n g r e a c t i o n . . 7 Eukaryotic c e l l receptors Receptors on mammalian c e l l s have been shown to include g l y c o p r o t e i n s , g l y c o l i p i d s , and p r o t e i n s (12,41,43). Information on the nature of the receptors i s l i m i t e d but there are a few examples i n the l i t e r a t u r e where the components of the eukaryotic receptor s i t e have been determined (11,12 22,74). Ofek and Beachey (74) have proposed that D-mannose-like residues on the surface of e r y t h r o c y t e s and other mammalian c e l l s serve as receptors f o r type I f i m b r i a e of Enterobacteriaceae. F r e t e r and others have shown that V. cholerae binds to at l e a s t two d i f f e r e n t g a s t r o i n t e s t i n a l receptors (36,52,53). One i s l o c a t e d on i n t e s t i -n a l brush border surfaces and i s L-fucose and mannose s e n s i t i v e . The second whose i n t e s t i n a l l o c a t i o n has not yet been determined, i s L-fucose r e s i s t a n t (36,53). Mycoplasma pneumoniae binds to s i a l i c a c i d residues on r e s p i r a t o r y e p i t h e l i a l c e l l s (20). Treatment of the e p i t h e l i a l c e l l s w i t h neuramini-dase decreases attachment by f i f t y percent (20). S i a l i c a c i d residues on a s a l i v a r y g l y c o p r o t e i n serve as the receptors f o r s a l i v a - i n d u c e d aggregation of S. sanguis (65). B a c t e r i a l adhesins B a c t e r i a l c e l l surface components i m p l i c a t e d i n mediating adherence include p o l y s a c c h a r i d e s , l i p o p o l y s a c c h a r i d e s , l i p o t e i c h o i c and t e i c h o i c a c i d s , p r o t e i n s and l i p o p r o t e i n s (11,12,41,42,43,91,92,100). Some have l e c t i n - l i k e p r o p e r t i e s and are present i n p i l i , f i m b r i a e , or s i m i l a r c e l l surface p r o t r u s i o n s (43,74,92). The nomenclature of b a c t e r i a l p r o t r u s i o n s i n v o l v e d i n adherence i s 8 c u r r e n t l y i n c o n f l i c t . P i l i , d escribed as short, r i g i d , proteinaceous appendages that cover the b a c t e r i a l c e l l s u rface, are r e s p o n s i b l e f o r b i n d i n g some b a c t e r i a to host t i s s u e (10,17,74,77,92). P i l i , have g e n e r a l l y been reserved f o r s t r u c t u r e s on Gram negative organisms. These s t r u c t u r e s have a l s o been c a l l e d f i m b r i a e (12,26,77) and, when present on Gram p o s i t i v e organisms, the term often used i s f i b r i l l a e . Fimbriae are found i n many species of Enterobacteriaceae, and are thought r e s p o n s i b l e f o r attachment of the b a c t e r i a to f u n g a l , p l a n t , and animal c e l l s (26,77). These c e l l s t r u c t u r e s have been c l a s s i f i e d i n t o four types according to t h e i r diameter, l e n g t h , adherence proper-t i e s and s e n s i t i v i t y of adherence to i n h i b i t i o n by sugars. The best studied fimbriae belong to Type I . They have been p u r i f i e d , shown to a g g l u t i n a t e e r y t h r o c y t e s and to adhere to t i s s u e c u l t u r e c e l l s , both r e a c t i o n s are i n h i b i t e d by mannose (7,26,77,92). Studies i n v o l v i n g f i f t y - n i n e s t r a i n s of Bacteroides melaninogenicus showed that only those s t r a i n s possessing f i m b r i a e - l i k e s t r u c t u r e s mani-fe s t e d hemagglutinating a c t i v i t i e s (75). V i r u l e n t s t r a i n s of N. gonorrhoeae possess p i l i which enable them to a t t a c h to a v a r i e t y of mammalian c e l l s (17,26,77,80). P i l i a t e d gonococci b i n d to human c e l l s i n greater numbers and at an increased r a t e of attachment over those v a r i a n t s that l a c k p i l i (80). Buchanan and Pearce, 1976, i s o l a t e d and p u r i f i e d gonococcal p i l i and found that they could a g g l u t i n a t e human er y t h r o c y t e s (17); a r e a c t i o n i n h i b i t e d when a n t i - p i l i antiserum wasadded. Surface appendages c a l l e d f i b r i l s , form p a r t of a fuzzy coat on the surface of S^. m i t i s , S_. s a l i v a r i u s and S_. pyogenes (32,39,40). These s t r u c t u r e s are b e l i e v e d to be important i n the b i n d i n g of these organisms 9 to r e s p i r a t o r y e p i t h e l i a l c e l l s (32,39,40). Possession of p i l i or f i m b r i a e by an organism could enhance i t s b i n d i n g to a n e g a t i v e l y charged surface i n two ways. ( i ) They help to counteract r e p u l s i v e e l e c t r o s t a t i c f o r c e s (11,26,74,77,80), since p i l i a t e d b a c t e r i a have been shown to possess a lower d e n s i t y of negative surface charge and a more hydrophobic character (26,74). Increased hydrophobicity may a s s i s t the convergence of the two surfaces and enable a r e c e p t o r - l i g a n d bond to form. ( i i ) P i l i may possess l i g a n d s i t e s which i n t e r a c t d i r e c t l y w i t h receptors on the e u k a r y o t i c c e l l s u r f a c e . In t h e i r review, Ofek and Beachey, (7*4), 1980, reported that increased hydrophobicity of p i l i a t e d organisms such as Salmonella typhimurium and _E. c o l i , f a c i l i t a t e d t h e i r attachment to t i s s u e c u l t u r e c e l l s . A s i m i l a r c l a i m has been made f o r the attachment of gonococci to u r e t h r a l e p i t h e l i u m (80,93). However, other i n v e s t i g a t o r s f e e l that a more s p e c i f i c b i n d i n g between l e c t i n - l i k e adhesins on gonococcal p i l i and sugar moieties on e p i t h e l i a l c e l l s i s r e s p o n s i b l e (17,77). The biochemical nature of p i l i and other b a c t e r i a l adhesins The chemical composition of adhesins on b a c t e r i a l c e l l surfaces has only been determined i n a few cases. A plasmid-coded p r o t e i n , the K88 antigen, has been found on the surface of f i m b r i a e of some enteropathogenic s t r a i n s of IS. c o l i and i s b e l i e v e d r e s p o n s i b l e f o r adherence of these b a c t e r i a to the i n t e s t i n a l w a l l of p i g l e t s (54,61). Since adherence i s i n h i b i t e d by mannose (54,61, 91), and by methyl-a-D-mannopyranoside (7), a 1ec t i n - r e cep t or 10 i n t e r a c t i o n may be i n v o l v e d . Other enteropathogenic s t r a i n s of E_. c o l i have a heat l a b i l e antigen d i s t i n c t from the K88 antigen, thought to be associated w i t h t h e i r adherence (26,80). P r o t e i n s are i n v o l v e d i n other b a c t e r i a l - c e l l surface i n t e r a c t i o n s . Adhesion of M. pneumoniae to human c e l l s i s b e l i e v e d to be mediated by a high molecular weight p r o t e i n (20). When the b a c t e r i a l c e l l s were heat, or t r y p s i n - t r e a t e d , they l o s t t h e i r a b i l i t y to b i n d to t r a c h i a l e p i t h e l i a l c e l l s and to ery t h r o c y t e s (20). P r o t e i n A, a component of _S. aureus c e l l w a l l s , i s known to bind to the Fc re g i o n of IgG an t i b o d i e s (8). A u s t i n and Daniels (8), have presented evidence that s t a p h y l o c o c c i l o c a l i z e i n lung t i s s u e i n f e c t e d by i n f l u e n z a v i r u s , by a t t a c h i n g to c e l l s coated w i t h a n t i v i r a l IgG a n t i b o d i e s . A p r o t e i n i n the c e l l w a l l of S^. s a l i v a r i u s , mediates attachment to V. a l c a l e s c e n s (98,99). The attachment of ^ 3. pyogenes to e p i t h e l i a l c e l l s was o r i g i n a l l y thought to be r e l a t e d to the M p r o t e i n present on the f i b r i l l a e of these s t r e p t o c o c c i (32). However, recent evidence from the la b o r a t o r y of Beachey and colleagues (10,12), i n d i c a t e s that the adhesin i s l i p o t e i c h o i c a c i d . The t e i c h o i c a c i d i s l i n k e d to the f i b r i l l a e ( p o s s i b l y by the M p r o t e i n ) , and the l i p i d binds to hydrophobic areas on the e p i t h e l i a l c e l l s (12). This would e x p l a i n why treatment of s t r e p t o c o c c a l c e l l s w i t h t r y p s i n would destroy t h e i r a b i l i t y to bind to e p i t h e l i a l c e l l s . L i p o t e i c h o i c a c i d s bind to o r a l mucosal c e l l s , human p l a t e l e t s , e r y t h r o c y t e s , and a v a r i e t y of other mammalian c e l l s (100). The b i n d i n g s i t e on the er y t h r o c y t e appears to be a p r o t e i n (100). The l i p o t e i c h o i c a c i d present i n the s t r e p t o c o c c a l c e l l w a l l has been i m p l i c a t e d as a c l a s s of a b a c t e r i a l polymer that c o n t r i b u t e s to cohesion i n human dental plaque (43,74,100). Carbohydrates, present as capsules or i n f i b e r s , have been described as the adhesin i n some b a c t e r i a l a n d eukaryotic c e l l s (22,42). Already mentioned were the mucilaginous or h o l d f a s t m a t e r i a l produced by some marine b a c t e r i a (21,72). Costerton et a l (22), examined the surface coats of adherent b a c t e r i a i n n a t u r a l environments and i n the mammalian host and found that many had polysaccharide f i b r e s extending from t h e i r c e l l s u rfaces. These f i b e r s are the adhesins that b i n d the microorganisms to p l a n t , animal, or other b a c t e r i a l c e l l s . F i b e r s of d i f f e r e n t b a c t e r i a vary i n t h e i r composition and arrangement of sugars (22). Some examples where carbohydrates act as b a c t e r i a l adhesins are: ( i ) Glucans, synthesized by J^. mutans, which a t t a c h to the b a c t e r i a l c e l l w a l l v i a the enzyme g l u c o s y l t r a n s f e r a s e and bind the organism to the tooth p e l l i c l e (40,41,43). ( i i ) Polysaccharide f i b e r s on the surface of Bacteroides f r a g i l i s which mediate b i n d i n g to r a t p e r i t o n e a l c e l l s , b a c t e r i a and yeast (22). ( i i i ) Dextran, which i s i n v o l v e d i n the coaggregation of A. v i s c o s u s 15987 w i t h S_. sanguis or S^. mutans (15). ( i v ) An u n i d e n t i f i e d carbohydrate on the surface of j ^ . sanguis that enables i t to aggregate w i t h A. viscosus T14V (68). (v) Dextran, produced by s t r e p t o c o c c i i s considered to be the agent mediating t h e i r adherence to normal and damaged endo c a r d i a l t i s s u e (46,87). The e x i s t e n c e of b a c t e r i a l polysaccharide f i b e r s has only been d e t e r -mined r e c e n t l y from s t u d i e s of b a c t e r i a i n n a t u r a l environments, where the production of adherent f i b e r s confers a s e l e c t e d advantage to the micro-12 organisms. Under c o n t r o l l e d , noncompetitive, l a b o r a t o r y c o n d i t i o n s the energy necessary to produce these f i b e r s i s probably u t i l i z e d f o r other c e l l u l a r a c t i v i t i e s (22). Adherence p r o p e r t i e s of S_. aureus The v i r u l e n c e of aureus i s a c c r e d i t e d to i t s production of t o x i n s , enzymes and f r e e coagulase. Another property, shared by the m a j o r i t y of pathogenic s t r a i n s , i s the a b i l i t y to b i n d to and clump i n the presence of f i b r i n o g e n and i t s degradation products (2,18,47,48,49,50). A component on the sta p h y l o c o c c a l c e l l r e s p o n s i b l e f o r t h i s a c t i v i t y i s known as clumping f a c t o r , \ ( C l f ) :(29).: S t r a i n s of S_. aureus that are clumping f a c t o r p o s i t i v e , ( C l f + ) , h a v e a l s o been reported to b i n d to immobilized f i b r i n o g e n (67), * -Although some controversy e x i s t s as to whether C l f confers an increased p a t h o g e n i c i t y to the organism, s e v e r a l r e p o r t s i n the l i t e r a t u r e a t t e s t to i t s importance i n the c o l o n i z a t i o n of j3. aureus i n the mammalian host (1,3,44,45,46). Alami et a l (1), c l a s s i f i e d S_. aureus ..strains i n t o groups according to t h e i r f r e e coagulase and C l f a c t i v i t i e s and then examined each f o r i t s p a t h o g e n i c i t y f o r mice. They found that both f r e e coagulase and C l f were s i g n i f i c a n t to the pathogenesis of s t a p h y l o c o c c a l i n f e c t i o n s , w i t h C l f p l a y i n g a major r o l e i n the e a r l y stages of i n f e c t i o n . In another study, G o r r i l l et a l (44), reported that a f t e r intravenous i n j e c t i o n of mice w i t h a v a r i e t y of b a c t e r i a , only S^ aureus l o c a l i z e d i n the mouse kidney i n high numbers, the other b a c t e r i a t e s t e d were r a p i d l y removed by the host. The i n v e s t i g a t o r s a t t r i b u t e d t h i s t r a i t of the s t a p h y l o c o c c a l c e l l s to the presence of kidney lodgement property (KLP) (44), and suggested i t was synonymous w i t h C l f . A f t e r f u r t h e r i n v e s t i g a t i o n , they decided that KLP and C l f were two d i f f e r e n t p r o t e i n s on the s t a p h y l o c o c c a l c e l l w a l l (45). However, they d i d not i s o l a t e any s t r a i n of S. aureus that was K L P + and C l f and the p o s s i b i l i t y remains that the f a c t o r s are the same and that C l f bound to f i b r i n o g e n i n the kidney. Other i n v e s t i g a t o r s (55), found that mice, i n j e c t e d i n t r a p e r i t o n -e a l l y w i t h C l f + s t r a i n s of ^. aureus, succombed to a ~ t o x i n produced by the b a c t e r i a . The sta p h y l o c o c c a l c e l l s had clumped w i t h f i b r i n o g e n i n the mouse p e r i t o n e a l c a v i t y and were thence pr o t e c t e d from phagocytosis. S t r a i n s of C l f j5. aureus were not protected and were destroyed more r a p i d l y i n the mouse. Aly et a l (3), determined that S. aureus adhered more r e a d i l y to n a s a l mucosal c e l l s i n c a r r i e r s , o f t h i s microorganism, suggesting that adherence receptors are present on the c e l l s of these i n d i v i d u a l s but . not on those of n o n c a r r i e r s . Another p o s s i b i l i t y may be that c a r r i e r s are more prone to s k i n eruptions which would create a supply of f i b r i n o g e n and f i b r i n f o r adherent C l f + s t r a i n s of S. aureus. Staphylococcal e n d o c a r d i t i s Endocardial t i s s u e i s c o n s t a n t l y bathed w i t h blood, thus attachment by an organism to the endocardium and valves i s viewed as the i n i t i a l event i n the pathogenesis of b a c t e r i a l e n d o c a r d i t i s (5,26,81,87). I n d i v i d -u a l s p a r t i c u l a r l y s u s c e p t i b l e to the dis e a s e , are those w i t h c o n g e n i t a l or rheumatic heart disease, w i t h i n t r a c a r d i a c c a t h e t e r s , or p r o s t h e t i c valves (34,35). These c o n d i t i o n s predispose the endocardium and valves to scar t i s s u e and thrombi. Although S^. aureus w i l l adhere to normal v a l v e s , i t shows a gceater a f f i n i t y f o r damaged endocardium or thrombi (5,27,66]81), 14 and has been i s o l a t e d w i t h . i n c r e a s i n g , frequency.from cases-of b a c t e r i a l e n d o c a r d i t i s (34,35). Thrombi, composed of p l a t e l e t s and f i b r i n (5.27), may act as a n i d i s f o r C l f + S_. aureus. A f t e r b i n d i n g to f i b r i n and lodging i n a thrombus, f u r t h e r envelopment w i t h f i b r i n would make the s t a p h y l o c o c c a l c e l l d i f f i c u l t to e r a d i c a t e (27,89). This premise has been supported by s t u d i e s of s t a p h y l o c o c c a l e n d o c a r d i t i s produced e x p e r i -mentally i n the r a b b i t (34,35,38,66,89). Fi b r i n o g e n and f i b r i n F i b r i n o g e n , a s o l u b l e p r o t e i n w i t h a molecular weight of 340,000 dal t o n s , i s found i n the blood and lymph of v e r t e b r a t e s (13,25). I t i s a dimeric molecule composed of two sets of n o n i d e n t i c a l chains; alpha (Aa) , beta (3b), and gamma (.y) interconnected by d i s u l p h i d e bonds., (25). The p o l y m e r i z a t i o n of f i b r i n o g e n r e s u l t s i n the formation of f i b r i n , an e s s e n t i a l component of normal hemostasis. This event occurs when thrombin cleaves the N-terminal ends of the alpha and beta chains r e l e a s i n g f i b r i n o p e p t i d e s . The remainder of the f i b r i n o g e n molecule, the f i b r i n monomer, polymerizes w i t h adjacent monomers by l a t e r a l p a i r i n g and p a r t i a l overlapping to form an i n s o l u b l e polymer known as non-cross-l i n k e d f i b r i n . ( 2 5 ) . When the hemostatic f a c t o r X I I I i s present, the f i b r i n g e l i s s t a b i l i z e d and strengthened through i n t r o d u c t i o n of covalent bonds between g l y c i n e and l y s i n e residues on adjacent monomers. The c o v a l e n t l y s t a b i l i z e d , or c r o s s - l i n k e d f i b r i n , can be d i s t i n g u i s h e d from the non-cross-linked form by i t s i n s o l u b i l i t y i n 5 molar urea and by i t s greater r e s i s t a n c e to enzymatic d i s s o l u t i o n . 