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Cell surface of hydrophobic and hydrophilic strains of Streptococcus sanguis Ganeshkumar, Nadarajah 1985

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/ C e l l s u r f a c e s o f h y d r o p h o b i c and h y d r o p h i l i c s t r a i n s of S t r e p t o c o c c u s s a n q u i s By NADARAJAH GANESHKUMAR B.D.S, U n i v e r s i t y o f P e r a d e n i y a , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE !n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA October 1985 (§) Nadarajah Ganeshkumar In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date IS- fD • I9?5" ABSTRACT C e l l surfaces of aggregation, adherence or hydrophilic variants of Streptococcus sanguis were compared to c e l l surfaces of the parent str a i n with regard to their protein and antigenic constituents. C e l l surface molecules were released by digestion with mutanolysin. Extraction with SDS-BME, urea, lithium chloride and boi l i n g water did not s o l u b i l i z e any material which stained with s i l v e r nitrate in an SDS-polyacrylamide gel. The parent organism Sj_ sanguis 12 which aggregates in sal i v a , adheres to saliva coated hydroxyapatite (S-HA) and is hydrophobic was found to possess a prominently staining 160,000 MW protein. This protein was almost completely absent from strain I2na, a hydrophobic non-aggregating variant, and was* completely absent from the hydrophilic non-aggregating, non-adherent s t r a i n 12L. Trypsinization of stra i n 12 resulted in the coincident loss of the 160,000 MW protein and the a b i l i t y to aggregate in s a l i v a . Trypsin treatment reduced, but did not eliminate the hydrophobic character of the c e l l s . Boiling destroyed the a b i l i t y to aggregate but did not a l t e r hydrophobicity. C e l l wall digests of stra i n 12 contained a large number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of the hydrophilic strains contained no material that was v i s i b l e in a s i l v e r stained SDS-polyacrylamide gel. Electron microscopy of phosphotungstic acid stained c e l l s showed a thick capsular material spread evenly over the c e l l surface of the parent strain 12. This layer was thinner around i i the c e l l s of stra i n 12na and appeared patchy on hydrophilic strains. Electron microscopy of uranyl acetate stained c e l l s revealed that s t r a i n 12 had short f i b r i l l a r structures evenly distributed over the c e l l surface, and long f i b r i l s which were more concentrated at the end of the c e l l . The hydrophilic s t r a i n 12L lacked both types of f i b r i l s . Crossed immunoelectrophoresis confirmed that the major c e l l surface antigens were located in the 160,000 MW region. C e l l wall digests of st r a i n 12 and 12na inhibited adherence of strain 12 to S-HA by 36% and 19% respectively. The digests of hydrophilic s t r a i n 12L were not inhibitory. The inh i b i t o r y a c t i v i t y was sensitive to heat and SDS. This thesis i s dedicated to the memory of my • father. i v ACKNOWLEDGEMENTS I gratefully acknowledge the guidance, support, encouragement and patience of Dr. B. C. McBride and the fi n a n c i a l support of the University of B r i t i s h Columbia and Medical Research Council of Canada. My sincere thanks to Drs. A. J. Warren, R. E. W. Hancock and G. D. Spiegelman for serving on my committee and their guidance. I wish to give special thanks to Dr. E. Jane Morris and Meja Song who shared their experience and for their constant encouragement. I record my sincere thanks to Heather Merilees, Janet Boyd and Umadatt Singh for their support. I also wish to thank Andre Wong for his work with the electron microscope and Warren Schmidt and Bruce McCaughey for their photography. I take this oppurtunity to express my gratitude to my mother, Kumar, Saratha, Nanthan, Ravi, Uma and Eswary for their unending support and confidence. I thank my friends for their encouragement sp e c i a l l y Mahesh for typing the thesis. v TABLE OF CONTENTS Abstract 1 Ac knowledgements Li s t of Tables v 11 L i s t of Figures I Introduction Materials and Methods Bacteria , Culture conditions Saliva Adherence assay Hydrophobicity Isolation of hydrophilic variants . .... Salivary aggregation Preparation of c e l l walls C e l l wall analysis Mutanolysin digestion Whole c e l l digestion C e l l wall digest Trypsin digestion '. SDS -Polyacrylamide gel electrophoresis.(SDS-PAGE) ... Electron microscopy Immunological procedures Immunoelectrophoresis Western blot Preparation of IgG v i Adsorbed antiserum 46 HPLC fractionation"., ....46 Protein assay 47 Azocoll assay 47 Results 48 Properties of £L sanquis 48 Isolation of hydrophilic variants 48 Salivary aggregation 55 Adherence to S-HA. . 55 Electron microscopy 59 Whole c e l l digests 62 C e l l wall digests , 64 C e l l modification 68 Fractionation of c e l l walls 75 Crossed immunoelectrophoresis 82 Adherence i n h i b i t i o n 83 Discussion 86 Bibliography 96 vi i LIST OF TABLES TABLE PAGE I C l a s s i f i c a t i o n of S_^  sanguis 16 II Adherence properties of S. sanguis 49 III Hydrophobic and salivary aggregating properties of : S . sangui s 51 IV Chemical characterization of c e l l walls of S. sanguis 54 V Adherence of sanguis to S-HA 58 VI Effect of c e l l surface modification on hydrophobicity and aggregation of S. sanguis 12 69 VII Effect of c e l l wall digests on the adherence of S. sanguis 12 to S-HA 84 v i i i LIST OF FIGURES FIGURE PAGE 1 Growth c h a r a c t e r i s t i c s of hydrophobic and hydrophilic strains of S^ sanquis . .. 53 2 Adherence of S^ sanguis, to increasing amounts of hexadecane. 56 3 Adherence of S_^  sanquis variants to increasing amounts of hexadecane 57 4 Electron micrographs of S_^  sanquis stained with phosphotungstic acid 60 5 Electron micrographs of S_^  sanquis stained with uranyl acetate 61 6 SDS-PAGE of whole c e l l s digested with mutanolysin. ... 63 7 SDS-PAGE of mutanolysin digests of SDS extracted c e l l walls 65 8 SDS-PAGE of mutanolysin digests of SDS extracted c e l l walls 67 9 Effect of trypsin on S_^  sanquis surface hydrophobicity 70 10 SDS-PAGE of mutanolysin digests of whole c e l l s of S. sanguis 12 obtained after treatment with trypsin 72 11 SDS-PAGE of S_j_ sanguis 12 whole c e l l s treated with trypsin . 7 3 12 SDS-PAGE of material released from whole c e l l s of S. sanquis 12 treated with trypsin 74 13 Crude c e l l wall digest of S_^  sanguis 12 fractionated on HPLC 76 14 SDS-PAGE of HPLC fractions of crude c e l l wall digest of S. sanguis 12. 77 15 Rocket immunoelectrophoresis of crude c e l l wall digests of S^ sanguis 12 79 i x 16 W e s t e r n b l o t s o f HPLC f r a c t i o n s o f c r u d e c e l l w a l l d i g e s t s o f s a n q u i s 12 80 17 C r o s s e d i m m u n o e l e c t r o p h o r e s i s of c r u d e c e l l w a l l d i g e s t s o f s a n g u i s 12 81 x INTRODUCTION Colonization of a host is a complex process which is i n i t i a t e d when the microorganism attaches to an immobilized surface. If attachment f a i l s to occur, the host defense mechanisms rapidly remove the organism. The importance of binding in the establishment of bacteria was not f u l l y appreciated u n t i l recently and consequently l i t t l e was known about the nature of these interactions. The situation has changed in the past decade and there is now a considerable l i t e r a t u r e dealing with the phenomenon of adherence. The study of microbial adherence has progressed from the purely descriptive to the molecular analysis of adherence-receptor interactions. Together with this change in emphasis has come an appreciation of the complexity of adherence. This is not surprising given the complex nature of the bacterial surface and the heterogenous nature of the surfaces with which the bacteria interact (36). In t h i s section of the thesis, I w i l l discuss the general ch a r a c t e r i s t i c s of adherence including such things as mechanisms and the b a c t e r i a l constituents involved in binding and methods of study. This w i l l be followed by a detailed description of adherence interactions in the oral cavity with a par t i c u l a r emphasis on Streptococcus sanguis and i t s interactions with salivary derived tooth p e l l i c l e . The appendages or molecules on the ba c t e r i a l surface responsible for mediating attachment are c a l l e d adhesins (6). 1 The e n t i t i e s on the surface to which adhesins bind are known as receptors, The process of adhesion may involve more than one type of adhesin-receptor interact ion (28,54,56,151). Attachment of an organism to a surface w i l l modify the environment and this may have either a positive or a negative effect on the host. An example of a positive effect is seen in the symbiotic relationship between the nitrogen f i x i n g bacteria and leguminous plants which i s i n i t i a t e d by a selective attachment of the bacteria to root hair of the plant (26). Another example is provided by the indigenous human f l o r a which serves to protect the host from invading pathogenic organisms (47,48). The negative effects.of adherence are evident in studies of a number of pathogenic organisms which have the a b i l i t y to sel e c t i v e l y adhere to host tissue (72). Enteropathogenic Escherichia c o l i possess multiple adhesins mediating attachment to i n t e s t i n a l epithelium (122,123). Gonorrheal infections are i n i t i a t e d when Neisseria qonorrhoae attaches to urinary epithelium via a fimbria! adhesin (66,149). Biofouling is an important and costly i n d u s t r i a l problem created when microbes adhere to surfaces that are in contact with moving l i q u i d s (12). Hulls of ships and the lumen of pipes are two examples which annually cost industry b i l l i o n s of do l l a r s . If we are to develop a thorough understanding of adherence i t w i l l be necessary to elucidate mechanisms involved in the 2 process. Despite many studies we s t i l l know r e l a t i v e l y l i t t l e about the s p e c i f i c nature of adhesin-receptor interactions. What is obvious though i s that adherence is a complex process which w i l l d i f f e r depending on the particular organism under study. Many organisms w i l l possess a number of d i f f e r e n t adhesins responsible for binding to different substrates. The mechanism involved, may d i f f e r in each case. The molecular basis of adherence has been postulated to involve ionic (66,132), l e c t i n - l i k e (13,52,56,103) or hydrophobic interactions (6,28). The ionic and hydrophobic bonds are thought to be r e l a t i v e l y nonspecific whereas the l e c t i n - l i k e interactions are highly s p e c i f i c . This i s a rather narrow view point given that ionic or hydrophobic regions can be organized in such a way that they w i l l interact s p e c i f i c a l l y with receptor molecules (6). Adhesion can be described as a state of i r r e v e r s i b l e attachment of bacteria to a surface (73). Marshall et a l . (93) have postulated that the bacterium i n i t i a l l y binds to a receptor via a nonspecific reversible interaction before a permanent bond is established. Doyle et a l . (28) further hypothesized that subsequent to the i n i t i a l adhesion, other nonspecific interactions may s t a b i l i z e or contribute toward the process of i r r e v e r s i b l e attachment leading to adhesion. Bacterial c e l l surfaces are complex e n t i t i e s composed of a variety of d i f f e r e n t constituents. Any of these components may be capable of acting as an adhesin, provided they are situated 3 so that they can interact with the receptor. The composition and organization of the c e l l surface varies markedly from species to species and indeed from str a i n to strain making i t important to c l e a r l y define the s t r a i n being studied. In fact some of the confusion surrounding adherence studies originates because experiments are being done with different strains having d i f f e r e n t surface properties (52). The growth environment can have a marked eff e c t on the surface structures of the c e l l (74). Rosan and his colleagues (140) have demonstrated that growth rate, growth pH and carbohydrate source w i l l influence the a b i l i t y of S_j_ sanguis to bind to saliva derived tooth p e l l i c l e . A largely unexplored problem is the effect of an organism's natural environment on the elaboration of surface constituents. A number of these components are discussed in d e t a i l in the following paragraphs. A variety of proteins are now recognised as c e l l wall constituents of Gram negative and Gram posit i v e organisms. The functions of these proteins include providing structure (145,146), f a c i l i t a t i n g transport of nutrients (124), wall assembly (124) and adherence (44,151). Group A streptococci have a protein associated with their c e l l wall c a l l e d M-protein. It has a molecular weight between 5-150 kD depending on the method used to release i t from the c e l l wall (94). It enhances virulence of streptococci by protecting i t from phagocytosis (80). It is also postulated to play a role in attachment of the organism to pharyngeal epithelium (35). 4 In some cases the proteins are formed into well defined structures which can be visualized in the electron microscope. One well studied structure is ca l l e d "fimbria" (30) (Latin for thread, fiber and fringe) (71). Fimbriae are proteinacious non-flagellar surface appendages that radiate outwards in a f a i r l y r i g i d and filamentous fashion. They were recognised to mediate haemagglutination reactions frequently observed with members of enterobacteriaciae (30). Bacterial fimbriae are smaller than f l a g e l l a and they are widely distributed among Gram negative bacteria. They are also now recognised as being present on a number of Gram positive species. C e l l surface appendages in the Gram positive organisms have acquired such names as f i b r i l s (61,62), f i b r i l l a e (73), filamentous or fuzzy coat (104) depending on their appearence in the electron microscope. Functions attributed to fimbriae are adherence (15,39,44,151), twitching m o t i l i t y (67), and p e l l i c l e formation (117). Haemagglutination among Gram negative organisms is an example of a well studied adherence phenomenon which can frequently be attributed to fimbriae. Haemagglutination reactions mediated by fimbriae can be d i f f e r e n t i a t e d on the basis of i n h i b i t i o n by simple sugars (30,31), size of fimbriae (125), species of bacteria (44), phage s p e c i f i c i t y and source of red blood c e l l s (126). Haemagglutinating fimbriae can be dif f e r e n t i a t e d as D-mannose sensitive and D-mannose resistant types depending on whether the reaction is inhibited by the sugar (31). 5 Fimbrial adhesins o f non-invasive enteropathogenic strains of c o l i are known t o s e l e c t i v e l y attach t o the small i n t e s t i n a l mucosal epithelium (44) and thus promote diarrheal disease in humans and domestic animals. The fimbrial adhesins involved in human disease are known as the colonization factor antigens {CFA/I (38), CFA/II (37)} and those involved in pigl e t s and calves as the K-88 (123) and K-99 (122) antigens respectively. Each of these fimbrial antigens are d i s t i n c t molecular e n t i t i e s . This demonstrates the heterogeneity among fimbrial adhesins within a single species of microorganisms. Only homologous antisera can neutralize the a c t i v i t y of these , colonization factors in vivo and protect or block attachment to host tissues in v i t r o (44). The role of fimbrial adhesins in •virulence has been studied by comparing the i n f e c t i v i t y of fimbriated and nonfimbriated variants in vivo. The virulence of enteropathogenic c o l i (44) and gonorrhoae (156) i s very much reduced or eliminated in animals infected with nonfimbriated strains'of the bacteria. Antibodies and p u r i f i e d fimbriae i n h i b i t adhesion to these tissues in in v i t r o experiments (129). Fimbriae l i k e structures have been implicated in the binding of S_^  pyogenes to mucosal epithelium and erythrocytes (35). Similar components are associated with adherence of S.  sanguis to saliva-coated tooth p e l l i c l e (42,55). Actinomyces  viscosus possesses two antigenically d i s t i n c t fimbriae, one mediates binding to sal i v a coated tooth p e l l i c l e and the other 6 is responsible for coaggregation with sanguis 34 (14,15 , 1 9 ) . The coaggregation reaction i s sensitive to lactose (98), binding to p e l l i c l e is is not affected by this sugar. Fimbriae isolated from gram-negative organisms have been shown to be made up of protein subunits having molecular weight in the range of 15-25 kD (73,126). These subunits contain 44-56% nonpolar aminoacids suggesting that they are strongly hydrophobic. The aminoacid sequence from the amino terminals i s highly conserved among d i f f e r e n t species (131). Fimbrial subunits from gram-positive organisms are not as well characterized. A_;_ viscosus fimbriae can be p u r i f i e d and visualized microscopically (164) but they do not breakdown into subunits which can be studied by electrophoretic and chromatographic techniques. This suggests the p o s s i b i l i t y that subunits are linked by covalent bonds which are not amenable to dissociation by detergents or other chaotropic agents (159,164). Corynebacterium fimbriae on the other hand can be dissociated into