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Fractionation and partial characterization of the hemagglutinating and bacterial aggregating adhesins… Boyd, Janet Doreen 1984

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FRACTIONATION AND PARTIAL CHARACTERIZATION OF THE HEMAGGLUTINATING AND BACTERIAL AGGREGATING ADHESINS OF BACTEROIDES GINGIVALIS By JANET DOREEN BOYD B.Sc, The University of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FU^IIXUVEOT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FAOJLTY OF GRADUATE STUDIES DEPARTMENT CF MICROBIOLOGY Vfe accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1984% ©!Janet Doreen Boyd, 1984 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 a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r s c h o l a r l y 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 p u b l i c a t i o n 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 MicrobioloG^ The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date aiOAujL A, /98^ ABSTRACT In order to characterize the surface components responsible for hemagglutination and bacterial aggregation, the outer membrane complex of Bacteroides gingivalis W12 was isolated. It was found to contain both hemagglutinating and bacterial aggregating activity. Examination of the membrane material by biochemical analysis, SDS-polyacrylamide gel elec-trophoresis and immunological means revealed that the crude outer mem-brane preparation contained three major proteins and a lipopolysaccharide (LPS) population which displayed size heterogeneity. At least two mem-brane proteins as well as the LPS were found to be antigenically active - using immun-blot analysis. Using gel chromatography and an LPS disaggregating buffer the membrane material was separated into two fractions. An accompanying separation of the two adherence- activities was observed. The fir s t mem-brane fraction, containing mostly protein and carbohydrate material was found to contain the bacterial aggregating activity. This fraction also contained a"smooth" IPS population. The second membrane fraction con-sisted of "rough"type LPS, protein and loosely bound li p i d and was found to contain the hemagglutinating activity. Further investigation revealed that bacterial aggregating activity was blocked by the presence of D-galactose (14 mM) , D-glucosamine (23 mm) and N-acetylglucoscmine (23 mm) and by dialysed iitmune rabbit serum. Dialysed non-immune rabbit serum did not block this activity. Bacterial i i aggregation was not removed by chloroform-methanol extraction or destroyed by proteolytic treatment. Pronase treatment was found in fact to enhance bacterial aggregating activity on a dry weight basis. Based on these observations i t appeared that the bacterial aggregating adhesin was either the capsular polysaccharide or "smooth" type LPS. Hemagglutinating activity was blocked by the presence of N-acetyl-galactosamine (23 mM) and bovine brain ganglioside (312 ug/ml) . As well, dialysed immune and non-immune serum were found to inhibit this activity, while /5-mercaptoethanol and dithiothreitol were found to enhance bacterial aggregation. Both protease digestion and chloroform-methanol extraction destroyed hemagglutinating activity which suggested that the hemagglutinin was either a protein or a lipid-protein complex. i i i LIST OF TABLES Table T i t l e Page I. Heinagglutinating and B a c t e r i a l Aggregating A c t i v -i t i e s of Crude and Fractionated Membranes 33 I I . Biochemical Analysis of Outer Membrane Fractions. . . 34 I I I . I n h i b i t i o n of Hemagglutinating and B a c t e r i a l Aggregating A c t i v i t y 42 IV. E f f e c t of Immune and Non-Immune Serum on Hemagg-l u t i n a t i o n and B a c t e r i a l Aggregation 44 V. Effe c t of Neuraminidase Treatment of Red Blood C e l l s on Hemagglutination A c t i v i t y 46 VI. E f f e c t of Chloroform-Methanol Extraction on Hemagglutination and B a c t e r i a l Aggregation 47 VII. E f f e c t of Proteolytic Treatment on Hemagglutination and B a c t e r i a l Aggregation 49 i v LIST OF FIGURES Figure T i t l e Page 1. Electron micrograph of B. g i n g i v a l i s W12 stained with ruthenium red 26 2. Electron micrograph of negatively stained B. g i n g i v a l i s W12 27 3. Electron micrograph of crude membrane material. . . . 2 8 4. Elution p r o f i l e of outer membrane on a column of Sephadex G100 equilibrated with desoxycholate buffer 31 5. SDS-PAGE of membrane fractions stained with Coamassie B r i l l i a n t Blue 36 6. SDS-PAGE of membrane fractions stained with s i l v e r s t a i n 37 7. SDS-PAGE of membrane fractions stained f o r LPS 39 8. Western b l o t of SDS-PAGE of membrane fractions. . . . 4 0 9. Elution p r o f i l e of Pronase digested pool 1 membrane on a Sephadex G100 column 51 v ACMO^EIXSFJyENTS I g r a t e f u l l y acknowledge the f i n a n c i a l support of the Medical Research Council and Dr. B. C. McBride throughout the course of t h i s study. I also wish to thank Andre Vfong f o r h i s work with the electron microscope and Warren Schmidt and Bruce McCaughey f o r t h e i r photography. The guidance, support, encouragement and patience of Dr. B. C. McBride was greatly appreciated and valued as was that of the other members of h i s lab. This thesis i s dedicated to David, Megan and Hamish. v i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES i v LIST OF FIGURES V A(^ KNOWIMX3EMENTS v i I. INTRODUCTION 1 I I . MATERIALS AND METHODS 14 A. Bacteria 14 B. Media and Growth Conditions 14 C. Iso l a t i o n of Outer Membrane 15 D. Fractionation of the Outer Membrane 16 E. A n a l y t i c a l Methods 17 F. Electrophoresis 18 G. B a c t e r i a l Aggregation Assay 19 H. Hemagglutination Assay 19 I. Hemagglutination and Ba c t e r i a l Aggregation I n h i b i t i o n Assays 20 J . I n h i b i t i o n by Immune and Non-Immune Rabbit Serum . . 21 K. Neuraminidase Treatment of Red Blood C e l l s . . . . 21 L. Eff e c t of Pronase Digestion on Membrane A c t i v i t i e s . . 22 M. Serology 23 v i i Page N. Electron Microscopy 23 I I I . RESULTS 25 A. Electron Microscopy 25 B. Iso l a t i o n and Separation of Outer Membranes . . . . 29 C. Separation and Localization of Membrane A c t i v i t i e s . 29 D. Biochemical Characterization of Membrane Fractions . 32 E. SDS-Polyacrylamide Gel Electrophoresis 35 F. Immunological Characterization of Membrane Fractions 38 G. I n h i b i t i o n of Hemagglutination and B a c t e r i a l Aggregation ' 41 H. Effe c t of Immune and Non-Immune Serum on Aggregating A c t i v i t y 43 I. E f f e c t of Neuraminidase Treatment of Red Blood C e l l s . 43 J . E f f e c t of Chloroform-Methanol Extraction an Aggregating A c t i v i t y 45 K. Ef f e c t of Pronase Treatment on Aggregating A c t i v i t i e s . 48 L. I s o l a t i o n of Aggregating A c t i v i t i e s from Culture Supernatant s 52 IV. DISCUSSION 54 V. REFERENCES . 67 v i i i INTRODUCTICN In many b a c t e r i a l diseases attachment to host tissues i s a pre-r e q u i s i t e f o r colonization and expression of pathogenic p o t e n t i a l . Such w e l l studied examples as V i b r i o cholerae, Escherichia c o l i and group A streptococci (102, 3, 105) are among t h i s group. An examination of these examples gives an in d i c a t i o n of some of the methods by which bacteria can attach to host tissues. In the case of E. c o l i , i n i t i a l colonization of the i n t e s t i n a l t r a c t by animal s p e c i f i c enterotoxigenic E. c o l i i s mediated by s p e c i f i c f i m b r i a l ( p i l i ) antigens, predominantly K88 and K99 (11, 39, 42). Other factors appear to be involved as w e l l since K88 negative strains can be enteropathogenic i n p i g l e t s (70). Attachment p i l i from enterotoxigenic E . e o l i pathogenic f o r humans are he a t - l a b i l e , surface antigens serolog-i c a l l y d i s t i n c t from K88 and K99 antigens and are designated CFA I and CFA I I (22, 21). Attachment fimbriae or p i l i are d i s t i n c t from so-c a l l e d common p i l i i n a l l of these st r a i n s i n that the former hemagglu-tina t e red blood c e l l s i n a mannose re s i s t a n t fashion while the l a t t e r mediate mannose sensitive hemagglutination (11, 23, 24). Neisseria gonorrhoeae i s found to adhere r e a d i l y i n v i t r o t o human c e l l s i n t i s s u e culture (111), to human f a l l o p i a n tube organ c u l -ture (12, 112) and to red blood c e l l s (9). In human f a l l o p i a n tube organ cultures, fimbriated gonococci shew four f o l d greater adhesion t o the mucosal surface than the non-fimbriated variants of the same s t r a i n (113). 1 However, penetration of the mucosal surface by the non-fimbriated variants implies that fimbriation i s not e s s e n t i a l f o r invasion and that d i f f e r e n t s t r u c t u r a l determinants on the bacteria must be involved i n t h i s interac-t i o n (114). Recent studies have shown that a ncn-fimbrial factor i s the primary determinant of the interaction between gonocccci and polymorpho-nuclear leucocytes (46, 107) and that the leucocyte association factor i s a protein e x h i b i t i n g a molecular weight of 28-29,000 daltons (46). In group A streptococci i t has been demonstrated that the M protein, an antigen associated with surface projections, mediates binding of Streptococcus pyogenes to e p i t h e l i a l surfaces (20). However further i n -vestigation revealed that l i p o t e i c h o i c acids can be located with M protein on the fimbriae of group A streptococci and that the r o l e of fimbriae i n e p i t h e l i a l c e l l binding i s due to t h e i r content of l i p o t e i c h o i c acids rather than M protein (5, 4). More recent studies have shown that l i p o -t e i c h o i c acid i s the major c e l l w a l l component which determines the sur-face hydrophobicity of group A streptococci (68). E^jrthermore i t has been demonstrated that l i p o t e i c h o i c acids of serotype I I I strains of group B streptococci mediate the adherence of these organisms to human embryonic, f e t a l and adult buccal e p i t h e l i a l c e l l s (74). Adherence i s also an important factor i n the interaction of plants and bacteria. Lipopolysaccharide preparations from Agrobacterium  tumefaciens have been shown to be i n h i b i t o r y to tumour i n i t i a t i o n — f o r which b a c t e r i a l adherence to the plant tissue i s a prerequisite—when applied with or before the i n f e c t i n g bacteria (117). This suggests that lipopolysaccharide i s the major Agrobacterium camponent involved i n wound s i t e adherence. This study also indicates that the O-antigenic portion 2 of the molecule i s e s s e n t i a l l y as i n h i b i t o r y as the whole molecule while the l i p i d A portion i s e s s e n t i a l l y ncn-inhibitory. In the Rhizobium  t r i f o l i i - c l o v e r plant interaction a l e c t i n has been is o l a t e d from clover roots that s p e c i f i c a l l y agglutinates infectious but not non-infectious R. t r i f o l i i mutants. The agglutination i s i n h i b i t e d by 2-deoxyglucose and N-acetylgalactosamine which suggests that the former, which i s known to be present i n the capsular polysaccharide antigen of infectious R. t r i f o l i i , i s involved i n the root lectin-bacterium interaction (17). Examination of these diseases and interactions indicates that adherence of the causative bacteria to the host mucosal or tis s u e sur-face i s a prerequisite for the development of i n f e c t i o n and that i n the causative organisms, the presence of c e l l surface components which pro-mote adherence to the host surfaces has been p o s i t i v e l y correlated with virulence. Recent studies indicate that i n areas which contain a f l u i d flow—such as the mouth—the a b i l i t y of bacteria to attach to surfaces i s a very important determinant i n t h e i r a b i l i t y to colonize (29). I t has been suggested that i n the o r a l dental plaque ecosystem there are three types of adhesive interactions that are required f o r plaque forma-t i o n (89). The f i r s t type i s d i r e c t adherence to host surfaces such as enamel, cementum and e p i t h e l i a l c e l l s leading to colonization of these surfaces. The second type of adhesive interaction allows f o r expansion of the b a c t e r i a l mass by the retention of the progeny of the o r i g i n a l l y attached c e l l s . The f i n a l type of adherence involves the attachment of d i s s i m i l a r species to the plaque biomass and requires c e l l to c e l l i n t e r -action between d i f f e r e n t species r e s u l t i n g i n the c h a r a c t e r i s t i c hetero-geneity of human dental plaque. 