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Dextran mediated interactions of Actinomyces viscosus Bourgeau, Gene 1975

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DEXTRAN MEDIATED INTERACTIONS OF ACTINOMYCES VISCOSUS by GENE BOURGEAU BSc, University of Toronto, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Microbiology We accept this thesis as conforming to the required, standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s rep r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of MICROBIOLOGY The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 7/9/75 i i i ABSTRACT A c t i n o m y c e s v i s c o s u s was shown t o be o n l y the second o r a l o r g a n i s m c a p a b l e o f b e i n g a g g r e g a t e d by low amounts o f d e x t r a n (as few as 3 m o l e c u l e s o f d e x t r a n p e r b a c t e r i a l c e l l ) ; S t r e p t o c o c c u s mutans b e i n g the f i r s t . A g g r e g a t i o n was shown t o depend on d e x t r a n m o l e c u l a r w e i g h t and d e x t r a n c o n c e n t r a t i o n . Ions were r e q u i r e d f o r a g g r e g a t i o n t o o c c u r but t h e r e was no s p e c i f i c ion r e q u i r e m e n t . C o n c a n a v a l i n A and p h y t o h e m a g g l u t i n i n bo th i n h i b i t e d d e x t r a n i n d u c e d a g g r e g a t i o n as d i d the s i m p l e s u g a r s s u c r o s e , t r e h a l o s e , m e l e z i t o s e and mannose. The h e a t i n g o f c e l l s t o 100°C in d i s t i l l e d wa te r removed the a b i l i t y to a g g r e g a t e w h i l e h e a t i n g a t 100°C f o r up to one hour in the p r e s e n c e o f d i v a l e n t c a t i o n s lowered but d i d not e l i m i n a t e t h i s a b i l i t y . T r e a t m e n t o f A . v ? s c o s u s c e l l s w i t h v a r i o u s p r o t e a s e s and a d e x t r a n a s e p r e p a r a t i o n e l i m i n a t e d the a b i l i t y t o a g g r e g a t e v i a d e x t r a n . Dex t ran i n d u c e d a g g r e g a t i o n was not l i m i t e d t o one A . v i s c o s u s s t r a i n but was a p r o p e r t y d i s p l a y e d by s e v e r a l ATCC s t r a i n s and s e v e r a l f r e s h l y i s o l a t e d s t r a i n s . Dex t ran i n d u c e d a g g r e g a t i o n was a l s o shown to be a p o t e n t i a l means o f i n t e r b a c t e r i a l a g g r e g a t i o n in the o r a l c a v i t y s i n c e i t m e d i a t e s an i n t e r a c t i o n between A_. v i s c o s u s and both S t r e p t o c o c c u s mutans and S t r e p t o c o c c u s s a n g u i s . T h i s i n t e r a c t i o n was s i m i l a r in most r e s p e c t s t o t h a t i n v o l v i n g o n l y A . v i s c o s u s o r o n l y mutans . • ! V TABLE OF CONTENTS Page I. INTRODUCTION .. 1 II. MATERIALS AND METHODS .7-. A. Organisms 7 1. Actinomyces viscosus (15987), Actinomyces viscosus (19246) Actinomyces naeslundii (19039) and Actinomyces i s r a e l i i (27027) 7 j 2. Actinomyces viscosus (freshly isolated) 7 3. Streptococcus mutans and Streptococcus sanguis (freshly isolated) 1 . B. Growth Studies of Actinomyces viscosus 7 c. Cell Storage 7 D. Isolation of Dextran 7 1. Commercial dextran 7 2. Streptococcal dextrans 7 3. lkC labelled streptococcal dextrans l6_ 4. Analysis of the isolated streptococcal dextrans 10 a. Total carbohydrate measurement 10 b. Ketohexose measurement 10 c. Protein measurement 10 d. Thin layer chromatography 10 E. Analytical Procedures 10 1. Glucostat assay 10 2. Park-Johnson ferricyanide method 10. F. Enzymes 10 1. Dextranase 10 2. Proteases 11 a. Trypsin 11 b. a-chymotrypsin. 11 c. Subtilisin-BPN' 11 d. Subtilopeptidase-A 11 e. Pronase 11. 3. Neuraminidase 11 4. Hyaluronidase 11 G. Aggregation Assay Involving Actinomyces viscosus 11 H. Interbacterial Aggregation 12 V Page III. RESULTS 13 A. Dextran Induced Aggregation of Actinomyces viscbsus • 13 1. Effect of dextran concentration ••• 13 2. Effect of dextran molecular weight 13 3. Ion requirements 13 4. Cell modification 13 a. Enzymatic treatment 13 b. Heating 21 5. Eh 24 6. pH 24 7. Inhibitory compounds 24 8. Aggregation caused by compounds other than dextran 24 9. Aggregation by streptococcal dextran 28 10. Culture age and growth medium 28 11. Aggregation of other Actinomycetes 33 B. Interbacterial Aggregation 33 1. Interactions between streptococci and A. viscbsus 33 2. Sugars used by streptococci to mediate aggregation \; with A. viscbsus 33 3. Effect of c e l l concentration on interbacterial aggregation 36 4. Ion requirements 38 5. pH 38 6. Effect of c e l l age on interbacterial aggregation 38 7. Effect of exogenously added dextran on interbacterial aggregation 41 8. Cell modification 41 IV. DISCUSSION '. 