subunits having a molecular weight of 19 kD (76). Fimbriae l i k e structures in oral streptococci are variously known as f i b r i l s , fuzzy coat or f i b r i l l a e which has confused whether true fimbriae exists in these species . Recently Handley et a l , d i f f e r e n t i a t e d surface appendages among S_^  s a l i v a r i u s (61) and S_^  sanguis (62) on the basis of their appearence in electron micrographs. They d i f f e r e n t i a t e d f i b r i l s as surface structures having an unmeasurable width due to clumping and a length varying from 70-210 nm. The structures were subdivided 7 into long f i b r i l s having a length varying from 150-210 nm and short f i b r i l s having a length from 70-110 nm. In addition some of the strains had c h a r a c t e r i s t i c fimbriae i . e . : they had a defined width. These workers correlated the lack of d i f f e r e n t f i b r i l s with certain adhesive properties. However there are inconsistencies in their observations and the results should be interpreted with caution. Direct evidence for such correlation needs to be established. Among streptococci the term fuzzy coat has been used to describe ext r a c e l l u l a r surface appendages in S.  sanguis (104), Streptococcus mutans (108),, Streptococcus mitis (84) and Streptococcus pyogenes (84). The c e l l wall amphiphile l i p o t e i c h o i c acid (LTA)-which occurs in Gram po s i t i v e bacteria, and 1ipopolysaccharide (LPS), i t s counterpart found in Gram negative-organisms, have been shown to function as adhesins (4,5,89). These molecules share the hydrophobic properties imparted by fatty acids together with hydrophilic properties related to the presence of polar groups. Either hydrophobic or hydrophilic regions on the molecule could be involved in adherence. LTA, the c e l l wall amphiphile most commonly found among Gram positive bacteria (138,166) i s a polyglycerol phosphate (PGP) polymer covalently linked to g l y c o l i p i d . The l i p i d i s presumed to anchor the molecule to the outer surface of the cytoplasmic membrane. The PGP chain is 25-30 units long and protrudes through the matrix of the c e l l wall so that i t can be detected on, the outer surface of the c e l l . Unlike teichoic acid, LTA is exclusively of the PGP type 8 in which glycerol is linked in a 1,3 manner by phosphodiester bonds. The glycerol units in the polymer chain may be substituted with glycosyl and/or D-alanine esters to a variable extent. The g l y c o l i p i d consists of a mono or diglyceride unit containing C12 to C 1 8 fatty acids and a number of sugar substituents. The complete molecule has amphipathic properties due to the presence of the hydrophobic g l y c o l i p i d and the hydrophilic teichoic acid chain. During growth some LTA is excreted into the medium in either the acylated or the deacylated form (92). LTA associated with M-protein has been reported to mediate adhesion of group A streptococci to fibronectin on mucosal c e l l s (115). It is also implicated in mediating the adherence of serotype III strains of-group B streptococci to human embryonic, fetal, and adult e p i t h e l i a l c e l l s (109). The importance of LTA in the binding of group A streptococci has been disputed by some workers (153,155). The role of LTA in adherence of oral streptococci is not well understood. An important question is whether the l i p i d or the PGP region is the active s i t e of the molecule. Rolla (132) has suggested that the PGP region i s responsible for binding to saliva-coated tooth p e l l i c l e by ionic interaction through phosphate groups. On the other hand Beachey (6) has postulated that the l i p i d portion of the molecule extends from the surface of the c e l l where i t i s able to interact with hydrophobic regions on the receptor c e l l . 9 In Aqrobacter ium tumefaciens (89) , the 0 antigen of bacterial LPS interacts with polygalacturonic acid residues exposedby wounding of plant c e l l walls. LPS is also involved in the binding of Rhizobium t r i f o l i to clover root hair (25,26). Carbohydrates are a prominent constituent of the bacterial c e l l wall. They can exist as t i g h t l y integrated e n t i t i e s associated with l i p i d (LPS), phosphate (teichoic acid), proteins (glycoproteins) or as independent substances. In some cases they exist as polymers on the c e l l surface. In this configuration they are referred to as capsules. Costerton et a l . (23) have given the name "glycocalyx" to the carbohydrate which surrounds the c e l l . There is extensive variation in carbohydrate composition between species. This c h a r a c t e r i s t i c has been exploited in the serotyping of many organisms (79). The adherence properties of the c e l l wall carbohydrate have not been well studied with the exception of LPS and some capsular material. In the case of the l a t t e r there has been extensive work done on the e x t r a c e l l u l a r glucan synthesized by S. mutans (48,60). The glucan mediates binding to the tooth but more importantly i t is responsible for l i n k i n g one S^ mutans to another. The organism possesses a c e l l surface protein which s p e c i f i c a l l y binds glucan. The glucan i s synthesized by the ext r a c e l l u l a r enzyme glucosyl transferase. E x t r a c e l l u l a r long chain polysaccharides generated by the c e l l surface polymerases of aquatic bacteria act as adhesives in their habitat. The 10 extracellular polymers form a continous "coat" around the c e l l s , giving an appearence of. f i b r i l s , blebs, droplets or holdfasts in electron microscopic examination (22). A galactose residue on the surface of sanquis 34 recognises and binds to a receptor on the surface of A^ viscosus. It is d i f f i c u l t to know whether to c a l l this carbohydrate an adhesin or receptor as the A.  viscosus is thought to bind to the streptococci via a galactose s p e c i f i c l e c t i n (98). A number of diff e r e n t methods have been employed in an attempt to gain a better understanding of adherence. The technique used has depended on whether one i s , (a) characterizing the adherence reaction, (b) ide n t i f y i n g and iso l a t i n g the adhesin or receptor, (c) studying in vivo relevance of the adhesin, or ; Cd) studying regulation and expression of the genes involved. A t r a d i t i o n a l approach to characterizing adherence reactions has involved chemical or enzymatic modification of sp e c i f i c c e l l surface.components. For example Weerkamp et a l . ( 1 5 7 ) , implicated s a l i v a r i u s c e l l surface protein in coaggregation of Fusobacter1urn nucleatum with protease treated streptococci. McBride et a l . (95) and subsequently Levine et a l . (83) demonstrated that sanguis reacted with salivary glycoproteins via a terminal s i a l i c acid which could be removed by neuraminidase. Inhibition of adherence reactions with receptor analogues has been helpful in characterizing a number of reactions. One of the most studied examples is the mannose 1 1 sensitive haemagglutination adhesin in gram negative bacteria (30,31). Another example is the lactose sensitive coaggregation of viscosus and S. sangui s 34 (98). The i s o l a t i o n and characterization of many adhesins has proven to be a d i f f i c u l t task. The greater successes have occured when adherence has been mediated by fimbriae (14,15,19,30,31,42,43,44), probably because these structures can be visualized microscopically and separated from c e l l wall by physical procedures such as ultracentrifugation (39). Isolation of non-adhering mutants has been the choice of many workers (42,43,75,157). Comparison of the c e l l walls of mutant and parent strains has helped in the i d e n t i f i c a t i o n of adhesins when the gene product i s d i r e c t l y involved in adherence. However adhesins can be carbohydrates, amphiphiles or post tr a n s l a t i o n a l l y modified proteins. In these cases there can be a more general modification of the c e l l surface making i t d i f f i c u l t to identify the s p e c i f i c molecule involved in adherence. Some mutants are unable to incorporate c e l l surface molecules including the adhesin into the c e l l wall (97). In other cases transport and assembly of adhesins i s blocked (161). Antiserum raised against the parent s t r a i n has been used to study the adhesins. Mutants lacking the adhesin were used to absorb antiserum in order to obtain p u r i f i e d antisera agagainst the adhesin (14,19,39). The absorbed antiserum has been useful in the i d e n t i f i c a t i o n of adhesins by immunoelectrophoretic procedures. The function of the adhesins has been assessed by 12 determining the adherence i n h i b i t i o n e f f e c ts of specific-antiserum. The i n s t a b i l i t y of surface structures has allowed many investigators to enrich for non-adhering strains (15, 55,163 ). Iii some cases d i f f i c u l t i e s have arisen because the strains have turned out to be phase variants which have the a b i l i t y to revert to the parent phenotype. Hagblom et a l . , (59), have shown that the genes coding for the fimbrial adhesins of gonorrhoae undergo gene rearrangement with the subsequent loss of the fimbriae. It has been-shown that type 1 fimbriation of E_^  c o l i is reversible and under tr a n s c r i p t i o n a l control by a regulatory element (33). Orndroff et a l (12(D), characterized the genes responsible for fimbriation in a c l i n i c a l i solate of c o l i . Four genes including the p i l i n gene were associated with p i l i n production. Two genes were involved in p i l i n assembly. A gene encoding for a 23 kD polypeptide was involved in regulation of expression of fimbriae because mutants lacking this protein exibited a forty f o l d increase in p i l i n production leading to hyperpiliation in t h i s organism (121). These findings were similar to those reported for Fim mutants of E c o l i K-12 (125). Fim mutants do not express fimbriae but are genetypically similar to the parent str a i n in that they could revert to the wild phenotype at a higher frequency suggesting a role for some regulatory element. 1 3 Much of the work on E_;_ c o l i has been f a c i l i t a t e d by the greater understanding of recombinant technology as applied to this organism. As similar technology in Gram positive organisms develops, i t should be possible to see whether such regulation exists in these organisms. Murchison et a l . (105) isolated a tota l of 117 mutants of S. mutans and observed that the majority of mutants were lacking in the a b i l i t y to mediate more than one of the adherence reactions seen in these organisms. This observation suggests that there are some closely associated genes responsible for adherence. Mutants have been p a r t i c u l a r l y informative in establishing the role of adhesins in i n i t i a l adherence and subsequent colonization in vivo. Afimbriate mutants of c o l i (44) and N.  gonorrhoae (129,149) were unable to bind to their respective e p i t h e l i a l surfaces. Consequently they w i l l not be able to colonize and could be cleared by host defence mechanisms before they could induce their pathological e f f e c t s . In the following section a detailed discussion of S.  sanguis w i l l be presented. Streptococcus sanguis i s the name given to the species of alpha haemolytic streptococci o r i g i n a l l y isolated from the blood of patients with subacute b a c t e r i a l endocarditis (165). The organisms were once grouped together with other oral streptococci as Streptococcus viridans (9,40). The primary habitat of S_^  sanguis is the tooth surface where i t colonizes in large numbers and forms an important part of the indigenous oral f l o r a (48). It was demonstrated by Carlsson et 14 a l , (10), that sanquis appear in the mouth only after the eruption of teeth and begins colonization of teeth long before other streptococci appear on the surface. sanguis hydrblyses arginine and esculin and produces glucan from sucrose (40). These metabolic c h a r a c t e r i s t i c s d i f f e r e n t i a t e S_^  sanquis from other o r a l , alpha haemolytic streptococci. This organism can also be di f f e r e n t i a t e d from other oral streptococci by colo n i a l morphology on Mitis Salivarius agar. Typical sanguis colonies are small, hard and firmly attached to the agar. A small moat forms around the colony. (60). The c l a s s i f i c a t i o n of strains of S_^  sanguis has been confused by c o n f l i c t i n g serological and biochemical data. Even today some authors c l a s s i f y certain strains of S_^  sanguis 298 as Streptococcus mitior (137). The confusion surrounding the state of S^ mitior derives from the fact that i t has been c l a s s i f i e d by negative c r i t e r i a , i.e: because an is o l a t e does not possess a certain c h a r a c t e r i s t i c i t is presumed to be S_^  mitior. The characterization of the d i f f e r e n t S^ sanguis serotypes is summarised in Table I. Five serotyping antigens have been i d e n t i f i e d (134). Serotype I lacks b antigen and the heterogeneous group which i s considered by some to be S_^  mitior lacks antigens a and e. Serotype antigen a was i d e n t i f i e d as LTA (141). Antigen b i s a polysaccharide containing glucose, rhamnose and phosphorus (1) but was la t e r determined to contain glucose: rhamnose: N-acetyl glucosamine (1.4: 2.5: 1.0) (116). 15 TABLE I C l a s s i f i c a t i o n of S. s a n g u i s C h a r a c t e r i s t i c s S e r o t y p i n g R e f e r e n c e s I I I H e t e r o g e n e o u s A n t i g e n s C o n s t a n t a a, b - 134 V a r i a b l e c,d,e c , d, e b,c ,d 1 35 136 C e l l w a l l c o n s t i t u e n t s Rhamnose h i g h h i g h low 1 47 Phos p h o r u s low low h i g h 21 R i b i t o l low low h i g h 20 G l u c o s e + + - 63 G a l a c t o s e + - • + 40 LTA + + — 141 A c i d from I n u l i n + + - 20 A r g i n i n e + + - 40 E s c u l i n + + - 63 S u c r o s e + + v-% GC 41-43 44-46 43-44 24 ( g r o u p l ) ( g r o u p l I I ) ( g r o u p l l ) B i o t y p e I I I I 41 R e a c t i o n w i t h + +/- -g r o u p H (t'ypel) ( t y p e l / I I ) ( t y p e l l ) 127 a n t i s e r a 16 Serotype I and II contain r e l a t i v e l y large amounts of rhamnose, no r i b i t o l and low levels of phosphorus in their c e l l wall (20,63). This group is also designated as Biotype I (41). Galactose is found in Serotype I but not in Serotype II strains (20,63). The heterogeneous group had low levels of rhamnose and larger amounts of r i b i t o l and phosphorus (Biotype.II) in their c e l l wall. N u t r i t i o n a l l y serotypes I and II were similar to but d i s t i n c t from the heterogeneous group. The %GC content provided convincing evidence that the heterogeneous group was d i s t i n c t from serotype I and II (24). Also serotype I and II have peptidoglycan linked by d i - or t r i - alanyl cross bridges while the heterogeneous group contained a l a n y l - l y s y l cross bridges (135). The various str a i n designations that have been reported in the l i t e r a t u r e are summarized at the bottom of Table I. In the following sections of t h i s chapter, the general ch a r a c t e r i s t i c s of the oral cavity as they affect microbial adherence w i l l be discussed. This w i l l be followed by a detailed discussion of the adherence properties of S_^  sanguis . Three types of surfaces exist in the oral cavity. They are shedding surfaces of keratinized or non-keratinized epithelium and nonshedding mineralised tissue (teeth). The chemical composition of the e p i t h e l i a l surfaces w i l l be modified by keratinization and probably d i f f e r from one ecosystem to another thus providing a multitude of potential 17 receptors. Oral surfaces are subjected to a number of modifying influences including the effect of s a l i v a , crevicular f l u i d and ingested food. Saliva secreted by major and minor salivary glands continuously rinses oral surfaces to remove nonadhering organisms. Crevicular f l u i d plays the same role in the gingival crevice. Both f l u i d s supply nutrients to the indigenous microflora and remove waste products. • The food we ingest can a l t e r the pH and depending on i t s composition favours the colonization of certain organisms (58). Fermentation of carbohydrates leads to the production of acids and the selection of; an acidogenic f l o r a . The most profound effect is caused by sucrose which in addition to being fermented, is converted to high molecular weight glucans which bind S_^  mutans to the teeth. The indigenous f l o r a of the oral cavity is a complex mixture of bacteria. Socransky et a l . (148) has estimated that at least 300 species w i l l be eventually be i d e n t i f i e d . Moore et a l . (101) have i d e n t i f i e d over 264 species in the gingival crevice alone. These organisms are arranged into unique ecosystems on the basis of their a b i l i t y to adhere and other metabolic c h a r a c t e r i s t i c s . They modify their environment by consuming 0 2, a l t e r i n g pH and elaborating enzymes which may change the nature of the bacterial.receptors on immobilized surfaces. 