15 Clumping f a c t o r Although there have been some re p o r t s of _S. aureus clumping w i t h s o l u b l e f i b r i n monomers (2,49,50,63), l i t t l e i s known of the i n t e r a c t i o n of the organism w i t h a matrix of non-cross-linked or c r o s s - l i n k e d f i b r i n . The c e l l w a l l component of S_. aureus c a l l e d C l f has not been pr o p e r l y c h a r a c t e r i z e d , nor i s i t known i f the same substance i s i n v o l v e d i n clumping with f i b r i n o g e n and i n clumping or b i n d i n g w i t h f i b r i n . McNeil (69,70), i n v e s t i g a t e d the nature of clumping f a c t o r on i n t a c t s t a p h y l o c o c c a l c e l l s . Other i n v e s t i g a t o r s (16,29,56,84,94), have attempted to i s o l a t e " C l f " and l e a r n more of i t s p h y s i c a l , chemical and a n t i g e n i c p r o p e r t i e s . L i t t l e i n f o r m a t i o n has been gained from these s t u d i e s and i t remains to be proven i f the substance they e x t r a c t e d from s t a p h y l o c o c c a l c e l l s was C l f . Several i n v e s t i g a t o r s ' i s o l a t e d a substance they c a l l e d C l f from the supernatant of mechanically d i s r u p t e d c e l l s (29,56). Their p r e p a r a t i o n contained a p r o t e i n that was destroyed by a u t o c l a v i n g or by p r o t e o l y t i c enzymes. The p r o t e i n had no demonstrable a c t i v i t y w i t h f i b r i n o g e n but could ab sorb or n e u t r a l i z e a n t i b o d i e s found i n a n t i — S . aureus antiserum that i n h i b i t e d fibrinogen-induced clumping of s t a p h y l o c o c c a l c e l l s . Immuno-e l e c t r o p h o r e t i c a n a l y s i s f a i l e d to demonstrate i f the substance had a n t i g e n i c p r o p e r t i e s . Another group ex t r a c t e d " C l f " w i t h phenol from whole c e l l s or from the supernatant of crushed c e l l s (56,84). Latex beads, coated w i t h t h e i r e x t r a c t , a g g l u t i n a t e d i n a n t i - S . aureus antiserum and. absorbed clump-ing i n h i b i t i n g a n t i b o d i e s . However, the e x t r a c t f a i l e d to r e a c t v i s i b l y i n agarose gels when tested a g a i n s t a n t i - S . aureus.antiserum or against a n t i -serum from r a b b i t s immunized w i t h the e x t r a c t . An assay was developed 16 by one group (56) which showed that the e x t r a c t could adsorb f i b r i n o g e n , however, phenol e x t r a c t s from C l f S^. aureus c e l l s or from C l f + c e l l s that had been t r y p s i n - t r e a t e d or autoclaved, a l s o demonstrated f i b r i n o g e n adsorbing a c t i v i t i e s . Other i n v e s t i g a t o r s (16,94), ex t r a c t e d s t a p h y l o c o c c a l c e l l s w i t h a c i d and then p u r i f i e d the product by i s o e l e c t r i c f o c u s i n g . The e x t r a c t was used to coat erythrocytes which then clumped i n f i b r i n o g e n . L i t t l e i n f o r m a t i o n was given of the biochemical or a n t i g e n i c p r o p e r t i e s of t h e i r e x t r a c t . Except f o r s t u d i e s of Duthie, 1955 (30), who examined the a c t i v i t i e s of some c o c c i w i t h f i b r i n o g e n , l i t t l e i s known of the i n t e r a c t i o n s of b a c t e r i a , other than S_. aureus w i t h f i b r i n o g e n or f i b r i n . R e s u l t s of s t u d i e s reported i n t h i s t h e s i s attempt to: ( i ) C h a r a c t e r i z e the i n t e r a c t i o n s of S_. aureus and f i b r i n . ( i i ) I d e n t i f y the s t a p h y l o c o c c a l C l f . ( i i i ) Develop an assay f o r s o l u b l e C l f . ( i v ) I d e n t i f y other b a c t e r i a which i n t e r a c t w i t h f i b r i n o g e n and f i b r i n . I I MATERIALS AND METHODS B a c t e r i a and c u l t u r a l c o n d i t i o n s S^  aureus-1, clumping f a c t o r p o s i t i v e ( C l f + ) , was i s o l a t e d from the blood of a p a t i e n t w i t h i n f e c t i v e e n d o c a r d i t i s . S. aureus-5, a mutant obtained from the parent s t r a i n , was free coagulase p o s i t i v e and clumping f a c t o r negative ( C l f ). Streptococcus Group C 12388F _S. aureus D25904 (free coagulase negative and C l f + ) , and A. vis c o s u s 15987, were obtained from the American Type Cult u r e C o l l e c t i o n . B a c t e r o i d e s , P e p t o s t r e p t o c o c c i , p-."~~aeruginosa, S. s a n g u i s . S_. m i t i s , S^. mutans, _S. s a l i v a r i u s , S t a p h y l o c o c c i , E_. c o l i , V e i l l o n e l l a a l c a l e s c e n s and Fusobacterium nucleatum, were human i s o l a t e s maintained i n la b o r a t o r y stock c u l t u r e . Inocula were kept at -70°C i n growth medium supplemented w i t h 7% ( v o l / v o l ) dimethyl s u l f o x i d e . The s t a p h y l o c o c c i and pseudomonas were grown i n a modified t r y p t i c a s e soy b r o t h (MTSB) c o n s i s t i n g of 3.4% t r y p t i c a s e peptone, 0.5% NaCl, 0.3% phytone peptone, and 0.25% K^HPO^. R a d i o a c t i v e l y l a b e l l e d c e l l s were prepared by growing the b a c t e r i a overnight i n 14 MTSB c o n t a i n i n g 0.05 pCi of [ C] glucose per ml (Radiochemical Center, Amersham, England; 295 mCi/mmol). V. a l c a l e s c e n s was grown i n a medium c o n t a i n i n g : 0 . 5% t r y p t i c a s e , 0.3% yeast e x t r a c t , 1.5% sodium l a c t a t e and 0.1% Tween 80 ( v o l / v o l ) ; pH was adjusted to 7.5. The s t r e p t o c o c c i , A. v i s c o s u s , and .E. c o l i were grown i n the medium of Gibbons and F i t z g e r a l d (40) enriched w i t h 0.3% yeast 18 e x t r a c t . To obta i n r a d i o a c t i v e l y l a b e l l e d c e l l s , the b a c t e r i a were 3 grown i n medium supplemented w i t h l y C i of [ H] thymidine per ml. (Radiochemical Center, Amersham, England, 26 mCi/mmol). _F. nucleatum and P e p t o s t r e p t o c o c c i were grown i n a medium of 1.7% t r y p t i c a s e , 0.3% yeast e x t r a c t , 0.5% NaCl, and 0.25% K^HPO^. Bacteroides were grown i n the same medium supplemented w i t h 5 ug of hemin per ml. A l l anaerobic b a c t e r i a were grown at 37°C i n an anaerobic glove box, (Coy Mfg., Ann Arbor, Michigan) c o n t a i n i n g an atmosphere of N 2:H 2:C0 2 (85:10:5). B a c t e r i a r e q u i r e d f o r clumping and adherence assays were grown f o r 18 h, harvested by c e n t r i f u g a t i o n , washed twice w i t h 0.005M phosphate-buffered (pH 7.0) 0.14M' sodium c h l o r i d e s o l u t i o n , (PBS) and resuspended i n the same b u f f e r , unless noted otherwise. Mutagenic procedures jS. aureus-1 c e l l s were mutagenized i n T r i s - m a l e i c b u f f e r (0.05M) pH 6.0 using N-methyl-N'-nitro-N-nitrosoguanidine at a co n c e n t r a t i o n of 100 ug/ml. A c u l t u r e of c e l l s i n exponential phase was exposed to the mutagen f o r 30 min, then washed twice w i t h MTSB and f i n a l l y resuspended i n a small amount of T r i s - m a l e i c b u f f e r . This c e l l sus-pension was mixed w i t h media and incubated at 37°C u n t i l the c u l t u r e had reached an of 0.8. C e l l s were harvested by c e n t r i f ugation, washed twice w i t h PBS and resuspended i n PBS. Mutants l a c k i n g the a b i l i t y to clump w i t h f i b r i n o g e n were enriched i n the f o l l o w i n g manner. F i b r i n o g e n , f i n a l c o n c e n t r a t i o n of 1.0 mg/ml, was added to the c e l l suspension and the mixture was 19 shaken at room temperature f o r 20 min. Clumped c e l l s were allowed to s e t t l e and the supernatant, c o n t a i n i n g unclumped c e l l s , was removed. Fresh human f i b r i n o g e n was added to the supernatant and the enrichment procedure repeated s e v e r a l times. An inoculum was taken from the supernatant, added to f r e s h media, and the c u l t u r e allowed to grow at 37 C to A 6 6 Q of 5.0. The enrichment procedure was repeated once more except that t h i s time the nonclumping s t a p h y l o c o c c i from the supernatant were p l a t e d onto mannitol s a l t agar p l a t e s . Colonies were subcultured onto MTSB and were test e d f o r t h e i r a b i l i t y to bind to f i b r i n and clump w i t h f i b r i n o g e n . C. P r e p a r a t i o n of f i b r i n o g e n Human f i b r i n o g e n was i s o l a t e d from outdated human plasma according to the method of Blomback and Blomback (13). The percentage of c l o t t a b l e p r o t e i n was 96%. P u r i t y of the prepar-a t i o n was confirmed by e l e c t r o p h o r e s i s i n SDS 10% polyacrylamide g e l (PAGE). Gels were s t a i n e d w i t h Coomassie B r i l l i a n t blue (R250 Bio-Rad, Richmond, C a l i f . ) ajid revealed a s i n g l e high molecular weight p r o t e i n . Carbohydrate a n a l y s i s , (24) demon-s t r a t e d mannose, g a l a c t o s e , and glucosamine to be the only sugars present. The s i a l i c a c i d content (96) was 0.8% wt. of f i b r i n o g e n . D. I s o l a t i o n of f i b r i n o g e n chains F i b r i n o g e n was reduced and a l k y l a t e d to o b t a i n polypeptide chains of: alpha ( a ) , beta (B), and gamma (y) by the method of Shinada and Hampton (88). B r i e f l y , l y o p h i l i z e d f i b r i n o g e n (300 mg) and NaCl (525 mg) were d i s s o l v e d i n 10 ml of deaerated guanidine 20 h y d r o c h l o r i d e at 6.0M pH 8.5. The pH was immediately adjusted to 8.5 w i t h 1.0M NaOH and the s o l u t i o n incubated at 40°C f o r 1 h. A f t e r adding d i t h i o t h r e i t o l (38.2 mg i n 0.5 ml deaerated 1^0), and a d j u s t i n g the pH to 8.8, the s o l u t i o n was f l u s h e d w i t h N 2 gas f o r 5 min. I t was then t i g h t l y capped and l e f t at room temperature f o r 1.5 h. Iodoacetic a c i d (228.7 mg) i n 3 ml H^ O was added and the pH immediately adjusted to 8.3 w i t h 2.OH NaOH. E x a c t l y 15 min l a t e r , g l a c i a l a c e t i c a c i d was added to give a f i n a l c o n c e n t r a t i o n of 50% v o l / v o l . The S-carboxymethylated f i b r i n o g e n was a p p l i e d to a G-10 Sephadex column (2.5 x 20 cm) which had been e q u i l i b r a t e d w i t h 50% deaerated a c e t i c a c i d and wrapped i n aluminum f o i l . The flow r a t e was 30 ml/h and the elu'ant was monitored at A „ o n ZoU. The f i r s t f r a c t i o n that e l u t e d was d i a l y z e d against d i s t i l l e d water and l y o p h i l i z e d . Approximately 150 mg of l y o p h i l i z e d m a t e r i a l was d i s s o l v e d i n 5 mis of 8M urea c o n t a i n i n g 0.025M sodium acetate pH 4.8 and a p p l i e d to a CM-cellulose column (2.5 x 15 cm), (Bio-Rad.). The s t a r t i n g b u f f e r (250 ml) of 8M urea and 0.025M sodium acetate pH 4.8 was followed by a l i n e a r gradient of 1 £ 0.025H sodium acetate pH 4.8 and 1 £ 0.175M sodium acetate pH 5.5 i n 8.0M urea. E l u t i o n was performed at room temperature and monitored f o r absorbance at 280 ym. P r i n c i p a l ^280 f r a c t i ° n s were c o l l e c t e d , d i a l y z e d against d i s t i l l e d water and adjusted to pH 8.5 w i t h 1.0.M NH^OH. The f r a c t -ions were concentrated by u l t r a f i l t r a t i o n w i t h a PM-10 membrane (Amicon). E. P r e p a r a t i o n of f i b r i n v i a l s Both c r o s s - l i n k e d and non-cross-linked f i b r i n were prepared 21 i n g l a s s s c i n t i l l a t i o n v i a l s . Human f i b r i n o g e n , 500 yg i n 350 y l PBS was added to each v i a l and mixed gently w i t h 2 u n i t s of thrombin i n 50 y l PBS. To prepare c r o s s - l i n k e d f i b r i n , 0.0025M •  Ca.Cl and 0.0125M c y s t e i n e were added to the f i b r i n o g e n s o l u t i o n i n each v i a l . The v i a l s were placed at room temperature on a r e c i p r o c a l shaker (80 strokes per ml) f o r 2 h and then l e f t to s i t at 4°C overnight. The f i b r i n matrix was washed twice w i t h 20 ml of PBS and then shaken at room temperature w i t h PBS f o r 20 min. V i a l s were drained and l e f t to dry at 37°C f o r 3 h. The presence of c r o s s - l i n k e d or non-cross-linked f i b r i n was confirmed by assaying t h e i r d i s s o l u t i o n i n 8M urea and by a n a l y s i s on SDS 10% PAGE (4, 97) F. Clumping t i t r e of b a c t e r i a 1. Tube d i l u t i o n method: C e l l s from an overnight c u l t u r e were washed and resuspended i n PBS to a standard absorbance of 4.0 at 660 nm. The c e l l sus-9 pension was approximately equal to 6.4 x 10 ( c e l l s ) per ml as determined by microscopic count, using a Petroff-Hausser counting chamber. Human f i b r i n o g e n 1%, was s e r i a l l y d i l u t e d i n g l a s s tubes w i t h PBS. An equal volume (0.1 ml) of the c e l l suspension was added to each tube. PBS was s u b s t i t u t e d f o r f i b r i n o g e n i n the c o n t r o l tube. Tubes were shaken f o r 20 min i n a s l a n t e d rack at room temperature. The clumping t i t r e was the r e c i p r o c a l of the conce n t r a t i o n of f i b r i n o g e n i n the l a s t tube showing v i s u a l l y d e tectable clumping. 2. M i c r o t i t r e method: This method e s s e n t i a l l y f o l lowed that of L e a v e l l e et a l (59). 22 A c a l i b r a t e d p i p e t t e dropper was used to d e l i v e r 0.025 ml PBS to each w e l l of a type u m i c r o t i t r e p l a t e , (Cooke Engineering Co., A l e x a n d r i a , Va.). S e r i a l 2 - f o l d d i l u t i o n s of 2.0% human f i b r i n o g e n i n PBS were made w i t h a 0.025 ml m i c r o t i t r e d i l u t e r . An equal amount of a b a c t e r i a l suspension, of 4.0, was added to each w e l l and the m i c r o t i t r e p l a t e placed on a r e c i p r o c a l shaker f o r 20 min at 25°C. A c o n t r o l w e l l c o n t a i n i n g c e l l s but no f i b r i n o g e n , was always included i n the assay to determine i f the b a c t e r i a self-aggregated. The end p o i n t was the l a s t w e l l c o n t a i n i n g clumped c e l l s . G. Adherence of b a c t e r i a to f i b r i n R a d i o a c t i v e l y l a b e l l e d b a c t e r i a were added to v i a l s c o n t a i n i n g c r o s s - l i n k e d or non-cross-linked f i b r i n . Unless sta t e d otherwise, g approximately 6.4 x 10 c e l l s (Aggg of 1.0) i n a volume of 400 ] i l PBS pH 7.0 were added to each v i a l which was then shaken on a r e c i p r o c a l shaker f o r 60 min at room temperature. Nonadherent b a c t e r i a were removed by washing the f i b r i n l a y e r twice w i t h 30 ml of PBS. The v i a l s were allowed to d r a i n f o r 10 min and 10 ml of s c i n t i l l a t i o n f l u i d was added. The amount of c or H was deter-mined i n a s c i n t i l l a t i o n counter. A sample of r a d i o a c t i v e l y l a b e l l e d c e l l s was a l s o counted m i c r o s c o p i c a l l y using a P e t r o f f -Hausser counting chamber to r e l a t e r a d i o a c t i v e counts and b a c t e r i a l numbers. Experiments were performed i n t r i p l i c a t e and were repeated on at l e a s t two separate occasions. 23 H. C h a r a c t e r i z a t i o n of the b i n d i n g receptors of S^. aureus 1. M o d i f i c a t i o n of whole c e l l s Whole c e l l s were tre a t e d w i t h v a r i o u s p h y s i c a l and chemical agents to determine i f any treatment a f f e c t e d the adherence of a u r e u s to f i b r i n . Unless s t a t e d otherwise, c e l l s were suspended i n PBS pH 7.0, k,rn of 1.0. oo(J a. A u t o c l a v i n g Whole c e l l s were autoclaved f o r 20 min at 121°C. b. S o n i c a t i o n A suspension of c e l l s was sonicated f o r 5 min at 4°C w i t h a microtipped b i o s o n i c probe ( B r o n w i l l S c i e n t i f i c Inc., Rochester, N.Y.). M i c r o s c o p i c examination showed that the c e l l s remained i n t a c t w i t h t h i s procedure. c. A c e t y l a t i o n Washed c e l l s were suspended i n 10 ml of 0.05M borate b u f f e r pH 8.5 Ag^Q of 1.0. Three ml of an a c e t i c anhydride-benzene mixture (1:2 v o l / v o l ) were added dropwise over a t h i r t y min time p e r i o d w i t h constant s t i r r i n g i n an i c e bath. The pH was maintained at 8-9 by adding IN NaOH. At the end of t h i s r e a c t i o n c e l l s were c e n t r i f u g e d , washed three times w i t h PBS and resuspended i n PBS. A c o n t r o l contained g l a c i a l a c e t i c a c i d i n s t e a d of a c e t i c anhydride. d. SDS Staphylococcal c e l l s were suspended i n PBS c o n t a i n i n g 1% sodium dodecyl sulphate (SDS) f o r 1 h at 37°C. C e l l s were washed f i v e times w i t h PBS, then shaken w i t h Bio-Beads SM-2 (Bio-Rad), to remove r e s i d u a l SDS. 24 e. NaOH, HC1 Staphylococcal c e l l s were t r e a t e d w i t h NaOH or HC1 at 0.2N ( f i n a l concentration) at 60°C f o r 60 min. Controls i n PBS without HC1 or NaOH were he l d at 60°C f o r 60 min. C e l l s were washed f i v e times w i t h PBS and resuspended i n the same b u f f e r . f. Proteases Whole c e l l s of S^. aureus were incubated at 37°C w i t h each of the f o l l o w i n g p r o t e a s e s : t r y p s i n , a-chymotrypsin, s u b t i l i s i n , and p r o n a s e , ; a t 1 mg/ml. C e l l s were washed 5 t i m e s w i t h PBS, 4°C, t o remove t h e enzyme. To determine i f any protease remained, c e l l s were suspended i n PBS, and were incubated w i t h A z o c o l l (20 mg) f o r 2 h at 37°C i n a shaking water bath, (5.1). I n s o l u b l e m a t e r i a l was removed by f i l t e r i n g and the amount of dye r e l e a s e d , d e t e r m i n e d by assaying the supernatant, s p e c t r o p h o t o m e t r i c a l l y at'520 nm. C e l l s were judged f r e e of protease i f no dye was s o l u b i l i z e d during the 2-h .incubation 2. M o d i f i c a t i o n of c e l l w a l l s a. T r i c h l o r a c e t i c a c i d e x t r a c t i o n C e l l w a l l s were e x t r a c t e d w i t h 5% t r i c h l o r a c e t i c a c i d (TCA), f o r 48 h at 4°C,or 10% TCA f o r 90 min at 60°C, and then washed 5 times w i t h PBS. b. Phenol e x t r a c t i o n C e l l w a l l s were mixed with 90% phenol f o r 15 min at 37°C, and were then washed 5 times w i t h PBS. 25 c. Sodium_j>eriodate treatment C e l l w a l l s were suspended i n 0.1 M acetate b u f f e r , pH 4.5, con t a i n i n g 0.01M sodium periodate and incubated i n the dark at room temperature f o r 20 h. Cont r o l c e l l w a l l suspensions were incubated i n the same b u f f e r but without sodium p e r i o d a t e . Walls were washed 3 times i n PBS to remove the periodate. d. Formamide C e l l w a l l s (1.5 ml) at krrr. of 4.0 i n H„0, were mixed w i t h 660 I 1 ml formamide, ;.and were thenheated i n an o i l bath at 150°C f o r 15 min. Formamide was removed by washing the w a l l s w i t h PBS. ' Other m o l l i f i c a t i o n procedures of ceMl'walls were performed-as described f o r whole c e l l s . I . I n h i b i t o r s of the S_. aureus f i b r i n b i n d i n g r e a c t i o n J3. aureus c e l l s were incubated w i t h a number of compounds f o r 30 min at 25°C i n a shaking water bath p r i o r to adding the c e l l s to the f i b r i n matrix. The c e l l s were incubated w i t h the f i b r i n f o r 60 min and then assayed f o r adherence. J . I s o l a t i o n and s o l u b i l i z a t i o n of c e l l w a l l s 1. I s o l a t i o n of w a l l s C e l l s were harvested from MTSB and were washed twice i n d i s t i l l e d water. Approximately 9 g (wet wt) of c e l l s , suspended i n 30 ml d i s t i l l e d H^ O were mixed w i t h 10 g glass beads (75-150 y, Bio-Rad), and the c e l l s disrupted-by s t i r r i n g at maximum speed f o r 2 h i n a M i n i - m i l l (Gifford-Wood Co.). The g l a s s beads were removed by f i l t r a -a t i o n on a coarse s i n t e r e d g l a s s f i l t e r . The whole c e l l s and c e l l 26 w a l l s were separated from the f i l t r a t e by c e n t r i f u g a t i o n at 40,000 x g f o r 15 min. Whole c e l l s were deposited as a p e l l e t of unbroken yellow organisms overlayered w i t h white c e l l w a l l s . The upper l a y e r of w a l l s was removed and washed 4 times w i t h d i s t i l l e d H^O. The p u r i t y of the w a l l p r e p a r a t i o n was checked by phase co n t r a s t micro-scopy and w i t h a Gram s t a i n . 2. S o l u b i l i z a t i o n of w a l l s A 5 ml suspension of c e l l w a l l s (0.2 g dry w t ) , i n PBS was digested f o r 2 h at 37°C w i t h 0.75 mg l y s o s t a p h i n . H y d r o l y s i s was measured o p t i c a l l y by f o l l o w i n g the decrease i n absorbance at 660 nm. Approximately 60-80% of the dry wt of S^ . aureus c e l l w a l l s were s o l u b i l i z e d i n t h i s procedure. The supernatant was separated from p a r t i c u l a t e m a t e r i a l by c e n t r i f u g i n g 3 times at 30,000 x g f o r 20 min. K. A n a l y s i s of s o l u b i l i z e d c e l l w a l l f r a c t i o n s 1. Phosphorus, p r o t e i n , and hexose content Phosphorus content of c e l l w a l l s and the s o l u b i l i z e d f r a c t i o n was determined by the method of Chen et a l (19). P r o t e i n was determined by the method of Lowry et a l (64), using bovine serum albumin (BSA) as a standard. The amount of hexoses present i n c e l l w a l l s and the s o l u b i l i z e d f r a c t i o n was determined by the anthrone assay (83), (glucose standard). 