3 Direct adherence of o r a l bacteria to host tissues has been studied i n a number of b a c t e r i a l species. These studies show strong corre l a t i o n between observed i n v i t r o or i n vivo adherence a b i l i t i e s and the natural d i s t r i b u t i o n of the b a c t e r i a l s t r a i n s i n the o r a l cavity, strongly im-plying that b a c t e r i a l adherence plays an important r o l e i n the determin-ation of host and tissue tropisms. For example, strains of Streptococcus  f a e c a l i s and serum requiring diptheroids which na t u r a l l y colonize the r a t tongue dorsum were found to adhere much better to the tongues of ra t s than to human tongues, while the converse was true with st r a i n s of Streptococcus s a l i v a r i u s and Streptococcus sanguis which are prominent on the human tongue (28). As w e l l S. pyogenes which binds w e l l to human buccal or pharyngeal e p i t h e l i a l c e l l s adsorbed i n higher numbers to human buccal e p i t h e l i a l c e l l s than to r a t e p i t h e l i a l c e l l s (28) . An e a r l i e r study showed that other streptococcal species have d i f f e r e n t trop-isms; Streptococcus m i t i o r was shown to adhere better than S. s a l i v a r i u s to human buccal mucosa and teeth while S. s a l i v a r i u s adhered with higher a f f i n i t y to the tongue dorsum (53). Streptococcus sanguis serotype one strains were shown to adhere better to s a l i v a coated hydroxyapatite than serotype two strains and better than Streptococcus mutans, S. mit i o r and S. s a l i v a r i u s (2). As indicated i n these studies d i f f e r e n t species and strains of bacteria adhere with varying a f f i n i t i e s d i r e c t l y to o r a l surfaces such as teeth and e p i t h e l i a l c e l l s . These bacteria i n turn can serve as a surface to which other bacteria, those not able to adhere as w e l l d i r e c t l y to o r a l t i s s u e s , can attach. This type of adhesion, mentioned above— attachment t o the b a c t e r i a l plaque of d i s s i m i l a r s p e c i e s — i s thought to 4 account for the majority of adherence reactions i n the o r a l c a v i t y due to the thickness of plaque and the l i m i t e d p e l l i c l e surface. In 1970 i t was demonstrated that many pairs of o r a l bacteria w i l l aggregate when mixed together i n v i t r o (27). Since that time the s p e c i f i c nature of agg-regation between many pairs of bacteria has been studied and many methods of i n t e r b a c t e r i a l aggregation observed. One of the most d i s t i n c t i v e sets of interacting bacteria i s Bacterionema matruchotii and S. sanguis which adhere to each other to form a structure which morphologically resembles a corn cob (50) . The s p e c i f i c nature of the reaction i s demonstrated i n that only serotype one strains of S. sanguis form corn cobs with B. matruchotii. The lo c a l i z e d surface "fuzz" observed on S. sanguis c e l l s i n the area of interaction with Bacterionema i s apparently not s u f f i c i e n t alone f° r com cob production since a mutant which does not adhere also has surface "fuzz" (50). The nature of the aggregation reaction between S. sanguis and S. mutans to Actinomyces viscosus has been more f u l l y defined (8). These two species bind together vigourously to form large microbial aggregates. This reaction appears to be mediated by dextran since glucose grown streptococci w i l l not aggregate with A. viscosus unless f i r s t mixed with high molecular weight dextran whereas sucrose grown streptococci adhere to A. viscosus without added dextran. A l a t e r study (66) demonstrated that A. viscosus T14V and S. sanguis 34 coaggregate by a mechanism that i s dextran independent and appears to require the interaction of a protein or glycoprotein on A. viscosus and a carbohydrate on S. sanguis. Another w e l l characterized interaction i s the binding of 5 S. s a l i v a r i u s HB to V e i l l o n e l l a alcalescens VI i n which a c e l l wall com-ponent of S. s a l i v a r i u s has been i d e n t i f i e d which mediates the reaction (116). This protein antigen can be s p e c i f i c a l l y adsorbed from s o l u b i l i z e d S. s a l i v a r i u s c e l l w a l l preparations by V. alcalescens but not by non-aggregating V e i l l o n e l l a parvula. In t h i s interaction there i s no cor-r e l a t i o n between the presence of fimbriae on S. s a l i v a r i u s and the a b i l i t y to aggregate V e i l l o n e l l a . V e i l l o n e l l a alcalescens also forms associations with S. mutans or S. sanguis implanted together i n germ-free r a t s (67). Electron micro-scope examination of the teeth showed V e i l l o n e l l a growing i n dense micro-colonies surrounded by c e l l s of S. mutans while V e i l l o n e l l a implanted by i t s e l f showed l i t t l e a b i l i t y to attach to cleaned teeth. A more recent study has demonstrated sim i l a r r e s u l t s (65). C l e a r l y i n t h i s association the a b i l i t y of V e i l l o n e l l a to attach to S. mutans of f e r s the V e i l l o n e l l a survival benefits. By extension i t i s easy to see how the a b i l i t y of an organism to attach to host tissues, or lacking that a b i l i t y , to attach to previously established organisms enables the bacteria to colonize a given s i t e i n the mouth, a necessary f i r s t step f o r s u r v i v a l and expres-sion of possible pathogenic p o t e n t i a l . The buildup of b a c t e r i a l plaque due to adherence of bacteria as described above has pathogenic consequences i n that i t can lead to caries and to diseases of the gums and supporting tissues of the tooth such as g i n g i v i t i s and pe r i o d o n t i t i s . Anaerobic gram negative b a c i l l i make up 75% of the c u l t i v a b l e microflora isol a t e d from deep g i n g i v a l pockets of patients with advanced p e r i o d o n t i t i s (94, 98). Nearly h a l f of these b a c i l l i are black pigmented Bacteroides species, many of which are now 6 designated Bacteroides g i n g i v a l i s . When these bacteria were f i r s t i s o -lated i n 1921 they were given the name Bacterium melaninogenicum (80), however l a t e r they were assigned to the genus Bacteroides and c l a s s i f i e d B. melaninogenicus (85). Although biochemical characterization—fermen-t a t i v e and pr o t e o l y t i c c a p a b i l i t i e s — h a d indicated heterogeneity within t h i s group of bacteria, B. melaninogenicus has long been recognized as a single species (88, 13). However i n 1970 Holdeman and Moore described three d i f f e r e n t subspecies of B. melaninogenicus on the basis of bio-chemical c h a r a c t e r i s t i c s including the production of v o l a t i l e f a t t y acids. The three new subspecies were B. melaninogenicus subspecies melaninogenicus, intermedius and asaccharolyticus (37). Subsequently the asaccharolytic subspecies was placed i n a separate species, B. asaccharolyticus, because of differences i n DNA base r a t i o s and energy y i e l d i n g metabolism path-ways (25). Most recently i t has been found that although strains of B. asaccharolyticus from o r a l and non-oral sources share a number of properties (25), they d i f f e r from each other by a number of c r i t e r i a . Oral strains strongly agglutinate sheep erythrocytes by d i r e c t hemagg-l u t i n a t i o n while those of non-oral o r i g i n do not (97). A capsular struc-ture has been shown by electron microscope to surround strains of o r a l o r i g i n while those of non-oral o r i g i n possess a ruthenium red staining structure that i s morphologically very d i f f e r e n t and d i s t i n c t from that of o r a l strains (57). Ant i g e n i c a l l y , o r a l and non-oral strains of B. asaccharolyticus have been shown to be unrelated (83). Oral st r a i n s also d i f f e r genetically from non-oral s t r a i n s , sharing l i t t l e DNA base sequence homology (14). As w e l l , non-oral i s o l a t e s have a higher G + C r a t i o compared with the G + C r a t i o of o r a l B. asaccharolyticus (14). 7 F i n a l l y , o r a l s t r a i n s produce phenylacetic a c i d whereas non-oral strains do not (62, 64, 44). Based upon t h i s evidence Coykendall et a l (14) pro-posed that o r a l s t r a i n s be placed i n a new species, B. g i n g i v a l i s ; the new c l a s s i f i c a t i o n was confirmed by van Steenbergen et a l (110). B. g i n g i v a l i s i s found i n low numbers, i f present at a l l i n healthy and g i n g i v i t i s s i t e s i n the o r a l c a v i t y (92, 101, 104, 19, 123). In the healthy g i n g i v a l sulcus less than 2% of the c u l t i v a b l e microflora i s black pigmented Bacteroides (95) while i n chronic g i n g i v i t i s , B. melan- inogenicus makes up 8% of the c u l t i v a b l e microflora (101). Most of these i s o l a t e s are subspecies intermedius and only occasionally are strains of B. asaccharolyticus i d e n t i f i e d among the predardnant i s o l a t e s (101). In early p e r i o d o n t i t i s the B. melaninogenicus/B. asaccharolyticus group comprises up to 22% of the t o t a l i s o l a t e s (16), while i n advanced perio-d o n t i t i s strains resembling B. asaccharolyticus (now B. g i n g i v a l i s ) are the most predominant of the Bacteroides species encountered i n most patients (94, 108). This species may make up to 67% of the c u l t i v a b l e f l o r a i n the deep periodontal pockets, with the degree of c l i n i c a l i n -flammation co r r e l a t i n g s i g n i f i c a n t l y with the percentage of B. g i n g i v a l i s present (108). Oral B. asaccharolyticus s t r a i n s are the predominant i s o l a t e i n most pockets with r a p i d l y progressing bone loss i n humans (104, 108,113) and i n experimental periodontal lesions i n monkeys (46, 99). That t h i s organism has the p o t e n t i a l to be an important pathogen has been demonstrated i n a number of studies. B. g i n g i v a l i s has been shown to play an e s s e n t i a l r o l e i n the pathogenesis of transmissible, mixed, anaerobic infections produced by mixtures of indigenous o r a l i s o -lates i n animal models (56, 103, 106). When black pigmented Bacteroides 8 strains are present i n such mixtures, t y p i c a l transmissible infections can be produced; however when these bacteria are deleted from the mixture, the other bacteria are no longer pathogenic, in d i c a t i n g a key r o l e f o r black pigmented Bacteroides strains i n these infections. Besides the Bacteroides st r a i n s many other bacteria are present i n these mixtures (26, 63), and while these other bacteria alone do not produce i n f e c t i o n , neither normally do the pigmented Bacteroides species alone. One example of the synergystic re l a t i o n s h i p necessary for i n f e c t i o n has been examiried and has revealed that the i n f e c t i v i t y of B. g i n g i v a l i s i s dependent on the production of succinate by K l e b s i e l l a pneumoniae (63). As w e l l as being involved i n infections i n animal models, B. g i n g i v a l i s has been shown to possess a number of pot e n t i a l virulence factors. I t has a c e l l bound collagenase and a c e l l associated proteo-l y t i c a c t i v i t y towards Azocoll and casein (64). As w e l l , most asaccha-r o l y t i c strains of black pigmented Bacteroides produce f i b r i n o l y s i n (82), although these st r a i n s were not c l a s s i f i e d further as B. asaccharolyticus or B. g i n g i v a l i s . Besides pr o t e o l y t i c enzymes, black pigmented Bacteroides species produce many substances that can be damaging to host tissues including phospholipase A (10), deoxyribonuclease (86) and hemolytic a c t i v i t y (36). B. g i n g i v a l i s produces endotoxin with the p o t e n t i a l to stimulate bone resorption i n v i t r o (33), as w e l l as hydrogen s u l f i d e , ammonia, and various v o l a t i l e f a t t y acids (36) of which butyrate and propionate have been shown to have growth i n h i b i t o r y e f f e c t s on cultured human g i n g i v a l f i b r o b l a s t s (93). There i s also a preliminary report of production of a cholera-like t o x i n by B. gi n g i v a l i s , ( 6 4 ) . As a periodontopathic organism the adherence patterns and mechan-9 isms of B. g i n g i v a l i s are of no l i t t l e significance