18 The most studied ecosystems of the oral cavity include the tongue, buccal surfaces, tooth and gingival crevice. The tongue is a r e l a t i v e l y aerobic environment which is thought to be the area of the most rapid c e l l p r o l i f e r a t i o n (4.9,157). Streptococcus s a l i v a r i u s i s found here and not oh the tooth probably because of i t s a b i l i t y to attach to tongue epithelium. Buccal surfaces are a sparsely populated aerobic ecosystem colonized by a r e s t r i c t e d number of organisms (46). The area between the tooth and the gum, known as the gingival crevice supports the growth of a s t r i c t anaerobic population. The gingival crevice is r e l a t i v e l y isolated from other oral ecosystems (47,48,57). Organisms have the p o s s i b i l i t y of binding to nonkeratinized epithelium or to the subgingival regions of the tooth. Food i s supplied by the crevicular f l u i d , which i s a nutrient r i c h serum transudate which seeps into the crevice from the gingiva. Not unexpectedly the area i s colonized by a unique and frequently anaerobic f l o r a . The p a r t i c u l a r species that are present r e f l e c t the state of health of the gingival tissues. In the healthy state the predominant organisms are gram-positive facultative rods and c o c c i . In the diseased state there i s a change to gram-negative anaerobic rods. One of the unique sites in the oral cavity i s the tooth. It has a surface which does not change after eruption and which cannot be repaired by the host once i t has been damaged. The exposed surface of the tooth is enamel which i s primarily 19 composed of hydroxyapatite, a complex calcium phosphate s a l t . The mineral surface therefore has the properties of calcium and phosphate ions and of hydroxyl groups with bound water (3,133). Hydroxyapatite is amphoteric due to both phosphate groups and calcium ions being exposed on i t s surface and therefore both acidic and basic components bind to t h i s material (7). Since many more phosphate groups than calcium ions are exposed, hydroxyapatite c r y s t a l s have a net negative charge. The dental enamel is covered by a thin membranous film termed the acquired p e l l i c l e (82,99). The p e l l i c l e is generally less than a micron thick and is thought to be formed by the selective adsorbtion of salivary constituents (64). Bacterial accumulations are commonly referred to as dental plaque. Plaque consists of dense masses of bacteria embedded in an amorphous matrix which is thought to contribute to i t s structural i n t e g r i t y (47,48). The matrix consists of b a c t e r i a l l y synthesized polymers and components derived from s a l i v a , crevicular f l u i d and the d i e t . Plaque tends to form most rapidly on protected areas of the teeth but in time w i l l cover a l l smooth surfaces as well. The bacterial composition of the dental plaque d i f f e r s with plaque age and with various stages of the dental diseases associated with the tooth. Mature dental plaque consists of gram-positive filaments (42%), Fusobacterium (4%), Neisseria (5%), S^ sanguis (15%), S. mitis (15%) and small percentages of L a c t o b a c i l l i , V e i l l o n e l l a and Bacteroides. S. mutans comprises 20 of 0-50% of bacteria cu l t i v a b l e from dental plaque and i t is the only niche for this organism in the oral cavity (48,57,60). C e l l p r o l i f e r a t i o n and surface interactions are overtly involved in the development of dental plaque. Three types of c e l l to surface interactions are possible. C e l l s can absorb s e l e c t i v e l y to the acquired p e l l i c l e on the tooth surface. Subsequently, by means of homotypic c e l l - c e l l interactions, c e l l of one species may accumulate and maintain their position in the growing plaque. Heterotypic c e l l - c e l l interactions leading to coaggregation between species are also important. With regard to the l a t t e r i t has been shown in in v i t r o studies that there are many combinations of oral organisms which can coaggregate when mixed together (45,47,48,75). McBride et a l . (96) have demonstrated that coaggregation supports colonization' of V e i l l o n e l l a in gnotobiotic rats infected with Streptococcus . In summary i t can be seen that oral microorganisms have the p o s s i b i l i t y of binding to host tissue or to other bacteria. The interaction between the microorganism and the surface to which i t i s attached can be attributed either to host synthesized polymers (e.g. saliva) or to b a c t e r i a l l y derived polymers (e.g. glucans) (60). Regardless of the mechanism the phenomenon of adherence i s characterized by a high degree of s p e c i f i c i t y . It is t h i s s p e c i f i c i t y which accounts in part for the development of unique microflora within s p e c i f i c ecosystems. The complexity of these systems i s compounded by the a b i l i t y of bacteria to modify both their surroundings and the 21 surfaces of their c e l l . Adhesion to the tooth surface is studied using an in v i t r o adherence model which consists of adding increasing numbers of radiolabelled bacteria to a fixed number of hydroxyapatite beads (HA). The hydroxyapatite beads are f i r s t treated with human saliva (S-HA) to form the in v i t r o equivalent of the acquired p e l l i c l e . Buffer treated hydroxyapatite beads are used as a control in these experiments. These experimental p e l l i c l e s have similar properties t o a naturally acquired p e l l i c l e (18,50) . The model system can be used to study the kinetics of binding. The number of adherent c e l l s and the number of free c e l l s can be estimated by s c i n t i l l a t i o n spectrometry (18,50). The graph (binding isotherm) obtained by p l o t t i n g the number- of free c e l l s (U) at equilibrium against the bound c e l l s (B) w i l l demonstrate the kinetics of adherence. In a homogeneous population of binding s i t e s , the binding isotherm (B vs U) w i l l show saturation k i n e t i c s . This can be altered by modifying the environment. Such adherence patterns can be f i t t e d to the Langmuir adsotbtion isotherm, U/B = K/N +(1/N)U, where U is free c e l l s at equilibrium; B i s bound c e l l s ; K i s the dissociaion constant and N is the maximum number of binding s i t e s on the S-HA beads. A plot of U/B vs U allows the estimation of the dissociation constant K.and i t s reciprocal, the a f f i n i t y constant 1/K from the x intercept. The theoretical number of binding sites (N) on the S-HA beads can be estimated from the slope. There is a r e l a t i v e l y high c o e f f i c i e n t of variation in 22 these numbers. Although the Langmuir isotherm was developed to describe adsorption equilbrium in very simple molecular systems (81), some useful information can be obtained regarding the interactions involved in microbial adsorption. A mathematical representation of the Langmuir isotherm i s the Scatchard equation: {B/U = K(N-B)}. AS w i l l be discussed l a t e r , the shapes of Scatchard plots provide information related to the nature of the interaction between the bacteria and the receptor. The presence of adsorbed salivary components on hydroxyapatite beads has been shown to a l t e r the s e l e c t i v i t y of bacterial adsorption (18). Binding and Langmuir isotherms were used to compare the adherence of di f f e r e n t species of oral bacteria to S-HA and HA. Certain species of bacteria (e.g. S.  sanquis, S.mitis, A. viscosus ) adhered better to S-HA than others (e.g. S_^  mutans, S. s a l i v a r i u s ) . These adherence patterns closely resembled the percentages of these bacteria isolated from human dental plaque. Significant differences were seen in the number of S.  sanquis adhering in the presence of s a l i v a (.18). Analysis of the Langmuir isotherm revealed a 36 fold increase in the number of binding s i t e s in S-HA when compared to the buffer coated control. This observation confirmed that s a l i v a was acting as a modulator of the ecology of the teeth. Binding to HA was found to be r e l a t i v e l y nonspecific whereas binding S-HA was a s p e c i f i c reaction and therefore s e l e c t i v e . There was competition for 23 binding sites on S-HA within a species of streptococci. This confirmed the e a r l i e r observations of Liljemark et a l , (85) that there was no competitive binding between species of oral streptococci to S-HA. This showed that b a c t e r i a l adhesins were recognising s p e c i f i c receptors on the salivary constituents adsorbed to HA and demonstrates that d i f f e r e n t bacterial species do have unique adhesins recognising s p e c i f i c S-HA receptors. S. sangui s is an oral bacterium which i s found in large numbers on the tooth surfaces and in the gingival crevice of humans. It may make up 15% of the microorganisms in coronal plaque and 8% in gingival crevice (48). The a b i l i t y of salivary constituents to form aggregates with a variety of oral streptococci is well documented and has been extensively reviewed by Bowden et al'. (8) and Gibbons et a l . (45,48). Such interactions may promote the homotypic bacterial aggregation in the plaque matrix as well as causing the removal of unattached bacteria by masking b a c t e r i a l adhesins and blocking attachment to immobilized receptors (17,158). It i s also thought that unattached aggregates of bacteria are more readily removed by host defence mechanisms from the oral cavity. Salivary constituents bound to b a c t e r i a l c e l l surfaces may also promote heterotypic b a c t e r i a l aggregation thus contributing to the species d i v e r s i t y seen in plaque (36,60). A number of salivary constituents have been implicated in the aggregation of S_^  sanguis. These include lysozyme (78) immunoglobulin A (igA) (86) and mucinous glycoproteins (68,83). 24 Salivary mucins involved in aggregation carry blood group r e a c t i v i t y , although the bacterial receptor sites are apparently d i s t i n c t from those responsible for the A and B antigenic determinants (51,70). Others have shown that streptococcal aggregation could also be mediated by nonmucinous glycoproteins (32). But this may not be the case in S^ sanquis. IgA and other salivary constituents have been found to form complexes with mucins as well with high molecular weight nonmucinous glycoproteins (16). However, at least in the case of the association between IgA and agglutinins in parotid s a l i v a , aggregation of S_^  mutans i s apparently due to the high molecular weight portion of the complex rather than the IgA (118). The aggregation of S^ sanguis in s a l i v a can be d i f f e r e n t i a t e d from that of other streptococci by i t s i n a b i l i t y to react with neuraminidase treated saliva (83,95). Aggregation appears to be caused by one or more s i a l i c - a c i d containing high molecular weight mucinous glycoproteins (65,68) as well as by a s i a l i c - a c i d containing monomeric mucin with a molecular weight of 200-250,000 (128). Murray et a l . (106) have isolated a s i a l i c - a c i d binding l e c t i n from S^ sanguis. It has been shown that nonimmune serum and crevicular f l u i d can cause neuraminidase sensitive aggregation of certain strains of S_^  sanguis. Some strains aggregated by a neuraminidase sensitive reaction in both s a l i v a and serum whereas others reacted only with s a l i v a (102). This raises the p o s s i b i l i t y that certain strains may possess more than one s i a l i c - a c i d 25 r e c o g n i s i n g a d h e s i n . S. s a n g u i s b i n d s t o t o o t h s u r f a c e i n t h e o r a l c a v i t y , f o r m i n g an i m p o r t a n t component of human d e n t a l p l a q u e ( 4 8 , 1 5 4 ) , A d h e r e n c e of S_^  s a n g u i s t o t h e t o o t h s u r f a c e i s b e l i e v e d t o be m e d i a t e d by s a l i v a r y g l y c o p r o t e i n s , w h i c h form a p e l l i c l e c o a t i n g t h e t o o t h enamel ( 1 7 ) . F o r e a c h s a l i v a r y r e c e p t o r t h e r e must be a complementary b a c t e r i a l a d h e s i n a l t h o u g h l i t t l e i s known about the n a t u r e or v a r i e t y of s u c h components. C o m p l e x i t y a t t h e c e l l u l a r l e v e l i s e x e m p l i f i e d by t h e a b i l i t y of some o r g a n i s m s t o b i n d t o a number of d i f f e r e n t s u r f a c e s ( 1 3 , 1 5 7 ) . Weerkamp e t a l . (157-160)•showed t h a t S. s a l i v a r i u s p o s s e s s a v a r i e t y of d i s t i n c t a d h e s i n s , e a c h r e s p o n s i b l e f o r m e d i a t i n g a t t a c h m e n t t o a d i f f e r e n t s u r f a c e . A d h e r e n c e n e g a t i v e m utants were i s o l a t e d w h i c h had s e l e c t i v e l y l o s t t h e c a p a c i t y t o b i n d t o e p i t h e l i a l c e l l s and t o a t t a c h t o S-HA; o t h e r s r e t a i n e d t h e s e p r o p e r t i e s but were u n a b l e t o c o a g g r e g a t e w i t h V e i l l o n e l l a  a l c a l e s e e n s . S e l e c t i v e e n z y m a t i c m o d i f i c a t i o n removed an a d h e s i n r e s p o n s i b l e f o r c o a g g r e g a t i o n w i t h F u s o b a c t e r i u m n u c l e a t u m , b u t l e f t t h e o t h e r a d h e s i n s f u n c t i o n a l . A c t i n o m y c e s v i s c o s u s has a t l e a s t two t y p e s of f i m b r i a l a d h e s i n s ( 1 3 ) , one s p e c i f i c f o r a d h e r e n c e t o S_^  s a n g u i s and t h e o t h e r s p e c i f i c f o r a r e c e p t o r i n t h e s a l i v a r y p e l l i c l e . O r g a n i s m s t h a t have a number of d i f f e r e n t a d h e s i n s a r e a t an e c o l o g i c a l a d v a n t a g e b e c a u s e t h e y a r e n o t d e pendent on a d h e r i n g t o a s i n g l e s u r f a c e and t h e y s h o u l d not be a s v u l n e r a b l e t o a g e n t s w h i c h i n t e r f e r e w i t h a s p e c i f i c a d h e s i n o r r e c e p t o r . 26 Complexity at the molecular level is exemplified by the number of reactions which appear to be involved in binding of S.  sanguis to S-HA (28 , 52,54,56,103,111,112). Kinetic analysis of the binding of S^ sanguis C5 has shown that there are a small number of high a f f i n i t y neuraminidase-sensitive salivary receptors and a larger number of lower a f f i n i t y neuraminidase-insensitive receptors (56). Binding at both si t e s is sensitive to reagents which interfere with the formation of hydrophobic bonds. Morris and McBride (103), compared S_^  sanguis 12 and S_^  sanguis 12na (a variant d e f i c i e n t in saliv a r y aggregation) to show that adherence to S-HA involved both a neuraminidase-sensitive receptor and another receptor sensitive to prolonged incubation at pH 5.0 (37°C). Doyle et a l . (28) postulated that binding i s dependent on the formation of l e c t i n l i k e or ionic bonds which are s t a b i l i z e d by hydrophobic interactions. Scatchard analysis of adsorbtion isotherms suggested that binding of S_^  sanguis to S-HA e x i b i t s c h a r a c t e r i s t i c s of positive cooperativity (103,1.11). These studies leave l i t t l e doubt that adherence i s a complex process involving a minimum of two types of cell-to-surface interactions. The complexity is compounded by differences between strains. This i s exemplified by a comparison of the effect of neuraminidase on the binding of S^ sanguis 12 and S^ sanguis C-5. The binding of strain 12 i s completely destroyed by the treatment of sal i v a with neuraminidase (103), whereas the enzyme only affects binding of stra i n C-5 to the 27 h i g h a f f i n i t y s i t e s ( 5 2 , 5 6 ) . In t h e p a s t few y e a r s t h e r e has been c o n s i d e r a b l e i n t e r e s t i n the r o l e of h y d r o p h o b i c i t y i n t h e a d h e r e n c e o f b a c t e r i a t o a v a r i e t y of s u r f a c e s (55,97,100,112,115,119,144,162,163). Much of t h i s i n t e r e s t has r e s u l t e d from t h e d e v e l o p m e n t of s i m p l e a s s a y s f o r m e a s u r i n g ' c e l l s u r f a c e h y d r o p h o b i c i t y ( 8 8 , 1 1 9 , 1 4 2 ) . The most common t e c h n i q u e i n v o l v e s m e a s u r i n g t h e d e g r e e of a d h e r e n c e of b a c t e r i a t o v a r i o u s h y d r o c a r b o n s ( e , g . h e x a d e c a n e ) . The d e c r e a s e i n t u r b i d i t y of a c e l l s u s p e n s i o n i n t h e p r e s e n c e and t h e absence of t h e t e s t h y d r o c a r b o n as q u a n t i t a t e d by s p e c t r o p h o t o m e t r y i s t a k e n as t h e measure of c e l l s u r f a c e h y d r o p h o b i c i t y ( 1 4 2 ) . The most commonly u s e d h y d r o c a r b o n i s hexadecane. - H y d r o p h o b i c i t y i s a l s o • d e t e r m i n e d by m e a s u r i n g a g g r e g a t i o n i n i n c r e a s i n g c o n c e n t r a t i o n s of ammonium s u l p h a t e (88) and b i n d i n g t o p h e n y l - o r o c t y l - S e p h a r o s e ( 1 1 9 ) . C a r e s h o u l d be t a k e n i n c o m p a r i n g r e s u l t s from d i f f e r e n t methods as t h e y do n o t n e c e s s a r i l y measure t h e same p r o p e r t y . Hexadecane b i n d i n g and ammonium s u l p h a t e a g g r e g a t i o n measure t o t a l c e l l s u r f a c e h y d r o p h o b i c i t y whereas b i n d i n g t o d e r i v a t i z e d S e p h a r o s e measures l o c a l i s e d a r e a s of h y d r o p h o b i c i t y . H y d r o p h o b i c i n t e r a c t i o n s a p p e a r t o p l a y an e s s e n t i a l r o l e i n t h e b i n d i n g of s t r e p t o c o c c i t o e p i t h e l i a l .surf a c e s ' ( 6 , 1 53) and t o s a l i v a r y p e l l i c l e (53,54,55,112,119,162,163). M u t a n t s w i t h a r e d u c e d a b i l i t y t o b i n d t o h e x a d e c a n e do n o t b i n d as w e l l a s t h e p a r e n t s t r a i n t o p e l l i c l e ( 5 5 , 1 