2. A f f i n i t y chromatography Sepharose 4B beads were a c t i v a t e d w i t h cyanogen bromide e s s e n t i a l l y according to the method of Porath et a l (79). Cyanogen bromide, (300 mg per ml packed beads), .was added to beads suspended i n H^O, and the pH maintained at 11.0 by a d d i t i o n of 6 N NaOH. When the pH s t a b i l i z e d (20-30 min), the Sepharose beads were washed on a coarse s i n t e r e d glass f i l t e r w i t h 30 times t h e i r volume of i c e - c o l d -H 20. P u r i f i e d human f i b r i n o g e n (0.5 mg/ml), i n 0.1M borate b u f f e r pH 9.0 was added to an equal volume of packed wet beads. The beads and f i b r i n o g e n were r o t a t e d at 4°C f o r 18 h. The fibrinogen-coupled Sepharose beads were then washed f r e e of uncoupled f i b r i n o g e n and were suspended i n 0.1M borate b u f f e r pH 9.0. The f i b r i n o g e n beads were assayed f o r t h e i r a b i l i t y to bind J3. aureus c e l l s by incu b a t i n g w i t h an equal volume (0.2 ml) of r a d i o a c t i v e l y l a b e l l e d S_. aureus c e l l s f o r 30 min i n a shaking water bath at 37°C. A f t e r washing the beads 3 times w i t h PBS, the number of r a d i o - l a b e l l e d c e l l s bound to the beads was determined i n a s c i n t i l l a t i o n counter. I f a minimum of 40% of the S^ . aureus c e l l s bound to the f i b r i n o g e n beads, the remaining beads were judged s u i t -able f o r a f f i n i t y chromatography. The fibrinogen-Sepharose column (1.25 x 25 cm) was e q u i l i b r a t e d w i t h PBS at room temperature, then a pr e p a r a t i o n of l y s o s t a p h i n -s o l u b i l i z e d c e l l w a l l s was a p p l i e d . The column was elu t e d w i t h PBS and the eluant monitored at 280 nm. When the &280 r e a ^ n S s nac^ returned to zero, the f r a c t i o n c o n t a i n i n g C l f a c t i v i t y was el u t e d w i t h 2.0 M NaCl i n ^ 0 . The f r a c t i o n s were d i a l y z e d overnight i n d i s t i l l e d H^ O and l y o p h i l i z e d . 3. Gel e l e c t r o p h o r e s i s SDS PAGE was performed, according to the method of Ames (4) and of Weber et a l (97), w i t h 10% polyacrylamide s l a b g e l s i n the pre-sence of . SDS. P r o t e i n s were s t a i n e d w i t h Coomassie b r i l l i a n t 28 blue and carbohydrates were s t a i n e d w i t h S c h i f f s reagent (101). 4. Immunological procedures Two methods f o r the immunization of r a b b i t s w i t h S_. aureus -1 and aureus"-5 were f o l l o w e d : a. New Zealand white r a b b i t s were i n j e c t e d subcutaneously at 1 week i n t e r v a l s w i t h 4.0 mg wet wt b o i l e d c e l l s or 8.0 mg wet wt c e l l w a l l s suspended i n PBS. C e l l suspensions were s t e r i l i z e d by b o i l i n g f o r 1-2 h. The i n j e c t i o n s were repeated 3 times, and a n t i s e r a were c o l l e c t e d 2 weeks a f t e r the f i n a l i n j e c t i o n . b. Rabbits were i n j e c t e d i n t r a v e n o u s l y every 4 days w i t h S_. aureus -1 or S_. aureus -5, ( b o i l e d 15 min) according to the f o l l o w i n g p r o t o c o l : C e l l s at k,,n 1.0 Day 1 0.2 ml 660 Day 4 0.5 ml Day 8 1.0 ml Day 12 1.5 ml C e l l s at A,,_ 2.0 Day 16 0.5 ml 660 Day 20 1.0 ml A n t i s e r a were c o l l e c t e d 1 week a f t e r the l a s t i n j e c t i o n . To o b t a i n absorbed antiserum, a c e n t r i f u g e d p e l l e t of b a c t e r i a l c e l l s was added to an equal volume of antiserum and shaken at 37°C f o r 2 h and then l e f t overnight at 4°C. C e l l s were removed by c e n t r i f u g a t i o n and the antiserum stored at -70°C. Immunoelectrophoresis of l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l f r a c t i o n s was done i n 1% agarose gels (Bio-Rad, -m = 0.13), w i t h sodium b a r b i t o l b u f f e r 0.1M pH 8.6 c o n t a i n i n g 0.6% sodium a z i d e . A current of lOV/cm was a p p l i e d at room temperature f o r 4 h. To o b t a i n a n t i - f i b r i n o g e n , a n t i - a l p h a , beta and gamma a n t i s e r a , r a b b i t s were i n j e c t e d subcutaneously w i t h 400 yg of the appro-p r i a t e p r o t e i n i n 0.5 ml PBS and 0.5 ml Freund's adjuvant ( D i f c o ) . F o u r a d d i t i o n a 1 . i n j e c t i o n s were made bi-weekly, x^ith 0.5 ml of p r o t e i n only. A n t i s e r a were c o l l e c t e d 2 weeks a f t e r the l a s t i n j e c t i o n . Double immunodiffusion techniques were followed (76), to deter-mine antibody content of the a n t i s e r a . Soluble clumping f a c t o r assay a. F i b r i n o g e n - c e l l clumping i n h i b i t i o n t e s t Determination of fibrinogen-S_. aureus c e l l - c l u m p i n g i n h i b i t i o n by a n t i b o d i e s present i n the antiserum from r a b b i t s immunized w i t h S^. aureus C l f + c e l l s , was performed according to the method of Rotter et a l (84). To i n s u r e u n i f o r m i t y of the st a p h y l o c o c c a l c e l l s used i n the assay, a stock c u l t u r e of c e l l s i n PBS at A,,_ OOU of 20.0 was stored i n 2 ml a l i q u o t s at -70°C. The suspension was d i l u t e d w i t h PBS to of 4.0 when needed f o r an assay. Antiserum, i n a s e r i a l two-fold d i l u t i o n i n PBS, was mixed w i t h an equal volume (0.1 ml) of the s t a p h y l o c o c c a l c e l l suspen-s i o n . Tubes were he l d f o r 2 h at room temperature and then 0.2 ml of f i b r i n o g e n (500 yg/ml) was added. A f t e r 5 min of r a p i d shaking at room temperature, the tubes were immediately checked to determine i f the c e l l s had clumped. The clumping i n h i b i t i n g 30 t i t r e of the antiserum t e s t e d , was recorded as the highest d i l u t i o n which i n h i b i t e d f i b rinogen-induced clumping. Controls i n the assay included c e l l s suspended i n f i b r i n o g e n , PBS, and nonimmune antiserum, b. Soluble clumping f a c t o r assay F r a c t i o n s i s o l a t e d from l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l s of S_. aureus by a f f i n i t y chromatography, were assayed f o r t h e i r a b i l i t y to absorb or n e u t r a l i z e c l u m p i n g - i n h i b i t i n g a n t i b o d i e s . An equal volume of the f r a c t i o n (0.1 ml) was added to s e r i a l l y d i l u t e d anti-S_. aureus antiserum and shaken f o r 30 min.at room temperature. S_. aureus -1 c e l l s (0.1 ml) were added and the mixture l e f t undisturbed f o r 2 h at the same temperature. Human f i b r i n o g e n 1% (0.1 ml) was added, the tubes were shaken r a p i d l y f o r 5 min and then assayed f o r clumped c e l l s . I f clumping occurred i n tubes c o n t a i n i n g antiserum which p r e v i o u s l y had showed i n h i b i t i o n i n the f i b r i n o g e n - c e l l clumping assay, the substance was b e l i e v e d to have combined w i t h the clumping i n h i b -i t i n g a n t i b o d i e s present i n the antiserum. When p a r t i c u l a t e m a t e r i a l such as whole c e l l s or c e l l w a l l s were assayed, they were removed from the antiserum by c e n t r i f u g -a t i o n before J3. aureus -1 c e l l s were added to the assay system. L. Hemagglutination assay Tanned red blood c e l l s (RBC) were coated w i t h f i b r i n o g e n according to the method of S w i t a l s k i (94) and the assay performed i n m i c r o t i t r e p l a t e s . Twenty-five y l of a 10% v/v RBC suspension i n PBS were added to 100 y l of s o l u b i l i z e d c e l l w a l l s or whole c e l l s which had 31 been s e r i a l l y d i l u t e d i n PBS c o n t a i n i n g 0.25% g e l a t i n . Controls con s i s t e d of normal RBC or RBC coated w i t h bovine albumin (0.05%). Results were read twice, once a f t e r shaking at room temperature f o r 10 min, and again a f t e r shaking at room temperature f o r 18 h. M« .Interactions of other b a c t e r i a w i t h _ f i b r i n and f i b r i n o g e n Other b a c t e r i a were tested f o r t h e i r a b i l i t y to bind to f i b r i n and to clump i n f i b r i n o g e n . Binding to f i b r i n and f i b r i n o g e n -induced clumping were measured as described p r e v i o u s l y w i t h S_. aureus • N. Adsorption of f i b r i n o g e n The f o l l o w i n g assay was used to determine i f b a c t e r i a could bind to f i b r i n o g e n , but not clump i n i t s presence. Organisms test e d were washed twice i n PBS and were then suspended i n the same b u f f e r to an A.,, of 4.0. The organisms were mixed w i t h an equal volume oou (0.5 ml) of 0.2% f i b r i n o g e n i n a glass tube and the mixture shaken at room temperature f o r 40 min. F i b r i n o g e n was replaced with PBS i n the c o n t r o l s . The c e l l s were examined both v i s u a l l y and micro-s c o p i c a l l y to determine i f they had clumped. I f no clumping was observed, the mixture was c e n t r i f u g e d i n an Eppendorf microfuge (Brinkman) and the c e l l p e l l e t was washed twice w i t h PBS and then resuspended i n PBS. The c e l l s were d i s t r i b u t e d i n t o 3 tubes and d i l u t e d such that the t u r b i d i t y of the c e l l s at A,,» was 1.0, 2.0, 660 and 4.0. An equal volume (0.2 ml) of S^. aureus of 4.0 was added to each tube and the tubes shaken at RT f o r 20 min and.then checked f o r clumped c e l l s . Several of the b a c t e r i a l s t r a i n s t e s t e d autoagglutinated; hence i t was not always p o s s i b l e to evaluate adsorption of f i b r i n o g e n and subsequent clumping of the c e l l s by _S. aureus by d e t e c t i o n of micro-32 scopic aggregation. Consequently, attachment and clumping by S_. aureus was determined by examination of Gram-stained samples of the mixtures. 0. E l e c t r o n microscopy C e l l s of S^ . aureus were checked f o r the presence of p i l i by negative s t a i n i n g w i t h a 2% (wt/vol) s o l u t i o n of phosphotungstic a c i d i n d i s t i l l e d water, (adjusted to pH 7.2 w i t h KOH). Observations were made w i t h a P h i l i p s EM300 e l e c t r o n microscope. P. Chemicals Trypsin (twice c r y s t a l l i z e d ; 12,000 U/mg), s u b t i l i s n BPN 1 type I I I (10.4 U/mg), g a n g l i o s i d e type I I I , l y s o s t a p h i n (270 U/mg), N-methyl-N'- n i t r o s o g u a n i d i n e , bovine serum albumin, and sugars were from Sigma Chemical Co., St. L o u i s , Mo. Pronase (45 KU/mg) was from Calbiochem, La J o l l a , C a l i f , ; a-chymotrypsin (49 U/mg) from Worthington Biochemical Corp, Freehold, New Jersey, ;and Freund's adjuvant was purchased from D i f c o . Other biochemicals used were of reagent grade and purchased from F i s h e r S c i e n t i f i c Co., P i t t s b u r g h . RESULTS A. C h a r a c t e r i z a t i o n of the S^. aureus - f i b r i n b i n d i n g r e a c t i o n 1. Adherence of S. aureus to f i b r i n Previous s t u d i e s i n our l a b o r a t o r y (67), had determined the parameters of fibrinogen-induced b i n d i n g and clumping of S. aureus. Both r e a c t i o n s appeared to be mediated by a c e l l bound m a t e r i a l , clumping f a c t o r ( C l f ) , which was unrelated to f r e e coagulase, a so l u b l e substance released by jS. aureus i n plasma. Free coagulase, a para-c o a g u l a t i n g agent, has an enzymatic a c t i o n on f i b r i n o g e n and was deter-mined to be a n t i g e n i c a l l y d i s t i n c t from C l f (29). The separateness of the two substances was f u r t h e r s u b s t a n t i a t e d when i s o l a t e s of jS. aureus were obtained that were C l f - and f r e e coagulase p o s i t i v e . The present study i n v e s t i g a t e d the a c t i v i t i e s of S. aureus w i t h a matrix of c r o s s - l i n k e d or non-cross-linked f i b r i n . An assay was develop-14 . . . . ed which determined b i n d i n g of C- ^3. aureus to f i b r i n - c o a t e d s c i n t i l l a -t i o n v i a l s . Results showed that every s t r a i n of S^. aureus examined that was C l f + , had the a b i l i t y to bind to f i b r i n . I s o l a t e s of j>. aureus that were f r e e coagulase- and Cl f + , bound to f i b r i n and i n d i c a t e d that free coagulase was not involved i n f i b r i n - b i n d i n g a c t i v i t i e s . To f a c i l i t a t e the c h a r a c t e r i z a t i o n of C l f , a mutant, j ^ . aureus-5, was obtained from the parent s t r a i n . This mutant lacked the a b i l i t y to bind to f i b r i n and to bind t o , and clump w i t h f i b r i n o g e n , and was c a l l e d C l f - . I t shared w i t h S. aureus-1 a l l other c h a r a c t e r i s t i c s examined 33 34 such as, c o l o n i a l morphology, fermentation of mannitol, production of free coagulase and hemolysis of red blood c e l l s (RBC) (Table I ) . A n a l y s i s of whole c e l l s and c e l l w a l l s by SDS 10% PAGE showed that each s t r a i n possessed the same major p r o t e i n bands (unpublished data). 2. Parameters of the j>. a u r e u s - f i b r i n b i n d i n g r e a c t i o n a. C e l l numbers versus b i n d i n g to f i b r i n The i n f l u e n c e of b a c t e r i a l c e l l numbers on the f i b r i n b i n d i n g r e a c t i o n i s described i n F i g . 1. The number of JS. aureus c e l l s that bound to 500 yg of c r o s s - l i n k e d f i b r i n increased i n a l i n e a r manner 7 9 w i t h the number (5.0 x 10 - 10 range) of c e l l s added. When the 9 number of c e l l s added exceeded 10 , a p l a t e a u was reached suggesting that the b i n d i n g receptor s i t e s of the c r o s s - l i n k e d f i b r i n were saturated w i t h c e l l s . This phenomenon d i d not occur when non-cross-l i n k e d f i b r i n was incubated w i t h the same number of c e l l s . Receptor s i t e s of the non-cross-linked f i b r i n d i d not appear to be saturated 9 w i t h c e l l s even when c e l l numbers exceeded 6.0 x 10 . L i n e a r i t y was observed between the number of c e l l s added and the number that bound, 9 . . . however, when the inoculum s i z e exceeded 10 c e l l s , microscopic examin-a t i o n revealed c e l l : c e l l i n t e r a c t i o n s i n a d d i t i o n to c e l l : f i b r i n i n t e r -a c t i o n s . These former i n t e r a c t i o n s may have produced erroneous r e s u l t s . When S. aureus-5 was incubated w i t h c r o s s - l i n k e d or non-cross-l i n k e d f i b r i n , the same number of c e l l s bound, regardless of the s i z e of the inoculum added. I n d i c a t i o n s are, that t h i s b i n d i n g was of the n o n - s p e c i f i c k i n d and was the same as that observed when S^. aureus bound i n low numbers to glass s c i n t i l l a t i o n v i a l s . Table I Comparison of S. aureus-1 and S. aureus-5 Property Organism jS. aureus-1 S_. aureus-5 Mannitol fermentation a) aerobic + + b) anaerobic + + Production of c e l l - f r e e coagulase + + Yellow c o l o n i e s on blood agar p l a t e s + ± Cream c o l o n i e s on BAP ± + Hemolysis + + Clumping w i t h f i b r i n o g e n a) s l i d e t e s t + -b) tube t e s t + c) m i c r o t i t r e assay + -Binding to f i b r i n + Figure 1. Influence of c e l l numbers on the _S. aureus-f i b r i n b i n d i n g r e a c t i o n . Each r e a c t i o n contained: 14 f i b r i n ; c r o s s - l i n k e d or non-cross-lmked, 500 yg and C S^. aureus-1 i n 400 y l PBS. The v i a l s were shaken gently at room temperature f o r 60 min. Unbound c e l l s were removed by gently washing w i t h PBS. S c i n t i l l a t i o n f l u i d 14 was added to the washed v i a l s and the C measured. 0 0 0 = non-cross-linked f i b r i n and- C l f + S^. aureus-1 B = c r o s s - l i n k e d f i b r i n and C l f + j>_. aureus-1. 1 • t = non-cross-linked f i b r i n and C l f J3_. aureus-5. 38 A standard inoculum of 6.4 x 10 c e l l s , determined m i c r o s c o p i c a l l y w i t h a Petroff-Hauser counting chamber, was chosen f o r a l l subsequent experiments, unless noted otherwise. This number of c e l l s was suspended i n 400 u l of PBS, r e s u l t i n g i n an absorbance at of 1.0. When g 6.4 X 10 c e l l s were added to c r o s s - l i n k e d f i b r i n , an average of 35% of them bound, and when added to non-cross-linked f i b r i n , an average of 43% bound. b. R e l a t i o n s h i p of time of in c u b a t i o n to adherence The e f f e c t of incu b a t i o n time on the number of jS. aureus c e l l s that bound to f i b r i n i s shown i n F i g . 2. Adherence of c e l l s to f i b r i n i n both systems was very r a p i d ; 10% of the added c e l l s had bound to non-cross-linked f i b r i n a f t e r 5 min in c u b a t i o n and 4% to c r o s s - l i n k e d f i b r i n i n the same i n c u b a t i o n time. In both systems the number of c e l l s b i n d i n g to the f i b r i n matrix increased w i t h time u n t i l a pl a t e a u was reached w i t h an i n c u b a t i o n time of 60 min f o r non-cross-linked f i b r i n and 120 min f o r c r o s s - l i n k e d f i b r i n . At every i n c u b a t i o n time examined, c e l l s c o n s i s t e n t l y bound i n greater numbers to non-cross-linked f i b r i n . c. E f f e c t of pH The number of S. aureus c e l l s b i n d i n g to f i b r i n was i n v e s t i g a t e d over a pH range of 4 to 10 and included the f o l l o w i n g b u f f e r s : 0.1M Na acetate pH 4.0, 5.0, 0.1M c i t r a t e pH 5.0, 6.0, 0.1M c i t r a t e phosphate pH 6.0, 7.0, 0.005M phosphate-buffered 0.14M sodium c h l o r i d e s o l u t i o n (PBS) pH 7.0, 0.1M borate pH 8.0, 9.0, 0.1M b o r i c acid-borax pH 9.0, 10.0, 0.1M b o r i c a c i d - NaOH pH 10.0. 39 Figure 2. E f f e c t of time on the j>. a u r e u s - f i b r i n b i n d i n g r e a c t i o n . Each r e a c t i o n mixture contained: f i b r i n ; c r o s s - l i n k e d or no n - c r o s s - l i n k e d , 500 ug, and 1 4 C _S. aureus-1, 6.4 x 1 0 8 c e l l s i n 400 y l PBS. V i a l s were shaken at room temperature. 40 41 As seen i n F i g . 3, b i n d i n g of c e l l s to f i b r i n occurred at pH 4.0, however, the high a c i d i t y of the environment caused c e l l : c e l l aggregat-ions. The number of c e l l s that bound to both c r o s s - l i n k e d and non-cross-l i n k e d f i b r i n was lowest at pH 5.0, then increased to a maximum at pH 7.0 and remained constant to pH 10.0. d. Temperature The e f f e c t of temperature on the b i n d i n g r e a c t i o n was determined by performing the assay at 4°C, 25°C and 37°C ( F i g . 4). The number of c e l l s that bound to c r o s s - l i n k e d f i b r i n was approximately the same at 4°, 25°, or 37°C. Although the number of c e l l s that adhered to non-cross-linked f i b r i n was greater at 37°C than at 4°C or 25°C, r e s u l t s were more con-s i s t e n t at 25°C. For t h i s reason, 25°C was chosen f o r the standard assay c o n d i t i o n . 