and, as indicated e a r l i e r , are probably among the most important factors i n the a b i l i t y of the bacteria to colonize and thus express i t s pathogenic p o t e n t i a l . Slots and Gibbons (98)studied the adherence of black pigmented Bacter-oides to a number of o r a l surfaces. They demonstrated that an o r a l s t r a i n of B. melaninogenicus subspecies asaccharolyticus (now B. g i n g i v a l i s ) could adhere i n high numbers to buccal and crevicular e p i t h e l i a l c e l l s and to a number of gram p o s i t i v e organisms present i n dental plaque, whereas i t adhered i n r e l a t i v e l y low numbers to untreated and s a l i v a treated hydroxyapatite. I t was found however, that c l a r i f i e d whole s a l -i v a i n h i b i t e d v i r t u a l l y completely the adherence to hydroxyapatite and to buccal e p i t h e l i a l c e l l s , while normal human serum strongly i n h i b i t e d attachment to c r e v i c u l a r e p i t h e l i a l c e l l s . However neither serum nor s a l i v a had i n h i b i t o r y e f f e c t s towards the attachment of B. g i n g i v a l i s to gram pos i t i v e organisms, suggesting that i n the o r a l c a v i t y the a b i l i t y of B. g i n g i v a l i s to adhere to established gram po s i t i v e bacteria i s of prime consideration i n i t s a b i l i t y to colonize. Adherence of black pigmented Bacteroides species to erythrocytes, which can be used as a measure of i t s a b i l i t y to bind to e p i t h e l i a l c e l l s i n general was demonstrated by Okuda and Takazoe (78), and was subsequently shown to be a s p e c i f i c property of B. g i n g i v a l i s (97, 110). I t was sug-gested by Okuda and Takazoe (78) that the hemagglutinating a c t i v i t y of these bacteria was due to p i l i and that p i l i were only present on those bacteria e x h i b i t i n g hemagglutinating a c t i v i t y . P a r t i a l l y p u r i f i e d p i l i preparations prepared by Slots and Gibbons (98) from a s t r a i n of B. g i n g i v a l i s again demonstrated strong hemagglutinating a c t i v i t y , how-ever p i l i were observed on a l l s t r a i n s of bacteria including those 10 lacking hemagglutinating a c t i v i t y . A more recent study has also indicated that p i l i preparations from B. g i n g i v a l i s contain hemagglutinating a c t i -v i t y , however again t h i s study also demonstrated p i l i on the surface of a l l Bacteroides species, not only B. g i n g i v a l i s - ( 7 7 ) . The c e l l surface morphology of B. g i n g i v a l i s has been examined i n a number of studies. Capsular material staining with ruthenium red located external to a t y p i c a l gram negative outer membrane has been observed by electron microscopy by a number of groups (58, 120, 61, 60, 57, 77, 121). Morphologically t h i s capsule appears as a t h i n electron dense layer, i n contrast to non-oral st r a i n s of B. asaccharolyticus and subspecies of B. melaninogenicus which e x h i b i t a much f i n e r , l e s s dense external layer (57, 121, 77, 120). The capsule of B. g i n g i v a l i s has been shown to be a n t i g e n i c a l l y active and d i s t i n c t from the large molec-ular s i z e polysaccharide (capsular) antigen obtained from non-oral B. asaccharolyticus and other Bacteroides species (58, 83, 57). A second antigenic f r a c t i o n from the c e l l surface has been i s o l a t e d by Mansheim et a l (60) and i d e n t i f i e d as lipopolysaccharide (LPS). This LPS has been found to be markedly d i f f e r e n t from the t y p i c a l LPS of f a c u l t a t i v e gram negative organisms. I t has almost no b i o l o g i c a l a c t i v i t y as assessed by the Schwartzman reaction, the limulus lysate assay and the chick embryo l e t h a l i t y t e s t when compared with LPS frcm f a c u l t a t i v e organisms (60, 61). Chemical analysis reveals the absence of two core sugars, 2-keto-3-deoxyoctonate and heptose (35, 60, 61). Furthermore, chrom-atographic analysis of the f a t t y acids reveals the absence of ^-hydroxy-myristic a c i d which i s known to be the predominant f a t t y a c i d i n the l i p i d A portion of the LPS of f a c u l t a t i v e organisms (54). 11 As previously mentioned, p i l i have been demonstrated on the surface of B. g i n g i v a l i s i n a number of studies (78, 98, 121). A crude p i l i prep-aration from B. g i n g i v a l i s has been found to be immunogenic i n rabbits and immunodiffusion of the antisera obtained against p i l i preparations from both hemagglutinating and non-hemagglutinating Bacteroides produced p r e c i p i t i n l i n e s only with p i l i preparations from hemagglutinating B. g i n g i v a l i s (79). In contrast, a number of str a i n s of B. g i n g i v a l i s have been examined by electron microscopy and were a l l found to be lacking p i l i (S. C. Holt personal communication to B. C. M). Examination of the c e l l surface ccmponents of B. g i n g i v a l i s respon-s i b l e f o r adherence functions i s f a c i l l i t a t e d by i s o l a t i o n and character-i z a t i o n of the outer membrane and capsule of the bacteria. Outer mem-branes are rou t i n e l y i s o l a t e d with r e l a t i v e ease from gram negative bac-t e r i a due to t h e i r unusual behaviour towards detergents and pH compared to other membranes (90, 91) . The presence of LPS and a r e l a t i v e l y simple protein composition makes i d e n t i f i c a t i o n of outer membranes easier. Although there have been a large number of suggestions for the separation of inner and outer membranes of gram negative bacteria, a preponderance of studies have used the modification of the method of Miura and Mizushima (69) described by Osborn e t a l (81). In t h i s procedure sphero-plasts prepared by lysozyme and EDTA treatment were lysed by sonic o s c i l -l a t i o n and the inner and outer membranes separated by sucrose density centrifugation. Mansheim and Kasper have developed a method f o r the i s o l a t i o n of the outer membrane complex of B. melaninogenicus subspecies asaccharo-l y t i c u s using r e l a t i v e l y gentle physical techniques (58) . Using an LPS 12 disaggregating buffer and column chromatography the outer membrane can be separated into two fra c t i o n s , one containing a protein-polysaccharide-l i p i d complex made up of the large molecular s i z e polysaccharide capsule described e a r l i e r , with associated proteins and l i p i d s . The other f r a c t i o n contains the disaggregated LPS component of the membrane (58). Since B. g i n g i v a l i s adherence reactions, such as the a b i l i t y to adhere to gram po s i t i v e organisms or to agglutinate red blood c e l l s , are presumably mediated by components of the outer membrane complex, iden-t i f i c a t i o n and characterization of these components w i l l contribute a great deal t o the understanding of the pathogenesis of t h i s organism. The purpose of t h i s study was to describe the i s o l a t i o n and character-i z a t i o n of membrane components containing hemagglutinating and b a c t e r i a l aggregating a c t i v i t y . 13 MATERIALS AND METHODS Bacteria B. g i n g i v a l i s W12 was i s o l a t e d from a periodontal l e s i o n of a patient at the University of B r i t i s h Columbia periodontal c l i n i c . The sample was streaked onto a freshl y poured laked blood agar plate (Difco Blood Agar Base with 5% laked human blood added). Af t e r seven days incu-bation i n an anaerobic glove box (Coy Manufacturing, Ann Arbor, Michigan) containing an atmosphere of N ^ ^ i C C ^ (85:10:5) at 37° C, black colonies, were picked and subcultured to the same media u n t i l pure. I d e n t i f i c a t i o n of t h i s i s o l a t e as B. g i n g i v a l i s was based on: (1) formation of black colonies on laked blood agar (38), (2) obligate anaerobic growth (38), (3) absence of fermentation of sugars (38), (4) production of butyric (37) and phenylacetic a c i d (62, 64, 44), (5) presence of hemagglutinating a c t i v i t y (97). S. mutans LM7 and C6715, S. sanguis 10556, 10557 and 10558, S. s a l i v a r i u s HB and HBV5 (115), A. viscosus and S. m i t i o r were from our culture c o l l e c t i o n . Media and Growth Conditions A l l s t r a i n s were stored frozen at -70° C i n growth medium supple-mented with 7% dimethyl sulfoxide. B. g i n g i v a l i s W12 was cultured i n a medium consisting of brain heart infusion (37 g), yeast extract (3 g), trypticase peptone (10 g), hemin (5 mg) and d i s t i l l e d ^ 0 to 1 l i t r e . 14 Cultures were incubated f o r 48 hours i n an anaerobic glove box as des-cribed. Large volumes of c e l l s required for membrane i s o l a t i o n were grown i n a 20 l i t r e carboy f i l l e d to the top with freshly prepared medium. Afte r inoculation the carboy was t i g h t l y stoppered and incubated f o r 48 hours i n an aerobic incubator at 37° C. A l l other organisms used were grown overnight i n trypticase soy broth supplemented with yeast extract (3 g/1). Isolation of Outer lVksmbrane The method used f o r i s o l a t i o n of the outer membrane was a s l i g h t modification of the technique developed by Mansheim and Kasper (58). C e l l s grown f o r 48 hours were pelleted by centrifugaticn at 10,000 g at 4° C and washed three times with 0.15 M NaCl. Pelleted organisms from 20 l i t r e s of culture were suspended i n approximately 750 ml of a buffer containing 0.05 M sodium phosphate, 0.15 M sodium chloride and 0.01 M EDTA adjusted to pH 7.4. The suspension was incubated f o r 30 minutes at rocm temperature, sheared by passage through a 26 guage needle with manual pressure and mixed i n a Waring blender for 10 seconds. The mix-ture was then centrifuged at 10,000 g for 20 minutes, the supernatant .. recovered and centrifuged at 80,000 g for 2 hours. The yellowish g e l -l i k e translucent p e l l e t s were resuspended i n d i s t i l l e d H 20 and the two centrifugations were repeated. The p e l l e t was suspended i n d i s t i l l e d H^ O and l y o p h i l i z e d . This was the crude outer membrane material. "Outer membrane material" was also isol a t e d from the culture super-natants. Culture supernatants were collected from c e l l s grown for 48 or 96 hours as above by centrifuging the supernatants 2 to 3 times at 15 10,000 g to remove c e l l s and other debris. The c e l l - f r e e supernatant was then centrifuged at 80,000 g f o r two hours. The pelleted material was resuspended i n d i s t i l l e d H 20 and the centrifugations repeated. The p e l l e t s were then resuspended i n d i s t i l l e d water and l y o p h i l i z e d . This material was not fractionated further. Fractionation of the Outer Membrane Lyophilized outer membrane material (45 mg) was dissolved i n 1.0 ml of a buffer containing 0.05 M glycine, 0.001 M EDTA and 0.5% sodium desoxycholate (Fischer) a t pH 9.0. The pH was then raised to 11 to c l a r i f y the suspension and then adjusted to 9.0. The suspension remained clear when the pH was lowered and the sample was then immediately chroma-tographed on a 1.2 cm x 75 cm column of Sephadex G100 equilibrated with sodium desoxycholate buffer. One ml fractions were collected and moni-tored fo r protein a t and f o r carbohydrate by the anthrone procedure. The void volume material (pool 1) was collected and concentrated to a volume of 5.0 mis with an Amicon u l t r a f i l t r a t i o n c e l l (Amican Cor-poration, Lexington Mass.) with a PM-30 membrane. This material was separated from the desoxycholate buffer by p r e c i p i t a t i o n with 0.2 M NaCl and 80% ethanol. The precipitated material was pelleted by c e n t r i f u g -ation at 17,000 g f o r 15 minutes. The p e l l e t was redissolved i n d i s t i l l e d H 20 and the p r e c i p i t a t i o n and centrifugation repeated. The remaining pr e c i p i t a t e was dissolved i n d i s t i l l e d H 20, dialysed against d i s t i l l e d ILjO for 24 hours to remove excess NaCl and l y o p h i l i z e d . A second pool of material from the column (pool 2) was treated s i m i l a r l y to pool 1 except that a UM-05 membrane was used to concentrate 16 t h i s f r a c t i o n . For seme studies pool 1 material was further p u r i f i e d by proteo-l y t i c digestion. Pool 1 material (15 mg) was suspended i n 1.0 ml of T r i s buffer (0.05 M, pH 7.3) and incubated f o r 18 hours with 7.5 mg of Pronase (Calbiochem, Lot 300114, B grade). A further 7.5 mg of Pronase was then added and the mixture incubated f o r another 18 hour period. The undigested material was separated from the digested material and Pronase by column chromatography on a 1.2 x 75 cm Sephadex G100 column e q u i l i -brated with 0.05 M T r i s (pH 9.0) with 2.0 M NaCl. Before application, the pH of the sample was raised to 9.0 and NaCl was added to r a i s e the concentration of NaCl to 2.0 M. Fractions (1.5 ml) were collected and the protein concentration and the anthrone r e a c t i v i t y monitored as previously described. The eluted material was collected i n three pools (Pr-1, Pr-2 and Pr-3), dialysed against d i s t i l l e d B^O overnight and l y o p h i l i z e d . A n a l y t i c a l Methods Protein was determined by a modification of the Lowry assay (87) using bovine serum albumin as a standard. Total sugars were measured by the phenol sulphuric a c i d method (18) using glucose as a standard. Hexoses were determined by the anthrone reaction (84) using glucose as a standard. Hexosamines were determined by the method of Elson and Morgan (48). The method of Dische and Schettles was used to determine methyl pentoses (43) using rhamnose standards while pentoses were deter-mined by the o r c i n o l method (34). Muramic ac i d was measured by a modif-i c a t i o n of the Elsan-Morgan procedure (48) using glucosamine standards. 