6 3 ) , nor a r e t h e y 28 s u c c e s s f u l i n c o l o n i z i n g t h e human o r a l c a v i t y ( 1 5 0 ) . The d a t a r e l a t i n g t h e l o s s of h y d r o p h o b i c i t y t o a d h e r e n c e s h o u l d be i n t e r p r e t e d w i t h c a u t i o n b e c a u s e i t has not been p r o v e d t h a t t h e s e m utants have not l o s t o t h e r a d h e s i n s c o i n c i d e n t w i t h t h e l o s s i n h y d r o p h o b i c i t y . A l t h o u g h t h e r e i s e v i d e n c e t o s u g g e s t t h a t h y d r o p h o b i c i t y may be i n v o l v e d i n a d h e r e n c e , p o s s e s s i o n of a h y d r o p h o b i c s u r f a c e does n o t mean t h a n an o r g a n i s m w i l l b i n d t o t h e s a l i v a r y p e l l i c l e . F o r example, S_^  s a l i v a r i u s HB b i n d s t o S-HA and i s s t r o n g l y h y d r o p h o b i c , but the mutant HB-7 wh i c h does no t b i n d t o S-HA i s as h y d r o p h o b i c as t h e p a r e n t s t r a i n ( 1 6 1 ) . The n a t u r e of t h e c o n s t i t u e n t s c o n f e r r i n g h y d r o p h o b i c i t y on t h e s t r e p t o c o c c i i s a m a t t e r of some c o n t r o v e r s y . T y l e w s k a e t a l . (153) w o r k i n g w i t h S_^  p y o g e n e s , showed t h a t l a b o r a t o r y v a r i a n t s w i t h and w i t h o u t M - p r o t e i n d i f f e r i n t h e i r c e l l s u r f a c e p r o p e r t i e s . M - p o s i t i v e s t r a i n s were more n e g a t i v e l y c h a r g e d and more h y d r o p h o b i c t h a n s t r a i n s l a c k i n g M - p r o t e i n . They c o n c l u d e d t h a t c e l l s u r f a c e p r o t e i n s a r e r e s p o n s i b l e f o r h y d r o p h o b i c i t y . The a m p h i p h i l i c m o l e c u l e l i p o t e i c h o i c a c i d (LTA) has a l s o been s u g g e s t e d t o c o n f e r h y d r o p h o b i c c h a r a c t e r i s t i c s on c e l l s u r f a c e s (6,100). M i o r n e r e t a l . (100 ) s u g g e s t e d t h a t LTA b i n d s t o M - p r o t e i n i n S^ pyo g e n e s v i a n e g a t i v e l y c h a r g e d p h o s p h a t e g r o u p s , l e a v i n g t h e f a t t y a c i d component t o e x t e n d t o t h e c e l l s u r f a c e where i t i s a b l e t o c o n t r i b u t e t o c e l l s u r f a c e h y d r o p h o b i c i t y , and i s a b l e t o i n t e r a c t w i t h h y d r o p h o b i c r e g i o n s on an e p i t h e l i a l c e l l . T h i s i s a p p a r e n t l y n ot t h e c a s e i n g r o u p D s t r e p t o c o c c i , where t h e l i p i d p o r t i o n i s b u r i e d w i t h i n t h e 29 c e l l envelope and therefore does not contribute to surface hydrophobicity ( 1 5 5 ) . Miorner et a l . (100) found that treatment of S_^_ pyogenes with protease led to a. reduction in hydrophobicity. This was interpreted to mean that protease was removing the proteins which served to anchor the LTA to the c e l l surface. In this view hydrophobicity results from a p r o t e i n - l i p i d complex. There is no difference in the LTA content of hydrophobic and hydrophilic strains of serotype C mutans (97). Hydrophobicity was not reduced by treatment of the c e l l s with proteolytic enzymes. U n t i l more evidence is available, i t would be unwise to preclude a contribution of either LTA or protein to hydrophobicity. Indeed i t is quite probable that both play a role and that the r e l a t i v e importance of one or the other compound varies with the organism in question. Certainly, i t can be imagined that the nature of the hydrophobic constituents mediating attachment to a c e l l membrane might be quite d i f f e r e n t from that of a comparable component mediating attachment to a salivary p e l l i c l e . In addition to characterizing the biochemical nature of adhesins, i t is necessary to have an understanding of how they are incorporated into the c e l l wall. C e l l surfaces are topographically complex e n t i t i e s possessing a variety of structural units. It is l i k e l y that the f i b r i l l a r or fimbriate structures which can act as adhesins possess hydrophobic domains consisting of nonpolar aminoacids or bound LTA (70). Alternatively, the adhesin could be composed of a number of 30 discrete subunits ( 1 6 0 ) , some responsible for l e c t i n - l i k e or ionic bonding and others responsible for forming hydrophobic bonds. The role of fimbriae in the adherence of S_^  sanguis has remained ambiguous u n t i l recently. Henriksen et a l . (67) demonstrated a correlation between polar fimbriae and twitching mot i l i t y ( a type of surface translocation) in S_^  sanguis. Fives-Taylor (42) postulated a role for peritrichous fimbriae present on S_^  sanguis FW213 in mediating adherence to S-HA. Recently these workers have (39) confirmed their e a r l i e r observations based on: (1) i s o l a t i o n of non-adherent nonfimbriated mutants, and (2) i n h i b i t i o n of adherence by a n t i - f i m b r i a l antibody. Hogg et a l (69), reported a role for peritrichous* surface f i b r i l l a r components in adherence to erythrocytes and blood group-reactive glycoproteins isolated from human saliva in S_;_ sanguis. Proteins in the c e l l walls of the streptococci play an important role in mediating attachment to e p i t h e l i a l surfaces (35,73,155,159), to salivary p e l l i c l e (60,87,97,110), and to other bacteria (157-160). Proteins with l e c t i n - l i k e a c t i v i t y have been isolated from S_^  sanguis ( 106,107). Liljemark and Bloomquist (87) have isolated a streptococcal c e l l surface protein that blocks adherence to S-HA. Large proteins found in the c e l l walls of the oral streptococci play a role in adherence. Adherence negative mutants of S_^  s a l i v a r i u s lacked certain high molecular proteins 31 (160 ), C e l l walls of the parent s t r a i n contain three proteins with molecular weights greater than 200,000. The proteins were solublized by digestion of the walls with either lysozyme or mutanolysin (159,160). S^ s a l i v a r i u s V5, a mutant unable to aggregate with V\_ alcalescens, was missing a 320,000 molecular weight protein. The protein binds s e l e c t i v e l y to alcalescens and induces aggregation. s a l i v a r i u s HB-7, a mutant unable to aggregate in saliva or bind to S-HA, was found to be missing two other high molecular weight proteins. The study reported here was undertaken to further characterize the c e l l surfaces of S_^  sanguis 12 and 12na, and to compare these with c e l l surfaces of hydrophilic variants isolated from both strains with the aim of iden t i f y i n g surface molecules involved in adherence. 32 MATERIALS AND METHODS Bacteria S. sanguis 12 (Biotype 1) and i t s non-salivary aggregating variant 12na have been described previously (95). Strain 12 was isolated from human dental plaque and 12na is a spontaneously occuring non-aggregating variant of 12. Bacterial cultures were stored frozen at -70°C in 10% g l y c e r o l . Rather than continuously subculturing from agar or broth, inoculum was obtained from the frozen stock culture. To ensure that the organisms retained their phenotypic c h a r a c t e r i s t i c s , cultures were routinely analyzed for their adherence and aggregation properties. Culture conditions Bacteria were grown overnight in trypticase soy broth supplemented with 0.3% yeast extract (TSBY) (BBL Microbiology Systems,Cockeysville,Md.) at 37°C. C e l l s were harvested by centrifugation and washed twice in 0.05M N-2-hydroxyethyl piperazine -N'-2-ethane sulphonic acid (HEPES) buffer, pH 7.2. Ce l l s for adherence assays were radiolabelled by the addition 1uCi of (methyl-1'-2'-3H) thymidine (The Radiochemical Centre, Amersham, England.) per ml to the culture medium. The bacteria were harvested and washed four times in HEPES buffer, and were sonicated for 60 s at an output of 5 (Sonifier, C e l l disruptor 350, Branson Sonic power Co., Conn.) to break chains. Bacteria were examined microscopically to ensure that only singles or 33 pairs were present. The bacteria were then washed two times in HEPES buffer and suspended in HEPES to an A 5 B 0 of 5.0. This absorbance corresponded to 1x10 1 0 cells/ml as determined by counting with a Petroff-Hauser c e l l counting chamber. Saliva Paraffin stimulated s a l i v a , pooled from a number of donors, was collected at 4°C and c l a r i f i e d by centrifugation at 17,000 xg for 10 min. The c l a r i f i e d s a l i v a was heated at 60°C for 30 min to destroy endogenous enzymes, centrifuged to remove particulate matter, and stored in aliquots at -20°C. When needed, sa l i v a was thawed and then centrifuged to remove sediment. Adherence assay Fines were removed from spheroidal hydroxyapatite beads (HA) (BDH Chemical Ltd,Poole, Eng.) by repeated washing in 500ml d i s t i l l e d water. After mixing with water the suspension was allowed to se t t l e for 2 min and the supernatant was discarded. This procedure was repeated f i v e times or u n t i l a l l the fines were removed. The beads were dried in an incubator and the dried beads (40mg) were placed in glass s c i n t i l l a t i o n v i a l s . C l a r i f i e d s aliva (0.5ml) was added and the mixture was shaken on a horizontal shaker (45 cycles/min) (Eberbach Corporation, Michigan) at room temperature for 2 hrs and then placed at 4°C overnight. 34 After incubation at 4°C, the unbound sa l i v a was removed by aspiration and the saliva-coated hydroxyapatite beads (S-HA) were washed twiced with a t o t a l of 20ml of d i s t i l l e d water and a t h i r d time with 10ml of HEPES buffer. Excess buffer was removed by aspiration. For examination of the effects of neuraminidase on adherence, the pH of the bead-saliva mixture was reduced to pH 5 by the addition of 0.25N HC1. Neuraminidase (Sigma Chemical Co., St.Louis, Mo.; type VI from Clostridium perfringens) was dissolved in 0.05 M acetate buffer (pH5) at a concentration of 50 ug/ml. 1Oul of the enzyme was added to v i a l s . 10 ul of 0.5% sodium azide was included to prevent microbial growth. The v i a l s containing the mixture were incubated overnight at 37°C. Controls were treated s i m i l a r l y , except that neuraminidase was not included. Beads were washed as described above. Bacterial suspension (1.0ml) containing 1x10 1 0 c e l l s was added to each v i a l containing S-HA and the v i a l s were shaken on a horizontal shaker at 45 cycles/min for 2 hrs. For adherence i n h i b i t i o n assays, S-HA beads were preincubated with the in h i b i t o r (0.5ml) for one hr prior to the addition of c e l l s . Subsequently 0.5ml of c e l l s (2X10 1° cells/ml) were added and treated as described above. A buffer control was included. Unattached bacteria were removed by aspiration and the bacteria-S-HA complex was washed three times with a t o t a l of 30ml of HEPES buffer. The washed beads were then transferred to 35 new p l a s t i c s c i n t i l l a t i o n v i a l s and dried overnight at 60°C. Following drying, the beads were mixed with 0.1ml of d i s t i l l e d water and 0.5ml of NCS Tissue s o l u b i l i s e r (The Radiochemical Centre, Amersham, Eng.) and incubated at 60°C overnight. S c i n t i l l a t i o n f l u i d (10ml) (Toluene : methanol: l i q u i f l u o r ; 6:4:0.42) was added to each v i a l and the r a d i o a c t i v i t y was monitored in a Tracor analytic l i q u i d s c i n t i l l a t i o n counter. Experiments were done in t r i p l i c a t e . Hydrophobic i t y Bacterial hydrophobicity was measured by determining the number of bacteria adhering to hexadecane as described by Rosenberg et a l . , (142) with minor modifications. Bacteria grown overnight in TSBY medium were washed'in HEPES buffer to give an absorbance at A u 3 6 of 0.5. Hexadecane (O.iml)was added to a 18x150mm tube containing 3ml of the bacterial suspension. The suspension was mixed vigorously on a vortex mixer for 60 s and then was allowed to stand for 20 min. The aqueous phase of the suspension was c a r e f u l l y removed with a pasteur pipette and i t s absorbance at 436nm was measured. A control without hexadecane was always included. Experiments were done in t r i p l i c a t e . 36 Isolat ion of hydrophilie var iants Variants of S. sanguis 12 and I2na with reduced a b i l i t y to bind to hexadecane were isolated by an enrichment procedure described by Rosenberg et a l , (143). Cells were grown in TSBY broth overnight, washed twice in HEPES buffer and adjusted to an A q 3 6 of 0.5 under s t e r i l e conditions. The hexadecane assay was performed as described above under s t e r i l e conditions. A portion of the aqueous phase was c a r e f u l l y removed with a s t e r i l e pasteur pipette and inoculated into s t e r i l e TSBY broth. Following incubation overnight at 37°C, c e l l s were then harvested and washed and the hexadecane assay was repeated. The procedure was continued u n t i l four hexadecane enrichments had been completed. The aqueous phase was then removed with pasteur pipette and spread on TSBY agar. The morphology of the colonies was examined microscopically. Morphological variants were subcultured and hydrophobicity measured as described above. Hydrophilic variants of s t r a i n 12 were obtained by inoculating the organism into 10ml of TSBY broth containing 0.4ml of hexadecane. The suspension was mixed vigorously on a rotary mixer (Super-Mixer, Cole-Parmer, Chicago,111.) for 60 s and then incubated overnight at 37°C. The aqueous phase was a s e p t i c a l l y removed and inoculated into a second tube of TSBY containing 0.4ml of hexadecane. The procedure was repeated twice more before spreading on TSBY agar. The morphology of colonies 37 was examined microscopically. Morphological variants subcultured and hydrophobicity measured as described were e a r l i e r . Salivary aggreqation Saliva (0.1ml) was s e r i a l l y d i luted in HEPES buffer in 12x75mm agglutination tubes. An equal volume of bacteria ( A 6 6 0 =3.0) in HEPES buffer was added and the mixture was shaken on a horizontal shaker (90 cycles/min) at room temperature for 5 min. The aggregation t i t e r was expressed as the highest d i l u t i o n of saliva which gave macroscopically v i s i b l e aggregation. A control without saliva was included in the assay. Preparation of c e l l walls C e l l walls were prepared e s s e n t i a l l y as described previously (95). Washed c e l l s (25g wet weight) suspended in HEPES buffer were mixed with 30g of glass beads (75-100 urn) (Sigma Chemical Co.,St.Louis, Mo.) and s t i r r e d overnight in a Minimill (Gifford Wood Co.,) at 4°C. Intact c e l l s and c e l l walls were separated from glass beads by f i l t r a t i o n through a sintered glass f i l t e r . The c e l l walls in the f i l t r a t e were separated from soluble c e l l u l a r material by centrifugation at 12,000xg for 15min. The majority of whole c e l l s were separated by gently resuspending the upper c e l l wall layer of the p e l l e t . C e l l walls were p u r i f i e d further by repeated centrifugation at 3000xg for 5 min. The whole c e l l s formed a firm p e l l e t , whereas the walls did not sediment or 38 formed a soft pe l l e t that was removed with the supernatant. The low speed centrifugations were repeated u n t i l there were no unbroken c e l l s in the preparation when visualized by phase contrast microscopy, This was designated as crude c e l l wall preparat ion. A portion of the crude c e l l wall was extracted with 3% SDS for 3 hrs at room remperature on a rotating tube mixer (Labquake, Labindustries, C a l i f . ) . The suspension was centrifuged and the p e l l e t was washed seven times with a t o t a l of 210 ml of HEPES buffer to remove residual SDS. This preparation was designated a clean -cell wall preparation. C e l l wall analysis Protein-was extracted employing the method of Nesbitt et a l , ( 1 l 0 ) . lOOmg of crude c e l l walls were suspended in 5ml of d i s t i l l e d water and placed in a bo i l i n g water bath for 30 min. The suspension was centrifuged at 12,000xg for 10 min. The pe l l e t was resuspended in d i s t i l l e d water and the process repeated. The p e l l e t was then sequentially extracted with 3% SDS and 5M L i C l . Both extractions were carried out for 30 min in a bo i l i n g water bath. Between the two treatments the walls were extracted for 30 min in a b o i l i n g water bath with d i s t i l l e d water. After extracting with 5M L i C l , c e l l walls were extracted with d i s t i l l e d water twice for 30 min followed by extraction with 8M urea at 4°C overnight. 