3. P h y s i c a l and chemical treatment of S. aureus C e l l s of S. aureus were t r e a t e d w i t h various p h y s i c a l and chemical agents to determine i f any treatment modified or destroyed the c e l l w a l l component r e s p o n s i b l e f o r adherence of the c e l l to f i b r i n (Table I I ) . a. P h y s i c a l treatment Clumping f a c t o r was r e s i s t a n t to heating at 60°C f o r 1 h or 100°C f o r 30 min. Heating S. aureus at 100°C f o r 1 h or 121°C f o r 20 min, caused a 70 percent r e d u c t i o n i n numbers that bound to c r o s s - l i n k e d f i b r i n and a 90 percent r e d u c t i o n of those bound to non-cross-linked f i b r i n . S o n i c a t i n g the c e l l s at maximum i n t e n s i t y f o r 5 min d i d not a l t e r t h e i r b i n d i n g a c t i v i t y which suggested that the adhesin was not Figure 3. E f f e c t of pH on the j3.. aureus-f i b r i n b i n d i n g r e a c t i o n . Each r e a c t i o n mixture contained: f i b r i n ; c r o s s - l i n k e d or non-cross-linked 500 ug and 14 8 C - J3. a u r e u s - 1 , 6 . 4 x 10 c e l l s i n 4 0 0 u l of the f o l l o w i n g b u f f e r s : 0*1 M sodium ac e t a t e , pH 4*0 , 5*0 , 0 « 1 M c i t r a t e , pH 5 - 0 , 6 - 0 , 0 - 1 M c i t r a t e phosphate pH 6 - 0 , 7-0 0 -005 M phosphate b u f f e r e d , 0 - 1 4 M NaCl, pH 7 - 0 , 8 - 0 , 0 -1 M borate pH 8 - 0 , 9 - 0 , 0 -1 M b o r i c acid-borax pH 9 - 0 , 10-0 0 -1 M b o r i c a c i d - NaOH pH 10-0 Reaction time was 60 min at room temperature 44 Figure 4. E f f e c t of temperature on the S^. aureus-f i b r i n binding reaction. Each reaction mixture contained: f i b r i n ; c ross-linked or non-cross-linked 500 ug and 14 8 C - _S. aureus-1, 6-4 x 10 c e l l s i n 400 y l PBS, Mixtures were incubated for 60 min at 4°C, 25°C, 37°C. 45 o x • o c .0 (A 3|2.0 £ 3 (0 (ft 1.5 O 1.0 o ~o 0 u o o° o° -O 0 o o o • o 4 o o 4 o • o 4 0 • 0 o o „o • 0 4 0 o 4 o L°l cross-1 inked GI non-cross-linked o Oo o'o o o o o ( o ( o p. 4 25 37 Temperature °C 46 associated w i t h l o o s e l y bound appendages. E l e c t r o n microscopic a n a l y s i s of whole c e l l s confirmed t h i s premise as there were no p i l i or s i m i l a r s t r u c t u r e s observed p r o t r u d i n g from the c e l l w a l l s . b. Chemical Treatment As seen i n Table I I , e x t r a c t i o n of the c e l l s w i t h SDS caused an increase i n the numbers that bound to c r o s s - l i n k e d and n o n - c r o s s - l i n k e d f i b r i n . However, microscopic examination of the c e l l s revealed that many had auto-agglutinated which could r e s u l t i n f a l s e l y elevated numbers of those bound to f i b r i n . Treatment of j>. aureus w i t h 0.2N a c i d or 0.2N base f o r 1 h, caused a decrease of at l e a s t 75% i n the numbers that bound to f i b r i n . E i t h e r the clumping f a c t o r was s o l u b i l i z e d or destroyed by these treatments or the environment was a l t e r e d i n such a manner that b i n d i n g i n t e r a c t i o n s between the c e l l and f i b r i n molecules were no longer f e a s i b l e . A c e t y l a t i o n of f r e e amino groups on S. aureus c e l l s was performed w i t h a c e t i c anhydride. This procedure caused a 75% r e d u c t i o n i n the number of c e l l s that bound to f i b r i n . A c e t i c anhydride treatment of whole c e l l s a l s o e l i m i n a t e d t h e i r a b i l i t y to bind to f i b r i n o g e n (67). Several d i f f e r e n t hypothesis could e x p l a i n why b l o c k i n g free amino groups of S^. aureus c e l l s r e s u l t e d i n t h e i r l o s s of b i n d i n g a b i l i t i e s . One p o s s i b i l i t y i s that the amino groups were d i r e c t l y i n v o l v e d i n the b i n d i n g i n t e r a c t i o n w i t h f i b r i n . Or, amino groups adjacent to the bindu-ing r e c e p t o r s , when blocked, posed a s t e r i c hindrance to the r e a c t i o n between the c e l l and the f i b r i n molecule. Another p o s s i b i l i t y i s that blockage of f r e e amino groups on the c e l l surface had r e s u l t e d i n an increased net negative charge of the c e l l , c r e a t i n g a greater e l e c t r o s t a t i c Table I I E f f e c t of p h y s i c a l and chemical treatment on The S. a u r e u s - f i b r i n b i n d i n g r e a c t i o n Treatment Conditions C e l l s bound to f i b r i n C r o s s - l i n k e d Non-cross-linked None 100 100 Heat 60°C, 60 min 100 100 100°C, 10 min 111 118 30 min 89 91 60 min 30 10 121°C, 20 min 27 11 So n i c a t i o n 5 min 70 132 A c e t y l a t i o n 30 min 60 8 SDS 1%; l h ; 37°C 175 143 NaOH 0.2M; l h ; 60°C 25 14 HC1 0.2M; l h ; 60°C 11 26 Expressed as a percentage of c o n t r o l c e l l s ( i n PBS) bound to f i b r i n . 48 b a r r i e r between i t s e l f and the f i b r i n molecule. P r o p i n q u i t y between the two surfaces was no longer favourable and the formation of bonds between them l e s s l i k e l y to form. 4. Protease treatment The r e s u l t s i n Table I I I i n d i c a t e d that treatment of c e l l s w i t h protease, abrogated t h e i r b i n d i n g a b i l i t i e s . Although the percentage of c e l l s bound to f i b r i n was reduced by 75% a f t e r treatment f o r 1 h w i t h pronase, up to 4 h of treatment w i t h a-chymotrypsin, t r y p s i n , and s u b t i l i s n was r e q u i r e d to produce the same e f f e c t . I f the c e l l w a l l component mediating adherence, i s a p r o t e i n , the r e s u l t s suggest that i t i s e i t h e r r e s i s t a n t to protease a c t i o n , or that i t i s protected i n the c e l l w a l l environment. 5. I n h i b i t o r s of the b i n d i n g r e a c t i o n An i n d i r e c t a n a l y s i s of the f i b r i n - b i n d i n g f a c t o r was attempted by i n c u b a t i n g S. aureus w i t h a v a r i e t y of compounds and then assaying the c e l l s f o r b i n d i n g a b i l i t i e s . Each compound was incubated w i t h S. aureus f o r 30 min at room temperature and then the mixture was added to the f i b r i n l a y e r . The substances examined were incubated w i t h the c e l l s to determine i f any s t r u c t u r e could act as an analogue of the b i n d i n g receptor on the f i b r i n molecule, and bind to the adhesins on the staphylo-c o c c a l c e l l . Subsequently, b i n d i n g of the c e l l s to the f i b r i n l a y e r would be impaired. Other substances were included i n the study to assess the importance of ions on the S. a u r e u s - f i b r i n b i n d i n g r e a c t i o n . Urea was t e s t e d since previous studies showed that i t prevented c e l l s b i n d i n g to f i b r i n o g e n (67). 0 49 Table I I I E f f e c t of protease treatment on the S. aureus-f i b r i n b i n d i n g r e a c t i o n C e l l bound to f i b r i n % Time (h) C r o s s - l i n k e d f i b r i n non-cross l i n k e d f i b r i n Enzyme 1 1 None 100 100 Pronase S u b t i l i s n 27 46 33 26 39 21 a-Chymotrypsin 1 4 79 25 70 8 t r y p s i n 1 4 80 11 81 7 Results expressed as a percentage of the number of untreated c e l l s bound to f i b r i n . n Concentrations of proteases were 2 mg/ml, 50 The r e s u l t s of c e l l s , b i n d i n g to the f i b r i n a f t e r i n c u b a t i o n w i t h the t e s t m a t e r i a l were expressed as a percentage of the number of c e l l s b i n d i n g to f i b r i n a f t e r p r e i n c u b a t i n g i n PBS. The r e s u l t s of the c o n t r o l c e l l s i n PBS were assigned the value of 100%. Although pretreatment of j>. aureus c e l l s w i t h 8 M urea f o r 1 h at 37°C had no apparent e f f e c t on the c e l l s ' a b i l i t y to bind to f i b r i n , (unpublished d a t a ) , i n c l u s i o n of urea at 4.0 M i n the r e a c t i o n mixture of the c e l l s and f i b r i n , caused a 60% decrease i n the number of c e l l s bound to f i b r i n (Table IV). This trend continued as the co n c e n t r a t i o n of urea i n the r e a c t i o n mixture increased, perhaps owing to a denaturation of the f i b r i n molecule. A d d i t i o n of the c h e l a t i n g agent EDTA, had no e f f e c t on the b i n d i n g r e a c t i o n , suggesting that d i v a l e n t c a t i o n s were not in v o l v e d i n the i n t e r -a c t i o n of S. aureus and f i b r i n . None of the p r o t e i n s or g l y c o p r o t e i n s t e s t e d s u b s t a n t i a l l y decreased the number of c e l l s bound to f i b r i n . Since g a n g l i o s i d e s were e f f e c t i v e i n i n h i b i t i n g the b i n d i n g r e a c t i o n , hydrophobic bonds were p o s s i b l y involved i n the i n t e r a c t i o n between jS. aureus and the f i b r i n molecule. A l t e r n a t i v e l y , g a n g l i o s i d e s may have competed w i t h S. aureus f o r the same receptor s i t e s on the f i b r i n molecule v i a a s u g a r - l e c t i n type of i n t e r -a c t i o n . As i s shown i n Table IV a l l monosaccharides included i n the study, caused a decrease i n the percentage of c e l l s bound to f i b r i n . However, the i n h i b i t i o n was not r e s t r i c t e d to a s p e c i f i c sugar and i t remains to be proven i f any sugar i s d i r e c t l y i n v o l v e d i n the b i n d i n g receptor s i t e s . To determine what e f f e c t the co n c e n t r a t i o n of NaCl had on the b i n d i n g r e a c t i o n , i t was added (0.14 - 2.0 N, f i n a l c o n c e n t r a t i o n ) , to the S>. aureus-f i b r i n mixture. As s e e n i n F i g . 5, the number of c e l l s that bound to f i b r i n , sharply decreased when the co n c e n t r a t i o n of NaCl i n the r e a c t i o n mixture was 1 M. At 2.0 M NaCl, the percent of c e l l s b i n d -ing to c r o s s - l i n k e d f i b r i n was e l i m i n a t e d . To determine i f NaCl (2.0 M), would cause bound c e l l s to desorb from f i b r i n , the s a l t was added to the c e l l - f i b r i n mixture a f t e r the c e l l s had incubated w i t h the f i b r i n f o r 60 min. The NaCl mixture was shaken at 25°C f o r 30 min, then the f i b r i n l a y e r r i n s e d 3 times w i t h PBS and assayed f o r the number of c e l l s that remained bound to the f i b r i n . C o n t r o l s were shaken f o r 30 min w i t h PBS. Results (unpublished) showed that at l e a s t 40% of the adherent c e l l s desorbed from c r o s s - l i n k e d f i b r i n and 65% desorbed from non-cross-linked f i b r i n . There was no desorbtion of c e l l s from f i b r i n a f t e r shaking f o r 30 min w i t h PBS. 6. E f f e c t of a n t i s e r a on the j ^ . a u r e u s - f i b r i n b i n d i n g r e a c t i o n a. A n t i s e r a prepared against f i b r i n o g e n and a,g and y chains A n t i s e r a prepared against f i b r i n o g e n and the i n d i v i d u a l a, 3 and y chains were used to determine i f any an t i b o d i e s present i n the sera would bind to the f i b r i n and subsequently i n h i b i t the b i n d i n g of j>. aureus. The antiserum (200 y l ) was added to the f i b r i n l a y e r and incubated f o r 60 min at 37°C. Cont r o l s contained PBS or serum from a nonimmunized animal. The f i b r i n l a y e r s were washed w e l l w i t h PBS, and then S. aureus c e l l s were added and assayed f o r adherence. Although a n t i - f i b r i n o g e n , a n t i - a l p h a , a n t i - b e t a , and anti-gamma sera a l l i n h i b i t e d b i n d i n g of S. aureus to c r o s s - l i n k e d f i b r i n the i n h i b i t i o n appeared to be n o n s p e c i f i c i n nature, and the a n t i b o d i e s present i n the a n t i s e r a probably d i d not compete w i t h S. aureus f o r the same receptors Table IV E f f e c t of i n h i b i t o r s on the _S. aureus-f i b r i n b i n d i n g r e a c t i o n C e l l s bound to f i b r i n 1 % I n h i b i t o r Concentration cross l i n k e d n o n - c r o s s - l i n k e d None 100 100 NaCl 1-OM 27 50 2-OM 0 29 Urea 2-OM 117 133 4-OM 42 42 6-OM 25 8 8-OM 17 4 EDTA 0-05M 120 .107 BSA 2 mg/ml 87 124 G e l a t i n 2 mg/ml 127 85 F e t u i n 2 mg/ml 93 87 Gangliosides 200yg/ml 50 74 Glucose 0-01M 71 65 Galactose 0-01M 71 65 N-acetyl glucosamine 0-01M 57 87 N-acetyl galactosamine O'OIM 64 65 Expressed as a percentage of c e l l s ( i n PBS) that bound to f i b r i n i n the absence of any i n h i b i t i n g compound. 53 Figure 5. The e f f e c t of NaCl on the j>. aureus-f i b r i n b i n d i n g r e a c t i o n . Reaction mixtures contained: f i b r i n ; c r o s s - l i n k e d or no n - c r o s s - l i n k e d , 500 ug and 14 8 C S^. aureus, 6*4 x 10 c e l l s i n 400 u l phosphate b u f f e r c o n t a i n i n g 0*14 - 2*0 M NaCl. The mixtures were incubated at room temperature f o r 60 min. 55 on the f i b r i n molecule but rat h e r posed a s t e r i c hindrance to the sta p h y l o c o c c a l c e l l . The a n t i s e r a d i d not i n h i b i t the adherence of j5. aureus to non-cross-linked f i b r i n . b. A n t i s e r a r a i s e d against aureus-1 and _S. aureus-5 P r i o r to adding S. aureus-1 c e l l s to f i b r i n , they were incubated f o r 30 min at 37°C w i t h anti-JS. aureus-1 antiserum, a n t i - S . aureus--5 antiserum or nonimmune serum. The c e l l s were washed 3 times w i t h PBS to remove any an t i b o d i e s not bound to the c e l l s . I f the an t i b o d i e s present i n the a n t i s e r a bound to the same receptors on the j>. aureus-1 c e l l w a l l s that mediated adherence to f i b r i n , some i n s i g h t i n t o the nature of these receptors might be gained. As seen i n Table V, in c u b a t i o n of " c e l l s i n a n t i - S . aureus-1 or i n anti-J3. aureus-5 sera, caused a decrease i n the numbers that bound to c r o s s - l i n k e d or non-cross-linked f i b r i n . When a n t i - S . aureus-1 antiserum had been absorbed w i t h S. aureus-1 or S. aureus-5 c e l l s p r i o r to incubat-ion w i t h J3. aureus-1 c e l l s i n the assay, i t r e t a i n e d the a b i l i t y to i n h i b i t b i n d i n g of the c e l l s to f i b r i n . There was no decrease i n the b i n d i n g a c t i v i t i e s of S. aureus-1 c e l l s a f t e r i n c u b a t i o n i n nonimmune serum. When the c e l l s had incubated i n the a n t i s e r a , they tended to clump spontaneously, and although washing the c e l l s i n PBS helped to disperse the clumps, i t d i d not e l i m i n a t e them completely. Thus, i t was impossible to determine i f jS. a u r e u s - f i b r i n b i n d i n g was i n h i b i t e d by s p e c i f i c a n t i b o d i e s present i n the a n t i s e r a or i f the decrease i n numbers of c e l l s bound to f i b r i n was caused by a removal of clumped c e l l s from the f i b r i n , by the washing procedure. 56 Other f a c t o r s such as s t e r i c hindrance by a n t i b o d i e s or n o n s p e c i f i c opsonins present i n the a n t i s e r a , may have a l s o caused the same i n h i b i t i n g e f f e c t . B. I s o l a t i o n and c h a r a c t e r i z a t i o n of C l f The parameters of fibrinogen-induced b i n d i n g of J3. aureus had previous-l y been c h a r a c t e r i z e d (67), and, i n t h i s study, the J3. a u r e u s - f i b r i n b i n d -ing r e a c t i o n was examined. Was the same c e l l w a l l component involved i n both of these a c t i v i t i e s , and i f so, was i t a l s o synonomous w i t h C l f ? An attempt to answer these questions was made by i s o l a t i n g and c h a r a c t e r i z i n g C l f from c e l l w a l l s of S. aureus. 1. P r e p a r a t i o n of c e l l w a l l s Before C l f could be i s o l a t e d from c e l l w a l l s , i t was f i r s t necessary to o b t a i n c e l l w a l l s which r e t a i n e d t h e i r clumping a c t i v i t i e s i n f i b r i n o -gen. I n i t i a l l y , an attempt was made to break c e l l s by u l t r a s o n i c a t i o n or by homogenization i n a French press. Neither of these methods was e f f e c t i v e i n d i s r u p t i n g the s t a p h y l o c o c c a l c e l l . To produce c e l l s more s u s c e p t i b l e to breakage, p e n i c i l l i n was added to the JS. aureus growth medium. Unfortunately, c e l l s grown i n the presence of p e n i c i l l i n l o s t t h e i r a b i l i t y to clump w i t h f i b r i n o g e n , i n d i c a t i n g a p o s s i b l e l i n k a g e between the C l f and c e l l w a l l peptidoglycan. S t i r r i n g at maximum i n t e n s i t y w i t h glass beads i n a m i n i - m i l l proved to be the most e f f e c t i v e procedure f o r d i s r u p t i n g the organism. G e n e r a l l y , i t was p o s s i b l e to break 50% of S. aureus c e l l s a f t e r s t i r r i n g f o r two h, however, there was considerable v a r i a t i o n from one c e l l p r e p a r a t i o n to another. C e l l w a l l s were e a s i l y separated from unbroken c e l l s by d i f f e r e n t i a l c e n t r i -f u g a t i o n . Table V E f f e c t of a n t i s e r a on the a u r e u s - f i b r i n b i n d i n g r e a c t i o n Antiserum jS. aureus, C l f + bound to f i b r i n Cross--linked non-cross-linked None (PBS) 100 100 A n t i - S . aureus-1 45 30 An t i - S . aureus-5 88 38 Nonimmune serum 91 110 Results expressed as a percentage of c e l l s incubated i n PBS p r i o r to adding them to f i b r i n . 58 2. Adherence p r o p e r t i e s of c e l l w a l l s C e l l w a l l s from the parent^ J3. aureus-1, r e t a i n e d t h e i r a b i l i t y to clump i n f i b r i n o g e n and, as determined m i c r o s c o p i c a l l y , to b i n d to f i b r i n . Walls from the mutant s t r a i n j3. aureus-5, d i d not possess e i t h e r of these p r o p e r t i e s . The m i c r o t i t r e assay, used to determine the clumping t i t r e of c e l l s i n f i b r i n o g e n was r a p i d , d i d not r e q u i r e r a d i o a c t i v e l y l a b e l l e d c e l l s , and was thus chosen f o r stu d i e s w i t h i s o l a t e d c e l l w a l l s . 3. M o d i f i c i c a t i o n The r e s u l t s of c e l l w a l l m o d i f i c a t i o n procedures and clumping of these c e l l w a l l s i n f i b r i n o g e n , c o r r e l a t e d w i t h s i m i l a r s t u d i e s done w i t h whole c e l l s (67), which suggested that the clumping f a c t o r i n the c e l l w a l l p r e p a r a t i o n was the same as that i n whole c e l l s . Although c e l l w a l l s were more s e n s i t i v e to heat and protease, (Table VI),, than were whole c e l l s , a l l other m o d i f i c a t i o n s produced s i m i l a r e f f e c t s i n both systems. In a d d i t i o n , c e l l w a l l s were t r e a t e d w i t h phenol, TCA, sodium p e r i o d a t e , and formamide and were then assayed f o r clumping a c t i v i t y . A f t e r c e l l w a l l s were e x t r a c t e d w i t h 5% TCA at 4°C f o r 48 h, no change i n t h e i r clumping t i t r e was observed, however, e x t r a c t i o n w i t h 10% TCA at 60°C f o r 90 min e l i m i n a t e d a l l clumping a c t i v i t i e s (Table V I ) . C o n t r o l c e l l s heated at 60°C f o r 90 min r e t a i n e d t h e i r a b i l i t y to clump i n f i b r i n o -gen. E x t r a c t i o n of c e l l w a l l s w i t h formamide caused them to self-aggregate and thus r e s u l t s of t h e i r clumping i n f i b r i n o g e n were d i f f i c u l t to i n t e r -p r e t . Many p r o t e i n s were removed from the w a l l s a f t e r e x t r a c t i o n w i t h formamide as was determined by g e l e l e c t r o p h o r e s i s (unpublished data). 