17 Glucuronic a c i d was measured by the method of Blumenkrantz and Asboe-Hansen (7) using glucuronic a c i d as a standard. Chloroform-methanol extractable l i p i d was measured using a modification of the procedure described by B l i g h and Dyer (6) as outlined by Mansheim and Kasper (58). Nucleic acid content was estimated by the r a t i o of u l t r a v i o l e t l i g h t absorption a t 280 nm compared with that at 260 nm (51) . Electrophoresis SDS-polyacrylamide g e l electrophoresis was performed using slab gels 1.5 mm thick. A 30% acrylamide, 0.8% bis-acrylamide stock solution was used to prepare a running g e l of 10 or 12.5% acrylamide and a stacking ge l of 3% acrylamide containing SDS a t 0.1%. Samples were boiled f o r 5 minutes i n a s o l u b i l i z a t i o n mix containing 4% SDS before application. Gels were electrophoresed at 40 milliamps constant current per g e l f o r approximately 2h hours, and stained with Coomassie B r i l l i a n t Blue, or s i l v e r stained using a modification of the procedure of Oakley et a l (76). Gels were stained f o r IPS with the method of Tsai and Frasch (109). For electrophoretic b l o t transfer of material a f t e r SDS-poly-acrylamide g e l electrophoresis, the g e l was sandwiched between two sheets of n i t r o c e l l u l o s e paper which had been wetted b r i e f l y with d i s t i l l e d H 20 and then soaked i n 0.4% wt/vol SDS, 1.24% i r i s , 0.76% glycine at 60° C f o r 30 minutes. The n i t r o c e l l u l o s e sandwich was then placed between two stacks of 3M Whatman f i l t e r paper which had been wetted i n the trans-fe r buffer (glycine 14.4 g/1, T r i s 3.025 g/1, methanol 200 ml/1, pH 8.3). The e n t i r e stack was placed i n the holding cassette of the Bio-Rad Trans Blo t c e l l and the c e l l f i l l e d with the transfer buffer. A current of 18 4 milliamps was applied overnight at room temperature. Staining of antigen-antibody complexes using antibody raised i n rabbits to whole c e l l s of B. g i n g i v a l i s was carried out using the Bio-Rad Immun Blo t (GAR-HRP) Assay K i t . B a c t e r i a l Aggregation Assay B a c t e r i a l aggregation by the outer membrane fractions of B. g i n g i - v a l i s was measured by suspending the membrane fractions i n d i s t i l l e d rJ^O to a concentration of 8.0 mg/ml dry weight. The b a c t e r i a l c e l l s t o be used i n the assay were harvested a f t e r overnight incubation and washed three times i n Hepes buffer (0.05 M, pH 7.2) plus 10 mM CaCl 2. The bacteria were suspended to an A^^^ of three. The b a c t e r i a l suspension (0.1 ml) was mixed with an equal volume of s e r i a l l y two f o l d d i l u t e d mem-brane and incubation was carried out a t room temperature with shaking f o r 30 minutes. Since aggregates formed with the membranes were generally much smaller than aggregates formed with whole B. g i n g i v a l i s , evaluation of the degree of aggregation was made with a L e i t z stereomicroscope at 20 times magnification. The t i t r e of the membrane material was taken as the recipr o c a l of the l a s t d i l u t i o n showing aggregation of the t e s t bacteria. Hemagglutination Assay Hemagglutinating a c t i v i t y of the membrane fractions was measured by suspending the membrane material i n d i s t i l l e d to a concentration of 8.0 mg/ml dry weight. S e r i a l l y two f o l d d i l u t e d membrane material (0.1 ml) was mixed with an equal volume of 2.5% washed formalinized 19 human red blood c e l l s i n phosphate buffered saline (0.05 M, pH 7.2) i n mi c r o t i t r e plates and incubated a t room temperature f o r 60 minutes. The t i t r e of the membrane material was taken as the reciprocal of the l a s t membrane d i l u t i o n showing complete hemagglutination. Hemagglutination and Ba c t e r i a l Aggregation I n h i b i t i o n Assays A number of substances were tested for t h e i r a b i l i t y to block the hemagglutinating and b a c t e r i a l aggregating a c t i v i t i e s of B. g i n g i v a l i s W12 membrane. The materials t o be tested were dissolved i n d i s t i l l e d FtjO t o the concentrations indicated. These solutions were then s e r i a l l y two f o l d d i l u t e d with d i s t i l l e d B^O (0.05 ml) and mixed with an equal volume of membrane. The concentration of membrane used was twice the highest d i l u t i o n of membrane which gave cl e a r hemagglutination i n a pre-liminary t i t r a t i o n of membrane with red blood c e l l s . A si m i l a r p r e l i m i -nary t i t r a t i o n of b a c t e r i a l aggregating a c t i v i t y was also car r i e d out to determine the concentration of membrane to be used i n the b a c t e r i a l aggregation i n h i b i t i o n assay. The mixtures of membrane and i n h i b i t o r y substances were incubated f o r 30 minutes at room temperature with gentle shaking and then 0.1 ml of either 2.5% formalinized human red blood c e l l s i n phosphate buffered sali n e (0.05 M, pH 7.2) or S. m i t i o r c e l l s suspended to A 6 6 0 of three i n Hepes buffer (0.05 M, pH 7.2) plus CaCl 2 was added. Incubation was then c a r r i e d out for a further 30 or 60 minutes and the t i t r e of i n h i b i t i o n of the substances was determined. The i n h i b i t o r y t i t r e of the sustances was taken to be the rec i p r o c a l of the highest d i l u -t i o n of the material which produced a blocking of the hemagglutination or b a c t e r i a l aggregation reaction. 20 Acid hydrolysed bovine type three ganglioside was assayed for i t s i n h i b i t o r y a c t i v i t y using t h i s assay. Acid hydrolysis of t h i s material was performed as follows. Ganglioside was suspended to 5 mg/ml i n 0.05 N HC1 and heated for 90 minutes at 80° C. The hydrolysed material was then dialysed against d i s t i l l e d B^ O f o r 24 hours, l y o p h i l i z e d and resus-pended to a concentration of 20 mg/ml for use i n the i n h i b i t i o n assay. Two control treatments were also included; i n the f i r s t , ganglioside was dissolved i n 0.05 N HC1 as above, but not heated, while i n the second treatment, ganglioside was dissolved i n d i s t i l l e d H^ O to 5.0 mg/ml and heated at 80° C f o r 90 minutes. The control treatments were then dialysed, l y o p h i l i z e d and assayed for i n h i b i t o r y a c t i v i t y as above. In h i b i t i o n by Immune and Non-Immune Rabbit Serum Immune and ncn-immune rabbit serum was dialysed against d i s t i l l e d fl^O f o r 36 hours. Crude B. g i n g i v a l i s membrane suspended i n d i s t i l l e d H^ O to a concentration of 8.0 mg/ml dry weight was mixed with an equal volume of dialysed serum and incubated with gentle shaking at room temper-ature f o r 60 minutes. Then 0.1 ml of each of the mixtures was s e r i a l l y two f o l d d i l u t e d and assayed f o r hemagglutdLnating or b a c t e r i a l aggregating a c t i v i t y as previously described. A control mixture was prepared by mixing equal volumes of membrane and d i s t i l l e d H^ O and incubating and assaying as above. Neuraminidase Treatment of Red Blood C e l l s Formalinized human red blood c e l l s (25%) were washed once i n phosphate buffered s a l i n e and then resuspended to 2.5% i n 0.05 M acetate 21 buffer (pH 5.2) or phosphate buffered saline (0.051 M, pH 7.2). Neuram-inidase (Sigma Type VT) was dissolved i n 0.1 ml acetate buffer giving an approximate concentration of 8 units/ml. The enzyme suspension was added to 4.9 ml of 2.5% red blood c e l l s i n acetate buffer giving a f i n a l concentration of 0.16 units/ml. Control suspensions of 2.5% red blood c e l l s i n 5.0 ml acetate or phosphate buffers were also prepared. The suspensions were incubated for 18 hours at 37° C with gentle agitation a f t e r which the c e l l s were washed f i v e times i n phosphate buffered saline. A suspension of red blood c e l l s that had not been incubated or treated with enzyme was also washed at the same time. Crude membrane at 8.0 mg/ml dry weight was s e r i a l l y two f o l d d i l u t e d and tested f o r hemagglutinating a c t i v i t y against each set of red blood c e l l s . E ffect of Pronase Digestion on Membrane A c t i v i t i e s The e f f e c t of Pronase digestion on the hemagglutinating and bac-t e r i a l aggregating a c t i v i t i e s of pool 1 and pool 2 membrane was examined as follows. Membrane was suspended to 5.0 mg/ml dry weight i n d i s t i l l e d ^ 0 and divided into four aliquots. One of the aliquots was mixed with an equal volume of 0.05 M Hepes buffer (pH 7.2) alone (sample ( i ) ) . The second ali q u o t was mixed with an equal volume of Pronase dissolved i n Hepes buffer to a concentration of 2.5 mg/ml for incubation a t 4° C (sample ( i i ) ) . The t h i r d aliquot was mixed as above with an equal volume of Pronase suspension (sample ( i i i ) ) . The f i n a l aliquot was mixed with an equal volume of Pronase at 5.0 mg/ml i n Hepes buffer. The Pronase suspension had been boiled at 100° C f o r 10 minutes before addition to the membrane sample (sample ( i v ) ) . The f i f t h sample contained 2.5 mg/ml 22 Pronase i n Hepes buffer mixed with an equal volume of d i s t i l l e d H20. A l l of the samples except sample ( i i ) were incubated for 18 hours a t 37° C. Sample ( i i ) was incubated at 4° C f o r 18 hours. A f t e r incubation fresh Pronase was added to samples ( i i ) , ( i i i ) and (v) to bring the f i n a l concentration of Pronase i n these samples to 2.5 mg/ml. The samples were a l l incubated f o r a further 18 hours at 37° C and 4° C. Each sample was then s e r i a l l y two f o l d d i l u t e d as described and tested f o r hemagglutin-ating and b a c t e r i a l aggregating a c t i v i t y . Serology Antiserum was prepared i n New Zealand white female rabbits by intravenous injections of whole B. g i n g i v a l i s W12 three times per week for two weeks. A booster i n j e c t i o n was given i n the fourth week and antiserum was co l l e c t e d i n the f i f t h week. Each i n j e c t i o n consisted of q 1.0 ml of approximately 5 x 10 0 2 - k i l l e d organisns/ml i n 0.15 M NaCl. The t i t r e of the antiserum was determined by mixing s e r i a l l y d i l u t e d antiserum with crude membrane (2.0 mg/ml dry weight) and assaying for clumping of the membranes. Antiserum was stored at -20° C. Electron Microscopy Membrane samples were prepared f o r electron microscopy by a modif-i c a t i o n of the method of Kasper and S e l l e r (45). Crude membrane was fix e d f o r 1 hour at 4° C i n 2.5 % phosphate buffered gluteraldehyde (pH 7.2). This procedure was followed by a b r i e f wash with 1 M phos-phate buffer and additional f i x a t i o n for 30 minutes i n aqueous 2% osmium tetroxide. The p e l l e t s were then dehydrated i n increasing concentrations 23 of ethanol t o 70%, exposed to 5% uranyl acetate i n 70% ethanol and dehydrated further to 100% ethanol. The p e l l e t s were embedded i n Epon A r a l d i t e f o r 2 days. Thin sections were stained with uranyl acetate and lead c i t r a t e and examined with a P h i l i p s 300 electron microscope. Whole c e l l s were prepared f o r negative staining as follows. C e l l s were fix e d f o r 1 hour at 4° C i n 2.5% phosphate buffered gluteraldehyde (pH 