39 Freeze dried clean c e l l walls dried to constant weight in a desiccator over P 2 0 5 were used for estimation of rhamnose, to t a l hexose, protein and phosphorus. Rhamnose was measured by the Dische and Shettles technique (27), phosphorus was determined by the method of Chen et al,(11). Total hexoses were measured by the phenol sulphuric acid method (29), with glucose as the standard; protein concentrations were measured by the method of Lowry et al(90) with bovine serum albumin as the standard. Mutanolysin digest ion Whole c e l l s and c e l l walls of hydrophobic and hydrophilic strains were s o l u b i l i z e d using mutanolysin, a streptococcal muramidase from Streptomyces globi sporus (Sigma Chemical Co.,St.Louis, Mo.). Some mutanolysin preparations were found to contain low levels of protease a c t i v i t y as determined by Azocoll assay. Phenyl methyl sulphonyl flu o r i d e (1 mM) was found to in h i b i t the protease a c t i v i t y and was included in the reaction mixture. Digestion was ca r r i e d out at 37°C for 72 hrs in the presence of 0.02% sodium azide. Whole c e l l digestion Washed c e l l s suspended in 5ml of HEPES buffer containing 0.02% sodium azide at an A 6 6 0 of 3.0 were mixed with 250 units of mutanolysin and incubated overnight at 37°C. Digestion was monitored by measuring the reduction of tu r b i d i t y at 660nm. 250 units of mutanolysin were added every 24 hrs u n t i l the t u r b i d i t y 40 had been reduced by 80%. Usually the digestion was carried out for 72 hrs to obtain satisfactory s o l u b i l i z a t i o n . A control without mutanolysin was included in the experiment. Undigested material was removed by centrifugation.The soluble digest was dialysed overnight against 6L of d i s t i l l e d water at 4°C , lyophilized, resuspended to the o r i g i n a l volume and retained for electrophoretic analysis. C e l l wall digest C e l l walls (5ml) were suspended at an A 6 6 0 of 10.0 in HEPES buffer supplemented with 0.02% sodium azide. Mutanolysin (500 units) and PMSF (1 mM) was added and the mixture was incubated overnight. Digestion was monitored by measuring the reduction of turbidity at- 660 hm. An additional 500 units of mutanolysin was added every 24 hrs u n t i l the t u r b i d i t y was reduced by 80%. The digestion was continued for 72 hrs to obtain s a t i s f a c t o r y s o l u b i l i z a t i o n . The digestion mixture was centrifuged to remove undigested material. The soluble digest was dialysed against 6L of d i s t i l l e d water overnight at 4°C, l y o p h i l i z e d and resuspended to i t s o r i g i n a l volume and retained for electrophoretic analysis. In some cases the c e l l wall digests were dialysed against HEPES buffer (21) overnight at 4°C. 41 Trypsin digest ion Whole c e l l s of S. sanguis 12 were suspended in HEPES buffer (A 6 6o=5.0). The c e l l suspension (2ml) was incubated at 37°C for one hour with 0.01-1.0 mg/ml of trypsin (Sigma Chemical Co., Mo.; type XI from bovine pancreas). The reaction was stopped by the addition of enough trypsin i n h i b i t o r (Sigma Chemical Co., Mo.; type 1-5 from soybean) to give a f i n a l concentration of 0.5mg/ml. Control c e l l s were treated with trypsin inactivated by heating in boiling water for 30 min. After incubation with the enzyme, the c e l l s were recovered by centrifugation. Supernatants were retained for electrophoretic analysis. Trypsin treated c e l l s were washed in HEPES buffer and their adherence to hexadecane -was measured. The remaining c e l l s were digested overnight with mutanolysin as described in the previous section. SDS -Polyacrylamide gel .electrophoresis.(SDS-PAGE) SDS-PAGE was performed in a 7.5% polyacrylamide gel with a 3% stacking gel by the method described by Laemmli (77). The samples were boiled in SDS-BME for 5 min before application to the gel. The gels were stained for proteins with s i l v e r nitrate (113). Molecular weight standards were myosin (200,00), j3-galactosidase (116,250), phosphorylase B (92,500), bovine serum albumin (66,200) and ovalbumin (45,000) obtained from Bio-Rad Laboratories, C a l i f . 42 Electron microscopy Cells washed in HEPES buffer were negatively stained with 2% (wt/vol) phosphotungstic acid adjusted to pH 7.2 with KOH or with 5% uranyl acetate in 70% alcohol. Observations were made with a P h i l i p s EM 300 electron microscope. Immunological procedures Rabbit antisera were raised against formalinized whole c e l l s of S_j_ sanquis. C e l l s supended in HEPES buffer were mixed with 0.5% formalin overnight at 4°C. The c e l l s were then recovered by centrifugation and washed seven times with HEPES buffer. F i n a l l y the c e l l s were resuspended in HEPES buffer ( A 6 6o = 1'0) and were stored in 1ml aliquots at -20°C. A 0.5ml c e l l suspsension was injected intravenously at weekly intervals for four weeks. After two weeks of rest, the rabbits were given another 0.5ml of c e l l s and one week la t e r were bled from the ear or heart. If more serum was required rabbits were given a booster of 0.5ml of c e l l s and bled during the following week. Immunoelectrophoresis A l l immunoelectrophoretic techniques were done in 1.2% agarose with 0.08M T r i s , 0.025M t r i c i n e , 0.02% sodium azide buffer (pH 8.6). Gels were cast on Gelbond fi l m (Marine Colloids d i v i s i o n , FMC Corporation) to a thickness of 0.15cm. The cathodic end of 43 the gel was prepared from agarose without serum. It measured 2cm in width. The gel immediately adjacent to this was formed from agarose containing i0% antiserum. For rocket immunoelectrophoresis antigens were placed in 4mm holes in the cathodic end and electrophoresed into the antiserum containing agarose at 400V for 18 hrs. Electrophoresed s t r i p s were dried by blotting with paper towels and then stained with s i l v e r n i t r a t e ( 1 6 7 ) . For crossed immunoelectrophoresis digests were f i r s t separated by 7.5% SDS-PAGE. Appropriate s t r i p s were cut from the polyacrylamide gel and embedded at the cathodic end of the gel. The antigens were then electrophoresed into the gel containing the antiserum (400V for 18 hrs). Duplicate samples were applied to the SDS-PAGE. The samples not used for immunoelectrophoresis were visualized by s i l v e r staining and saved for comparison with the agarose gel. The polyacrylamide component of the gel was removed and the remaining gel was dried and stained with s i l v e r as before. Western blot F o r western blot a n l y s i s , samples were electrophoresed in 7.5% SDS-PAGE. Appropriate s t r i p s were cut from the gel and placed between two sheets of n i t r o c e l l u l o s e paper which had been wetted by immersion in d i s t i l l e d water and then soaked in 0.4% (wt/vol) SDS, 1.24% T r i s , 0.76% glycine at 60°C f o r 30 min. The n i t r o c e l l u l o s e enclosed gel was sandwiched between two stacks of 44 3M. Whatman f i l t e r paper which had been wetted in the transfer buffer (glycine 14.4g/l, T r i s 3.025g/l, methanol 200ml/l pH 8.6). The entire stack was placed in a holding cassette and transferred to the Bio-Rad Trans blot c e l l which was f i l l e d with transfer buffer. A current of 25V was applied overnight followed by a current of 60V for 2 hrs. . Following transfer of the antigens the n i t r o c e l l u l o s e sheets were soaked in 3% bovine serum albumin for 30 min to saturate a l l remaining binding s i t e s . The sheets were washed twice with TTBS (20mM T r i s , 500mM NaCl, 0.05% Tween-20, pH 7.5) and then incubated for 2 hrs with IgG prepared from rabbit antiserum. The procedure i s described in the following section. The unbound IgG was removed by washing with TTBS. The sheets were incubated next with goat anti-rabbit IgG coupled to horse radish peroxidase for 2 hrs. Unbound antiserum was removed by washing with TTBS. Bio-Rad GAR-HRP substrate was added according to instructions in the Bio-Rad technical b u l l e t i n supplied with the Bio-Rad Immun-Blot (GAR-HRP) assay k i t . Preparation of IgG Protein A-Sepharose CL-4B (300mg) (Sigma Chemical Co., St. Louis, Mo.)was swollen overnight at 4°C in phosphate buffered saline (PBS), pH 7.0 and poured into a 15x65 mm column. The column was washed with glycine (0.1M glycine, 0.5M NaCl, pH 2.5) buffer and borate buffer (0.1M borate, 0.5M NaCl, pH 8.4) and f i n a l l y equilbrated with borate buffer. 45 Antiserum (3ml) adjusted to pH 8.4 with d i l u t e NaOH was slowly loaded onto the column. Unbound constituents were removed by washing the column with borate buffer u n t i l no protein could be detected in the eluate. Bound immunoglobulin was eluted with 5ml of glycine buffer. The eluate was neutralised with d i l u t e NaOH and dialysed against two changes of 2L of PBS at 4°C. The IgG fraction was stored at -20°C. Adsorbed antiserum Adsorbed antiserum was prepared as described previously (34) with minor modifications. Rabbit antiserum (2ml) was mixed with 40mg of c e l l s and incubated at 4°C for 30 min. Ce l l s were removed by centrifugation and the supernatant was absorbed again with 40mg of c e l l s at 37°C for 30 min. This procedure was repeated three times. The adsorbed antiserum was used for immunoelectrophoretic analysis. HPLC fractionation Crude c e l l wall digests of S_^  sanguis 12 were fractionated on a molecular sieving (10-500 kD) protein Pak 300 column (Waters Associate, Milford, Mass.) (7.5mmx30cm) in a Waters High performance l i q u i d chromatograph (HPLC). Samples (0.1ml) were applied to the column by an automatic inje c t i o n system. A t o t a l of 28 fractions were eluted with 0.05M HEPES, 0.1M NaCl buffer (pH 7.2) at a flow rate of 0.5ml/min. The eluate was monitored at 280nm. Following elution of the sample another sample was 46 automatically applied to the column and the elution repeated, fractions (0.5ml) being collected in the same set of tubes. A tota l of 3.5ml was fractionated by this procedure. The fractions were dialysed against 2L of HEPES buffer overnight at 4°C. Each fraction was l y o p h i l i z e d and resuspended in 3.5ml of HEPES buffer. Protein assay Protein was determined by the Bio-Rad method (Bio-Rad Laboratories, C a l i f . ) using bovine serum,albumin as the standard. Azocoll assay Mutanolysin or c e l l s treated with trypsin were suspended in HEPES buffer. The sample was incubated with 1Omg Azocoll at 37°C for one hr. After one hr, the unreacted substrate was removed by centrifugation and the supernatant was examined spectroscopi.cally at 520nm for the presence of s o l u b i l i z e d chromophore. 47 RESULTS P r o p e r t i e s of S. s a n g u i s S t r e p t o c o c c u s s a n g u i s 12 i s a h y d r o p h o b i c o r g a n i s m which b i n d s t o hexadecane, a g g r e g a t e s i n s a l i v a and a d h e r e s t o S-HA. S t r a i n 12na, a s p o n t a n e o u s l y a r i s i n g v a r i a n t i s a g g r e g a t i o n d e f i c i e n t but r e t a i n s i t s a b i l i t y t o b i n d t o h e x a d e c a n e and S-HA. I t was a p p a r e n t t h a t s t r a i n 12na was not a s t a b l e v a r i a n t and would o c c a s i o n a l l y r e v e r t t o i t s p a r e n t p h e n o t y p e . T h i s was o n l y a p a r t i a l r e v e r s i o n i n w h i c h t h e s t r a i n would r e g a i n some of i t s s a l i v a r y a g g r e g a t i n g p r o p e r t i e s ( T a b l e I I ) . The n o n - a g g r e g a t i n g v a r i a n t has been g i v e n t h e d e s i g n a t i o n l 2 n a / 2 and the p a r t i a l r e v e r t a n t i s r e f e r r e d t o as 1 2 n a / l . B e c a u s e of t h i s i n s t a b i l i t y c u l t u r e s were a l w a y s m o n i t o r e d t o e n s u r e t h a t t h e y p o s s e s s e d th e d e s i r e d p h e n o t y p e . S a l i v a r y a g g r e g a t i o n i s s e n s i t i v e t o n e u r a m i n i d a s e t r e a t m e n t of s a l i v a . T h i s i s t r u e f o r s t r a i n 12- and 12na/l i n d i c a t i n g t h a t l i k e t h e p a r e n t , t h e p a r t i a l r e v e r t a n t a g g r e g a t e s v i a a t e r m i n a l s i a l i c a c i d r e s i d u e . I s o l a t i o n o f h y d r o p h i 1 i c v a r i a n t s In o r d e r t o d e t e r m i n e i f h y d r o p h o b i c f o r c e s were i m p o r t a n t i n a d h e r e n c e t o S-HA, i t was d e c i d e d t o i s o l a t e h y d r o p h i l i c s t r a i n s of S^ s a n g u i s and compare t h e i r a d h e r e n c e p r o p e r t i e s and c e l l w a l l c o m p o s i t i o n w i t h t h e h y d r o p h o b i c s t r a i n s . H y d r o p h i l i c v a r i a n t s o f S_^  s a n g u i s 12 and 12na were i s o l a t e d by e n r i c h i n g f o r o r g a n i s m s t h a t d i d not adhere' t o 48 TABLE II Adherence properties of S. sanguis Strain % adsorbed Salivary Adherence to aggregating to hexadecane t i t e r S-HA 12 91 256 + 12na 89 4 + a 12na/l 92 8 N.D 12na/2 90 0 N.D a N.D Not Determined. 49 h e x a d e c a n e . B a c t e r i a l s u s p e n s i o n s were mixed w i t h hexadecane and t h e o r g a n i s m s from the lower aqueous phase removed and c u l t u r e d . A f t e r r e p e a t i n g t h i s e n r i c h m e n t f o u r t i m e s , t h e c u l t u r e was s p r e a d on TSBY a g a r . The e n r i c h m e n t p r o c e d u r e l e d t o t h e i s o l a t i o n of f o u r s t r a i n s of s a n g u i s 12na w h i c h formed m o r p h o l o g i c a l l y d i s t i n c t c o l o n i e s on TSBY a g a r . T h e s e c o l o n i e s were d e s i g n a t e d 12naAL, 12naAD, 12naBL and 12naBD. A and B d e n o t e s t r a i n s i s o l a t e d from two d i f f e r e n t e n r i c h m e n t s . D i f f u s e (D)and L a r g e (L) i n d i c a t e s t h e a p p e a r a n c e of c o l o n i e s on TSBY a g a r v i e w e d under a l i g h t m i c r o s c o p e . The p r o c e d u r e d i d not y i e l d m o r p h o l o g i c a l v a r i a n t s of S.  s a n g u i s 12, i n d e e d t h e r e was no g r o w t h when t h e aqueous p h a s e was c u l t u r e d . T h e r e f o r e h e x a d e c a n e was i n c l u d e d i n t h e b r o t h d u r i n g g r o w t h . F o l l o w i n g i n c u b a t i o n o v e r n i g h t a t 37°C t h e e n r i c h m e n t was r e p e a t e d . A f t e r r e p e a t i n g t h i s p r o c e d u r e f o r a t o t a l o f f o u r t i m e s a c o l o n y was i s o l a t e d w h i c h showed r e d u c e d a d h e r e n c e t o h e x a d e c a n e . T h i s s t r a i n was d e s i g n a t e d 12L. The c e l l s u r f a c e h y d r o p h o b i c i t y o f t h e c o l o n i a l v a r i a n t s was a s s e s s e d i n t h e h exadecane a s s a y ( T a b l e I I I ) . As i n d i c a t e d p r e v i o u s l y t h e p a r e n t s t r a i n s were s t r o n g l y h y d r o p h o b i c . The c o l o n i a l v a r i a n t s o f 12na e x h i b i t e d a marked r e d u c t i o n i n t h e i r a b i l i t y t o b i n d t o h e x a d e c a n e . The p e r c e n t b i n d i n g v a r i e d f r o m 25% f o r 12naAD t o 41% f o r 12naBD. The h y d r o p h o b i c i t y of v a r i a n t 12L was r e d u c e d t o 44%. A l l h y d r o p h i l i c s t r a i n s r e t a i n e d some a b i l i t y t o b i n d t o h e x a d e c a n e . A t t e m p t s t o i s o l a t e a h y d r o p h i l i c v a r i a n t t h a t was 50 TABLE I I I H y d r o p h o b i c and s a l i v a r y a g g r e g a t i n g p r o p e r t i e s of S. s a n g u i s S t r a i n % a d s o r b e d s a l i v a r y t o a g g r e g a t i n g h e x a d e c a n e t i t e r 12 .98 256 12na 97 4 12L 44 12naAL 33 12naAD 25 12naBL 31 12naBD 41 51 u n a b l e t o b i n d t o were u n s u c c e s s f u l . G i b b o n s e t a l . ( 5 5 ) , o b s e r v e d t h a t n o n n y d r o p h o b i c m u t a n t s i s o l a t e d by a s i m i l a r e n r i c h m e n t p r o c e d u r e a l s o a d h e r e d t o h e x a d e c a n e t o some d e g r e e . H y d r o p h o b i c and h y d r o p h i l i c s t r a i n s e x h i b i t e d s i m i l a r g r o w t h c h a r a c t e r i s t i c s when c u l t u r e d i n TSBY ( F i g . 1 ) . In t h e e x p e r i m e n t d e s c r i b e d h e r e c e l l s r e a c h e d t h e l o g a r i t h m i c phase o f g r o w t h w i t h i n 60 min and c e a s e d t o i n c r e a s e i n numbers a f t e r f o u r h o u r s . M i c r o s c o p i c e x a m i n a t i o n r e v e a l e d - t h a t c e l l s were s i m i l a r i n s i z e b ut t h a t 12na and h y d r o p h i l i c s t r a i n s t e n d e d t o form l o n g c h a i n s o f 6-14 c o c c i , whereas s t r a i n 12 formed s h o r t c h a i n s of 2-4 c o c c i . C e l l w a l l s o f S^ s a n g u i s 12, 12na and 12L were c h a r a c t e r i z e d w i t h r e g a r d t o t h e i r c h e m i c a l c o m p o s i t i o n t o d e t e r m i n e i f t h e r e were marked d i f f e r e n c e s between t h e s t r a i n s . SDS e x t r a c t e d c e l l w a l l s d r i e d t o a c o n s t a n t w e i g h t were a n a l y z e d f o r p r o t e i n , h e x o s e , rhamnose and p h o s p h o r u s ( T a b l e I V ) . The w a l l s of t h e p a r e n t s t r a i n 12 c o n t a i n e d a p p r o x i m a t e l y 5% more p r o t e i n t h a n 12na and t h e h y d r o p h i l i c 12L. P e p t i d o g l y c a n r e a c t s i n t h e p r o t e i n a s s a y and t h u s would mask d i f f e r e n c e s between s t r a i n s . The hexose, rhamnose and p h o s p h o r u s c o n t e n t s were s i m i l a r f o r a l l t h r e e s t r a i n s . The h i g h l e v e l s o f rhamnose and low l e v e l s of p h o s p h o r u s c o n f i r m t h a t t h e o r g a n i s m b e l o n g s t o S_^  s a n g u i s B i o t y p e I . A p p r o x i m a t e l y 67% of t h e c e l l w a l l d r y 52 F i g u r e 1. Growth c h a r a c t e r i s t i c s o f h y d r o p h o b i c and h y d r o p h i l i c s t r a i n s o f S^ s a n g u i s . 250ml o f TSBY medium was i n o c u l a t e d w i t h 3ml o f o v e r n i g h t grown c u l t u r e and growth was m o n i t o r e d a t 660nm. 53, Time(hr) 53a C h e m i c a l TABLE IV c h a r a c t e r i z a t i o n S. s a n g u i s of c e l l w a l l s of Component a C o m p o s i t i o n b 12 c 1 2na d 1 2L P r o t e i n 21.1 16.7 16.6 Hexose 23.5 23.0 23.9 Rhamnose 22.2 22.5 25.3 P h o s p h o r o u s 0.7 0.7 0.5 a P e r c e n t c o m p o s i t i o n of f r e e z e d r i e d c e l l w a l l s . b S t r a i n 12 a d h e r e s t o S-HA and a g g r e g a t e s i n s a l i v a . c S t r a i n 12na a d h e r e s t o S-HA but do n o t a g g r e g a t e . d S t r a i n 12L do n o t a d h e r e t o S-HA o r a g g r e g a t e . 