59 Table VI E f f e c t of chemical, p h y s i c a l and enzymatic treatment on the fibrinogen-induced clumping of S. aureus c e l l w a l l s 1 Clumping F i n a l c o n c e n t r a t i o n of f i b r i n o g e n ug/ml Treatment Condition 500 250 125 63 30 15 7.5 0 None + + + + + + — — Heat 60°C, 90 min + + + + + + - -100°C, 15 min + + - - - - - -T r y p s i n 2 mg/ml, 37°C, 2 h - - - - - - - -Periodate 0.01M, pH 4.5,R.T. 20h + + + + - - - -Phenol 90%, 37°C, 15 min + + + + + + - -TCA 5%, 4°C, 48h + + + + + + - -10%, 60°C, 90 min - - - - - - - -"""Fibrinogen 1% was s e r i a l l y d i l u t e d i n PBS i n m i c r o t i t r e p l a t e s ( f i n a l v o l . 0.025 ml). S. aureus c e l l w a l l s A,, n of 1.0, modified as described, were added The mixture was shaken f o r 30 min at room temperature and then the w e l l s were assayed f o r clumped c e l l s . 60 Neither phenol nor sodium periodate treatment s i g n i f i c a n t l y a l t e r e d the a b i l i t y of the c e l l w a l l s to clump i n f i b r i n o g e n . 4. S o l u b i l i z a t i o n of c e l l w a l l s A f t e r i t was determined that i s o l a t e d c e l l w a l l s of C l f + JS. aureus-1 clumped i n f i b r i n o g e n , an attempt was made to s o l u b i l i z e them and to compare the s o l u b i l i z e d f r a c t i o n w i t h that from the C l f mutant S_. aureus-5. I n i t i a l l y , c e l l w a l l s were e x t r a c t e d w i t h 10% TCA at 4°C and then t r e a t e d w i t h lysozyme, since lysozyme treatment alone produced no e f f e c t . However, t h i s treatment f a i l e d to s o l u b i l i z e the w a l l s . Therefore l y s o s t a p h i n was chosen and was found to s o l u b i l i z e 80% of the dry wt of the c e l l w a l l s a f t e r 40 min incu b a t i o n at 37°C ( F i g . 6 ) . Soluble m a t e r i a l was separated from p a r t i c u l a t e matter by c e n t r i f u g a t i o n . The p a r t i c u l a t e m a t e r i a l d i d not clump when mixed w i t h f i b r i n o g e n . 5. C h a r a c t e r i z a t i o n of s o l u b i l i z e d c e l l w a l l s a. P r o t e i n , phosphorus and hexose The p r o t e i n , phosphorus and hexose content of the c e l l w a l l s of _S. aureus-1 and JB. aureus-5 were very s i m i l a r (Table V I I ) . The t o t a l recovery of p r o t e i n of the c e l l w a l l s was only 52% w i t h S. aureus-1 and 77% w i t h S. aureus-5, but was approximately 100% of t h e i r s o l u b i l i z e d w a l l s . The method used to determine p r o t e i n content d i d not account f o r peptides i n peptidoglycan. Walls of both organisms contained s i m i l a r amounts of phosphorus. Although the hexose content of the w a l l s of both s t r a i n s appeared low, the assay system used d i d not q u a n t i t a t e sugars such as those found i n c e l l w a l l t e i c h o i c a c i d . Figure 6. S o l u b i l i z a t i o n of J3. aureus c e l l w a l l s w i t h l y s o s t a p h i n . 5 ml S_. aureus c e l l w a l l s (0»2 g dry wt.) were incubated at 37°C wi 0.75 mg l y s o s t a p h i n . At the designated times, samples were removed and the o p t i c a l d e n s i t y determined. 62 Table V I I P r o t e i n , phosphorus and hexose content of l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l s and c e l l w a l l s Dry Weight P r o t e i n Phosphorus Hexose Organism mg : % i l mg % mg % mg % S. aureus-1 A. C e l l w a l l s 18.3 100 9.5 52 0.44 0.21 B. Lysostaphin-s o l u b i l i z e d 24.3 100 24.5 100 0.67 3 0.13 0.5 A. C e l l w a l l s 16.7 100 12.9 77 0.53 3 0.31 2 S. aureus-5 B. Lysostaphin-s o l u b i l i z e d 11.5 100 11.3 98 0.42 4 0.03 0.3 1A11 values were correct e d f o r p r o t e i n , phosphorus, and hexose content of l y s o s t a p h i n . 1 : LValues expressed as a percent of the dry weight. b. Immunoelectrophoresis The antigens present i n the s o l u b i l i z e d c e l l w a l l s of both S. aureus s t r a i n s were examined by Immunoelectrophoresis ( F i g . 7 and 12). A n t i s e r a were r a i s e d against whole c e l l s or w a l l s of each s t r a i n . As depicted i n F i g . 7 and 12, antigens, (a,b,c) were present i n s o l u b i l i z e d w a l l s of JS. aureus-1. S o l u b i l i z e d w a l l s of S. aureus-5 contained only two a n t i -gens (a,b) when reacted w i t h a n t i - S . aureus-5 serum ( F i g . 12) or w i t h a n t i - S ^ aureus-1 serum ( F i g . 7). When a n t i - S ^ aureus-1 was absorbed w i t h c e l l s of JS. aureus-5. the C l f s t r a i n , only one antibody could be detected which formed a p r e c i p i t a t i o n arc w i t h antigen C of s o l u b i l i z e d JS. aureus-1 c e l l w a l l s ( F i g . 7). The absorbed antiserum d i d not react w i t h S^. aureus-5 s o l u b i l i z e d w a l l s . 6. I s o l a t i o n of, C l f To i s o l a t e C l f from s o l u b i l i z e d c e l l w a l l s of C l f + s t r a i n s , a s e l e c t i v e b i n d i n g of the s o l u b l e C l f onto fibrinogen-coupled sepharose beads was attempted. The f i b r i n o g e n a f f i n i t y column was el u t e d w i t h PBS and NaCl and monitored at ^ 2QQ' ^ s ^s s n o w n i n F i g . 8, a large p r o t e i n peak #1- el u t e d i n the PBS wash. A second, smaller peak #2, was el u t e d w i t h 2.0 M NaCl. The f r a c t i o n that e l u t e d w i t h 2.0 M NaCl, was present i n s o l u b i l i z e d c e l l w a l l s of the C l f + s t r a i n but was not present i n s o l u b i l i z e d w a l l s of the C l f s t r a i n ( F i g . 9). A summary of t h i s method used to i s o l a t e s o l u b l e clumping f a c t o r i s depicted i n F i g . 10. 65 Figure 7 . Immunoelectrophoretic a n a l y s i s of s o l u b i l i z e d c e l l w a l l s of J3. aureus - 1 and jS. aureus - 5 against a n t i - S . aureus - 1 and anti-j>. aureus - 1 absorbed w i t h S. aureus - 5 c e l l s . The antigen w e l l s contained 25 y l o f : 1, 3 , l y s o s t a p h i n - s o l u b i l i z e d S. aureus - 1 c e l l w a l l s 2 l y s o s t a p h i n - s o l u b i l i z e d j>. aureus - 5 c e l l w a l l s The antiserum troughs contained 75 u l o f : A. anti-jS. aureus - 1 B. a n t i - S . aureus - 1 absorbed w i t h S. aureus - 5 c e l l s . 66 67 Figure 8. F i b r i n o g e n - a f f i n i t y chromatography of S. aureus-1 s o l u b i l i z e d c e l l w a l l s . E l u t i o n p r o f i l e of l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l s of j>. aureus-1, measured at 280 nm. Peak 1 e l u t e d w i t h PBS and peak 2, w i t h 2 M NaCl. 68 69 Figure 9. F i b r i n o g e n - a f f i n i t y chromatography of S. aureus-5 s o l u b i l i z e d c e l l w a l l s . E l u t i o n p r o f i l e of l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l s of S. aureus-5, measured at 280 nm. Peak 1 e l u t e d w i t h PBS . Figure 10. P r o t o c o l f o r the i s o l a t i o n of s o l u b l e clumping f a c t o r from S. aureus. ISOLATION of SOLUBLE CLUMPING FACTOR S.aureus 9 g wet wt 7 2 " — i Suspend in 30 mis H 0 0 i M i n i - m i l l 2 h I Centrifuge 40 K xg 15 min Supernatant Cell walls Whole cells Wash with H^O 4x J Solubi l ize with Lysostaphin 0*75 mg in PBS 2h 3 7 a C I Centr i fuge 30 K x g 20 min Supernatant Pellet Fi br inogen—aff in i ty column i PBS I Fraction 1 NaCl 2 M i Fraction 2 Clumping Factor 73 7. C h a r a c t e r i z a t i o n of C l f a. Gel e l e c t r o p h o r e s i s The l y s o s t a p h i n - s o l u b i l i z e d w a l l s of aureus-1 and S^. aureus-5 and the f r a c t i o n s that e l u t e d from the f i b r i n o g e n a f f i n i t y column were ana-lyzed by SDS 10% PAGE and st a i n e d w i t h Coomassie blue and p e r i o d a t e - S c h i f f reagent F i g 11 (a,b). A mixture of reference p r o t e i n s (Bio-Rad), c o n t a i n -ing myosin (molecular weight 200,000), g-galactosidase (116,500), phosphorylase B (94,000), bovine serum albumin (68,000), and ovalbumin (43,000), were used to estimate molecular weights (unpublished data). The major p r o t e i n s present i n the l y s o s t a p h i n - s o l u b i l i z e d f r a c t i o n s of the two s t r a i n s showed some d i f f e r e n c e s . Walls from S. aureus-1 had s i x major p r o t e i n bands whereas those from S^. aureus-5 had nine. Of p a r t i c u l a r i n t e r e s t was a p r o t e i n , l a b e l l e d C ( F i g . 11), molecular weight a p p r o x i -mately 90,000, which was found i n preparations of w a l l s from the C l f + s t r a i n , S^. aureus-1, but was l a c k i n g i n those of the C l f s t r a i n , J5. aureus-5. This p r o t e i n was the only one to s t a i n p o s i t i v e l y w i t h p e r i o d a t e - S c h i f f reagent. I n t e r e s t i n t h i s p r o t e i n increased a f t e r the a n a l y s i s of the f r a c t i o n s , e l u t e d from the f i b r i n o g e n a f f i n i t y column, had been completed. The SDS PAGE a n a l y s i s of peak #1 ( F i g . 8), which had elu t e d w i t h PBS from the column, revealed an i d e n t i c a l p r o t e i n p r o f i l e to the pr e p a r a t i o n of j>. aureus-1 s o l u b i l i z e d w a l l s , except that p r o t e i n C was absent (lane 2, F i g . 11). A n a l y s i s of peak #2, the f r a c t i o n that e l u t e d from the column w i t h 2M NaCl, showed that i t contained a s i n g l e p r o t e i n , molecular weight approximately 90,000, that s t a i n e d p o s i t i v e l y w i t h p e r i o d a t e - S c h i f f (lane 3, Fig. 1 1 ) . I n d i c a t i o n s were, that the ( g l y c o ) p r o t e i n present i n peak #2, was the same ( g l y c o ) p r o t e i n C, present i n S. aureus-1 s o l u b i l i z e d w a l l s , that had absorbed to the f i b r i n o g e n a f f i n i t y column and had desorbed w i t h 2 M NaCl. A n a l y s i s of ^ . aureus-5 s o l u b i l i z e d w a l l s showed that p r o t e i n C was absent from preparations both p r i o r t o , and a f t e r a f f i n i t y chromatography. No substance could be detected to desorb from the a f f i n i t y column w i t h 2M NaCl. The unique c h a r a c t e r i s t i c s of ( g l y c o ) p r o t e i n C, namely t h a t : ( i ) I t was the only p r o t e i n observed to s t a i n p o s i t i v e l y w i t h p e r i o d a t e - S c h i f f . ( i i ) I t was present as a major c o n s t i t u e n t of the s o l u b i l i z e d w a l l s of the C l f + s t r a i n but was l a c k i n g i n those of the C l f s t r a i n . ( i i i ) I t was the only p r o t e i n observed to s e l e c t i v e l y absorb to a f i b r i n o g e n a f f i n i t y column, l e d one to b e l i e v e that i t might be synonomous w i t h C l f . Further i n f o r m a t i o n of the c h a r a c t e r i s t i c s of p r o t e i n C was attempted w i t h Immunoelectrophoresis and assays to determine i t s a c t i v i t i e s w i t h f i b r i n o g e n and w i t h a n t i - C l f a n t i b o d i e s . b. Immunoelectrophoresis The a n t i g e n i c p r o p e r t i e s of the m a t e r i a l i n peak #2, were examined w i t h a n t i s e r a prepared against c e l l w a l l s of C l f + s t r a i n s and of C l f s t r a i n s . A s i n g l e p r e c i p i t a t i o n a r c , s i m i l a r to that produced w i t h antigen C of C l f + s o l u b i l i z e d c e l l w a l l s , was detected upon e l e c t r o p h o r e s i s of peak #2 w i t h C l f + w a l l antiserum ( F i g . 12). There was no detec t a b l e r e a c t i o n of peak #2 w i t h C l f w a l l antiserum. c. . Measurement- of the a c t i v i t y of. s o l u b l e C l f Attempts were made to determine i f the f r a c t i o n i s o l a t e d from C l f + s o l u b i l i z e d c e l l w a l l s (peak #2), possessed f i b r i n o g e n clumping f a c t o r a c t i v i t y . When the s o l u b l e f r a c t i o n was mixed w i t h f i b r i n o g e n , no d e t e c t -able p r e c i p i t a t e was produced. E i t h e r an aggregate had formed but was not 75 Figures 11a and l i b SDS 10% PAGE a n a l y s i s of s o l u b i l i z e d c e l l w a l l f r a c t i o n s of S. aureus-1 and _S. aureus-5. A 25 u l p o r t i o n of each sample was a p p l i e d to an SDS, 10% p o l y -acrylamide g e l system. P r o t e i n s were s t a i n e d w i t h Coomassie b r i l l i a n t b lue R250, and carbohydrates w i t h periodate S c h i f f reagent. Coomassie Stained Lane 1, l y s o s t a p h i n - s o l u b i l i z e d S. aureus-1, ( C l f + ) c e l l w a l l s p r i o r to a f f i n i t y chromatography Lane 2, l y s o s t a p h i n - s o l u b i l i z e d _S. aureus-1 c e l l w a l l s a f t e r a f f i n i t y chromatography, peak #1, f i g . 8. Lane 3, _S. aureus*-1 c e l l w a l l f r a c t i o n e l u t e d from a f f i n i t y column w i t h 2 M NaCl, peak #2, f i g . 8. Lane 4, l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l s of S. aureus-5 ( C l f ) peak / / l , f i g . 9. Lane 5, j>. aureus-5 c e l l w a l l f r a c t i o n e l u t e d from a f f i n i t y column w i t h 2 M NaCl, f i g . 9. P e r i o d a t e - S c h i f f . Lane 3, j>. aureus-1 c e l l w a l l f r a c t i o n that e l u t e d from a f f i n i t y column w i t h 2 M NaCl, peak #2, f i g . 8. 76 1 2 3 4 3 II 1II II 11 mi II II i C o o m a s s i e P e r i o d a t e S c h i f f r 78 Figure 12. Immunoelectrophoretic a n a l y s i s of l y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l f r a c t i o n s of j>. aureus-1 and _S. aureus-5. i Antigen w e l l s contained 25 p i amounts o f : (1) Soluble' clumping f a c t o r , peak #2, f i g . 8. (2) L y s o s t a p h i n - s o l u b i l i z e d S. aureus-1, C l f + c e l l w a l l s . (3) L y s o s t a p h i n - s o l u b i l i z e d S. aureus-5, C l f c e l l w a l l s . Antiserum troughs contained 75 p i o f : A, B anti--S. aureus-1 antiserum C anti-S,- aureus-5 antiserum 79 80 v i s i b l e , o r , the f r a c t i o n contained clumping f a c t o r i n a monovalent form which was incapable of forming v i s i b l e aggregates w i t h f i b r i n o g e n . A method developed by S w i t a l k s i (94) to detect s o l u b l e C l f by a g g l u t i n a t i o n of fibrinogen-coated tanned red blood c e l l s (RBC), was followed. Although the RBC always a g g l u t i n a t e d when mixed w i t h ,S. aureus C l f + whole c e l l s , they d i d not give c o n s i s t e n t r e s u l t s when mixed w i t h s o l u b l e C l f . At times the RBC clumped spontaneously. Con t r o l RBC and RBC coated w i t h albumin a l s o clumped on occasion w i t h whole c e l l s or s o l u b l e C l f . Rotter et a l (84), experienced the same d i f f i c u l t i e s w i t h coated RBC, and thus i t was concluded that the fibr i n o g e n - c o a t e d RBC assay was not an e f f e c t i v e assay f o r d e t e c t i n g s o l u b l e C l f . An attempt was made to determine i f s o l u b i l i z e d C l f , i s o l a t e d from 14 jS. aureus-1 c e l l s r a d i o l a b e l l e d w i t h C, could be detected adhering to immobilized f i b r i n o g e n or to f i b r i n . U n fortunately, r e s u l t s from t h i s assay were not c o n c l u s i v e . Since methods which demonstrated f i b r i n o g e n C l f a c t i v i t y d i r e c t l y , were not e f f e c t i v e when a p p l i e d to the i s o l a t e d s o l u b l e C l f , an i n d i r e c t assay was developed. The assay determined i f a substance could absorb s p e c i f i c a n t i b o d i e s i n anti-JS. aureus-1 C l f + , antiserum (anti-S^ aureus-1) which i n h i b i t e d fibrinogen-induced clumping of S. aureus C l f + c e l l s . Antiserum prepared against S. aureus-1 c e l l w a l l s , but not S. aureus-5 C l f w a l l s ( a n t i - S . aureus-5) contained clumping i n h i b i t i n g a n t i b o d i e s (CIA) which i n h i b i t e d the S. aureus"f i b r i n o g e n clumping r e a c t i o n . Other i n v e s t i g a t o r s reported s i m i l a r f i n d i n g s (29, 56, 84). As t a b l e V I I I shows, JS. aureus-1 c e l l s , incubated i n a n t i - S ^ aureus-1 f o r 2 h, d i d not clump when f i b r i n o g e n was added to the assay system. Clumping w i t h f i b r i n o g e n was not i n h i b i t e d when the same C l f + c e l l s were incubated i n 81 nonimmune serum or i n antiserum r a i s e d against S. aureus-5, C l f c e l l s . Presumably, CIA were not present i n anti-jS. aureus-5. When anti-S.aureus-was absorbed w i t h Clf"*" c e l l s , and then assayed, i t no longer i n t e r f e r e d w i t h fibrinogen-induced clumping of C l f + c e l l s . I t appeared that the antib o d i e s had been absorbed by the C l f + c e l l s . The assumption i s corroborated w i t h r e s u l t s of a n t i - S ^ aureus-1 that had been absorbed w i t h C l f + S. aureus c e l l s , b o i l e d f o r 60 min or tr e a t e d w i t h t r y p s i n . Both of these treatments destroyed the b i o l o g i c a l a c t i v i t y of C l f on whole c e l l s or c e l l w a l l s . Antiserum absorbed w i t h these t r e a t e d C l f + c e l l s r e t a i n e d the CIA, which suggested that b o i l i n g or treatment w i t h t r y p s i n a l s o destroyed the a n t i g e n i c p r o p e r t i e s of C l f on whole c e l l s . When anti-S_. aureus-1 was extr a c t e d w i t h C l f c e l l s , the CIA were not removed (Table V I I I ) and i n h i b i t e d the C l f + j ^ . aureus f i b r i n o g e n clumping r e a c t i o n The preceeding r e s u l t s i n d i c a t e d that there were s p e c i f i c a n t i - C l f a n t i -bodies present i n a n t i - S . aureus-1 serum which only reacted w i t h S. aureus c e l l s . S o l u b i l i z e d c e l l w a l l f r a c t i o n s , i n c l u d i n g s o l u b l e C l f i n peak #2, were assayed i n the same system to determine i f any s o l u b l e m a t e r i a l could absorb or remove the CIA from a n t i - S ^ aureus-1. The so l u b l e t e s t m a t e r i a l was incubted w i t h a n t i - S . aureus-1 f o r 30 min at room temperature, then, S. aureus C l f + c e l l s were added and, 2 h l a t e r , f i b r i n o g e n was added. A f t e 5 min i n c u b a t i o n , the tubes were examined f o r clumped c e l l s . As seen i n Table IX, s o l u b l e C l f (peak #2), reacted w i t h CIA which thus enabled the C l f + c e l l s to clump w i t h the added f i b r i n o g e n . L y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l s of C l f + S. aureus s t r a i n s , a l s o appeared to react w i t h the a n t i bodies .in the antiserum, however, the j3. aureus - f i b r i n o g e n aggregates were not as large as those observed i n antiserum absorbed w i t h s o l u b l e clumping f a c t o r . L y s o s t a p h i n - s o l u b i l i z e d c e l l w a l l s of the C l f s t r a i n Table V I I I Influence of clumping i n h i b i t i n g a n t i b o d i e s on fibrinogen-induced clumping of j>. aureus C l f a c t i v i t y 1 Serum Pr e p a r a t i o n 1:8 .1:16 1:32 1:64 1:128 1:256 C l f + antiserum + + C l f " antiserum + + + + + + Nonimmune + + + + + + C l f + antiserum absorbed w i t h C l f c e l l s or w a l l s + + + + + + C l f + antiserum absorbed w i t h C l f c e l l s or w a l l s + + C l f + antiserum absorbed w i t h b o i l e d C l f c e l l s + + C l f + antiserum absorbed w i t h t r y p s i n t r e a t e d C l f c e l l s + + S e r i a l l y d i l u t e d antiserum was incubated w i t h C l f c e l l s f o r 2 h at room temperature. Fibrinogen was added, the tubes shaken, and then assayed f o r the presence of clumped c e l l s . 