7.2). This was followed by two washes i n phosphate buffer. A drop of the washed, resuspended bacteria was placed on the top of a collodion carbon coated g r i d and stained with 2% phosphotungstic a c i d for 30 sec-onds. The g r i d was examined with a P h i l i p s 300 electron microscope at 60 kV. Whole c e l l s were stained with ruthenium red following the proce-dure of Woo e t a l (120), except that 3.0% v o l / v o l gluteraldehyde was used and f i x a t i o n was c a r r i e d out f o r 1 hour a t 4° C. 24 RESULTS Electron Microscopy B. g i n g i v a l i s W12 was examined by electron microscopy a f t e r s t a i n -ing f o r capsular polysaccharide with ruthenium red (Figure 1). The c e l l s demonstrated t y p i c a l trilaminar inner and outer membranes separated by a t h i n layer of peptidoglycan. A t h i n electron dense layer of capsular material located external t o the outer membrane of the c e l l was seen which was i n close association with the outer membrane. Fibrous and more dif f u s e material extended f o r long distances from the c e l l s and i n some cases was seen to connect i n d i v i d u a l c e l l s . Generally t h i s material was seen to be associated d i r e c t l y with the c e l l s . Whole c e l l s were also examined by negative staining with phospho-tungstic acid (Figure 2). The c e l l s showed a variable number of f i b r i l l a r "appendages" apparently attached to the surface of the c e l l . F i b r i l l a r material was also seen i n the spaces between c e l l s apparently not attached to any p a r t i c u l a r c e l l . The amount of f i b r i l l a r material seen on any one c e l l was quite variable, as was the length and thickness of the i n -d i v i d u a l f i b e r s . The f i b e r s d i d not appear to have a r i g i d structure and some seemed to branch towards the t i p s . Crude membrane material from B. g i n g i v a l i s W12 was also examined by electron microscopy. Membrane fragments made up of single tri l a m i n a r structures were observed (Figure 3). Some fragments were present i n long open ended segments while other fragments had joined ends to form closed 25 Figure 1. Electron rnicrograph of B. g i n g i v a l i s W12 stained with ruthenium red. Bar represents 0.5/x. 26 Figure 2. Electron ndcrograph of negatively stained B. g i n g i v a l i s W12. Ear represents 0.5 27 Figure 3. Electron micrograph of crude outer membrane from B. g i n g i v a l i s W12. Bar represents 1.0 28 c i r c u l a r structures. Diffusely stained (capsular) material was also seen i n the membrane preparation. Structures resembling fimbriae were never observed. Iso l a t i o n and Separation of Outer Membranes Using the procedure described e a r l i e r , one percent of the dry weight of B. g i n g i v a l i s W12 was recovered as crude outer membrane. Further f r a c t i o n a t i o n of the membranes was achieved by chromatography on a Sephadex G100 column equilibrated with 0.5% sodium desoxycholate buffer. The e l u t i o n p r o f i l e showed two d i s t i n c t peaks of material e l u t i n g from the column (Figure 4) . Pool 1 material eluted a t the void volume of the column (58). This peak contained most of the membrane protein as shown by the 0D2go p r o f i l e . The second peak of material e l u t i n g from the column (Pool 2) contained more carbohydrate material and less protein than pool 1. Material was also present i n a t h i r d f r a c t i o n , pool 3, but the amount of material present i n t h i s f r a c t i o n varied from experiment to experiment and was not studied further. Of the material applied to the column approximately 40% was recovered i n the eluant; of the material recovered, 45.5% was recovered i n pool 1 and 55.5% was i n pool 2. Separation and Localization of Membrane A c t i v i t i e s Throughout the i s o l a t i o n and separation of B. g i n g i v a l i s outer membranes, the hemagglutinating and b a c t e r i a l aggregating a c t i v i t i e s of the membranes were monitored. Crude outer membranes were found to have both hemagglutiriating and b a c t e r i a l aggregating a c t i v i t y . The l a t t e r included the a b i l i t y to aggregate several gram p o s i t i v e o r a l organisms: 29 Figure 4. Elution p r o f i l e of an outer membrane preparation of B. gingivalis-Wl2 on a Sephadex G100 column e q u i l i -brated with desoxycholate buffer. The sample was ap-p l i e d and eluted with the same buffer. The eluant was monitored f o r protein at and reacted with anthrone reagent f o r carbohydrate (Ag20^' (protein) A, 9 f l (carbohydrate) ;  30 0.9 V O L U M E (m l ) S. mutans LM7, S. mutans C6715, S. m i t i o r , S. sanguis 10556, 10557 and 10558, S. s a l i v a r i u s HB and HBV5 and A. viscosus (data not shown) . S. m i t i o r was chosen for further investigation as i t showed the l e a s t amount of self-aggregation i n the assay. After separation of the crude outer membrane material on the Sephadex G100 column, pool 1 and pool 2 were tested f o r hemagglutinating and b a c t e r i a l aggregating a c t i v i t y . The r e s u l t s are summarized i n Table I. The r a t i o s of hemagglutination t i t r e s to b a c t e r i a l aggregation t i t r e s indicated an enrichment f o r b a c t e r i a l aggregating a c t i v i t y i n pool 1 and f o r hemagglutinating a c t i v i t y i n pool 2. Since t h i s data appeared to indicate that these two a c t i v i t i e s were mediated by d i f f e r e n t membrane components, the biochemical compo-s i t i o n of each of the fractions was analysed. Biochemical Characterization of Membrane Fractions Crude membrane, pool 1 and pool 2 were characterized biochemically by colourimetric assays and the r e s u l t s summarized i n Table I I . Pool 1 contained 51% protein by weight while pool 2 contained 11.7%. In other experiments pool 2 was found to contain only 5% protein, however t h i s p a r t i c u l a r separation (Figure 4) involved larger volumes of material which appeared to r e s u l t i n some contamination of pool 2 with protein. The other p r i n c i p a l biochemical difference between the two fractions was that pool 2 contained a larger amount of chloroform-methanol extractable l i p i d (22% by weight) compared to pool 1 (5% by weight). Other cons t i t u -ents were generally equivalent. 32 TABLE I Hemagglutinating and B a c t e r i a l Aggregating A c t i v i t i e s of Crude and Fractionated Membranes Membrane Hemagglutination B a c t e r i a l Aggregation Patio Fraction T i t r e (HA) T i t r e (BA) HA:BA Crude 1024 512 2:1 Pool 1 2 128 1:64 Pool 2 128 2 64:1 33 TABLE I I Biochemical Analysis of Outer Membrane Fractions Percentage of Dry Weight of Membrane Fractions Substance Crude Pool 1 Pool 2 Protein 22.1 51.1 11.7 Hexose 32.1 20.2 18.9 Total Sugars 35.8 25.9 27.9 Methyl Pentoses 2.7 4.5 4.0 Pentose 1.6 1.3 2.1 Hexosamines ND* Glucuronic Acid ND ND ND Muramic Acid ND Chlcroform-Methanol Extractable L i p i d 20.0 5.0 21.6 Nucleic Acid 4.0 ND* not detectable i n quantities of l e s s than 2-3%. Assay samples con-tained 0.5-1.5 mg dry weight of membrane. 34 SDS-Polyacxylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed on the membrane fract i o n s . A 10% acrylamide g e l stained f o r proteins with Coomassie B r i l l i a n t Blue (Figure 5) revealed that crude membrane material (Lane B) possessed three major protein bands, as w e l l as a small number of minor bands. Pool 1 material (Lane C) appeared to contain the same three pro-t e i n bands as the crude membrane f r a c t i o n , however the position of the two lower bands had sh i f t e d downwards s l i g h t l y r e l a t i v e to the bands i n the crude membrane f r a c t i o n . Pool 2 (Lane D) contained one major protein band which appeared to correspond to the lowest molecular weight major protein seen i n both pool 1 and crude membrane. However i n the pool 2 f r a c t i o n the apparent molecular weight of t h i s protein had increased s i g n i f i c a n t l y . A s i m i l a r g e l (10% acrylamide) stained with s i l v e r n i t r a t e showed b a s i c a l l y the same banding pattern as the Coomassie stained gel (Figure 6). Comparison of the membrane bands with standards of known molecular weight (Lane A) revealed that the three major protein bands seen i n crude and 4 4 pool 1 membrane had molecular weights of 2.22 x 10 , 4.15 x 10 and 4 6.9 x 10 . The minor protein bands seen with Coomassie staining were much more v i s i b l e with the s i l v e r s t a i n , and one band with a molecular 4 weight of 5.65 x 10 appeared to be more d i s t i n c t i n pool 1 (lane C) com-pared to crude membrane (Lane B). Again pool 2 (Lane F) contained only one protein band with a molecular weight of approximately 2.2 x 10^. The phenomenon of the same protein band appearing i n d i f f e r e n t positions i n di f f e r e n t fractions was not observed with s i l v e r n i t r a t e stained gels. A 12.5% acrylamide g e l was used to separate the iso l a t e d membrane 35 A B C D Figure 5. SDS-PAGE of crude membranes and fractions eluted from Sephadex G100. Acrylamide concentration was 10% and g e l was stained with Coomassie B r i l l i a n t Blue. Lane A molecular weight markers bovine serum albumin 66,000 daltons, ovalbumin 45,000, tryp-sinogen 24,000,/3-lactaglobulin 18,400, lysozyme 14,300. Lane B crude membrane. Lane C pool 1. Lane D pool 2. A l l membrane fractions contained 70 ug protein. 36 A B C D E F Figure 6. SDS-PAGE of crude membrane, fractions eluted from Sephadex G100 and Pronase digested pool 1. Acrylamide concentration was 10% and the g e l was s i l v e r stained. Lane A molecular weight markers. Lane B crude membrane. Lane C pool 1. Lane D Pr-1. Lane E Pronase. Lane F pool 2. A l l membrane fractions contained 7.0 ug protein. The same dry weight of pool 1 and Pr-1 (14 ug) was applied to the g e l . The Pronase sample also contained 7.0 ug protein. 37 fractions before staining with the lipopolysaccharide s t a i n (Figure 7). The fractions showed regularly repeating bands throughout the length of the g e l which i s c h a r a c t e r i s t i c of LPS preparations (109). Crude mem-brane (Lane B) material showed up to 25 d i s t i n c t bands of LPS material, a large proportion of which was at the low molecular weight end of the gel. Pool 1 material (Lane C) showed only trace amounts of the low mole-cular weight LPS material while i t d i d contain amounts of the high mole-cular weight material roughly equivalent to that present i n crude mem-brane. In contrast, pool 2 material (Lane F) contained primarily the low molecular weight material, and only trace amounts of the high mole-cular weight LPS seen i n pool 1 and crude membrane. Immunological Characterization of Membrane Fractions Analysis of antigens transfered by electrophoretic b l o t t i n g following SDS-polyacrylamide ge l electrophoresis (Figure 8) revealed that crude membrane contained three w e l l defined bands that reacted with anti-B. g i n g i v a l i s serum. The most predondnant of these was a band that 4 appeared to correspond to the 4.15 x 10 dalton protein. These three bands on the immun-blot stood out as d i s t i n c t and discrete bands against a background of d i f f u s e l y staining material. In the crude membrane f r a c -t i o n , material i n the upper t h i r d of the track stained as a dark d i f f u s e smear, while the material i n the lower ha l f of the track stained i n regularly repeating d i f f u s e bands resembling i n orientation and spacing those seen i n SDS-polyacrylamide gels of crude membrane stained for LPS. Pool 1 and pool 2 fractions had d i f f e r e n t staining patterns. Pool 1 material contained the d i f f u s e dark staining material i n the upper t h i r d 38 Figure 7. SDS-PAGE of crude membrane, fractions eluted from Sephadex G100 and Pronase digested pool 1. The acrylamide concentration was 12.5% and gel was stained f o r LPS. Lane A molecular weight markers. Lane B crude membrane. Lane C pool 1. Lane D Pr-1. Lane E Pronase. Lane F pool 2. A l l membrane fractions con-tained 18 ug dry weight. Pronase sample was as i n Figure 6. 39 A B C Figure 8. Western blot of SDS-PAGE of crude membrane and fractions eluted from a Sephadex G100 column. Antigens transfered to nitro-cellulose were reacted with anti-B. gingivalis serum. Lane A crude membrane 7.0 ug protein (32 ug dry weight). Lane B pool 1 7.0 ug protein (14 ug dry weight). Lane C pool 2 7.0 ug protein (63 ug dry weight). 4 —} -indicates position of 4.15 x 10 dalton protein. 