54 weight was accounted for in the assays described here. The remaining c e l l wall material was presumably in the form of l i p i d and complex polysaccharides which would not react in the hexose assay. Occassionally the parent strains were observed to self-aggregate after vigorous shaking. The hydrophilic is o l a t e s never exhibited such c h a r a c t e r i s t i c s . The effect of increasing the amount of hexadecane on the binding of c e l l s to the organic layer was studied (Fig. 2 and 3). The parent strains 12 and I2na were bound maximally when 0.1 ml of hexadecane was mixed with 3 ml c e l l suspension (Fig. 2). In contrast i t can be seen that increasing the amount of hexadecane with the hydrophilic strains led to an increase in the number of bound c e l l s (Fig. 3). Maximal binding occurred at 0.3 ml of hexadecane. Salivary aggregation None of the hydrophilic isolates showed any tendency to aggregate when mixed with saliva (Table I I I ) . None of these strains have ever reverted to a salivary aggregating phenotype. Adherence to S-HA. The a b i l i t y of the hydrophilic strains to adhere to S-HA i s described in Table V. As shown previously the parent str a i n s 12 and 12na, both bind to S-HA, but str a i n 12 adheres by a s i a l i c - a c i d on the S-HA which can be removed with neuraminidase. 55 F i g u r e 2. A d h e r e n c e of s a n g u i s t o i n c r e a s i n g amounts of h e x a d e c a n e . 56. 80H 70 A c o 60H o CO •D < 50 H 40 H 30 20 H Legend A 12 O 12na 10 0.1 0.2 0.3 0.4 0.5 Volume of hexadecane(ml) 0.6 56a F i g u r e 3. A d h e r e n c e o f s a n q u i s v a r i a n t s t o i n c r e a s i n g amounts of h e x a d e c a n e . 57; c o o CO u < Legend A 12L O 12naAL • 12naAD O 12naBL V 12naBD 0.2 0.3 0.4 0.5 Volume of hexadecane(ml) 0.6 57h TABLE V a A d h e r e n c e of S. s a n g u i s t o S-HA 9 Number of c e l l s bound (X10 ) S t r a i n % a d s o r b e d t o h e x a d e c a n e S a l i v a N e u r a m i n i d a s e t r e a t e d s a l i v a 12 98 4.2 0.6 1 2na 97 6. 1 0.5 12L 44 0.7 0.6 12naAL 33 0.9 0.6 12naAD 25 0.7 0.6 12naBL 31 1.0 0.8 12naBD 41 1.0 1 . 1 a 10 10 c e l l s were i n c u b a t e d w i t h 40 mg of S-HA. 58 None of the h y d r o p h i l i c i s o l a t e s bound t o S-HA by a n e u r a m i n i d a s e s e n s i t i v e mechanism. D e s p i t e the g r e a t l y r e d u c e d a b i l i t y t o b i n d t o S-HA t h e h y d r o p h i l i c s t r a i n s were s t i l l c a p a b l e of b i n d i n g t o S-HA t o a l i m i t e d e x t e n t . I t i s i n t e r e s t i n g t o n o t e t h a t t h e e x t e n t of t h e b i n d i n g i s s i m i l a r t o t h a t o b s e r v e d when 12 and 12na b i n d t o n e u r a m i n i d a s e t r e a t e d S-HA. T h i s s u g g e s t s t h a t t h e h y d r o p h i l i c s t r a i n s have l o s t a s i a l i c - a c i d l e c t i n o r t h a t h y d r o p h o b i c c o n s t i t u e n t s not f o u n d i n t h e s e c e l l s a r e r e q u i r e d t o form a s t a b l e u n i o n w i t h S-HA. I t i s a l s o p o s s i b l e t h a t b o t h t h e l e c t i n and h y d r o p h o b i c c o n s t i t u e n t s r e q u i r e d f o r b i n d i n g a r e m i s s i n g . E l e c t r o n m i c r o s c o p y E l e c t r o n m i c r o g r a p h s of h y d r o p h o b i c and h y d r o p h i l i c s t r a i n s were examined t o l o o k f o r d i f f e r e n c e s i n c e l l s u r f a c e s t r u c t u r e s . C e l l s grown o v e r n i g h t i n b r o t h were n e g a t i v e l y s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d ( F i g . 4 ) . S t r a i n 12 had a t h i c k c o a t i n g of e x t r a c e l l u l a r m a t e r i a l s u r r o u n d i n g t h e e n t i r e c e l l s u r f a c e . 12na had a s i m i l a r s t r u c t u r e but t h e t h i c k n e s s o f t h e e x t r a c e l l u l a r l a y e r was v e r y much r e d u c e d . S t r a i n 12L had a s u r f a c e w hich had p a t c h y a r e a s of e x t r a c e l l u a r m a t e r i a l . Many a r e a s of t h e c e l l s u r f a c e s were d e v o i d of t h i s e x t r a c e l l u l a r m a t e r i a l . The p h o s p h o t u n g s t i c a c i d s t a i n p r o v i d e d l i m i t e d i n f o r m a t i o n on t h e n a t u r e o f t h e c e l l s u r f a c e m a t e r i a l . I n o r d e r t o t r y and v i s u a l i z e t h e s u r f a c e more c l e a r l y c e l l s were s t a i n e d w i t h u r a n y l a c e t a t e . As seen i n f i g . 5 ( a ) t h e h y d r o p h o b i c 59 F i g u r e 4. E l e c t r o n m i c r o g r a p h s of s a n g u i s . C e l l s were n e g a t i v e l y s t a i n e d w i t h p h o s p h o t u n g s ' t i c a c i d : ( a ) , 12; ( b ) , 12na; ( c ) , 12L. Bar r e p r e s e n t s 0.5um. 60 F i g u r e 5. E l e c t r o n m i c r o g r a p h s o f s a n g u i s . C e l l s were n e g a t i v e l y s t a i n e d w i t h u r a n y l a c e t a t e : ( a ) , 12; ( b ) , 12L. Bar r e p r e s e n t s 0.5um. 61 b 61a s t r a i n 12 had two l e n g t h s of f i b r i l s . The l o n g e r s t r u c t u r e s w h i c h were more p r e v a l e n t a t the p o l e s were e s t i m a t e d t o be 350-450nm l o n g . A c c o r d i n g t o H a n d l e y e t a l ( 6 2 ) , t h e s e might be c l a s s i f i e d as f i m b r i a e , a l t h o u g h t h e y d i d not have c o n s i s t e n t m e a s u r a b l e w i d t h . The s h o r t e r f i b r i l s w h i c h c o v e r e d t h e whole s u r f a c e , had t h e a p p e a r e n c e of a ' f u z z y c o a t ' and had a l e n g t h of a r o u n d 70nm. S t r a i n 12na a l s o p o s s e s s e d b o t h th e l o n g p o l a r f i m b r i a e and t h e s h o r t e r f i b r i l l a r c o a t b u t t h e d e n s i t y of t h e appendages a p p e a r e d t o be r e d u c e d . In c o n t r a s t , s t r a i n 12L ( F i g . 5b) was denuded of b o t h p o l a r f i m b r i a e and t h e f i b r i l l a r c o a t . The l o n g f i b r i l s were a r r a n g e d i n p a r a l l e l t o one a n o t h e r a p p r o x i m a t e l y 10-20 i n mumbers. P e r i t r i c h o u s f i b r i l s formed a d e nse mat and were t o o numerous t o c o u n t . I n c u b a t i o n o f c e l l s w i t h immune serum, non-immune serum and BSA d i d not improve t h e q u a l i t y of t h e e l e c t r o n m i c r o g r a p h s . Whole c e l l d i g e s t s In an a t t e m p t t o i d e n t i f y m o l e c u l e s w h i c h m i g h t c o n t r i b u t e t o a d h e r e n c e , a g g r e g a t i o n o r h y d r o p h o b i c i t y , c e l l s f r o m v a r i o u s s t r a i n s were s o l u b i l i z e d and f r a c t i o n a t e d by g e l e l e c t r o p h o r e s i s . Whereas a number of p r o t e i n s were r e l e a s e d f r o m t h e c e l l s u r f a c e o f S^ mutans LK by b o i l i n g i n SDS-BME ( 9 7 ) , no m a t e r i a l d e t e c t a b l e i n s i l v e r s t a i n e d g e l s c o u l d be e x t r a c t e d from h y d r o p h o b i c and h y d r o p h i l i c s t r a i n s of S^ s a n g u i s . C e l l s were t h e r e f o r e d i g e s t e d w i t h m u t a n o l y s i n and t h e s o l u b i l i z e d 6 2 Figure 6. SDS-PAGE of whole c e l l s digested with mutanolysin: Lane 1, molecular weight markers; Lane 2, control (whole c e l l s of 12 incubated without mutanolysin); Lane 3, 12; Lane 4, 12na; Lane 5, 12L; Lane 6, 12naAL; Lane 7, 12naAD; Lane 8, 12n'aBL; Lane 9, 12naBD. The 160,000 MW band i s indicated by the arrow.The molecular weight markers are myosin(200,000), /3-galactosidase (116,250), Phosphorylase B (92,250), Bovine serum albumin (66,200), and ovalbumin (45,000). 63 1 2 3 4 5 6 7 8 9 63* m a t e r i a l f r a c t i o n a t e d by SDS-PAGE> Whole c e l l d i g e s t s of S.  s a n g u i s 12 showed a p r o m i n e n t d i f f u s e p r o t e i n band w i t h a m o l e c u l a r w e i g h t o f 160,000 ( F i g 6 ) , w hich was seen f a i n t l y i n d i g e s t s of 12na and was c o m p l e t e l y a b s e n t from t h e h y d r o p h i l i c s t r a i n s . In i n s t a n c e s where 12na had r e g a i n e d some s a l i v a r y a g g r e g a t i n g a c t i v i t y t h e band a p p e a r e d more p r o m i n e n t . F i g . 6 shows t h a t s t r a i n 12 c o n t a i n s two o t h e r f a i n t b a nds, w i t h a m o l e c u l a r w e i g h t a r o u n d 200,000, w h i c h a r e a b s e n t from o t h e r s t r a i n s . A l t h o u g h bands were c o n s i s t e n t l y seen i n t h i s m o l e c u l a r weight range t h e y v a r i e d i n number and i n t e n s i t y between p r e p a r a t i o n s . In view of t h e s i m i l a r i t i e s between t h e h y d r o p h i l i c i s o l a t e s i t was d e c i d e d t o c o n f i n e t h e s t u d y t o s t r a i n 12L. The m u t a n o l y s i n d i g e s t i o n r e q u i r e d 72 h r s t o r e a c h a l e v e l of 80% r e d u c t i o n . A t t e m p t s were made t o r e d u c e t h i s i n c u b a t i o n p e r i o d by a l t e r i n g t h e pH. At pH 4.5, an optimum pH f o r t h e l y s i s o f A c t i n o m y c e s v i s c o s u s t h e r e was no l y s i s o f s a n g u i s 12. Optimum a c t i v i t y was f o u n d t o o c c u r a t pH 6.8 o r 7.2 i n e i t h e r T r i s or HEPES b u f f e r . U n f o r t u n a t e l y i t was n o t p o s s i b l e t o r e d u c e t h e t i m e r e q u i r e d f o r h y d r o l y s i s . C e l l w a l l d i g e s t s To p r o v i d e e v i d e n c e f o r t h e c e l l s u r f a c e l o c a t i o n o f t h e s e p r o t e i n s , d i f f e r e n c e s between t h e s t r a i n s were f u r t h e r e x a m i n e d i n m u t a n o l y s i n d i g e s t s f o l l o w e d by f r a c t i o n a t i o n i n SDS-PAGE ( F i g . 7 ) . A v a r i e t y o f o t h e r a g e n t s i n c l u d i n g u r e a , l i t h i u m 64 F i g u r e 7. SDS-PAGE of m u t a n o l y s i n d i g e s t s of SDS e x t r a c t e d c e l l w a l l s . SDS e x t r a c t e d c e l l w a l l s : Lane 1, 12; Lane 2, 12na; Lane 3, 12L. C e l l w a l l d i g e s t s c o n t a i n i n g r e s p e c t i v e l y 250, '75 and 1Oug p r o t e i n were a p p l i e d t o t h e g e l . P o s i t i o n o f bands r e f e r r e d t o i n t h e t e x t a r e i n d i c a t e d by s m a l l a r r o w s . M o l e c u l a r w e i g h t m a r k e r s a r e i n d i c a t e d by l a r g e a r r o w s . 65 65a chloride or b o i l i n g in d i s t i l l e d water proved to be i n e f f e c t i v e . A large number of bands were observed in both 12 and 12na; however the amounts were much less in the non-aggregating 12na and a number of bands were missing. In contrast the c e l l wall digests of the hydrophilic strain 12L were almost completely devoid of s i l v e r staining bands. In previous studies McBride et a l , have shown that adherence defective strains of mutans (97) and S. s a l i v a r i u s (159) lose high molecular weight c e l l wall proteins. The results here indicate that S^ sanguis behaves in a similar fashion. Strain 12na is missing high molecular weight proteins (MW 263,000, 250,000, 243,000, 220,000 and 190,000) as well as proteins of molecular weight 130,000,48,000 and 44,000. The 160,000 MW band which in seen in whole c e l l digests was also present in the SDS-extracted c e l l walls, and as expected was considerably weaker in 12na c e l l walls than in 12 c e l l walls. The amount of each sample applied to the gel (Fig. 7) was calculated to represent the material released from an equivalent quantity of c e l l walls. To be sure that the q u a l i t a t i v e differences in the protein p r o f i l e s of the three strains were not simply due to the amount of protein applied, samples of 12, 12na and 12L were adjusted to an equivalent protein concentration and electrophoresed (Fig. 8) . There was so l i t t l e protein in the digests of 12L c e l l walls that i t was not possible to electrophorese an equivalent amount of protein in the sample. The banding pattern was similar to that seen in F i g . 7. Strain 12 possessed a number of high 66 F i g u r e 8. SDS-PAGE of m u t a n o l y s i n d i g e s t s of SDS e x t r a c t e d c e l l w a l l s . SDS e x t r a c t e d c e l l w a l l s : Lane 1, 12; Lane 2, 12na; Lane 3, 12L. Lane 1 and 2 c o n t a i n e d 1Oug p r o t e i n . Lane 3 c o n t a i n e d l e s s t h a n 1Oug of p r o t e i n . I t c o u l d not be c o n c e n t r a t e d t o a l e v e l e q u i v a l e n t t o l a n e 1 and 2. P o s i t i o n o f bands r e f e r r e d t o i n t h e t e x t a r e i n d i c a t e d by s m a l l a r r o w s . M o l e c u l a r w e i g h t m a r k e r s a r e i n d i c a t e d by l a r g e a r r o w s . 67--1 2 3 67a m o l e c u l a r w e ight p r o t e i n s not seen i n s t r a i n - 1 2 n a . Thus t h e r e a p p e a r t o be b o t h q u a n t i t a t i v e and q u a l i t a t i v e d i f f e r e n c e s i n t h e p r o t e i n s p r e s e n t on c e l l w a l l s of t h e s e t h r e e s t r a i n s . D i f f e r e n c e s between s t r a i n s i n t h e g e l s c i a t i o n i n s u s c e p t i b i l i t y t o m u t a n o l y s i n s i n c e a l l w a l l p r e p a r a t i o n s were a p p r o x i m a t e l y 80% d i g e s t e d . In f a c t s t r a i n 12, f r o m w h i c h most, m a t e r i a l was r e l e a s e d , t e n d e d t o be r e s i s t a n t t o m u t a n o l y s i n d i g e s t i o n . C e l l modif i c a t i o n C e l l s were e n z y m a t i c a l l y , p h y s i c a l l y and c h e m i c a l l y m o d i f i e d t o c o n f i r m t h e l o c a t i o n of t h e p r o t e i n s o b s e r v e d i n whole c e l l and c e l l w a l l d i g e s t s of s a n g u i s 12 and t o p r o v i d e i n s i g h t i n t o t h e i r r o l e i n a d h e r e n c e . T r e a t m e n t of S_^  s a n g u i s 12 w i t h t r y p s i n a t a c o n c e n t r a t i o n of 10 ug/ml c a u s e d a d e f i n i t e but l i m i t e d r e d u c t i o n i n h y d r o p h o b i c i t y ( T a b l e V I ) . The e f f e c t o f i n c r e a s i n g t h e c o n c e n t r a t i o n of t r y p s i n i s s e e n i n F i g . 9. T h e r e was a s h a r p d r o p i n hexadecane b i n d i n g as t h e c o n c e n t r a t i o n o f t r y p s i n i n c r e a s e d from 50-250 ug p e r m l . I t r e a c h e d a minimum o f 49% a t 250 ug/ml and d i d n o t change w i t h f u r t h e r i n c r e a s e s . The h y d r o p h o b i c i t y was n o t s i g n i f i c a n t l y a f f e c t e d by any of t h e o t h e r t r e a t m e n t s , i n c l u d i n g b o i l i n g t h e c e l l s i n SDS ( T a b l e V I ) . A s i m i l a r r e s u l t was o b t a i n e d w i t h s t r a i n I2na. B o t h b o i l i n g and t r y p s i n t r e a m e n t d e s t r o y e d t h e a b i l i t y of t h e c e l l s t o a g g r e g a t e i n s a l i v a . 68 TABLE VI E f f e c t o f c e l l s u r f a c e m o d i f i c a t i o n and a g g r e g a t i o n of S. s a n g u i s 12. on h y d r o p h o b i c i t y % T r e a t m e n t A d s o r b e d t o h e x a d e c a n e S a l i v a r y a g g r e g a t i o n t i t e r None 1 SDS (room temp.) /-\ 91 256 82 256 \J 100 C, 15 min. 2 o SDS (100 C, 15 min 3 T r y p s i n ( l O u g / m l ) 84 .) 87 64 o 4 8 T r y p s i n (300ug/ml) A 51 0 H e a t - d e n a t u r e d t r y p s i n (1mg/ml) 86 256 1 C e l l s were i n c u b a t e d w i t h 3% SDS f o r 15 min. C e l l s were i n c u b a t e d w i t h 3% SDS. 3 T r y p s i n was i n a c t i v a t e d by t h e a d d i t i o n of 0.5 mg/ml t r y p s i n i n h i b i t o r . 4 T r y p s i n i n a c t i v a t e d by b o i l i n g f o r 30 min p r i o r t o i n c u b a t i o n w i t h c e l l s . 69 F i g u r e 9. Changes i n s u r f a c e h y d r o p h o b i c i t y of S. s a n q u i s i n c u b a t e d w i t h t r y p s i n . C e l l s were ' i n c u b a t e d w i t h enzyme f o r one h r . The a d s o r p t i o n t o hexadecane ( O ) t r y p s i n ; ( A ) i n a c t i v a t e d t r y p s i n . 70 90-i 1 1 1 1 i i ' 0 100 200 300 400 500 600 Trypsin(ug/ml) 7.0 & Heat i n a c t i v a t e d t r y p s i n had no e f f e c t on a g g r e g a t i o n . In o r d e r t o e l i m i n a t e t h e p o s s i b i l i t y t h a t r e s i d u a l t r y p s i n was i n t e r f e r i n g w i t h the r e a c t i o n , c e l l s were i n c u b a t e d w i t h a z o c o l l f o r 1 h r . No p r o t e o l y t i c a c t i v i t y was d e t e c t e d . T r y p s i n i n h i b i t o r w h i c h was u s e d t o s t o p t r y p s i n a c t i v i t y had no e f f e c t on t h e a g g r e g a t i n g a c t i v i t y . T r y p s i n - t r e a t e d c e l l s o f S_^  s a n g u i s 12 were d i g e s t e d w i t h m u t a n o l y s i n and examined by SDS-PAGE ( F i g . 1 0 ) . T r e a t m e n t w i t h 50ug/ml t r y p s i n f o r 1 h r , w h i c h l a r g e l y d e s t r o y e d s a l i v a r y a g g r e g a t i n g a c t i v i t y , r e s u l t e d i n t h e e l i m i n a t i o n o f t h e 160,000 MW p r o t e i n from t h e c e l l s . A r e d u c t i o n was a l s o seen i n bands o f m o l e c u l a r w e i g h t 72,000,52,000,37,000 and 35,000 ( F i g . 1 1 ) . However, t h e s i g n i f i c a n c e of t h e s e l o w e r m o l e c u l a r w e i g h t bands i s d o u b t f u l i n view of t h e f a c t t h a t c o r r e s p o n d i n g bands d i d n o t a p p e a r p r o m i n e n t i n m u t a n o l y s i n d i g e s t of c e l l w a l l s ( F i g . 