83 Table IX Assay f o r s o l u b l e clumping f a c t o r 1 P -i D i l u t i o n of C l f + antiserum Sample 1:8 1:16 1:32 1:64 1:128 1:256 PBS pH 7.0 S_. aureus C l f + c e l l s 1 1 or w a l l s j>. aureus C l f - c e l l s or w a l l s L y s o s t a p h i n - s o l u b i l i z e d C l f + w a l l s L y s o s t a p h i n - s o l u b i l i z e d C l f - w a l l s Soluble clumping f a c t o r """Sample was mixed w i t h antiserum f o r 30 min p r i o r to the a d d i t i o n of C l f + c e l l s . A f t e r 2 h in c u b a t i o n at room temperature, f i b r i n o g e n was added and 5 min l a t e r , the c e l l s were assayed f o r clumping. i:LWhen the antiserum was incubated w i t h whole c e l l s or w a l l s , they were removed by c e n t r i f u g a t i o n p r i o r to adding S_.. aureus-1 c e l l s . + + + + + + + + + - + + + + + + + + + + + + + + + 84 di d not appear to absorb or remove the a n t i b o d i e s . C. Reaction of other b a c t e r i a w i t h f i b r i n and f i b r i n o g e n Table X summarizes studies aimed at c h a r a c t e r i z i n g the f i b r i n - b i n d i n g and f i b r i n o g e n clumping a c t i v i t i e s of a number of d i f f e r e n t b a c t e r i a . 1. Clumping i n f i b r i n o g e n The a b i l i t y to clump i n the presence of f i b r i n o g e n was only observed w i t h s t r e p t o c o c c a l 'species of group C." Since A. viscosus and F. nucleatum a u t o - a g g l u t i n a t e d , fibrinogen-induced clumping of these organisms could not be determined. 2. Binding to f i b r i n S t r e p t o c o c c a l species of groups A and C, P e p t o s t r e p t o c o c c i , A. v i s c o s u s , and F. nucleatum bound to c r o s s - l i n k e d and non-cross-linked f i b r i n . Several C l f i s o l a t e s of S. aureus-1, and, o c c a s i o n a l l y _S. aureus-5, were found to bind i n low numbers to f i b r i n . A serum r e s i s t a n t s t r a i n of P_. aeruginosa : bound to non-cross-linked f i b r i n , whereas a serum s e n s i t i v e s t r a i n d i d not bind to e i t h e r form of f i b r i n . 3. Absorption of f i b r i n o g e n B a c t e r i a which d i d not clump i n f i b r i n o g e n were assayed f o r t h e i r a b i l i t y to absorb f i b r i n o g e n . Organisms that could absorb f i b r i n o g e n but not clump i n i t s presence, may have monovalent or "incomplete adhesins'' f o r f i b r i n o g e n which could bind to the molecule but not form a v i s i b l e c e l l aggregate. I f one assumes that the f i b r i n o g e n molecule i s b i v a l e n t , then f i b r i n o g e n , bound to such an organism's c e l l s u r f a c e , may have a free receptor s i t e a v a i l a b l e f o r an organism such as S. aureus-1, possessing • complete adhesins. The s t a p h y l o c o c c a l c e l l s could bind to the f r e e Table X Reaction of other b a c t e r i a w i t h f i b r i n and f i b r i n o g e n B a c t e r i a C e l l s bound to f i b r i n % Clumped i n Adsorbed c r o s s - l i n k e d non-cross-linked f i b r i n o g e n f i b r i n o g e n + 55 S. aureus C l f + 42 _S. aureus C l f JS. aureus, _free c o a g u l a s e } C l f S^. epidermidis J3. s a l i v a r i u s S. sanguis S t r e p t o c o c c i 40 Group C S. m i t i s -45 47 41 + + + + + + S. mutans S. pyogenes V e i l l o n e l l a a l c a i e s c e n s A. v i s c o s u s F. nucleatum 16 P e p t o s t r e p t o c o c c i 46 50 17 22 40 40 9 + + + + P. aeruginosa serum r e s i s t a n t serum s e n s i t i v e E. c o l i B. melaninogenicus a s a c c h a r o l y t i c u s 29 + B. f r a g i l i s Auto-agglutinated 86 f i b r i n o g e n s i t e s and draw the other b a c t e r i a together to produce a v i s i b l e c e l l aggregate. When S. aureus-1 was mixed w i t h b a c t e r i a i n the absence of f i b r i n o g e n , no clumping of c e l l s occurred. As seen i n Table X, s e v e r a l b a c t e r i a l species appeared to adsorb f i b r i n o g e n and clumped when S. aureus-1 c e l l s were added to the assay system. Every organism examined that bound to f i b r i n , a l s o absorbed or bound to f i b r i n o g e n . This was a l s o true of the reverse s i t u a t i o n except f o r S. epidermidis and some C l f mutants of jS. aureus which o c c a s i o n a l l y absorbed f i b r i n o g e n but d i d not bind to f i b r i n c o n s i s t e n t l y , or i n high numbers. An unknown c u l t u r a l c o n d i t i o n may have been res p o n s i b l e f o r the i n d u c t i o n of formation of an incomplete receptor which enabled the. organism to bind weakly on occasion w i t h f i b r i n and, or f i b r i n o g e n . Other b a c t e r i a ' t h a t absorbed or bound f i b r i n o g e n , may have bound to i d e n t i c a l f i b r i n o g e n receptors as those recognized by S. aureus-1, or they may have bound to an e n t i r e l y d i f f e r e n t moiety on the f i b r i n o g e n molecule. This b i n d i n g s i t e may have acted as an analogue of a substance or a c e l l surface receptor to which the organism normally adheres to i n i t s n a t u r a l environment. D. Summary 1. Comparison of the f i b r i n and f i b r i n o g e n b i n d i n g adhesins Results from t h i s study have i n d i c a t e d that the c e l l w a l l component of "S. aureus r e s p o n s i b l e f o r b i n d i n g to f i b r i n , i s i d e n t i c a l , to that mediating adherence to f i b r i n o g e n . The evidence f o r t h i s hypothesis Is summarized below: ( i ) A l l S. aureus s t r a i n s tested that, bound to f i b r i n . . . a l s o bound t o , or absorbed f i b r i n o g e n , ( i i ) M o d i f i c a t i o n t o whole c e l l s - w h i c h , a l t e r e d t h e i r 87 b i n d i n g a c t i v i t y w i t h f i b r i n were the same as those m o d i f i c a t i o n s which a l t e r e d t h e i r b i n d i n g to f i b r i n o g e n . ( i i i ) C e l l w a l l s i s o l a t e d from ^. aureus could bind to both f i b r i n and f i b r i n o g e n i n the same manner as could whole c e l l s . ( i v ) S t r a i n s of j ^ . aureus that bound to f i b r i n w i t h a hig h a v i d i t y were used to i s o l a t e a s o l u b i l i z e d c e l l w a l l component that bound s e l e c t i v e l y to immobilized f i b r i n o g e n . (v) Antibodies r a i s e d against jS_. aureus s t r a i n s that bound to f i b r i n and f i b r i n o g e n were s p e c i f i c and appeared to i n h i b i t b i n d i n g of c e l l s to f i b r i n and f i b r i n o g e n . 2. R e l a t i o n s h i p of C l f to f i b r i n and f i b r i n o g e n b i n d i n g , and to fibrinogen-induced clumping. Although every organism examined w i t h the a b i l i t y to clump i n f i b r i n o g e n a l s o bound to f i b r i n and immobilized f i b r i n o g e n , the reverse was not t r u e . I f the f i b r i n o g e n molecule and f i b r i n monomer were t r u l y b i v a l e n t and possessed at l e a s t two i d e n t i c a l b i n d i n g r e c e p t o r s , and i f both receptors were bound to an organism w i t h complete, f i b r i n o g e n r e c e p t o r s , c e l l aggregates could be produced. This appears to be the s i t u a t i o n w i t h aureus-1 (Model 1, F i g . 13). Only these s t r a i n s were capable of e l i c i t i n g the production of s p e c i f i c CIA. Other b a c t e r i a l s t r a i n s , i n c l u d i n g J3. aureus-5, may have incomplete r e c e p t o r s , which allows f o r a weak b i n d i n g of c e l l s to f i b r i n and f i b r i n o g e n but not formation of a c e l l aggregate w i t h f i b r i n o g e n . (Model 2, F i g . 13). Another p o s s i b i l i t y i s shown i n Model 3, F i g . 13, where C l f receptors 88 are present i n the c e l l w a l l but i n a c c e s s i b l e to the m a j o r i t y of f i b r i n or f i b r i n o g e n molecules. Thus, a f i b r i n o g e n molecule may b i n d to a receptor on one staphylococcal c e l l but, owing to the f i b r i n o g e n molecule's l i m i t e d s i z e or to i t s c o n f i g u r a t i o n when bound to a masked re c e p t o r , be unable to bind to any other staphylococcal c e l l . The organ-ism would then be able to absorb the f i b r i n o g e n molecule but not clump i n i t s presence. When S. aureus-1 c e l l s were added to jS. aureus-5 c e l l s that had absorbed f i b r i n o g e n , clumping of c e l l s occurred. However, i f the C l f possessed by S. aureus-5 was incomplete or i n a masked form, i t s a f f i n i t y f o r f i b r i n o g e n would conceivably be f a r l e s s than that of the complete, a c c e s s i b l e C l f possessed by j>. aureus-1. These complete adhesins should be more s u c c e s s f u l i n a competition f o r f i b r i n o g e n and may have removed the f i b r i n o g e n that had bound to S. aureus-5 c e l l s . Thus, i n the assay used to demonstrate absorption of f i b r i n o g e n by c e l l s , i t was impossible to determine i f JS. aureus-1 had bound to a f r e e s i t e on the bound, b i v a l e n t f i b r i n o g e n molecule, and caused clumping of S. aureus-5 c e l l s , or i f the f i b r i n o g e n had d i s s o c i a t e d from the bound c e l l s and was used to clump S. aureus-1 c e l l s . I f C l f e x i s t s as depicted i n models 2 and 3, than i t would e x p l a i n why S. aureus-5 c e l l s possessing e i t h e r form of the C l f would be unable to e l i c i t the formation of s p e c i f i c antibody and would not have a c e l l w a l l component that could be i s o l a t e d w i t h the c h a r a c t e r i s t i c s observed w i t h g l y c o p r o t e i n C. A f i n a l model to e x p l a i n the a b i l i t y of jS. aureus-5 to absorb f i b r i n o g e n but not clump, i s presented i n F i g . 13, Model 4. This model 89 suggests that S. aureus-5 c e l l s may possess complete C l f but i n i n s u f f i c ent numbers to procure the clumping of c e l l s . ure 13. Models of complete, incomplete f a c t o r of S. aureus. and masked clumping MODELS OF MODEL 1 Many, complete adhesins Fl BRINOGEN—IN DUCED CLUMPING OF S. q ureus 91 S. aureus — 1 Clf + O - O MODEL 2 S. aureus—5 Clf — Incomplete adhesins MODEL 3 S.aureus - 5 Clf — Recessed or masked adhesins MODEL 4 S.aureus —5 Clf — Insufficient number of adhesins DISCUSSION The i n t e r a c t i o n between S. aureus and f i b r i n has been studied and c h a r a c t e r i z e d , and the c e l l w a l l component mediating f i b r i n o g e n induced clumping has been i d e n t i f i e d . The r e s u l t s i n d i c a t e that the c e l l w a l l component, or "clumping f a c t o r " ( C l f ) , of S^. aureus which enables i t to clump w i t h f i b r i n o g e n i s the same f a c t o r involved i n the c e l l ' s i n t e r a c t i o n w i t h f i b r i n . S t r a i n s of S. aureus that were clumping f a c t o r p o s i t i v e ( C l f + ) c l u m p e d i n f i b r i n o g e n and bound to c r o s s - l i n k e d and non-cross-linked f i b r i n . Mutants, obtained from the parent C l f + s t r a i n lacked both p r o p e r t i e s and were c a l l e d clumping f a c t o r negative ( C l f ). Previous r e s u l t s i n d i c a t e d that C l f was a separate e n t i t y of the c e l l , u nrelated to f r e e coagulase. This premise was f u r t h e r s u b s t a n t i a t e d w i t h i s o l a t e s of S. aureus which produced only one of the substances. The b i n d i n g of S. aureus C l f + c e l l s to f i b r i n was maximal at *37°C, w i t h a pH optima of 6-8 and was independent of d i v a l e n t c a t i o n s . The i n t e r a c t i o n between the two surfaces occurred w i t h i n minutes, and once bound to the f i b r i n m a t r i x , c e l l s were d i f f i c u l t to remove. Sodium c h l o r i d e , 1 M i n h i b i t e d b i n d i n g and caused des o r b t i o n of the c e l l s from immobilized f i b r i n or f i b r i n o g e n . This procedure d i d not create any permanent damage to e i t h e r c o n s t i t u e n t but may have a l t e r e d t h e i r surface charge or i n t e r f e r e d w i t h i o n i c i n t e r a c t i o n s between them. Adherence of S^. aureus to f i b r i n - c o a t e d glass v i a l s was prevented by a high 92 93 concentration of urea, t h i s may have caused a conformational change i n the C l f or f i b r i n molecule and thus abrogated the b i n d i n g r e a c t i o n . An attempt to f i n d a compound that could mimic or act as an analogue of the molecules i n v o l v e d i n the b i n d i n g s i t e s of aureus or f i b r i n was i n c o n c l u s i v e . None of the g l y c o p r o t e i n s , p r o t e i n s or g l y c o l i p i d s t e s t e d had an appreciable i n h i b i t o r y e f f e c t on the f i b r i n - c e l l i n t e r a c t i o n . I n h i b i t i o n was observed when monosaccharides were incubated w i t h S. aureus and f i b r i n i n the assay system; however, the i n h i b i t i o n was not r e s t r i c t e d to a p a r t i c u l a r sugar. Gangliosides showed an i n h i b i t o r y e f f e c t but at a c o n c e n t r a t i o n that suggests i t was of a n o n s p e c i f i c nature. Gangliosides could i n t e r f e r e w i t h hydrophobic bonding between the f i b r i n molecule and j>. aureus C l f , but r e s u l t s of i n h i b i t i o n s t u d i e s w i t h albumin f a i l to support t h i s premise. Albumin, known to i n h i b i t many hydrophobic i n t e r a c t i o n s (12, 100) d i d not have any d e t e c t a b l e e f f e c t on the jS. aureus-f i b r i n b i n d i n g r e a c t i o n . A n a l y s i s of the i n t e r a c t i o n of f i b r i n and S_. aureus a f t e r m o d i f i -c a t i o n and treatment of whole c e l l s or i s o l a t e d c e l l w a l l s , suggests that the adhesin or C l f i s a p r o t e i n . I t i s r e s i s t a n t to moderate heat ( b o i l i n g 30 min but not 60 min or autoclaving) and to m i l d pH changes. Results of s o n i c a t i o n procedures and e l e c t r o n microscopic s t u d i e s suggest that C l f i s not a s s o c i a t e d w i t h surface p r o j e c t i o n s such as p i l i or a capsule, but i s r a t h e r an i n t e g r a l component of the c e l l w a l l , p o s s i b l y attached to peptidoglycan. Methods of e x t r a c t i o n of whole c e l l s w i t h l i p i d s o l v e n t s , detergents, or phenol were unsuccessful i n a l t e r i n g t h e i r C l f a c t i v i t y . Clumping a c t i v i t y of whole c e l l s was destroyed by e x t r a c t i o n w i t h TCA, HC1, or formamide. However, a substance w i t h b i o l o g i c a l a c t i v i t y 94 could not be e f f e c t i v e l y removed from the c e l l s by any of these e x t r a c t -ion procedures. The adhesin was a l s o i n a c t i v a t e d by p r o t e o l y t i c enzymes and a c e t y l a t i o n of amino groups. Clumping f a c t o r a c t i v i t y was more e a s i l y destroyed i n preparations of i s o l a t e d c e l l w a l l s than of whole c e l l s , which may i n d i c a t e that the f a c t o r i s masked i n whole c e l l s and, o r , p a r t i a l l y p rotected from the a c t i o n of heat and protease. Since C l f could not be p h y s i c a l l y removed or e x t r a c t e d from whole c e l l s or w a l l s , a method was devised to i s o l a t e i t from a p r e p a r a t i o n of s o l u b i l i z e d c e l l w a l l s . A n a l y s i s of the s o l u b i l i z e d c e l l w a l l p r e p a r a t i o n by SDS PAGE, revealed s i x Coomassie s t a i n i n g bands i n the p r e p a r a t i o n from the C l f + s t r a i n and nine bands i n that of the C l f s t r a i n . Of p a r t i c u l a r i n t e r e s t was a p r o t e i n , l a b e l l e d C i n F i g . 11, which had a molecular weight of approximately 90,000 daltons and that s t a i n e d p o s i -t i v e l y w i t h p e r i o d i c S c h i f f reagent. This p r o t e i n was only observed i n w a l l s of C l f + s t r a i n s . Immunoelectrophoretic a n a l y s i s of the s o l u b i l i z e d c e l l w a l l s demonstrated an antigen, l a b e l l e d C i n f i g . 12, present i n the C l f + s t r a i n and absent i n the C l f s t r a i n . Only antiserum prepared against w a l l s of the C l f + s t r a i n ( a n t i - S , aureus-1,antiserum) contained an a n t i -body that p r e c i p i t a t e d antigen C. When a n t i - S . aureus-1 antiserum was absorbed w i t h C l f c e l l s , only the antibody p r e c i p i t a t i n g antigen C could be detected i n the absorbed antiserum. The g l y c o p r o t e i n present i n l y p o s t a p h i n - s o l u b i l i z e d c e l l w a l l s of the C l f + s t r a i n was i s o l a t e d by a f f i n i t y chromatography using immobilized f i b r i n o g e n . Sodium c h l o r i d e , shown p r e v i o u s l y to desorb adherent S_. aureus c e l l s from f i b r i n o g e n and f i b r i n , was found to be e f f e c t i v e i n e l u t i n g a f r a c t i o n w i t h clumping f a c t o r a c t i v i t y . This f r a c t i o n was 95 not present when a p r e p a r a t i o n of s o l u b i l i z e d c e l l w a l l s of a C l f s t r a i n was a p p l i e d to the a f f i n i t y column. A n a l y s i s of the f r a c t i o n by SDS 10% PAGE, revealed a s i n g l e band that s t a i n e d p o s i t i v e l y w i t h Coomassie blue and w i t h p e r i o d i c S c h i f f and that had an i d e n t i c a l m o b i l i t y (molecular weight approximately 90,000 d a l t o n s ) , to that of g l y c o p r o t e i n C. Immunological p r o p e r t i e s of the f r a c t i o n were i d e n t i c a l to those of antigen C and a p r e c i p i t i n l i n e could only be demonstrated w i t h a n t i - S . aureus-fantiserum. Immunoelectrophoretic a n a l y s i s of a p u r i f i e d p r e p a r a t i o n of P r o t e i n A i n d i c a t e d that the C l f f r a c t i o n was not contaminated- w i t h t h i s c e l l w a l l component. M a t e r i a l i n .the C l f f r a c t i o n bound s e l e c t i v e l y to immobilized f i b r i n -ogen but could not be demonstrated to clump i n s o l u b l e f i b r i n o g e n . An assay was developed which showed that the f r a c t i o n n e u t r a l i z e d or absorbed s p e c i f i c clumping i n h i b i t i n g a n t i b o d i e s (CIA) present i n a n t i - S . aureus-1 antiserum. No other e x t r a c t or c e l l s t e s t e d i n t h i s assay, other than those of C l f + s t r a i n s could absorb these an t i b o d i e s and enable the S. a u r e u s - f i b r i n o g e n clumping r e a c t i o n to occur. In summary, a substance w i t h clumping f a c t o r a c t i v i t y could be i s o l a t e d from c e l l w a l l s of C l f + S^  aureus s t r a i n s but not from C l f s t r a i n s . I t e x h i b i t e d p r o p e r t i e s of a g l y c o p r o t e i n , molecular weight'of approximately 90,000 daltons, and was a n t i g e n i c . I n d i c a t i o n s are that antigen C, present i n s o l u b i l i z e d c e l l w a l l preparations of C l f + s t r a i n s , a n d the antigen i n the C l f f r a c t i o n are the same. The f r a c t i o n formed a p r e c i p i t a t e w i t h an antibody present i n anti-Si. aureus-1 antiserum and absorbed CIA. These a n t i b o d i e s are probably i d e n t i c a l . Further work i s i n progress to determine i f the f r a c t i o n w i l l e l i c i t production of a n t i b o d i e s when i n j e c t e d i n t o r a b b i t s . 