40 of the gel as w e l l as the 4.15 x 10 dalton antigen. The regular repeating bands i n the lower h a l f of the g e l were not present i n t h i s f r a c t i o n . Pool 2 material lacked the darkly staining d i f f u s e material i n the upper part of the track while the lower part of the track showed the repeating bands seen i n the crude membrane f r a c t i o n . I n h i b i t i o n of Hemagglutination and B a c t e r i a l Aggregation The s e n s i t i v i t y of each a c t i v i t y to interference by various sub-stances was examined and the r e s u l t s summarized i n Table I I I . As the r e s u l t s indicate none of the saccharides tested except N-acetylgalac-tosamine had any ef f e c t on hemagglutinating a c t i v i t y . N-acetylgalac-tosamine mixed with membrane to a f i n a l concentration of 5mg/ml (23 mM) produced i n h i b i t i o n of hemagglutination. B a c t e r i a l aggregating a c t i v i t y showed more s e n s i t i v i t y to saccharide i n h i b i t i o n . Galactose at a con-centration of 2.5 mg/ml (14 mM) and D-glucosamine (23 mM) and N-acetyl-glucosamine (23 mM) a t 5 mg/ml produced i n h i b i t i o n of b a c t e r i a l aggrega-t i o n . z^rercaptoethanol and d i t h i o t h r e i t o l a t concentrations of 30.5 ug/ml (0.4 mM) and 19.5 ug/ml (0.1 mM) respectively were found to enhance hemag-gl u t i n a t i n g a c t i v i t y , while they d i d not have any e f f e c t on b a c t e r i a l aggregation. Type I I I mixed bovine brain gangliosides were found to i n h i b i t hemagglutinating a c t i v i t y t o a concentration of 312 ug/ml while they i n h i b i t e d b a c t e r i a l aggregating a c t i v i t y t o a concentration of 2.5 mg/ml. To determine the significance of the s i a l i c component of bovine brain ganglioside on i t s i n h i b i t o r y a c t i v i t y , a c i d hydrolysis of the gang-41 TABLE I I I I i i h i b i t i o n of Hemagglutinating and Bac t e r i a l Aggregating A c t i v i t y Substance Effect on A c t i v i t y 0 Hemagglutination B a c t e r i a l Aggregation D-mannose D-glucose D-fucose D-ribose D-mannitol D-fructose lactose rhamnose D-galactose D-glucosamine N-acetylglucosaitdne colcminic acid dextran T250 /S-mercaptoethanol (100 mM) d i t h i o t h r e i t o l EDTA (1 irM) CaCl 2 (2 mM) bovine brain gangliosides ac i d hydrolysed acid treated, unhydrolysed heated i n d i s t i l l e d H 20 N-acetylgalactosamine 0 0 0 0 0 0 0 0 0 0 0 0 0 +(30.5) +(19.5) 0 0 -(312) 0 -(156) 0 -(5000) 0 0 0 0 0 0 0 0 (2500) •(5000) (5000) 0 0 0 0 0 0 (2500) (2500) (2500) 0 E f f e c t on a c t i v i t y designated as follows: enhancement of a c t i v i t y (+), i n h i b i t i o n of a c t i v i t y (-) , no e f f e c t on a c t i v i t y (0) . The bracketted numbers indicate the f i n a l concentration (ug/ml) of substance mixed with membrane which led to enhancement or i n h i b i t i o n of a c t i v i t y . A l l the t e s t substances were at an i n i t i a l concentration of 20 mg/ml unless otherwise indicated. 42 l i o s i d e was performed to remove these residues. The r e s u l t s indicated that while acid hydrolysis removed the i n h i b i t o r y a c t i v i t y of ganglioside i n hemagglutination, heat treatment i n d i s t i l l e d H^ O alone also removed the i n h i b i t o r y a c t i v i t y . Acid treated but unheated ganglioside retained i t s i n h i b i t o r y a c t i v i t y . In contrast, acid hydrolysis or heating i n d i s t i l l e d H^ O alone had no e f f e c t on the a b i l i t y of ganglioside to i n -h i b i t b a c t e r i a l aggregation. E f f e c t of Immune and Non-Immune Serum on Aggregating A c t i v i t y The i n h i b i t o r y e f f e c t s of non-irarrune and immune rabbit serum raised to whole B. g i n g i v a l i s W12 c e l l s was investigated. Preliminary t e s t s showed that both immune and non-immune serum interfered with both a c t i v i t i e s . To eliminate non-specific interference, possibly due to an ion mediated clumping of the membranes, both irtrnune and non-immune serum were dialysed against d i s t i l l e d H^ O f o r 36 hours. A p r e c i p i t a t e formed i n the serum upon d i a l y s i s , but t h i s was determined to have no e f f e c t on the subsequent r e s u l t s , and i t was removed by low speed centrifugation. Crude membrane material was preincubated with immune serum, non-inrtune serum or d i s t i l l e d H^ O and then assayed f o r hemagglutinating and b a c t e r i a l aggregating a c t i v i t y . The r e s u l t s are shown i n Table IV. As indicated, hemagglutinating a c t i v i t y was i n h i b i t e d by both immune and non-immune serum, while b a c t e r i a l aggregating a c t i v i t y was i n h i b i t e d only by immune serum. Effe c t of Neurairnjiidase Treatment of Red Blood C e l l s Formalinized human red blood c e l l s were treated with neuraminidase 43 TABLE IV Eff e c t of Immune and Non-Immune Serum on Hemagglutination and Ba c t e r i a l Aggregation Membrane Preincubated with: T i t r e of A c t i v i t y Hemagglutination B a c t e r i a l Aggregation 256 128 0 64 0 2 D i s t i l l e d tL^ O Non-Immune Serum Immune Serum 44 to determine the e f f e c t removal of s i a l i c a c i d residues from the surface of the c e l l s would have cn hemagglutination. Results are shown i n Table V. The r e s u l t s indicate that neuraminidase treatment increased hemagglutination by crude membrane two f o l d over a control set of red blood c e l l s incubated i n acetate buffer alone. The neuraminidase treated red blood c e l l s were then assayed to determine i f neuraminidase treatment rendered the hemagglutination a c t i v i t y , more sensitive to galactose i n h i b i t i o n . Using the assay previously des-cribed i t was found that galactose d i d not have any ef f e c t on hemagglutin-ation with neuraminidase treated or untreated red blood c e l l s . E f f e c t of Chloroform-Methanol Extraction on Aggregating A c t i v i t y As part of the biochemical characterization of the membrane, a chloroform-methanol extraction of each of the fractions was performed to determine the amount of loosely bound l i p i d present i n each f r a c t i o n . The material which was not extracted i n t o the chloroform phase was dried to remove the methanol, resuspended i n d i s t i l l e d t^O and l y o p h i l i z e d . This material was then suspended i n d i s t i l l e d E^O to a concentration of 8.0 mg/ml dry weight and assayed f o r hemagglutinating and b a c t e r i a l aggregating a c t i v i t y . The r e s u l t s are shown i n Table VI. A comparison of the t i t r e s and the HA:BA r a t i o s obtained with the extracted and non-extracted fractions indicated that chloroform-methanol extraction had s e l e c t i v e l y removed or inactivated the component necessary f o r hemagglutination while leaving the b a c t e r i a l aggregating a c t i v i t y v i r t u a l l y untouched. 45 TABLE V Effec t of Neuraminidase Treatment of Red Blood C e l l s on Hemagglutination A c t i v i t y Red Blood C e l l Treatment Hemagglutination T i t r e Untreated 1024 Acetate buffer treated 4096 Neuraininidase treated 8192 46 TABLE VI Effect of Chlorofom-Methanol Extraction on Heitagglutination (HA) and Ba c t e r i a l Aggregation (BA) Fraction Extracted Non--Extracted HA BA Ratio HA BA Ratio T i t r e T i t r e HA:BA T i t r e T i t r e HA:BA Crude 8 128 1:16 1024 512 2:1 Pool 1 2 128 1:64 2 128 1:64 Pool 2 8 8 1:1 128 2 64:1 47 E f f e c t of Pronase Treatment on Aggregating A c t i v i t i e s Pronase treatment of pool 1 and pool 2 membranes was c a r r i e d out to determine the s e n s i t i v i t y of the membrane a c t i v i t i e s to p r o t e o l y t i c action. The r e s u l t s are summarized i n Table V I I . The r e s u l t s appeared to indicate that incubation of pool 2 mem-brane with Pronase removed v i r t u a l l y a l l of the hemagglutinating a c t i -v i t y . The small amount of a c t i v i t y remaining was probably due to the protease i t s e l f , since a control consisting only of Pronase showed a hemagglutination t i t r e of 2-4. The low t i t r e s present i n those samples containing active Pronase was not due to p r o t e o l y t i c action on the red blood c e l l s during the one hour assay period. A separate experiment i n which red blood c e l l s were incubated with Pronase f o r one hour at room temperature and then washed extensively and assayed for t h e i r a b i l i t y to be hemagglutinated by crude membranes revealed that Pronase treatment d i d not reduce the t i t r e obtained compared to red blood c e l l s that had not been Pronase treated. Pronase that had been inactivated by b o i l i n g f o r 10. minutes di d not reduce the hemagglutinating t i t r e s i g n i f i c a n t l y . In contrast, Pronase treatment of pool 1 material d i d not reduce the a b i l i t y of t h i s material to aggregate S. m i t i o r . Treatment of pool 1 material with Pronase was then car r i e d out on a larger scale and the material remaining a f t e r p r o t e o l y t i c treatment was separated frcm the remaining Pronase and the digested material by chroma-tography on a Sephadex G100 column equilibrated with 0.05 M T r i s buffer (pH 9.0) plus 2M NaCl (Figure 9). The undigested material (Pr-1) eluted from the column at the void volume, while two other peaks eluted l a t e r . Lowry protein analysis of Pr-1 showed that the percentage of protein i n 48 TABLE, VII Effect of Pro t e o l y t i c Treatment on Hemagglutination and B a c t e r i a l Aggregation Bac t e r i a l Aggregation T i t r e 64-128 64-128 64 64 4 Hemagglutination T i t r e 256 4 8-16 256 2-4 Membrane Treatment Pool 1 Pool 1 + Pronase Pool 1 + Pronase (4° C) Pool 1 + Pronase (boiled) Pronase Pool 2 Pool 2 + Pronase Pool 2 + Pronase (4° C) Pool 2 + Pronase (boiled) Pronase 49 Figure 9. E l u t i o n p r o f i l e of Pronase digested pool 1 membrane on a Sephadex G100 column equilibrated with 0.05 M T r i s pH 9.0 plus 2.0 M NaCl. The eluant was monitored f o r protein at ( and reacted with anthrone reagent f o r carbohydrate (Ag20^" void volume pool was Pr-1. (protein) — A,„ n (carbohydrate) 50 0.8 — X280 VOLUME (ml) t h i s pool had been reduced by 90% compared to untreated pool 1 material. Polyacrylamide g e l electrophoresis of Pr-1 shown i n Figures 6 and 7, indicated that a l l the protein bands had been removed by Pronase t r e a t -ment. The g e l also indicated that Pr-1 lacked any bands that could be regarded as Pronase contaniinants. The material i n Pr-1 that stained with the LPS s t a i n remained unchanged a f t e r Pronase treatment compared to untreated pool 1 material. This material (Pr-1) was also assayed f o r i t s a b i l i t y to aggregate S. m i t i o r . Compared to the b a c t e r i a l aggregation t i t r e obtained with pool 1 suspended to 8.0 mg/ml dry weight—64—the t i t r e obtained with Pr-1 suspended t o the same concentration—4096—represented a 64-fold increase i n a c t i v i t y . Analysis of the antigenic pattern of Pr-1 by Trans-blot and GAR-HRP staining a f t e r SDS-polyacrylamide ge l electrophoresis revealed that i t had the same staining pattern as pool 1 except f o r the absence of the 4 protein bands, most notably that corresponding to the 4.15 x 10 d a l -ton protein. The absence of t h i s protein antigen from the Pr-1 sample was quite obvious as i t stained darkly i n the pool 1 preparation and was t o t a l l y absent from the Pr-1 preparation when the same weight of each sample was applied to the g e l . I s o l a t i o n of Aggregating A c t i v i t i e s from Culture Supematants An attempt was made to i s o l a t e membrane material from culture supematants i n the hope that overproduction and blebbing o f f of the outer membrane might provide an alternate and more productive source of hemagglutinating and b a c t e r i a l aggregating components. Supematants from 52 48 and 96 hour cultures were collected and 'membrane1 material harvested by high speed centrifugation as described. The amount of material recovered a f t e r l y o p h i l i z a t i o n was 9.3 mg/100 ml of 48 hour culture and 32 mg/100 ml of 96 hour culture. This material was then assayed f o r aggregating a c t i v i t y . The HAtitre of 48 hour culture supernatants was 2048, while the BA t i t r e was 512. The HA t i t r e of 96 hour culture super-natants was 8192 and the BA t i t r e was 2048. However when these culture supernatants were analysed by g e l electrophoresis they were seen to have markedly d i f f e r e n t p r o f i l e s compared to crude membrane material. There were a number of protein bands i n the supernatant material which were not present i n the prepared membrane material. These proteins were present i n much larger amounts than those common to both the supernatant material and the prepared membrane material. These fractions were also found to contain 5.5 to 6.5% nucleic a c i d compared to 4.0% found i n crude membrane material. 