7 ) . L i t t l e f u r t h e r change i n t h e g e l p r o f i l e c o u l d be seen i n m u t a n o l y s i n d i g e s t of c e l l s w h i c h had been t r e a t e d w i t h a h i g h e r c o n c e n t r a t i o n o f t r y p s i n (300 u g / m l ) . I t i s i m p o r t a n t t o n o t e t h a t w i t h 50 ug/ml of t r y p s i n , t h e 160,000 MW p r o t e i n i s l o s t from t h e whole c e l l d i g e s t . When t h e c o n c e n t r a t i o n o f t r y p s i n was r e d u c e d t o 10 ug/ml, t h e r e was a l s o a d e c r e a s e i n h e x a d e a c a n e b i n d i n g as w e l l as t h e l o s s of t h e 160,000 MW p r o t e i n . T h i s i n d i c a t e d t h a t some o f t h e s u r f a c e l o c a t e d p r o t e i n s c o n t r i b u t e d a t l e a s t i n p a r t t o t h e a d h e r e n c e t o h e x a d e c a n e by S^ s a n g u i s . C o n t r o l s i n c u b a t e d under t h e same c o n d i t i o n , but w i t h o u t t r y p s i n had t h e same g e l p r o f i l e a s 71 F i g u r e 10. SDS-PAGE of m u t a n o l y s i n d i g e s t s o f whole c e l l s o f S^ s a n g u i s 12 o b t a i n e d a f t e r t r e a t m e n t w i t h t r y p s i n : Lane 1, no t r y p s i n ; Lane 2, 50ug/ml t r y p s i n ; Lane 3, 1OOug/ml t r y p s i n ; Lane 4, 150ug/ml t r y p s i n ; Lane 5, 200ug/ml t r y p s i n ; Lane 6, 250ug/ml t r y p s i n ; Lane 7, 500ug/ml t r y p s i n ; Lane 8, 1OOOug/ml t r y p s i n . The 160,000 MW band i s i n d i c a t e d . M o l e c u l a r w e i g h t m a r k e r s a r e i n d i c a t e d by l a r g e a r r o w s . 72 1 2 3 4 5 6 7 8 72a F i g u r e 11. SDS-PAGE of s a n g u i s 12 whole c e l l s t r e a t e d w i t h t r y p s i n : Lane 1, m u t a n o l y s i n d i g e s t o f u n t r e a t e d c e l l s ; L ane 2, m u t a n o l y s i n d i g e s t of c e l l s t r e a t e d w i t h I0ug/ml t r y p s i n ; Lane 3, m u t a n o l y s i n d i g e s t o f c e l l s t r e a t e d w i t h 300ug/ml o f t r y p s i n ; Lane 4, s u p e r n a t a n t from 1Oug/ml t r y p s i n d i g e s t ; Lane 5, s u p e r n a t a n t f r o m 300ug/ml t r y p s i n d i g e s t . A r rows i n d i c a t e p o s i t i o n s o f m o l e c u l a r w e i g h t m a r k e r s . 7 3 7 3 ^ Figure 12. SDS-PAGE of material released from whole c e l l s of S_^  sanguis 12 treated with trypsin. C e l l s were incubated with • increasing concentration of trypsin and supernatants electrophoresed: Lane 1, 50ug/ml; Lane 2, I00ug/ml; Lane 3, 150ug/ml; Lane 4, 200ug/ml; Lane 5, 250ug/ml; Lane 6, 500ug/ml; Lane 7, I000ug/ml; Lane 8, no trypsin; Lane 9, 50ug/ml of heat inactivated trypsin. The 160,000 MW region i s indicated by the small arrow. Molecular weight markers are indicated by large arrows. 74, 7Aa untreated c e l l s . Material released into solution by treatment with 50 ug/ml of trypsin contained a number of high molecular weight components (Fig. 12), including several bands with molecular weight of 200,000 and a heavily stained band in the region of MW 160,000. In some cases these proteins were not seen after treatment with 300 ug/ml of trypsin, and were presumably degraded to lower molecular weight components (Fig. 11). Rocket immunoelectrophoresis with antiserum raised against whole c e l l s of str a i n 12 confirmed that trypsin released a number of molecules with antigenic a c t i v i t y . No remaining c e l l u l a r antigens could be found in mutanolysin digests of c e l l s after treatment with even the lowest (10 ug/ml) concentration of trypsin. Fractionation of c e l l walls In view of the complexity of the gel p r o f i l e s obtained from mutanolysin digests of sanguis 12 c e l l walls, i t was decided to fractionate these digests with the hope of separating the high molecular weight constituents. Samples were fractionated on a molecular sieving column by HPLC. Digests in HEPES buffer were applied to the column and eluted as shown in Fig. 13. The majority of the sample absorbing at A 2 8o eluted at the void volume indicating a maximum molecular weight of 500,000. This suggests that the digest i s composed of high molecular weight complexes which are not separated by digestion with mutanolysin. 75 F i g u r e 13. Crude c e l l w a l l d i g e s t o f S_^  s a n g u i s 12 f r a c t i o n a t e d on HPLC. D i g e s t were a p p l i e d o n t o a p r o t e i n pak 300 column and e l u t e d w i t h 0.05M HEPES, 0.1M N a C l , 0.02% sodium a z i d e b u f f e r pH 7.2 and a b s o r b a n c e of e l u a t e was m o n i t o r e d a t 280nm. f r a c t i o n numbers a r e i n d i c a t e d . S o l i d c i r c l e s r e p r e s e n t f r a c t i o n numbers. 76: 0.5 < i i i i I i 1 0 5 10 15 20 25 30 Fraction number F i g u r e 14. SDS-PAGE o f HPLC f r a c t i o n s o f c r u d e c e l l w a l l d i g e s t o f S^ s a n g u i s 12: Lane 1, f r a c t i o n 8; Lane 2, f r a c t i o n 9; Lane 3, f r a c t i o n 10; Lane 4, f r a c t i o n 11; Lane 5, f r a c t i o n 12; Lane 6, f r a c t i o n 13; Lane 7, f r a c t i o n 14; Lane 8, f r a c t i o n 15; Lane 9, f r a c t i o n 16; Lane 10, 12 c e l l w a l l d i g e s t . The 160,000 MW band i s i n d i c a t e d . M o l e c u l a r w e i g h t m a r k e r s a r e i n d i c a t e d by l a r g e arrows'. 77-1 2 3 4 5 6 7 8 9 1 0 775. m u t a n o l y s i n . S m a l l peaks e l u t e d ' a t f r a c t i o n s 10 and 14. A l l f r a c t i o n s were a n a l y z e d by 7.5% SDS-PAGE ( F i g . 1 4 ) . F r a c t i o n 7 c o n t a i n e d a s m a l l amount of s i l v e r s t a i n i n g m a t e r i a l which m i g r a t e d o n l y a v e r y s m a l l d i s t a n c e i n t o t h e g e l . F r a c t i o n s 8, 9 wh i c h formmed t h e v o i d volume c o n t a i n e d a number o f components h a v i n g m o l e c u l a r w e i g h t s r a n g i n g f r o m 200,000 t o 92,000. P r e s u m a b l y t h e y formed a l a r g e complex o r were p r e s e n t as m u l t i m e r s w h i c h were s e p a r a t e d i n t o s u b u n i t s by b o i l i n g i n SDS-BME. No p r o t e i n s c o u l d be seen a f t e r f r a c t i o n 16. The 160,000 MW p r o t e i n was s p r e a d t h r o u g h f r a c t i o n 8-11. Does t h i s i n d i c a t e t h a t t h e 160,000 MW p r o t e i n e x i s t s i n c o m b i n a t i o n w i t h a number of o t h e r c o n s t i t u e n t s w h i c h c a n be s e p a r a t e d on t h e b a s i s o f s i z e o r t h a t i t e x i s t s i n d i f f e r e n t m u l t i p l e s o f a b a s i c s u b u n i t ? F r a c t i o n s were a l s o a n a l y z e d by r o c k e t i m m u n o e l e c t r o p h o r e s i s ( F i g . 15). I t can be seen t h a t a l l t h e a n t i g e n s a r e e l u t e d i n f r a c t i o n s 8-16. F r a c t i o n s 8-11 c o n t a i n a number o f a n t i g e n s and m i r r o r t h e c o m p l e x i t y o b s e r v e d i n SDS-PAGE. F r a c t i o n s 14, 15, 16 p r e s e n t a s i m p l e p i c t u r e . Many of t h e s e f r a c t i o n s c o n t a i n a n t i g e n s w h i c h a p p e a r t o c r o s s - r e a c t w i t h one a n o t h e r . F r a c t i o n s 8-14 were e l e c t r o p h o r e s e d i n 7.5% SDS-PAGE and s u b j e c t e d t o w e s t e r n b l o t a n a l y s i s 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 whole c e l l s of s t r a i n s 12 and 12L and a g a i n s t t h e 160,000MW p r o t e i n ( F i g . 16). The 160,000 MW p r o t e i n a n t i s e r u m was k i n d l y p r o v i d e d by E. J . M o r r i s . 78 F i g u r e 15. R o c k e t i m m u n o e l e c t r o p h o r e s i s of c r u d e c e l l w a l l d i g e s t s o f s a n g u i s 12 f r a c t i o n a t e d on HPLC a g a i n s t a n t i - 1 2 serum: ( A ) , 12 c e l l w a l l d i g e s t . F r a c t i o n numbers a r e i n d i c a t e d . 79 F i g u r e 16. W e s t e r n b l o t s o f HPLC f r a c t i o n s o f c r u d e c e l l w a l l d i g e s t s of s a n g u i s 12 s e p a r a t e d by 7.5% SDS-PAGE. F r a c t i o n s a d s o r b e d t o n i t r o c e l l u l o s e were r e a c t e d w i t h : ( A ) , a n t i - 1 2 serum; ( B ) , a n t i - 1 2 L serum;- ( C ) , ' a n t i 160,000 MW p r o t e i n serum. HPLC f r a c t i o n s : Lane 1, f r a c t i o n 8; Lane 2, f r a c t i o n 9; Lane 3, f r a c t i o n 10; Lane 4, f r a c t i o n 11; Lane 5, f r a c t i o n 12; Lane 6, f r a c t i o n 13; Lane 7, f r a c t i o n 14. The 160,000 MW p r o t e i n i s i n d i c a t e d . 80* 802s F i g u r e 17. C r o s s e d i m m u n o e l e c t r o p h o r e s i s of c r u d e c e l l w a l l d i g e s t of S_;_ s a n g u i s 12. • D i g e s t s was s e p a r a t e d by 7.5% SDS-PAGE and e l e c t r o p h o r e s e d i n t o a g a r o s e c o n t a i n i n g : ( A ) , a n t i - 1 2 serum; ( B ) , a n t i - 1 2 serum a d s o r b e d w i t h 12L c e l l s . The 160,000 MW p r o t e i n i s i n d i c a t e d by an a rrow. The s i l v e r n i t r a t e s t a i n e d g e l i s r e p r e s e n t e d d i a g r a m a t i c a l l y b e n e a t h 17 b. 81-8 & C r o s s e d i m m u n o e l e c t r o p h o r e s i s C e l l w a l l d i g e s t s of Sj_ s a n g u i s 12 were a n a l y s e d by two d i m e n s i o n a l i m m u n o e l e c t r o p h o r e s i s ( F i g . 1 7 ) . Samples were s e p a r a t e d i n SDS-PAGE f o l l o w e d by e l e c t r o p h o r e s i s i n t o a g a r o s e c o n t a i n i n g a n t i s e r u m r a i s e d a g a i n s t whole c e l l s of S. s a n g u i s 12 ( F i g . 17A). A number of p r e c i p i t i n l i n e s c a n be s e e n . The s t r o n g e s t r e a c t i o n o c c u r r e d i n t h e r e g i o n o f t h e 160,000 MW p r o t e i n , where a maximum of 4 p r e c i p i t i n l i n e s c a n be s e e n . Some of t h e a n t i g e n s h a v i n g d i f f e r e n t m o l e c u l a r w e i g h t a p p e a r t o c r o s s - r e a c t w i t h one a n o t h e r . F a i n t p r e c i p i t i n l i n e s can be seen i n t h e r e g i o n of 200,000 MW. These a n t i g e n s a p p e a r t o be c r o s s - r e a c t i v e a s w e l l . In a d d i t i o n t h e r e a r e a number of f a i n t l y s t a i n i n g bands w i t h m o l e c u l a r w e i g h t s r a n g i n g from 116,000 t o 45,000. When a n t i - 1 2 serum was a d s o r b e d w i t h t h e h y d r o p h i l i c 12L c e l l s , t h e a n t i b o d i e s w h i c h r e a c t e d s t r o n g l y w i t h t h e a n t i g e n i n the 160,000 MW r e g i o n were not a d s o r b e d . T h i s p r o v i d e f u r t h e r e v i d e n c e t h a t t h e s e a n t i g e n s a r e not f o u n d on t h e c e l l s u r f a c e o f 12L. A s i m i l a r a b s o r b t i o n e x p e r i m e n t p e r f o r m e d w i t h whole c e l l s o f 12 removed a l l a n t i b o d i e s . A n t i s e r u m r a i s e d a g a i n s t 12L was f o u n d t o c o n t a i n a n t i b o d y r e a c t i v e w i t h t h e 160,000 MW a n t i g e n s and a number of o t h e r a n t i g e n s . These a n t i b o d i e s c o u l d be a d s o r b e d w i t h whole c e l l s o f 12. These r a t h e r s u r p r i s i n g r e s u l t s s u g g e s t t h a t s t r a i n 12L s t i l l s y n t h e s i z e s t h e s e h i g h MW p r o t e i n s , b u t c a n n o t i n c o r p o r a t e 82 them i n t o the c e l l w a l l . S i m i l a r r e s u l t s have been r e p o r t e d f o r A. v i s c o s u s (15) and S^ s a n g u i s FW213 ( 3 9 ) . A d h e r e n c e i n h i b i t i o n I f t h e c e l l w a l l d i g e s t s c o n t a i n e d the a d h e s i n s r e s p o n s i b l e f o r b i n d i n g t o S-HA, t h e y m i g h t ' b e a b l e t o b l o c k b i n d i n g of whole c e l l s of s t r a i n 12 by c o m b i n i n g w i t h t h e s a l i v a r y p e l l i c l e r e c e p t o r . Based on t h e b i o c h e m i c a l and i m m u n o l o g i c a l d a t a one would p r e d i c t t h a t t h e d i g e s t s o f 12L would not i n h i b i t b i n d i n g . C e l l w a l l d i g e s t s were i n c u b a t e d w i t h S-HA p r i o r t o t h e a d d i t i o n of c e l l s and t h e i r a b i l i t y t o i n h i b i t a t t a c h m e n t measured. As can be seen i n T a b l e VII d i g e s t s p r e p a r e d from 12 i n h i b i t e d b i n d i n g by 36%. A s i m i l a r d i g e s t o f 12na i n h i b i t e d 19%. S i m i l a r r e s u l t s were o b t a i n e d w i t h f i v e s e p a r a t e e x p e r i m e n t s . D i g e s t s o f t h e n o n - a d h e r e n t h y d r o p h i l i c 12L d i d not i n h i b i t b i n d i n g . The i n h i b i t o r y a c t i v i t y was h e a t s e n s i t i v e . B o i l i n g f o r 30 min d e s t r o y e d t h e a c t i v i t y . D i g e s t s p r e p a r e d from SDS e x t r a c t e d c e l l w a l l s had l i t t l e i n h i b i t o r y a c t i v i t y . The s e n s i t i v i t y t o SDS c o u l d r e f l e c t i n a c t i v a t i o n o r e x t r a c t i o n of t h e a d h e s i n . The i n h i b i t o r y a c t i v i t y of b o i l e d 12L d i g e s t c a n n o t be e x p l a i n e d a t t h i s t i m e . I n h i b i t o r y e f f e c t s s e e n w i t h d i f f e r e n t d i g e s t s c o r r e l a t e w e l l w i t h e a r l i e r o b s e r v a t i o n s on t h e a d h e r e n c e p r o p e r t i e s o f t h e s e s t r a i n s . But t h e i n h i b i t i o n was n o t as s t r o n g as one w o u l d e x p e c t . T h i s c o u l d be due t o t h e method o f p r e p a r i n g c e l l w a l l s . Whole c e l l s were b r o k e n w i t h g l a s s b e a d s , w h i c h might have 83 TABLE V I I E f f e c t of c e l l w a l l d i g e s t s on t h e a d h e r e n c e of S. s a n g u i s 12 t o S-HA. I n h i b i t o r % A d h e r e n c e u n h e a t e d h e a t e d HEPES b u f f e r 100 12 c r u d e c e l l w a l l 64 12na ' - ." • 81 12L " 102 1 06 1 06 75 a 10 10 c e l l s were i n c u b a t e d w i t h 40mg o f S-HA f o l l o w i n g p r e i n c u b a t i o n of S-HA w i t h t h e i n h i b i t o r . b i n h i b i t o r b o i l e d f o r 30 min. 84 removed many a d h e s i n s w h i c h would not s e d i m e n t w i t h t h e c e l l w a l l s . T h e r e f o r e t h e c e l l w a l l may be d e v o i d o f many of i t s p u t a t i v e a d h e s i n s . A l l f r a c t i o n s o b t a i n e d f r o m HPLC ( F i g . 13) f r a c t i o n a t i o n of c e l l w a l l d i g e s t s f r o m s t r a i n 12 were t e s t e d t o d e t e r m i n e i f t h e y c o u l d i n h i b i t b i n d i n g t o S-HA. T h e r e a p p e a r e d t o be a some i n h i b i t o r y a c t i v i t y i n f r a c t i o n s 7-11, but t h e a c t i v i t y was so low t h a t f u r t h e r s t u d y o f t h e s e f r a c t i o n s was d i s c o n t i n u e d . 85 DISCUSSION The i s o l a t i o n o f S^ s a n g u i s v a r i a n t s w i t h d e f e c t s i n a d h e r e n c e p r o p e r t i e s has made p o s s i b l e a c o m p a r a t i v e s t u d y of t h e c h e m i s t r y and m o r p h o l o g y o f t h e i r c e l l s u r f a c e s . These v a r i a n t s c o m p r i s e (1) s t r a i n s w h i c h r e t a i n t h e a b i l i t y of t h e p a r e n t ' s s t r a i n t o a d s o r b t o h e x a d e c a n e , but have l o s t t h e p a r e n t s a b i l i t y t o a g g r e g a t e i n s a l i v a , and show a l t e r e d c h a r a c t e r i s t i c s i n t h e i r a d h e r e n c e t o S-HA; and (11) S t r a i n s w h i c h do not a d s o r b t o h e x a d e c a n e , do n o t a g g r e g a t e i n s a l i v a , and show much r e d u c e d a d h e r e n c e t o S-HA. The a d h e r e n c e and a g g r e g a t i o n n e g a t i v e s t r a i n s d e s c r i b e d i n t h e e x p e r i m e n t s a r o s e s p o n t a n e o u s l y , and were not s e l e c t e d f o l l o w i n g m u t a g e n e s i s . Thus i t i s n o t p o s s i b l e t o say t h a t t h e y a r e m u t a n t s and a c c o r d i n g l y t h e y a r e r e f e r r e d t o as v a r i a n t s . I t i s p o s s i b l e t h a t s t r a i n 12na, w h i c h was a b l e t o r e g a i n some i t s a g g r e g a t i n g a c t i v i t y , i s a phase v a r i a n t a n a l o g o u s t o t h o s e d e s c r i b e d i n N e i s s e r i a ( 1 5 2 ) . S i m i l a r r e v e r s i o n was not o b s e r v e d among t h e i s o l a t e s of h y d r o p h i l i c s t r a i n s . A s t r o n g c o r r e l a t i o n was o b s e r v e d between t h e p o s s e s s i o n of a d i f f u s e l y s t a i n i n g p r o t e i n of MW 160,000 seen i n S D S - p o l y a c r y l a m i d e g e l s and t h e a b i l i t y t o a g g r e g a t e i n s a l i v a . T h i s p r o t e i n was f o u n d i n c e l l d i g e s t s of the p a r e n t s t r a i n 12, but was much r e d u c e d i n t h e v a r i a n t I2na. I t i s p a r t i c u l a r l y i n t e r e s t i n g t o n o t e t h a t t h e v a r i a n t I2na/1 which has p a r t i a l l y r e g a i n e d t h e a b i l i t y t o a g g r e g a t e c o n t a i n e d a s m a l l amount of t h i s p r o t e i n whereas s t r a i n !2na/2 which does not a g g r e g a t e 86 l a c k e d t h e p r o t e i n e n t i r e l y . The n o n - a g g r e g a t i n g h y d r o p h i l i c s t r a i n s were a l s o m i s s i n g t h i s p r o t e i n . The 160,000 MW p r o t e i n was f o u n d t o be m i s s i n g from whole c e l l s d i g e s t e d w i t h t r y p s i n . T r y p s i n t r e a t m e n t r e s u l t e d i n t h e l o s s of s a l i v a r y a g g r e g a t i n g a c t i v i t y , and has been shown p r e v i o u s l y t o e l i m i n a t e b i n d i n g t o S-HA ( 1 3 9 ) . I t has a l s o been r e p o r t e d (139) t h a t t r y p s i n d i g e s t i o n c a u s e d t h e l o s s of a 160,000 MW p r o t e i n , as w e l l as p r o t e i n s of 92,000 and 86,000. The i n v o l v e m e n t o f a c e l l s u r f a c e p r o t e i n i n s a l i v a r y a g g r e g a t i o n was f u r