96 Some of the c h a r a c t e r i s t i c s of C l f such as i t s moderate r e s i s t a n c e to heat and protease, i t s apparent a n t i g e n i c i t y , and i t s property of adherence, are shared by l i p o t e i c h o i c acids (LTA). Both substances are d i f f i c u l t to e x t r a c t from c e l l w a l l s and i s o l a t e i n a pure form (100). However, b i n d i n g r e a c t i o n s of l i p o t e i c h o i c acids are i n h i b i t e d by albumin; (10) i t can be ext r a c t e d from c e l l s w i t h c o l d 10% t r i c h l o r a c e t i c a c i d or phenol, and i s r e s i s t a n t to t r y p s i n (100). These p r o p e r t i e s are not shared by the c e l l w a l l component of j ^ . aureus e x h i b i t i n g C l f a c t i -v i t i e s . The p o s s i b i l i t y e x i s t s that C l f i s a la r g e peptide complex w i t h t e i c h o i c a c i d as a component. The novel method used i n t h i s study to e x t r a c t C l f from a preparat-i o n of s o l u b i l i z e d c e l l w a l l s , i s based on the f a c t o r ' s a b i l i t y to s e l e c t i v e l y adhere to immobilized f i b r i n o g e n . The method appears to be more s p e c i f i c , e f f i c i e n t and doesn't i n v o l v e damaging chemicals as do some of the e x t r a c t i o n methods reported i n the l i t e r a t u r e . Although s e v e r a l i n v e s t i g a t o r s have reported i s o l a t i n g a substance from s t a p h y l o c o c c a l c e l l s ' w i t h C l f a c t i v i t y (16, 29, 56, 84, 94) proof that the substance i s the C l f remains to be demonstrated. Duthie, (1954) (29), was f i r s t i n d e f i n i n g a method f o r C l f i s o l a t i o n . He ext r a c t e d a p r o t e i n , i n p a r t i c u l a t e form, from an auto-l y s a t e of S. aureus c e l l s . Although the p r o t e i n n e u t r a l i z e d clumping i n h i b i t i n g a n t i b o d i e s present i n antiserum prepared against J^. aureus c e l l s , i t had questionable a c t i v i t i e s w i t h f i b r i n o g e n . The assay he used to demonstrate absorption of f i b r i n o g e n l a c k s s p e c i f i c i t y . Many substances examined i n our l a b o r a t o r y by Duthie's assay system demon-s t r a t e d the a b i l i t y to absorb f i b r i n o g e n . These substances included non-JS. aureus^ b a c t e r i a l c e l l s and g l a s s . 97 Duthie hypothesized that a s i n g l e substance w i t h d i f f e r e n t s i t e s f o r absorbing and clumping f i b r i n o g e n was present on the s t a p h y l o c o c c a l c e l l w a l l and that the clumping r e a c t i o n occurred i n two stages. In the f i r s t stage, an ion-independent absorption of f i b r i n o g e n occurred over a pH range of 2-11. This was followed by clumping of the c e l l s , that r e q u i r e d , a minimum of 0.01 M sodium ions and had a pH optima of 5,5, Two methods were employed by Kato and Omori (56) to e x t r a c t a substance w i t h C l f a c t i v i t y from J 3 . aureus c e l l s . One method used a s a l t i n g out procedure to e x t r a c t a substance from the supernatant of crushed c e l l s ; the other method used a.phenol e x t r a c t i o n to remove a substance from whole c e l l s . The substances e x t r a c t e d by these methods could n e u t r a l i z e CIA from a n t i - S . aureus antiserum. This property of the e x t r a c t s was destroyed when they were autoclaved or t r e a t e d w i t h t r y p s i n . However, these treatments d i d not e l i m i n a t e t h e i r a b i l i t y to absorb f i b r i n o g e n . E x t r a c t s from C l f c e l l s or from t r y p s i n - t r e a t e d C l f + c e l l s a l s o demonstrated the a b i l i t y to absorb f i b r i n o g e n i n t h e i r assay system. When whole c e l l s of C l f + S^. aureus are t r e a t e d w i t h t r y p s i n or autoclaved they lose f i b r i n o g e n - a b s o r b i n g and clumping a c t i v i t i e s . They e x p l a i n t h i s discrepancy by suggesting that the clumping a c t i v i t y of s t a p h y l o c o c c a l c e l l w a l l s i s dependent on two substances. One substance lo c a t e d w i t h i n the c e l l i s t r y p s i n r e s i s t a n t , absorbs f i b r i n o g e n and can be e x t r a c t e d from crushed c e l l s of C l f + , C l f and t r y p s i n - t r e a t e d C l f + c e l l s . The other substance, bound to the c e l l surface of C l f + s t r a i n s , i s t r y p s i n s e n s i t i v e and absorbs f i b r i n o g e n and a g g l u t i n i n s . This sub-stance can be removed from the c e l l by phenol e x t r a c t i o n and can be separated by f i f t y percent acetone i n t o a p r e c i p i t a t e that absorbs a n t i b o d i e s , and a supernatant that absorbs f i b r i n o g e n . 98 Rotter and K e l l y (84) used the method of phenol e x t r a c t i o n to obt a i n a substance from staphylococcal c e l l s which they c h a r a c t e r i z e d w i t h s e r o l o g i c a l t e s t s . They coated l a t e x beads w i t h the e x t r a c t e d substance and found that a n t i - S . aureus C l f + antiserum absorbed w i t h C l f c e l l s or w i t h t r y p s i n - t r e a t e d C l f + c e l l s , a g g l u t i n a t e d the beads. A n t i -S. aureus C l f + antiserum which had been absorbed w i t h C l f + c e l l s f a i l e d to do so. The e x t r a c t d i d not e l i c i t the production of detectable a n t i b o d i e s when i n j e c t e d i n t o r a b b i t s and d i d not form a p r e c i p i t a t e w i t h C l f + anti-_S. aureus antiserum. They could not determine i f the e x t r a c t - l a t e x p a r t i c l e s clumped i n f i b r i n o g e n because untreated l a t e x beads and er y t h r o c y t e s clumped spontaneously i n a - f i b r i n o g e n s o l u t i o n . This observation was corrobor-ated i n our l a b o r a t o r y when s i m i l a r s tudies w i t h untreated e r y t h r o c y t e s or those coated w i t h albumin, were observed to clump i n f i b r i n o g e n . The phenol e x t r a c t obtained by Rotter and K e l l y contained a p r o t e i n which could absorb CIA but whether the p r o t e i n was the C l f remains to be determined. One could speculate that t h e i r e x t r a c t contained p r o t e i n A, t h i s would e x p l a i n the e x t r a c t ' s a b i l i t y to b i n d antibody, i t s s e n s i -t i v i t y to protease, but i n a c t i v i t y w i t h f i b r i n o g e n . However, the i n a b i l i t y of the e x t r a c t to e l i c i t antibody production or to p r e c i p i t a t e w i t h a n t i -bodies present i n a n t i - S . aureus antiserum i s not c o n s i s t e n t w i t h known immunogenic p r o p e r t i e s of p r o t e i n A, unless, as was proposed by these i n v e s t i g a t o r s , t h e i r e x t r a c t was not of a s u f f i c i e n t c o n c e n t r a t i o n to e l i c i t the immune response or to p r e c i p i t a t e w i t h a n t i - S . aureus a n t i b o d i e s . Attempts i n our l a b o r a t o r y to e x t r a c t C l f from whole c e l l s or i s o l a t e d c e l l w a l l s w i t h phenol were un s u c c e s s f u l . A f t e r phenol t r e a t -ment the c e l l s r e t a i n e d t h e i r a b i l i t y to clump i n f i b r i n o g e n . The phenol 99 e x t r a c t f a i l e d to show any a c t i v i t y w i t h f i b r i n o g e n or f i b r i n and d i d not demonstrate p r o t e i n when analyzed by SDS PAGE. Another method reported i n the l i t e r a t u r e , (16, 94) i n v o l v e d the e x t r a c t i o n of a substance from S. aureus c e l l s w i t h formic a c i d , and i t s p u r i f i c a t i o n by i s o e l e c t r i c f o c u s i n g techniques. The p u r i f i e d e x t r a c t was reported to have C l f a c t i v i t i e s since e r y t h r o c y t e s coated w i t h the e x t r a c t , clumped i n f i b r i n o g e n . I n d i c a t i o n s are, that previous attempts to i s o l a t e C l f from jS. aureus c e l l s have not been wholly s u c c e s s f u l . The assays used by i n v e s t i g a t o r s lacked s p e c i f i c i t y , and p r o p e r t i e s of e x t r a c t e d C l f f a i l e d to concur w i t h those observed f o r C l f w h i l s t i t was a component of i n t a c t c e l l s . I f one views C l f as a composite of two e n t i t i e s , w i t h one e n t i t y capable of b i n d i n g f i b r i n o g e n but both e n t i t i e s r e q u i r e d f o r clumping of c e l l s , an e x p l a n a t i o n f o r the observed d i s c r e p a n c i e s may e x i s t . The e x t r a c t s of some i n v e s t i g a t o r s may have contained a s i n g l e component of the C l f complex and thus would have lacked the f u l l complement of C l f p r o p e r t i e s . Another p o s s i b i l i t y may be that t h e i r C l f e x t r a c t contained p r o t e i n A, another S^. aureus c e l l w a l l component that shares some p r o p e r t i e s w i t h C l f . This may e x p l a i n why t h e i r e x t r a c t absorbed clumping i n h i b i t i n g a n t i b o d i e s (CIA), i n a n t i - S . aureus serum but f a i l e d to form a p r e c i p i t i n l i n e w i t h these a n t i b o d i e s . P r o t e i n A i s known to bind to the Fc region of IgG but would not p r e c i p i t a t e these s p e c i f i c CIA. Results from t h i s study i n d i c a t e that C l f i s a s i n g l e c e l l w a l l compo-nent which may however, e x i s t i n an incomplete form. The a f f i n i t y f o r f i b r i n o g e n or f i b r i n by t h i s form of C l f would not be equivalent to that of the complete adhesin, and may cause c e l l s possessing the former to 100 r a p i d l y r e l e a s e bound f i b r i n o g e n or to a l t e r i t s c o n f i g u r a t i o n when bound. In e i t h e r case, c r o s s - l i n k s between separate c e l l s , r e s u l t i n g i n c e l l aggregates, would not l i k e l y occur. B a c t e r i a other than S. aureus were a l s o observed to bind to f i b r i n or f i b r i n o g e n but not clump, i n d i c a t i n g that they too. may have possessed incomplete or masked adhesins. Or, they may have C l f i n numbers too low to e l i c i t formation of clumped c e l l s . A l t e r n a t i v e l y , these organisms may have adhesins on t h e i r w a l l s which merely mimic C l f a c t i v i t y . Duthie (30) observed that s t r e p t o c o c c i of groups A, C and G could absorb f i b r i n o g e n but not clump i n i t s presence. His r e s u l t s were corroborated i n t h i s study i n a d d i t i o n to observations that serum r e s i s t a n t s t r a i n s of Pseudomonas aeruginosa and P e p t o s t r e p t o c o c c i a l s o absorbed f i b r i n o g e n but d i d not clump. The assumption that f i b r i n or f i b r i n o g e n b i n d i n g i s p o s s i b l e w i t h b a c t e r i a that possess incomplete, masked, or inadequate numbers of C l f , or that possess adhesins analogous to C l f , suggests that assays that demonstrate b i n d i n g r e a c t i o n s are more s e n s i t i v e than those determining clumping of c e l l s . The l a t t e r a c t i v i t y i n d i c a t e s a requirement f o r p l e n t i f u l , complete C l f on tfhe b a c t e r i a c e l l surface and may be r e s t r i c t e d to a very few b a c t e r i a l species. The importance of C l f to the p a t h o g e n i c i t y of S. aureus i s p r e s e n t l y under some controversy. I t has been reported (55) that some v i r u l e n t s t r a i n s of J3_. aureus^ possess a capsule " i n v i v o " that mask C l f . The encapsulated s t r a i n s were shown to evade phagocytosis and thus remain i n the host longer than s t r a i n s without capsules. I t i s reasonable to assume that possession of C l f i s advantageous to a s t a p h y l o c o c c a l c e l l when i t i n i t i a l l y encounters a surface of the 101 host c o n t a i n i n g f i b r i n o g e n or f i b r i n . The c e l l can a t t a c h f i r m l y to the surface and r e s i s t removal by the host. Then, i f i t invades the host's t i s s u e s or c a v i t i e s and no longer r e q u i r e s C l f , i t may>produce a capsule w i t h d i f f e r e n t adherent p r o p e r t i e s , more s u i t a b l e i n the new environment. This adaptive mechanism could enable the b a c t e r i a to evade contact and engulfment by wandering phagocytes. .In h i s . review, Beachey (12) reports that many pathogenic b a c t e r i a w i t h adhesins e i t h e r shed or mask them w i t h a capsule when invading host t i s s u e . Is a knowledge of b a c t e r i a l adherence to a host's t i s s u e s purely of academic i n t e r e s t , or can i t be used to c o n t r o l i n f e c t i o n s by e i t h e r preventing b a c t e r i a l attachment or d i s r u p t i n g adherent c e l l s ? Both the host and invading microbe must have t h e i r attachment s i t e s f r e e and a c c e s s i b l e f o r a b i n d i n g i n t e r a c t i o n to occur. I f the host's receptor s i t e s are blocked by p u r i f i e d b a c t e r i a l adhesins, analogues of the adhesins, or a n t i b o d i e s d i r e c t e d against them, attachment of the b a c t e r i a could be prevented. Or, i f an agent administered to the host prevents formation of the b a c t e r i a l adhesin, and i s harmless to the host, an i n f e c t i o n caused by the b a c t e r i a may be avoided. There i s c u r r e n t l y some controversy over the proposal that a n t i b o d i e s d i r e c t e d against b a c t e r i a l a d h e s i n s o f f e r more p r o t e c t i o n to a host than do a n t i b o d i e s d i r e c t e d against other b a c t e r i a l surface antigens (12, 22, 92, 93). Immunoglobulin A (SIgA), found i n normal body s e c r e t i o n s may be e f f e c t i v e i n preventing b a c t e r i a l adherence, i n areas such as the o r a l c a v i t y . Results of s e v e r a l experimental s t u d i e s have i n d i c a t e d that s a l i v a r y SIgA, produced i n response to c e l l s and c e l l products of S. mutans, proved e f f e c t i v e i n preventing the attachment of t h i s organism 102 to the tooth surface (33, 95). Other workers (14, 95) a l s o found that SIgA diminished the numbers of S. mutans found i n d e n t a l plaque, thus decreasing the incidence of c a r i e s i n immunized animals. An attempt to develop an e f f e c t i v e vaccine f o r the prevention of human dent a l c a r i e s i s now i n progress (33). Several vaccines have been prepared from the a n t i g e n i c f i m b r i a e of enteropathogenic s t r a i n s of E. c o l i . When the vaccine was administered to pregnant sows, t h e i r o f f s p r i n g were protected against d i a r r h e a , a f t e r challenge w i t h i n f e c t i v e doses of E. c o l i . Other i n v e s t i g a t o r s , (28) have studied the p r o t e c t i v e r o l e of a n t i -bodies, conferred to a host, against organisms causing b a c t e r i a l endo-c a r d i t i s . Rabbits that were immunized w i t h S. mutans or S. sanguis and that produced a h i g h l e v e l of s p e c i f i c antibody i n t h e i r sera, were l e s s l i k e l y to develop s t r e p t o c o c c a l e n d o c a r d i t i s than animals w i t h lower antibody t i t r e s or those not immunized w i t h s t r e p t o c o c c i . Although these a n t i b o d i e s may have s p e c i f i c a l l y i n h i b i t e d adherence of the organism to host t i s s u e and thus prevented the sequel of e n d o c a r d i t i s , i t cannot be discounted that a g g l u t i n a t e d or opsonized b a c t e r i a are unquestionably more s u s c e p t i b l e to phagocytic a t t a c k . Analogues or substances that mimic molecules i n v o l v e d i n the b i n d i n g s i t e s of a host or bacterium have been used i n some studies to prevent b a c t e r i a l adherence " i n v i v o " . Aronson, 1979, (7) i n j e c t e d pathogenic s t r a i n s of E. c o l i w i t h methyl a-D-mannopyranoside i n t o the bladders of mice. Methyl a-D-mannopyranoside i s a known competitive i n h i b i t o r of the b i n d i n g i n t e r a c t i o n between E. c o l i and mannose residues on host receptors. (7, 26). The presence of t h i s compound r e s u l t e d i n a considerable reduct-ion i n the numbers of E. c o l i c e l l s that adhered to and c o l o n i z e d the bladder c e l l s . 103 Sublethal doses of a n t i b i o t i c s have been suggested as another means of preventing b a c t e r i a l adherence i n the host (11,12). Sub l e t h a l doses of p e n i c i l l i n were found to induce S. pyogenes to secrete LTA, r e s u l t i n g i n a l o s s of i t s a b i l i t y to adhere w i t h c e l l u -l a r LTA to e p i t h e l i a l c e l l s (12). Our studies demonstrated that c e l l s of S. aureus grown i n a medium supplemented w i t h a s u b l e t h a l dose of p e n i c i l l i n could not bind to f i b r i n or clump i n f i b r i n o g e n (unpublished o b s e r v a t i o n ) . Thus, a b e t t e r knowledge of the adherence p r o p e r t i e s of pathogenic b a c t e r i a may help prevent i n f e c t i o n s caused by them i n a plant or animal host. Further examination of the i s o l a t e d C l f may lead to a b e t t e r understanding of i t s r o l e i n the st a p h y l o c o c c a l c e l l and how i t r e l a t e s to the p a t h o g e n i c i t y of JS. aureus. 104 Alami, S.Y., F.C. K e l l y , and G.J. Race. 1968. P a t h o g e n i c i t y of st a p h y l o c o c c i (with s p e c i a l reference to the p e r s i s t e n c e of i n f e c t i o n i n mice). Amer. J . Path. 53:577-589. A l l i n g t o n , M.J. 1967. Fib r i n o g e n and f i b r i n degradation products and the clumping of st a p h y l o c o c c i by serum. B r i t . J . Haemat. 13: 550-567. A l y , R., H.I. S h i n e f i e l d , W.B. Straub, and H.I. Maibach. 1977. B a c t e r i a l adherence to na s a l mucosal c e l l s . I n f e c t . Immun. 17: 546-549. Ames, G.F.L. 1974. R e s o l u t i o n of b a c t e r i a l p r o t e i n s by p o l y a c r y l a -mide g e l e l e c t r o p h o r e s i s on s l a b s . J . B i o l . Chem. 249:634~644. A n g r i s t , A.A., and M. Oka. 1963. Pathogenesis of b a c t e r i a l endo-c a r d i t i s . J . Am. Med. Assoc. 183:249-252. Arbuckle, J.B.R. 1970. The l o c a l i z a t i o n of E s c h e r i c h i a c o l i i n p i g i n t e s t i n e . Med. M i c r o b i o l . _3:333-340. Aronson, M., 0. Medalia, L. S c h o r i , D. Mirelman, N. Sharon, and J. Ofek. 1979. Prevention of c o l o n i z a t i o n of the u r i n a r y t r a c t of mice w i t h E s c h e r i c h i a c o l i by b l o c k i n g of b a c t e r i a l adherence w i t h methyl a-D-mannopyranoside. J . I n f e c t . D i s . 139:329-332. A u s t i n , R.M., and C.A. D a n i e l s . 1978. The r o l e of p r o t e i n A i n the attachment of s t a p h y l o c o c c i to i n f l u e n z a - i n f e c t e d c e l l s . Lab. Invest. 