53 DISCUSSION Bacteria colonizing the oral cavity are often capable of binding to host tissue as well as to other bacteria (98, 115). S. salivarius, for instance, binds to human erythrocytes and buccal epithelial cells as well as to several other oral organisms (115). Furthermore i t was shown in S. salivarius that the adhesins mediating binding to host tissues were distinct from those reacting with other bacteria (115). The frac-tionation of B. gingivalis W12 outer membranes into two components with different binding properties demonstrated that this organism had simi-lar characteristics. From the view point of the organism, such a separa-tion of adhesive constituents makes sound ecological sense as interference with binding to one receptor does not preclude binding to an alternate receptor. B. gingivalis is an example of an organism which must often have to rely on binding to gram positive bacteria because binding to host tissue will frequently be inhibited by crevicular fluid (serum exudate) or saliva (98). The use of relatively gentle physical techniques allowed the outer membrane complex to be isolated in a morphological unit; electron micro-graphs showed intact membrane structures with a typical trilaminar arrangement. The isolated membranes also retained biological activity. A relatively simple protein composition, as determined by SDS-PAGE, and the presence of material which stained with an LPS stain indicated that the material isolated by this procedure was indeed outer membrane. The 54 absence of detectable amounts of muramic ac i d and the low amount of nuc-l e i c a c i d present suggested that the membrane preparations were generally free of cytoplasmic or c e l l w a l l contamination. Although separation of the hemagglutinating and b a c t e r i a l aggregating a c t i v i t i e s a f t e r column chromatography was observed, 100% resolution was not achieved i n t h i s experiment. Complete separation of a c t i v i t i e s was achieved i n experiments i n which s l i g h t l y smaller amounts of crude membrane were applied to the column. I t would appear that the amount of membrane applied t o the column i n the separation reported here was j u s t above the resolving capacity of the column. Biochemical studies of the two membrane fractions revealed that they had b a s i c a l l y the same composition as those i s o l a t e d by Mansheim and Kasper (58). Pool 1 consisted of a large amount of protein and carbohydrate material with l i t t l e chloroform-methanol extractable l i p i d , while pool 2 consisted mostly of loosely bound l i p i d , carbohydrate mate-r i a l and a small amount of protein. The d i s t r i b u t i o n of membrane mate-r i a l into pool 1 and pool 2 fractions following column chromatography as w e l l as the t o t a l amounts of carbohydrate and chloroform-methanol extractable l i p i d present i n the two pools varied s i g n i f i c a n t l y from the r e s u l t s obtained by Mansheim and Kasper (58). These differences could be due to s t r a i n variations i n the composition of the outer mem-brane complex or to the fa c t that the method used f o r the i s o l a t i o n of the outer membrane d i f f e r e d from Mansheim and Kasper by omitting the heating step.• I t was found that heating the c e l l s i n the sodium chloride, sodium phosphate, EDTA buffer resulted i n a loss of both aggregation a c t i v i t i e s . I t i s not unreasonable to assume that the material i s o l a t e d 55 by the modified procedure varied biochemically from the material isol a t e d by Mansheim and Rasper. Further information about the composition of the membrane f r a c -tions was obtained from SDS-polyacrylamide gel electrophoresis. The presence of LPS i n crude outer membranes was demonstrated by staining with the LPS st a i n of Tsai and Frasch (109). The staining pattern indicated that the LPS present i n the crude membrane exhibited size heterogeneity. This phenomenon has been demonstrated i n several other organisms (30, 40 49, 52), but has not been reported f o r B. g i n g i v a l i s . The observation of LPS size heterogeneity has l e d to the proposal that smooth type or-ganisms contain a mixture of LPS molecules, seme of which lack O-antigenic side chains while others contain varying numbers of covalently bound O-antigenic side chain units. Rough variants contain large amounts of the low molecular weight LPS and l i t t l e of the high molecular forms. Separation of crude membrane into pool 1 and pool 2 fractions using the LPS disaggregating buffer resulted i n the fractionation of the LPS com-ponent of the membrane, with the majority of the high molecular weight or smooth LPS remaining i n pool 1 and the low molecular weight or rough LPS e l u t i n g i n pool 2. This i s i n contrast to the previously reported d i s t r i b u t i o n of B. g i n g i v a l i s LPS af t e r disaggregation by sodium desoxy-cholate (58) i n which LPS was located only i n pool 2. I t i s reported (15) that rough and smooth LPS molecules are extracted i n d i f f e r e n t pro-portions depending on the extraction procedure used, so i t i s not su r p r i s -ing that the two types of LPS would separate upon treatment with sodium desoxycholate. The protein banding patterns of crude outer membranes seen i n s i l v e r 56 stained SDS gels are t y p i c a l of outer membrane preparations from other gram negative organisms (1, 55, 73) i n that there are only a few major peptide bands present; B. g i n g i v a l i s outer membranes contained three major peptides with molecular weights of 69,000, 41,500, and 22,200 d a l -tons. Five major peptide bands were reported to be present i n the outer membrane preparations of Mansheim and Kasper (58), three of which had molecular weights i n the range of those seen here. The i d e n t i t y and function of these peptides has not been determined. However, studies with a number of enteric bacteria have shown that the outer membranes contain proteins (porins) which act to make the outer membrane s e l e c t i v e l y permeable or impermeable to c e r t a i n substances (71, 72, 75) . The porins vary i n s i z e from s t r a i n to s t r a i n , but are generally major outer mem-brane polypeptides with molecular weights of 35,000 to 40,000 daltons. One can speculate that the 41,500 dalton peptide observed i n the outer membrane of t h i s organis, i s i n fact a porin. The 22,200 dalton peptide has a molecular weight i n a range consistent with i t being a fimbriae ( p i l i ) subunit (41). Polyacrylamide gels stained with Coomassie blue revealed the same three major peptide bands, however i n these gels the 22,200 dalton pep-t i d e was present i n s l i g h t l y d i f f e r e n t positions i n each of the three membrane preparations. The apparent changes i n molecular weight could be due to association of the peptide with LPS. This type of interaction has been suggested to a l t e r the apparent molecular weight of other outer membrane proteins (32). The electrophoretic mobility of outer membrane proteins can also be modified by d i f f e r e n t s o l u b i l i z a t i o n conditions (31). The temperature of s o l u b i l i z a t i o n and the presence of /S-mercaptoethanol 57 a f f e c t the m o b i l i t y of outer membrane proteins of Pseudcmonas aeruginosa (31). Immunological studies also indicated that pool 1 and pool 2 were made up of d i f f e r e n t membrane components. Immun-blots demonstrated that pool 1 contained several protein bands which reacted with antibody raised to whole c e l l s while pool 2 d i d not appear to contain any w e l l defined protein antigens. Both pools contained a n t i g e n i c a l l y active LPS; pool 1 contained p r i m a r i l y the high molecular weight forms and pool 2 the low molecular weight forms. The capsular polysaccharide of t h i s organism i s also a n t i g e n i c a l l y active (57, 58, 83), however because of the large 5 molecular s i z e , reported to be 7.2 x 10 daltons (58) i t probably d i d not enter the g e l . The fimbriae of B. g i n g i v a l i s are also reported to be a n t i g e n i c a l l y active (79), however i f the 22,200 dalton protein seen i n g e l electrophoresis i s a f i m b r i a l subunit i t was not observed to react with antiserum r a i s e d against whole c e l l s . P a r t i c u l a t e material containing hemagglutinating and b a c t e r i a l aggregating a c t i v i t y could be isolat e d from 48 and 96 hour culture supernatants by centrifugation. On a dry weight basis t h i s material contained more aggregating a c t i v i t y than the is o l a t e d membranes. I t was thought that t h i s material might serve as an alternate source of these membrane components, however ge l electrophoresis revealed the presence of peptide bands not seen i n the iso l a t e d membrane preparations which probably represented cmtendnation due to seme c e l l l y s i s . The a b i l i t y of t h i s organism to shed outer membrane fragments containing these agglu-t i n i n s may serve a protective function by binding antibody which would interf e r e with adherence. 58 Separation of b a c t e r i a l aggregating and hemagglutinating a c t i v i t i e s i n i s o l a t e d membranes implied that each was mediated by a d i f f e r e n t sur-face component. Further examination of the ch a r a c t e r i s t i c s of these two a c t i v i t i e s provided a better i n d i c a t i o n of the i d e n t i t y of the membrane components involved i n these a c t i v i t i e s . B a c t e r i a l aggregation was found to be sensitive to blocking by D-galactose, D-glucosamine and N-acetylglucosainine. As well, i t was blocked by mixed bovine brain gangliosides, possibly due to the galactose r e s i -dues i n the molecule. Gas l i q u i d chrcmatographic analysis of the sugar components of outer membrane preparations have demonstrated that D-galactose and D-glucosamine are present i n both the LPS (59) and the capsular polysaccharide (58) of B. g i n g i v a l i s . Both these components are present i n pool 1 membrane associated with the b a c t e r i a l aggregating a c t i v i t y . Antiserum raised to whole c e l l s also i n h i b i t e d the b a c t e r i a l aggre-gating a c t i v i t y while ncn-immune antiserum d i d not. The l a t t e r observa-t i o n has been previously reported (98). The fa c t that B. g i n g i v a l i s can adhere to b a c t e r i a l c e l l s i n the presence of ncn-immune serum and s a l i v a (98) i s es s e n t i a l for t h i s organism to be able to establ i s h i t -s e l f i n subgingival plaque. Once the organism has colonized periodontal pockets and the disease state i s established the host immune system recognizes and responds to the b a c t e r i a l antigens. Thus the a b i l i t y of immune serum to block b a c t e r i a l aggregation can be seen as the host's attempt to remove or l i m i t the spread of t h i s organism. However, as previously mentioned, the organism may be p a r t i a l l y able t o counteract t h i s interference by blebbing o f f b i t s of the outer membrane and "soaking" 59 up the antibody. A balance between host antibody production and bac-t e r i a l c e l l blebbing may account f o r the presence of periodontal pockets which remain a constant s i z e . I f i t was possible to t i p the balance to favour antibody production, perhaps by exogenous immunization with the b a c t e r i a l aggregation adhesin, one could perhaps envision the eradication of t h i s organism from periodontal s i t e s and immunity from further i n f e c t i o n . The a b i l i t y of irtmune serum to block the b a c t e r i a l aggregation reaction implied that the adhesin was a n t i g e n i c a l l y active. Since the capsular polysaccharide (58), LPS (59) and at l e a s t one protein compo-nent of t h i s pool have been demonstrated to be antigenic, from an immun-o l o g i c a l point of view any of these three components could be the adhesin. Further investigation revealed that b a c t e r i a l aggregation was not destroyed by Pronase digestion. In f a c t , the a c t i v i t y of the high mole-cular weight material remaining a f t