t h e r i m p l i c a t e d by t h e f a c t t h a t b o i l i n g d e s t r o y e d a g g r e g a t i n g a c t i v i t y . E v i d e n c e f o r a c e l l s u r f a c e l o c a t i o n f o r t h e 160,000 MW p r o t e i n was f o u n d i n t h e o b s e r v a t i o n t h a t ; (1) a band of 'comparable m o l e c u l a r w e i g h t was f o u n d i n SDS e x t r a c t e d c e l l w a l l , (2) t h e p r o t e i n was removed d u r i n g t r y p s i n d i g e s t i o n of whole c e l l s , and (3) t h e p r o t e i n r e a c t e d w i t h t h e a n t i s e r u m r a i s e d a g a i n s t whole c e l l s o f s t r a i n -12 when a n a l y z e d by c r o s s e d i m m u n o e l e c t r o p h o r e s i s f o l l o w i n g SDS-PAGE. A d s o r p t i o n of t h e a n t i s e r u m w i t h s t r a i n 12 but not s t r a i n 12L removed a n t i b o d i e s r e a c t i n g w i t h t h i s p r o t e i n . T h e s e o b s e r v a t i o n s s u p p o r t t h e view t h a t t h e 160,000 MW p r o t e i n i s l o c a t e d on t h e c e l l s u r f a c e and t h a t i t i s m i s s i n g from th e s u r f a c e of s t r a i n 12L. I t i s p o s s i b l e t h a t t h i s p r o t e i n c o u l d be t h e s i a l i c - a c i d b i n d i n g l e c t i n w h i c h i s r e s p o n s i b l e f o r n e u r a m i n i d a s e s e n s i t i v e s a l i v a r y a g g r e g a t i o n ( 1 0 2 ) , and w hich a c t s as an a d h e s i n m e d i a t i n g b i n d i n g t o S-HA ( 1 03 ). However a c o m p a r i s o n of SDS e x t r a c t e d 87 c e l l walls of 12 and I2na showed differences in at least eight other bands, a number of them with a molecular weight higher than 160,000. Based on the findings with s a l i v a r i u s (159,160) and S_j_ mutans (97) one would anticipate that i t i s the higher molecular weight proteins that are responsible for adherence. A s i a l i c acid binding l e c t i n from S_^  mitior has been shown to have have components of MW 85,000, 77,000 and 109,000 (P. A. Murray, M. J. Levine, L. A. Tabak and M. S. Reddy, J. Dent. Res. special issue 62: A789, 1983). Liljemark and Bloomquist (87) have i d e n t i f i e d a protein-containing adherence blocking component from S. sanguis with a MW between 70,000 and 90,000, while a galactose-binding l e c t i n i d e n t i f i e d by Nagata et a l . (107) had a MW of 20,000. Unfortunately, there is no correlation between the molecular weights of any of these putative adhesins and the molecular weights of proteins bands which were present in SDS extracted c e l l walls of 12 but not from 12na. It seems unlikely that the proteins seen in our c e l l wall preparations represent contaminating membrane or cytoplasmic components since comparable bands were not seen in c e l l wall digests of str a i n 12L. Appelbaum and Rosan (2) have reported a similar complexity in c e l l surface protein composition using radioiodination techniques. In l i g h t of the experience with other species of streptococci (97) i t was surprising to find that b o i l i n g in SDS-BME did not extract s i g n i f i c a n t amounts of proteins from whole c e l l s of S_^  sanguis. However this is consistent with the 88 o b s e r v a t i o n o f R e u s c h ( 1 3 0 ) , who f o u n d d i f f i c u l t y i n e x t r a c t i n g c e l l w a l l p r o t e i n s from s a n g u i s 34. W h i l e t h e a b s e n c e of s a l i v a r y a g g r e g a t i n g a c t i v i t y from s t r a i n 12na a p p e a r s t o i n v o l v e t h e s e l e c t i v e l o s s o f c e r t a i n c e l l s u r f a c e m o l e c u l e s , c e l l w a l l s f r o m v a r i a n t s w h i c h i n a d d i t i o n do n o t a d s o r b t o h e x a d e c a n e d i d not c o n t a i n any s i l v e r s t r a i n i n g p r o t e i n s . S i n c e t h e change from a h y d r o p h o b i c t o a h y d r o p h i l i c c e l l s u r f a c e i n v o l v e s . t h e l o s s o f most of t h e c e l l w a l l a s s o c i a t e d p r o t e i n s , i t i s t h e r e f o r e d i f f i c u l t t o a s c r i b e h y d r o p h o b i c i t y t o any p a r t i c u l a r m o l e c u l e . I t i s p o s s i b l e t h a t components c o n f e r r i n g h y d r o p h o b i c i t y may e x i s t as l a r g e c o m p l e x e s on t h e c e l l s u r f a c e . A l t h o u g h t r e a t m e n t w i t h t r y p s i n a f f e c t e d b o t h s a l i v a r y a g g r e g a t i o n and h y d r o p h o b i c i t y t h e f a c t t h a t b o i l i n g d e s t r o y e d a g g r e g a t i o n w i t h o u t c a u s i n g any r e d u c t i o n i n h y d r o p h o b i c i t y s u g g e s t s t h a t h y d r o p h o b i c i t y a s s o c i a t e d m o l e c u l e s may not be c l o s e l y a s s o c i a t e d w i t h a d h e s i n s r e s p o n s i b l e f o r s a l i v a r y a g g r e g a t i o n . T h i s a l s o e m p h a s i z e s t h a t s i m p l y p o s s e s s i n g a h y d r o p h o b i c s u r f a c e does n o t a l l o w an o r g a n i s m t o b i n d a g g r e g a t i o n i n d u c i n g s a l i v a r y m a c r o m o l e c u l e s . The i n a b i l i t y t o r e d u c e h y d r o p h o b i c i t y by b o i l i n g i n SDS s u g g e s t s t h a t h y d r o p h o b i c i t y i s a s s o c i a t e d w i t h m o l e c u l e s bound c o v a l e n t l y t o t h e c e l l w a l l . T h e i r i n s e n s i t i v i t y t o h e a t and t h e l i m i t e d r e d u c t i o n i n h y d r o p h o b i c i t y p r o d u c e d by t r e a t m e n t w i t h even t h e h i g h e s t c o n c e n t r a t i o n o f t r y p s i n s u g g e s t t h a t a n o n - p r o t e i n a c i o u s m o l e c u l e s u c h as LTA might be i n v o l v e d ( 1 0 0 ) . I t i s q u i t e p o s s i b l e b o t h LTA and p r o t e i n s c o n t r i b u t e t o 89 h y d r o p h o b i c i t y . T h e s e f i n d i n g s a r e i n c o n t r a s t t o r e s u l t s o b t a i n e d p r e v i o u s l y w i t h p yogenes (114) and mutans ( 9 7 ) , where a d h e r e n c e t o h e x a d e c a n e was e l i m i n a t e d by b o i l i n g and t r y p s i n t r e a t m e n t . However u n l i k e S_^  s a n g u i s , b o i l i n g removed c e l l s u r f a c e p r o t e i n s f r o m S_^  mutans ( M c B r i d e e t a l . , 1984). The f i n d i n g s t h a t h y d r o p h i l i c s t r a i n s o f s a n g u i s had l o s t most o f t h e c e l l w a l l a s s o c i a t e d p r o t e i n s s u g g e s t s t h a t t h e c e n t r a l mechanism i n v o l v e d i n e x p r e s s i o n , t r a n s p o r t or i n c o r p o r a t i o n o f t h e s e m o l e c u l e s has been a l t e r e d . T h e r e i s no e v i d e n c e t h a t t h e s e m o l e c u l e s a r e a s s o c i a t e d w i t h e x t r a c h r o m o s o m a l e l e m e n t s ( u n p u b l i s h e d o b s e r v a t i o n s ) . H y d r o p h i l i c s t r a i n s o f S_;_ mutans were a l s o f o u n d t o have l o s t a number of c e l l w a l l p r o t e i n s ( 9 7 ) , but i n t h i s c a s e t h e p r o t e i n s were* f o u n d i n t h e c u l t u r e s u p e r n a t a n t s i n d i c a t i n g t h e r e was a d e f e c t i n t h e c e l l ' s a b i l i t y t o i n c o r p o r a t e t h e s e m o l e c u l e s i n t h e c e l l w a l l . In t h e c a s e o f S. s a l i v a r i u s a d h e r e n c e n e g a t i v e mutants w h i c h were u n a b l e t o t r a n s p o r t a d h e s i n s have been f o u n d ( A. H. Weerkamp, p e r s o n a l c o m m u n i c a t i o n ) . U n f o r t u n a t e l y t h e l o s s of so many p r o t e i n s from t h e h y d r o p h i l i c S_^  s a n g u i s makes i t d i f f i c u l t t o i d e n t i f y t h e s p e c i f i c a d h e s i n s i n v o l v e d i n t h e complex S-HA a d h e r e n c e r e a c t i o n . B i o c h e m i c a l and i m m u n o l o g i c a l d i f f e r e n c e s between t h e s t r a i n s c o r r e s p o n d e d t o m o r p h o l o g i c a l d i f f e r e n c e s r e v e a l e d by e l e c t r o n m i c r o s c o p y . T h i s i s p a r t i c u l a r l y a p p a r e n t when t h e SDS-PAGE p r o f i l e s of c e l l w a l l d i g e s t s of s t r a i n 12 and 12L a r e compared t o e l e c t r o n m i c r o g r a p h s of t h e o r g a n i s m s . S_;_ s a n g u i s 12 90 has a complex c e l l w a l l p r o t e i n c o n t e n t which i s matched by t h e t o p o g r a p h i c a l c o m p l e x i t y o f i t s c e l l s u r f a c e . In c o n t r a s t s t r a i n 12L has v e r y few p r o t e i n s i n t h e c e l l w a l l and i s d e v o i d of any i d e n t i f i a b l e s u r f a c e s t r u c t u r e s . I t i s p o s s i b l e t h a t f i b r i l l a r s t r u c t u r e s seen on our s t r a i n s may be s i m p l y c o n d e n s e d s t a n d s of c o a t m a t e r i a l . N e g a t i v e s t r a i n i n g w i t h p h o s p h o t u n g s t i c a c i d d i d n o t r e v e a l any f i b r i l s even when an i n c u b a t i o n s t e p w i t h s p e c i f i c a n t i s e r u m was i n c l u d e d t o s t a b i l i z e c a p s u l a r m a t e r i a l ( 9 1 ) . N e v e r t h e l e s s , a number of w o r k e r s who have examined o t h e r s t r a i n s of S_^  s a n g u i s i n t h e e l e c t r o n m i c r o s c o p e have f o u n d e v i d e n c e f o r p e r i t r i c h o u s f i b r i l s (42,69) as w e l l as p o l a r f i m b r i a e (42,55,67) o r t u f t s ( 1 0 4 ) . The p o l a r f i m b r i a e have been p r o p o s e d t o m e d i a t e a d h e r e n c e t o B a c t e r i o n e m a m a t r u c h o t i i ( 1 0 4 ) , t o be r e s p o s i b l e f o r h y d r o p h o b i c i t y ( 5 5 ) , t o c a u s e t w i t c h i n g m o t i l i t y (43,67) and t o s t a b i l i z e (but not t o be e s s e n t i a l f o r ) b i n d i n g t o S-HA ( 4 3 ) . In c o n t r a s t p e r i t r i c h o u s f i b r i l s have been s u g g e s t e d t o be e s s e n t i a l f o r a d h e r e n c e t o S-HA (39) as w e l l as m e d i a t i n g h a e m a g g l u t i n a t i o n and s a l i v a - m e d i a t e d a g g r e g a t i o n ( 6 9 ) . S t r a i n s a n g u i s 12 and i t s v a r i a n t s do not h a e m a g g l u t i n a t e . However f i n d i n g s r e p o r t e d h e r e would s u p p o r t a r o l e f o r f i b r i l l a r s t r u c t u r e s i n h y d r o p h o b i c i t y and a d h e r e n c e t o S-HA. The i n a b i l i t y t o remove c e l l s u r f a c e c o n s t i t u e n t s by g e n t l e p r o c e d u r e s l e d t o t h e n e c e s s i t y of p r e p a r i n g c e l l w a l l s . W a l l s were made by r e l a t i v e l y h a r s h p h y s i c a l p r o c e d u r e s which may have 91 led to the selective loss of delicate surface structures. These structures would have remained in the supernatant together with cytoplasmic material during p u r i f i c a t i o n of the walls. Comparison of the amount of the 160,000 MW protein in whole c e l l digests versus c e l l wall digests supports the supposition that this material i s p a r t i a l l y lost during wall preparation. This also might explain why s o l u b i l i z e d c e l l walls of strain 12 caused only 36% i n h i b i t i o n of adherence. The whole c e l l s were not subjected to any harsh physical procedures and yielded substantially more of the 160,000 MW protein. The r e l a t i v e l y large number of cross-reacting components released by mutanolysin digestion could be attributable, at least in part, to release of molecules with d i f f e r i n g length of attached peptidoglycan. The fact that c e l l wall proteins appear to be covalently linked to the peptidoglycan would be consistent with this p o s s i b i l i t y . The necessity for prolonged incubation with mutanolysin could create a r t i f a c t s a r i s i n g from hydrolysis of the wall constituents by low levels of endogenous enzymes or contaminants in the mutanolysin. These p o s s i b i l i t i e s were examined by incubating c e l l s without mutanolysin and by treating mutanolysin with PMSF, but these experiments do not rule out the p o s s i b i l i t y that mutanolysin, activated an endogenous enzyme or liberated a substrate which became sensitive to hydrolysis when s o l u b i l i z e d . The adherence blocking a c t i v i t y exhibited by digests of strain 12 c e l l walls is believed to be due to the presence of 92 s p e c i f i c a d h e s i n s s o l u b l i z e d by m u t a n o l y s i n . The l i m i t e d e x t e n t of i n h i b i t i o n c o u l d be t h e r e s u l t of a number of f a c t o r s i n c l u d i n g : (a) l o s s of a d h e s i n s d u r i n g p r e p a r a t i o n of c e l l w a l l s ; (b) i n a c t i v a t i o n o f a d h e s i n s d u r i n g s o l u b i l i z a t i o n ; or (c) d e s t r u c t i o n of a complex r e q u i r e d t o f orm an e f f e c t i v e bond w i t h t h e r e c e p t o r . An example of t h e l a t t e r would be t h e s t a b i l i z i n g of s p e c i f i c l e c t i n - l i k e i n t e r a c t i o n s by h y d r o p h o b i c bonds as p r o p o s e d by D o y l e e t a l . ( 2 8 ) . I f t h e h y d r o p h o b i c and l e c t i n a d h e s i n s were on s e p a r a t e m o l e c u l e s , t h e y m i g h t be s e p a r a t e d d u r i n g s o l u b i l i z a t i o n and c o n s e q u e n t l y would n o t a c t i n a c o o r d i n a t e d f a s h i o n . T h i s might e x p l a i n why p u r i f i c a t i o n by HPLC was n o t s u c c e s s f u l . In f u t u r e f r a c t i o n s o b t a i n e d from HPLC s h o u l d be combined i n o r d e r t o l o o k f o r a s y n e r g i s t i c e f f e c t . I f t h e a s s u m p t i o n i s c o r r e c t t h a t t h e a d h e r e n c e - b l o c k i n g a c t i v i t y of t h e c e l l w a l l d i g e s t s i s due t o t h e p r e s e n c e of s o l u b i l i z e d a d h e s i n s which c a n e f f e c t i v e l y compete f o r s a l i v a r y r e c e p t o r s , t h e n c h a r a c t e r i z a t i o n of t h e s e m o l e c u l e s s h o u l d h e l p t o c l a r i f y t h e n a t u r e of t h e c h e m i c a l i n t e r a c t i o n s b i n d i n g t h e o r g a n i s m t o S-HA. In l i g h t of what i s known about t h e b i n d i n g of S. s a n g u i s , i t seems r e a s o n a b l e t o p r e d i c t t h a t a t l e a s t p a r t of t h e i n h i b i t o r y a c t i v i t y i s due t o a d h e s i n s w h i c h b i n d t o s p e c i f i c s u g a r s ( l e c t i n s ) . T h e s e c o n c e p t s s t i l l l e a v e u n r e s o l v e d t h e q u e s t i o n o f t h e c o n t r i b u t i o n of t h e h i g h - m o l e c u l a r - w e i g h t p r o t e i n s t o c e l l u l a r h y d r o p h o b i c i t y and t o t h e i r f u n c t i o n i n t h e f o r m a t i o n of h y d r o p h o b i c bonds. The o n l y i n f o r m a t i o n t h a t i s p r e s e n t i s , (a) 93 t h a t t h e p r o t e i n s a r e f o u n d i n c e l l w a l l s of h y d r o p h o b i c o r g a n i s m s and not i n h y d r o p h i l i c v a r i a n t s ; and (b) t h e y a d h e r e t i g h t l y t o h y d r o p h o b i c p h e n y l S e p h a r o s e beads (Data not shown). The s t r u c t u r a l o r g a n i z a t i o n o f s u c h m o l e c u l e s on t h e c e l l s u r f a c e i s a l s o open t o s p e c u l a t i o n . The p o s s i b i l i t y e x i s t s t h a t m o l e c u l e s c o n f e r r i n g h y d r o p h o b i c i t y a r e a s s o c i a t e d w i t h o t h e r a d h e s i n s i n a c e l l s u r f a c e complex. A l t e r n a t i v e l y i o n i c o r l e c t i n - l i k e a d h e s i n s t h e m s e l v e s have h y d r o p h o b i c domains. In t h e l a t t e r c a s e t h e l o s s of h y d r o p h o b i c i t y would mean a c o i n c i d e n t l o s s o f t h e a d h e s i n , whereas i n t h e f o r m e r i t would be p o s s i b l e t o l o s e s e l e c t i v e l y e i t h e r an a d h e s i n o r h y d r o p h o b i c i t y . T h i s s e l e c t i v e l o s s i s e x e m p l i f i e d i n s a n g u i s (103) or S.  s a l i v a r i u s (161) w h i c h r e t a i n t h e i r h y d r o p h o b i c i t y but l o s e • p a r t i c u l a r a d h e r e n c e c h a r a c t e r i s t i c s . I t seems l i k e l y t h a t t h e f i b r i l l a r s u r f a c e s t r u c t u r e s on S. s a n g u i s may c a r r y a number of m o l e c u l e s w h i c h f u n c t i o n as a d h e s i n s . L e c t i n - l i k e p r o t e i n a c i o u s a d h e s i n s may form complexes w i t h c e l l s u r f a c e LTA ( 1 1 4 ) . C. Mouton and 0. D e s b i e n s ( A b s t . Ann M e e t i n g . Am. Soc. M i c r o b i o l : J 5 , p92, 1982) d e m o n s t r a t e d the p r e s e n c e o f t e i c h o i c a c i d i n t h e p o l a r t u f t s u s i n g s e r o l o g i c a l t e c h n i q u e s . I f , as has been p o s t u l a t e d ( 1 0 0 ) , LTA i s . r e s p o n s i b l e f o r h y d r o p h o b i c i t y , i t i s p r e s u m a b l y not c l o s e l y a s s o c i a t e d w i t h t h e s i a l i c - a c i d b i n d i n g l e c t i n ( p o s s i b l y t h e 160,000 MW c e l l w a l l component) on t h e p e r i t r i c h o u s f i b r i l s w h ich m e d i a t e s s a l i v a r y a g g r e g a t i o n , s i n c e i t i s p o s s i b l e t o l o s e b o t h t h i s p r o t e i n and t h e a g g r e g a t i n g a c t i v i t y w i t h o u t a f f e c t i n g 94 a d s o r b t i o n t o h e x a d e c a n e . F u r t h e r s t u d y s h o u l d h e l p t o e l u c i d a t e t h e r o l e s p l a y e d by v a r i o u s m o l e c u l e s w h i c h make up t h e c e l l s u r f a c e . 95 BIBLIOGRAPHY 1. Appelbaum, B., and B. Rosan. 1978. 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