39:128-132. B a i e r , R.E. 1970. Surface p r o p e r t i e s i n f l u e n c i n g b i o l o g i c a l adhesion, p. 15-48. I n : R.S. Manly (ed.). Adhesion i n b i o l o g i c a l systems. Academic pr e s s , New York. 105 10.' Beachey, E.H., and I. Ofek. 1976. E p i t h e l i a l c e l l b i n d i n g of group A s t r e p t o c o c c i by l i p o t e i c h o i c a c i d on fi m b r i a e denuded of M p r o t e i n J . Exp. Med. 143:759-771. 11. Beachey, E.H. 1980. Preface. I n : E.H. Beachey (ed.), B a c t e r i a l adherence, receptors and r e c o g n i t i o n . Series B. V o l . 6. Chapman and H a l l , New York. 12. Beachey, E.H. 1981. B a c t e r i a l adherence: Adhesion-receptor i n t e r -a c t i o n s mediating the attachment of b a c t e r i a to mucosal sur f a c e s . J . I n f e c t . D i s . 143:325-345. 13. Blomback, B., and M. Blomback. 1956. P u r i f i c a t i o n of human and bovine f i b r i n o g e n . A r k i v . Kemi. 10:415-443. 14. Boweh, W.H. 1969. A vaccine against d e n t a l c a r i e s . A p i l o t e x p e r i -ment w i t h monkeys. (Macaca: iru>s) . Br. Dent. J . 126 :159-160 . 15. Bourgeau, G., and B.C. McBride. 1976. Dextran-mediated i n t e r b a c t e r i a l aggregation between d e x t r a n - s y n t h e s i z i n g s t r e p t o c o c c i and Actinomyces v i s c o s u s . I n f e c t . Immun. 13:1228-1234. 16. B r u c k l e r , J . , W. Schaeg, and H. B l o b e l . 1974. I s o l i e r u n g der "Clumping f a c t o r s " von Staphylococcus aureus. Z b l . Bakt. Hyg. I. Abst. O r i g . A. 228:465-473. 17. Buchanan, T.M., and W.A. Pearce. 1976. P i l i as a mediator of the attachment of gonococci to human e r y t h r o c y t e s . I n f e c t . Immun. 13: 1483-1489. 18. Carvalho, A.C.A., L.L. Ellman, and R.W. Cohman. 1974. A comparison of s t a p h y l o c o c c a l clumping t e s t and an a g g l u t i n a t i o n t e s t f o r d e t e c t i o n of f i b r i n o g e n degradation products. Am. J . C l i n . P a t h o l . 62:107-112. 19. Chen, P.S., T.Y. T o r i b a r a , and H. Warner. 1956. Microdetermination of phosphorous. Anal. Chem. 28:1756-1758. 20. C o l l i e r , A.M. 1980. Attachment of Mycoplasma pneumoniae to r e s p i r a -t o r y e p i t h e l i u m , pp.159-183. I n : E.H. Beachey ( e d . ) , B a c t e r i a l adherence, receptors and r e c o g n i t i o n . Series B, V o l . 6. Chapman and H a l l . New York. 21. Corpe, W.A. 1970. Attachment of marine b a c t e r i a to s o l i d s u r f a c e s , pp. 73-87. I n : R.S. Manly (ed.), Adhesion i n b i o l o g i c a l systems. Academic Press Inc., New York, 22. Costerton, J.W., G.G. Geesey, and K.J. Cheng. 1978. How b a c t e r i a s t i c k . S c i . Am. 238:86-95. 23. D a n i e l s , S.L. 1972. The adsorption of microorganisms onto s o l i d s u r f a c e s : a review. Dev. Ind. M i c r o b i o l . 13:211-253. 24. Dische, Z., and L.B. S h e t t l e s . 1948. A s p e c i f i c c o l o r r e a c t i o n of methylpentoses and a spectrophotometric micromethod f o r t h e i r determin-a t i o n . J . B i o l . Chem. 175:595-603. 25. R.F. D o o l i t t l e . 1981. Fib r i n o g e n and f i b r i n . S c i . Am. 245:126-135, 26. Duguid, J,P., and D.C. O l d . 1980. Adhesive p r o p e r t i e s of Entero-b a c t e r i a c e a e . p. 185-217. JEn: E.H. Beachey (ed.), B a c t e r i a l adherence, receptors and r e c o g n i t i o n . Series B, V o l . 6. Chapman and H a l l , New York. 27. Durack, D.T. 1975. Experimental b a c t e r i a l e n d o c a r d i t i s IV S t r u c t -ure and e v o l u t i o n of very e a r l y l e s i o n s . J . P a t h o l . 115:81-89. 28. Durack, D.T., B.C. G i l l i l a n d , and R.G. Pe t e r s d o r f . 1978. E f f e c t of immunization on s u s c e p t i b i l i t y to experimental Streptococcus mutans and Streptococcus sanguis e n d o c a r d i t i s . I n f e c t , Immun. 22:52-56. 29. Duthie, E.S. 1954. Evidence f o r two forms of sta p h y l o c o c c a l coagulase. J . Gen. M i c r o b i o l . 10:427-436. 30. Duthie, E.S. 1955. The a c t i o n of f i b r i n o g e n on c e r t a i n pathogenic c o c c i . J . Gen. M i c r o b i o l . 13:383-393. 31. E d i t o r i a l . 1974. B a c t e r i a l s t i c k i n e s s . Lancet 1_:716-717. 32. E l l e n , R.P., and R.J. Gibbons. 1972. M-protein a s s o c i a t e d adherence of Streptococcus pyogenes to e p i t h e l i a l s u r f a c e s : p r e r e q u i s i t e f o r v i r u l e n c e . I n f e c t . Immun. 5^:826-830. 33. Evans, R.T., F.G. Emmings, and R.G. Genco. 1975. Preve n t i o n of Streptococcus mutans i n f e c t i o n , of tooth surfaces by s a l i v a r y antibody i n i r u s monkeys. (Macaco f a s c i c u l a r i s ) . I n f e c t . Immun. 12:293-302. 34. Freedman, L.R., S. Arnold, and J . Valone. 1974. Experimental e n d o c a r d i t i s . J-n: W.W. Yates (ed.). Recent advances i n staphylococcal research. Ann. N.Y. Acad. S c i . 263:456-465. 35. Freedman, L.R., and J . Valone. 1979. Experimental i n f e c t i v e endo-c a r d i t i s . Progress i n c a r d i o v a s c u l a r d i s e a s e s . V o l . XXII _3:169-180. 36. F r e t e r , R., and G.W. Jones. 1976. Adhesive p r o p e r t i e s of V i b r i o  c h o l e r a e : nature of the i n t e r a c t i o n w i t h i n t a c t mucosal sur f a c e s . I n f e c t . Immun. 14.:246-256. 37. Fubara, E.S., and R. F r e t e r . 1973. P r o t e c t i o n against e n t e r i c b a c t e r i a l i n f e c t i o n by secretory IgA a n t i b o d i e s . J . Immunol.111:395-403. 38. G a r r i s o n , P.K., and L.R. Freedman. 1970. Experimental e n d o c a r d i t i s . I. Staphylococcus e n d o c a r d i t i s i n r a b b i t s r e s u l t i n g from placement of a polyethylene catheter i n the r i g h t side of the heart. Yale J . B i o l . Med. 42:394-410. 39. Gibbons, R.J. 1977. Adherence of b a c t e r i a to host t i s s u e , p.395-406. I n : D. Schlessinger (ed.)., M i c r o b i o l o g y . American s o c i e t y f o r micro-b i o l o g y , Washington, D.C. 40. Gibbons, R.J., and R.J. F i t z g e r a l d . 1964. Dextran-induced a g g l u t i n a t i o n of Streptococcus mutans and i t s p o t e n t i a l r o l e i n the formation of dent a l plaques. J . B a c t e r i o l . 98:341-346. 41. Gibbons, R.J., and J . van Houte. 1971. S e l e c t i v e b a c t e r i a l adherence to o r a l e p i t h e l i a l surfaces and i t s r o l e as an e c o l o g i c a l determinant. I n f e c t . Immun. _3:567-573. 42. Gibbons, R.J., and J . van Houte. 1975. B a c t e r i a l adherence i n o r a l m i c r o b i a l ecology. Ann. Rev. M i c r o b i o l . 29:19-44. 43. Gibbons, R.J., and J . van Houte. 1980. B a c t e r i a l adherence and the formation of d e n t a l plaques, pp.61-104. JEn: E.H. Beachey (ed.), Bacter-i a l a d h e r e n c e , ; receptors and r e c o g n i t i o n . Series B, V o l . 6. Chapman and H a l l , New York. 44. G o r r i l l , R.H., K.M. Klyhn, and E.M. McNeil. 1966. The i n i t i a t i o n of i n f e c t i o n i n the mouse kidney a f t e r intravenous i n j e c t i o n of b a c t e r i a . J . Path. Bact. 91;157-172. 45. G o r r i l l , R.H., and E.M. McNeil. 1968. The problem of staphylococcal lodgement i n the mouse kidney. J . Path. Bact. 96:431-441. 46. Gould, K., C.H. Ramirez-Ronda, R.K. Holmes, and J.P. Sanford. 1975. Adherence of b a c t e r i a to heart valves i n v i t r o . J . C l i n . Invest. 56:1364-1370. 47. Hawiger, • j . } D.K. Hammond, and S. Timmons. 1975. Human f i b r i n o g e n possesses b i n d i n g s i t e f o r s t a p h y l o c o c c i on Aa and BB polypeptide chains. Nature 258:643-645. 48. Hawiger, J . , D.K. Hammond, S. Timmons, and A.Z. Budzynski. 1978. I n t e r a c t i o n of human f i b r i n o g e n w i t h s t a p h y l o c c o c c i : Presence of a b i n d i n g r e g i o n on normal and abnormal f i b r i n o g e n v a r i a n t s and f i b r i n o g e n d e r i v a t i v e s . Blood. 51:799-812. 49. Hawiger-, J . , A. Ha'wiger, and M.G. Kc-enig. 1970. Staphylococcal clumping and f i b r i n o g e n and f i b r i n degradation products i n inflammatory exudates. Proc. Soc. Exp. B i o l . Med. 136:132-136. 50. H a w i g e r , J . , S. Niewiarowski, V. Gurewich,and D.P. Thomas. 1970. Measurement of f i b r i n o g e n and f i b r i n degradation products i n serum by s t a p h y l o c o c c a l clumping t e s t . J . Lab. C l i n . Med. 75:93-108. 51. Jackson, R.L., and G.R. Matsueda. 1970. Myxobacter AL-1 protease, pp. 591-599. I n : G.E. Perlmann and L. Lorand (ed.)^ Methods i n enzymology. V o l . XIX, Academic Press, Inc., New York. 52. Jones, G.W., G.D. Abrams, and R. F r e t e r . 1976. Adhesive p r o p e r t i e s of V i b r i o cholerae; Adhesion to i s o l a t e d r a b b i t brush border membranes and hemagglutinating a c t i v i t y . I n f e c t . Immun. 14:232-239. 53. Jones, G.W.,and R. F r e t e r . 1976. Adhesive p r o p e r t i e s of V i b r i o  c h o l e r a e : Nature of the i n t e r a c t i o n w i t h i s o l a t e d r a b b i t brush border membranes and human e r y t h r o c y t e s . I n f e c t . Immun. 14:240-245. 54. Jones, G.W., and J.M. R u t t e r . 1972. Role of the K88 antigen i n the pathogenesis of neonatal d i a r r h e a caused by E s c h e r i c h i a - c o l i i n p i g l e t s . I n f e c t . Immun. 6_: 918-927. 55. K a p r a l , F.A. 1966. Clumping of Staphylococcus aureus i n the p e r i t o n e a l c a v i t y of mice. J . B a c t e r i o l . 92:1188-1.195.-' 56. Kato, Y., and G. Omori. 1959. E x t r a c t i o n of bound coagulase from st a p h y l o c o c c a l c e l l s . Biken's J . 2^:321-332. 57. King, R.D., J.C. Lee, and A.L. M o r r i s . 1980. Adherence of Candida  a l b i c a n s and other Candida species to mucosal e p i t h e l i a l c e l l s . I n f e c t . Immun. 27:667-674. 58. Labree, E.H., H. Schneider, T.J. Manani, and S.B. Forma. 1964. E p i t h e l i a l c e l l p e n e t r a t i o n as an e s s e n t i a l step i n the pathogenesis of b a c i l l a r y dysentery. J . B a c t e r i o l . 88:1503-1518. 110 LeavgHe, D.E., B.F. Mertens, E.J.W. Bowie, and C.A. Owen. 1971. Staphylococcal clumping on m i c r o t i t r e p l a t e s . Am. J . C l i n . Path. 55:452-457. Lehner, T., S.J. Challacombe, and J . C a l d w e l l . 1975. An immuno-l o g i c a l i n v e s t i g a t i o n i n t o the .prevention of c a r i e s i n deciduous te e t h of rhesus monkeys. Arch. O r a l . B i o l . 20:305-310. Levine, M.M., M.B. Rennels, V. Daya, and T.P. Hughes. 1980. Hema-g g l u t i n a t i o n and c o l o n i z a t i o n f a c t o r s i n e n t e r o t o x i g e n i c and entero-pathogenic E s c h e r i c h i a c o l i that cause d i a r r h e a . J . I n f e c t . M s . 141:733-737. L i l j e m a r k , W.F., and R.J. Gibbons. 1972. The p r o p o r t i o n a l d i s t r i -b u t i o n and r e l a t i v e adherence of Streptococcus miteor ( m i t i s ) i n the human o r a l c a v i t y . I n f e c t . Immun, _6:852-859. L i p i n s k i , B., J . H a w i g e r , a n d J . J e l j a s z e w i c z . 1967. Staphylococcal clumping w i t h s o l u b l e f i b r i n monomer complexes. J . Exp. Med. 126: 979-989. Lowr^-, O.H., N.J. Rosebrough, A.L. F a r r , and R.J. R a n d a l l . 1951. P r o t e i n measurement w i t h the F o l i n phenol reagent. J . B i o l . Chem. 193:265-275. McBride, B.C., and M.T. Gisslow. 1977. Role of s i a l i c a c i d i n sa l i v a - i n d u c e d aggregation of Streptococcus sanguis. I n f e c t . Immun, JL8_: 35-40. McBride, B.C., A.W. M a r t i n , and J.G, S i l v e r . 1982.. B a c t e r i a l c o l o n i z a t i o n of endocardial thrombotic emboli i n r a b b i t s . I n f e c t . Immun. In Press. M a r t i n , A.W., . J . G . " S i l v e r a n d B.C. McBride. C h a r a c t e r i z a t i o n of the S. aureus-f i b r i n o g e n b i n d i n g r e a c t i o n (submitted). I l l 68. M c l n t i r e , F.C., A.E. V a t t e r , J . Baros, and J . Arnold. 1978. Mechanism of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34. I n f e c t . Immun. 21:978-988. 69. McNeil, E.M. 1966. The e f f e c t of c u l t u r a l c o n d i t i o n s on the product-ion of clumping f a c t o r (bound coagulase) by Staphylococcus pyogenes. J . Med. Lab. Tech.. 23:83-89. 70. McNeil, E.M. 1968. Bound coagulase or clumping f a c t o r ; an i n v e s t i -g a t i o n i n t o i t s nature by means of s e l e c t i v e d e s t r u c t i o n and a comparison w i t h s o l u b l e coagulase. J . Med. Lab. Tech. 25:357-369 71. Mardh, P.A., and L. Westran, 1976. Adherence of b a c t e r i a to v a g i n a l e p i t h e l i a l c e l l s . I n f e c t . Immun. 13:661~666. 72. M a r s h a l l , K.C., M. Stout, and R. M i t c h e l l . 1971. Mechanism of the i n i t i a l events i n the s o r p t i o n of marine b a c t e r i a to surfaces. J . Gen. M i c r o b i o l . 68:337-348. 73. Morgan, R.L., R.E. Isaacson, H.W. Moon, C.C. Brinton, and C.C. To. 1978. Immunization of s u c k l i n g p i g s against e n t e r o t o x i g e n i c E s c h e r i c h i a c o l i - i n d u c e d d i a r r h e a l disease by vaccinated dams w i t h p u r i f i e d 987 or K99 p i l i : p r o t e c t i o n c o r r e l a t e s w i t h p i l u s homology of vaccine c h a l l e n g e . I n f e c t . Immun. 22:771-777. 74. Ofek, I . , and E.H. Beachey. 1980. B a c t e r i a l adherence pp.503-532. I n : G.H. Stollerman (ed.), Advances i n i n t e r n a l medicine. V o l . 25. Year Book Medical P u b l i s h e r s , Chicago. 75. Okuda, K., and I. Takrazoe, 1974. Haemagglutinating a c t i v i t y of Bacteroides melaninogenicus, Arch. Oral B i o l . 19:415-416. 112 76. Ouchterlony, 0. 1968. Handbook of immunodiffusion and Immuno-e l e c t r o p h o r e s i s . Ann.Arbor Science Publ. Ann Arbor. 77. Pearce, W.A., and T.M. Buchanan. 1980. Structure and cell-membrane b i n d i n g p r o p e r t i e s of b a c t e r i a l f i m b r i a e , pp,289-344. I n : E.H. Beachey (ed.). B a c t e r i a l adherence, re c e p t o r s and r e c o g n i t i o n . S e r i e s B, Vo l . 6. Chapman and H a l l , New York. 78. Perlman, B.B.,and L.R. Freedman. 1971. Experimental e n d o c a r d i t i s . I I . Staphylococcal i n f e c t i o n of the a o r t i c valve f o l l o w i n g p l a c e -ment of a polyethylene catheter i n the l e f t side of the heart. Yale J . B i o l . Med. 44:206-213. 79. Porath, J . , R. Axen, and S. Ernback. 1967. Chemical c o u p l i n g of pr o t e i n s to agarose. Nature, 215:1491~1492. 80. Punsalang, A.P.Jr., and W.D. Sawyer. 1973. Role of p i l i i n the v i r u l e n c e of N e i s s e r i a e gonorrhoeae. I n f e c t . Immun. j5:255-263. 81. Ramirez-Ronda, C.H. 1978. Adherence of g l u c a n - p o s i t i v e and glucan-negative s t r e p t o c o c c a l s t r a i n s to normal and damaged heart v a l v e s . J . C l i n . Invest. 62:805-814. 82. Reed, W.P., and R.C. W i l l i a m s . 1978. B a c t e r i a l adherence: f i r s t step i n pathogenesis of c e r t a i n i n f e c t i o n s . J . Chron. D i s . 31:67-72. 83. Roc, J.H. 1955. The determination of sugar i n blood and s p i n a l , f l u i d w i t h the Anthrone reagent. J . B i o l . Chem. 212:335-343. 84. R o t t e r , J., and F.C. K e l l y . 1965. S e r o l o g i c a l r e a c t i o n s a s s o c i a t e d w i t h the clumping f a c t o r of Staphylococcus aureus. J . B a c t e r i o l . 91: 588-594. 85. Savage, D.C. 1969. M i c r o b i a l i n t e r f e r e n c e between indigenous yeast and l a c t o b a c i l l i i n the rodent stomach. J . B a c t e r i o l . 98:1278-1283. 113 86. Savage, D.C. 1972. S u r v i v a l on mucosal e p i t h e l i a , e p i t h e l i a l p e n e t r a t i o n and growth i n t i s s u e s of pathogenic b a c t e r i a , p.25?-57. In: Smith, H., and J.H. Pearse (ed.). M i c r o b i a l p a t h o g e n i c i t y i n man and animals. Cambridge U n i v e r s i t y Press, New York. 87. Scheld, W.M., J.A. Valone, and M.A. Sande. 1978. B a c t e r i a l adherence i n the pathogenesis of e n d o c a r d i t i s * J . C l i n . Invest. 6^:1394-1404. 88. Shinada, S., and J.W. Hampton. 1975. The separation of S-carboxy-methylated human f i b r i n o g e n chains w i t h a s i n g l e carboxymethyl-cellulose chromatography. Biochem. Biophys. Acta. 412:357-360. 89. S i l v e r , J.G., A.W. M a r t i n , and B.C. McBride. 1982. Prophylaxis ' of experimental - e n d o c a r d i t i s v .J,.Dent. Res, 61:320, 90. S l o t s . J . , and R.J. Gibbons. 1978. Attachment of Bacteroides  melaninogenicus subsp. a s a c c h a r o l y t i c u s to o r a l surfaces and i t s p o s s i b l e r o l e i n c o l o n i z a t i o n of the month and of p e r i o d o n t a l pockets. I n f e c t . Immun. 19:254-264. 91. Smith, H. 1977. M i c r o b i a l surfaces i n r e l a t i o n to p a t h o g e n i c i t y . B a c t e r i o l . Rev. 41:475-500. 92. Sugarman, B. 1980. Attachment of b a c t e r i a to mammalian su r f a c e s . I n f e c t . j?:132-141. 93. Swanson, J . , and G. King. 1978. p. 221-226. I n : G.F, Brooks et a l (ed.), Immunology of N e i s s e r i a e gonorrhoeae. American s o c i e t y f o r micro b i o l o g y , Washington. 94. S w i t a l s k i , L.M. 1976. I s o l a t i o n and p u r i f i c a t i o n of st a p h y l o c o c c a l clumping f a c t o r , p.413-425. I n : J . J e l j a s z e r v i e z (ed.), S t a p h y l o c o c c i and s t a p h y l o c o c c a l diseases. Gustav, F i s c h e r , V e r l a g , New York. 114 95. Taubman, M.A., and D.J. Smith. 1974 E f f e c t s of l o c a l immunization w i t h Streptococcus mutans on i n d u c t i o n of s a l i v a r y immunoglobulin A antibody and experimental d e n t a l c a r i e s i n r a t s . I n f e c t . Immun. 9: 1079-1091. 96. Warren, L. 1959. The t h i o b a r b i t u r i c a c i d assay of s i a l i c a c i d s . J . B i o l . Chem. 234:1971-1975. 97. Weber, K.^  and M. O.sborn. 1969. The r e l i a b i l i t y of molecular weight determination by dodecyl s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . J . B i o l . Chem. 244:4406-4412. 98. Weerkamp, A.H., and B.C. McBride. 1980. C h a r a c t e r i z a t i o n of the adherence p r o p e r t i e s of Streptococcus s a l i v a r i u s . I n f e c t . Immun. 29:459-468. 99. Weerkamp, A.H., and B.C. McBride. 1981. I d e n t i f i c a t i o n of a Streptococcus s a l i v a r i u s c e l l w a l l component mediating coaggregation w i t h V e i l l o n e l l a a l c a l e s c e n s VI. I n f e c t . Immun. 32:723-730. 100. Wicken, A.J. 1980. Structure and c e l l membrane-binding p r o p e r t i e s of •b a c t e r i a l l i p o t e i c h o i c a c i d s and t h e i r p o s s i b l e r o l e i n adhesion of s t r e p t o c o c c i to euk a r y o t i c c e l l s , pp. 137-158. I n : E.H. Beachey (ed-,) B a c t e r i a l adherence, receptors and r e c o g n i t i o n . Series B, Vo l . 6. Chapman and H a l l , New York. 101. Zacharius, R.M.,T.E. Z e l l , J.H. Morrison, and J . J . Woodlock. 1969. Gly c o p r o t e i n s t a i n i n g f o l l o w i n g e l e c t r o p h o r e s i s on acrylamide g e l s . Anal. Biochem. 30:148-152. S^vi^-, Q - C j A - u i - M « A X L ^ , Q - W A - (3>.c. p u / ^ - u ^ 199-7. 6. (VXo-s*^ , A- u)., Q-tfA- S'JU^, ^ B.C. mA^Lo. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095711/manifest

Comment

Related Items