e r p r o t e o l y t i c treatment and column chromatography was found to be greatly enhanced when compared to the same weight of undigested material. The enhancement was greater than could be accounted f o r as due simply to the removal of the protein component (50% of the dry weight) and was probably due to an unmasking of active s i t e s blocked by protein. The b a c t e r i a l aggregating a c t i v i t y was also r e s i s t a n t to chloro-form-methanol extraction, suggesting that the small amount of loosely bound l i p i d present i n t h i s f r a c t i o n played no part i n t h i s a c t i v i t y . Thus i t appeared that the active component was either the capsular polysaccharide or the LPS, both of which would be unaffected by these two treatments. Both of these components have been shown to be active i n adherence i n other systems (17, 117) . However i n t h i s case, the f a c t 60 that LPS was present i n both pool 1 and pool 2 while b a c t e r i a l aggregating a c t i v i t y was present only i n pool 1 would seem to indicate that e i t h e r LPS was not involved or that only smooth or high molecular weight LPS was active. Pool 1 material aggregated a number of gram p o s i t i v e bacteria (unpublished observation). The l i m i t e d number of components available i n t h i s f r a c t i o n suggests that the same adhesin i s involved i n a l l the reactions. I f t h i s i s the case, then the adhesin must recognize a recep-tor common to many gram p o s i t i v e organisms. In l i g h t of the uniqueness of the chemical composition of the c e l l walls of d i f f e r e n t bacteria, i t i s possible that the adhesin reacts with charged groups on the c e l l surface. Characterization of the hemagglutinin of B. g i n g i v a l i s was more d i f f i c u l t . Hemagglutination was blocked by N-acetylgalactosamine; none of the other sugars had i n h i b i t o r y a c t i v i t y . I t has been indicated i n other studies (98, 77) that t h i s sugar does not i n h i b i t hemagglutination. The concentration of N-acetylgalactosamine that produced i n h i b i t i o n of a c t i v i t y (23 mM) was lower than the concentrations—100 mM (98) and 50-100 mM (77)—used i n the other studies. The fact that i n h i b i t i o n was observed i n t h i s case could be due to s t r a i n v a r i a t i o n or to differences i n 4 t h e s e n s i t i v i t y to blocking of whole c e l l a c t i v i t y as compared to membrane a c t i v i t y . The amount of sugar that was needed to produce i n -h i b i t i o n was high when compared with the amounts of other materials, such as the S-containing components and the gangliosides, which affected membrane a c t i v i t y . I t i s possible that the blocking i s merely a non-s p e c i f i c phenomenon due to the high concentration of sugar used. On the 61 other hand, the blocking may be a s p e c i f i c phenomenon and the high con-centrations necessary due simply to the fact that monosaccharides are generally not very good i n h i b i t o r s , as they lack the three dimensional arrangements found i n larger molecules. Hemagglutination was also blocked by bovine brain gangliosides. The i n h i b i t o r y a c t i v i t y was not due to s i a l i c acid residues since acid hydrolysed ganglioside showed the same amount of i n h i b i t i o n as ganglio-side heated without acid. Loss of i n h i b i t o r y a c t i v i t y upon heating i n d i s t i l l e d H 20 alone could be due to the disruption or formation of a ganglioside ultrastructure such as a mic e l l a r arrangement, making the i n h i b i t o r y portion of the molecule less accessible. That s i a l i c acid was not involved was also suggested by the fact that colominic a c i d — a polymer of s i a l i c a c i d — d i d not i n h i b i t hemagglutination. Neuraminidase treatment of formalinized red blood c e l l s to remove s i a l i c a c i d residues an the surface of the red blood c e l l s demonstrated that these residues were not involved i n hemagglutination by outer mem-brane. In f a c t , neuraminidase treatment l e d to a two f o l d increase i n hemagglutination t i t r e r e l a t i v e to c e l l s treated i n an acetate buffer control. A s i m i l a r r e s u l t has been noted f o r the adherence of Eikenella  corrodens to neuraminidase treated buccal e p i t h e l i a l c e l l s (122). Enhanced hemagglutination could be due to two mechanisms. The majority of s i a l i c a c i d on the red blood c e l l surface i s linked to galactose and N-acetyl-galactosamine (119), and removal of these residues would expose these sugars. I f e i ther of these sugars were involved i n the hemagglutination reaction t h e i r increased a v a i l a b i l i t y would allow increased hemagglutination. As previously mentioned, N-acetylgalactosamine i n h i b i t e d hemagglutination 62 which suggested that t h i s sugar played a r o l e i n the hemagglutination reaction and i n the increased t i t r e seen with neuraminidase treated red blood c e l l s . The second p o s s i b i l i t y i s that removal of s i a l i c a c i d residues changed the e l e c t r o s t a t i c charge balance on the surface of the red blood c e l l , thus making hemagglutination easier. I t i s known that the negative charge on the surface of erythrocytes i s l a r g e l y due to s i a l y l groups on the c e l l membrane glycoprotein (119). Both immune and non-iitmune rabbit serum i n h i b i t e d hemagglutination. The l a t t e r r e s u l t has been previously demonstrated (98, 77), and i n agreement with one of these studies (77) i t was found that dialysed immune and non-immune serum retained i n h i b i t o r y a c t i v i t y . I t was further noted that there was no difference i n the i n h i b i t o r y t i t r e of immune versus non-immune serum. This is i n agreement with the r e s u l t s of Slots and Gibbons (98) which indicated that serum from adults with and without pe r i o d o n t i t i s had the same amount of i n h i b i t o r y a c t i v i t y . These r e s u l t s indicated that the i n h i b i t o r y a c t i v i t y of immune serum was due to the in h i b i t o r y components present i n non-immune serum. This implied that either no antibodies capable of blocking hemagglutination were formed or that antibodies were formed but that any blocking a c t i v i t y due to t h e i r presence was masked by the i n h i b i t o r y components common to both iirmune and non-immune serum. Hemagglutinating a c t i v i t y appeared to involve a protein ccmponent since Pronase treatment of pool 2 material t o t a l l y removed t h i s a c t i v i t y . Furthermore, enhancement of hemagglutinating a c t i v i t y by /S-mercaptoethanol and d i t h i o t h r e i t o l implied that a S-oDnteining protein was involved i n the reaction. Whether the protein ccmponent was involved d i r e c t l y i n the 63 adherence reaction or was required i n d i r e c t l y to maintain the correct s p a t i a l arrangement of the active binding component i s unknown. S e n s i t i v i t y of hemagglutination to p r o t e o l y t i c treatment i s i n contrast to previously reported r e s u l t s (98, 77) which indicated that hemagglutin-at i o n of whole c e l l s was r e s i s t a n t to a number of proteases including Pronase. I t i s not unexpected that a protein on the surface of an i n t a c t c e l l could be r e s i s t a n t to p r o t e o l y t i c a c t i v i t y while being sensitive to the same treatment when i n a more exposed state i n an outer membrane preparation. Similar differences i n s e n s i t i v i t y to p r o t e o l y t i c t r e a t -ment have been noted for the ccmponent mediating coaggregation between S. s a l i v a r i u s and V. alcalescens (116). The selective loss of hemagglutinating a c t i v i t y concarmitant with the removal of loosely bound l i p i d by cMorofom-methanol extraction could mean that a l i p i d ccmponent i s somehow involved d i r e c t l y or i n d i -r e c t l y i n t h i s a c t i v i t y . A l t e r n a t i v e l y , although the extraction procedure should not extract proteins (6), i t i s equally l i k e l y that a protein could be altered by the solvents such that i t was no longer functional as a hemagglutinin. The LPS ccmponent of t h i s pool d i d not appear to be involved i n hemagglutination as i t would be unaffected by both of the procedures which removed the binding a c t i v i t y . This was i n agreement with a recent study which demonstrated that neither LPS nor the capsular polysaccharide contained hemagglutinating a c t i v i t y (77). Thus i t appeared that the hemagglutinin was either a protein or a l i p i d - p r o t e i n complex. While previous studies (78, 79, 77, 98) have suggested that fim-briae ( p i l i ) are responsible f o r the hemagglutinating a c t i v i t y of 64 B. g i n g i v a l i s , these r e s u l t s should be viewed with caution. The sugges-t i o n of f i m b r i a l involvement i s based, i n part, on the a b i l i t y of par-t i a l l y p u r i f i e d p i l i preparations to cause hemagglutination (98, 78, 77) and the a b i l i t y of antiserum raised to p i l i preparations from hemagglu-t i n a t i o n p o s i t i v e organisms to block t h i s a c t i v i t y i n whole c e l l s (78). The p a r t i a l l y p u r i f i e d p i l i preparations used i n these studies would a l l appear to be mixtures of several membrane components. Electron microscopic examination of one preparation (98)< revealed a heterogeneous mixture of aggregated p i l i and globular material. I t was l a t e r observed that the o r a l i s o l a t e s of B. asaccharolyticus possessed a loosely adherent ruthenium red layer and produced large amounts of e x t r a c e l l u l a r membrane vesi c l e s which resembled the globular p a r t i c l e s seen by Slots and Gibbons (120). Outer membrane complex c h a r a c t e r i s t i c s such as these would make i t d i f f i c u l t to produce preparations that were not cxxitaminated with other outer membrane components. I t would appear to be d i f f i c u l t to conclusively assign hemagglutination to one component over another i n heterogeneous preparations such as these. As w e l l , the p i l i preparations used for production of antiserum for blocking hemagglutination (78) were again crude suspensions and probably a heterogeneous mixture of membrane components. I t i s possible that other outer membrane components which are known to be an t i g e n i c a l l y active, such as LPS and capsular polysaccharide were present as contami-nants and that the Ouchterlony reaction was detecting antibodies raised to these components. Secondly, although i t was demonstrated that serum from rabbits immunized with these preparations was able to in t e r f e r e with hemagglutination, controls using nm-immune serum were not included. 65 Previous studies (98) and experiments reported here have shown that non-immune rabbit serum alone i n h i b i t s hemagglutination. Thus, although there may be indications that fimbriae are involved i n t h i s a c t i v i t y , t h e i r r o l e has not been conclusively proven. This study has provided evidence both f o r and against f i m b r i a l involvement. The 22,200 dalton peptide present i n pool 2 the primary protein compo-nent of that pool had a molecular weight i n the range suggestive of a fimbriae subunit (41). However, although negatively stained c e l l s showed fimbriae-like structures when examined by electron microscopy, membrane preparations d i d not appear to contain fimbriae. Furthermore, procedures designed to remove fimbriae from c e l l s (unpublished results) d i d not a f f e c t hemagglutinating a c t i v i t y . In the present study i t was demonstrated that hemagglutination and b a c t e r i a l aggregation were mediated by d i f f e r e n t components of the outer membrane complex of B. g i n g i v a l i s W12. These components demonstrated d i f f e r e n t s e n s i t i v i t i e s to p r o t e o l y t i c treatment, blocking by sugar and other compounds and had d i f f e r e n t protein,loosely bound l i p i d and LPS compositions. The hemagglutinin appeared to be either a protein or l i p i d - p r o t e i n complex and the b a c t e r i a l aggregating ccmponent appeared to be the capsular polysaccharide, although there was a p o s s i b i l i t y that smooth type LPS could also be the active ccmponent. Further investigation should reveal the i d e n t i t i e s of these adhesins more precisely. 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