45 V. BIBLIOGRAPHY. 48 ACKNOWLEDGEMENTS I wish to thank Dr. B. C. McBride for his continuous help and guidance and also for his always present enthusiasm for new ideas and approaches. My thanks also go to everyone associated with the lab; they are outstanding people, both professionally and personally. I would also l i k e to thank G. Bourgeau for the beautiful job done in typing this thesis. 1 INTRODUCTION The oral cavity represents a complex and fascinating ecological picture. The oral surfaces present many different niches for microbial growth; the non-keratinized epithelium lining the gingival sulcus, the keratinized epithelium of the masticatory mucosa and also hydroxylapatite on the tooth surface. The tooth i t s e l f i s composed of exposed smooth surfaces and crevices and fissures. A l l of these surfaces are subject to the cleansing action of salivary flow and scrubbing activity when two opposing surfaces rub against one another. In order to remain in a suitable niche many organisms have developed specific mechanisms to allow them to adhere to surfaces i n the most favourable environment for their growth (65,71). The surface of a mammalian c e l l presents many different proteins, glycoproteins and glycolipids to the outside environment which serve as potential receptors for microbial organisms. Bacteria also have complex surface structures, many molecules of which may be used in an attachment role. There are an indeterminate number of salivary glycoproteins (58,59,83,96), not to mention enzymes such as neuraminidases and other glycosidases (32,55,95,97,98,110,125) which may modify these glycoproteins to better suit them for a function such as permitting adherence of bacteria to clean hydroxylapatite, to saliva coated hydroxylapatite (58,96,101,111,133) and to various tissue surfaces (45,46,61,64,65,79,102,103). Interbacterial aggregation can also be mediated by salivary glycoproteins (43,59,83), by type specific surface antigens (26,27,104,119) and by bacterial polysaccharides. Bacterial retention by purely mechanical means such as entrapment in crevices or impaction with food can also be important. The best studied means of bacterial adherence i s that which is mediated by bacterial polysaccharides. Adherence to hydroxylapatite has been shown to be mediated by complex polysaccharides (39,40,57,64,65,73) produced by various oral organisms such as Streptococcus sanguis and Streptococcus mutans (10-13,15,17,19,40,42,84,85,87,92,97,112,154). These extracellular polysaccharides can be either retained on the bacterial c e l l surface or released into the environment where they may bind to other bacteria, to hydroxylapatite or simply be washed away (17,39,40,42, 92,99,114,140,154). They not only provide a means for.adherence but they may control ion flow to and from the tooth surface and such gelatinous deposits appear to allow a low pH to persist at the tooth surface for many hours (20,79). Such polymers may also serve as nutrient sources(16,36,82,91) 2 when molecules that are more easily metabolized are no longer available. The bacterial polysaccharidesmost strongly associated with an adherence function is dextran. The dextran produced by S_. sanguis or S_. imi tans often has a molecular weight of up to 107-108 daltons. Dietary sucrose provides the substrate which the enzyme systems (dextransucrases) of these organisms utilize' to produce dextran. A concentration of sucrose greater than 0.5% appears necessary with 10% allowing maximum synthesis (39). The enzymes are constitutive and appear in c e l l bound and soluble forms. The dextrans themselves can be c e l l bound or soluble. The soluble dextran may adhere to enamel surfaces, to other bacteria or maybbe washed away. Gibbons and Banghart (39) showed that dextran forms non-specific precipitates with proteins but they only occurred at very high concentrations of both dextran and protein. The dextran-protein interaction may be an important means by which free dextran and salivary glycoproteins are incorporated into dental plaque. However, S^. mutans or A. viscosus are exquisitely sensitive to interactions with dextran since as few as 3 molecules of dextran per bacterial c e l l can induce c e l l aggregation. The various dextransucrases differ in their mode of action; some appear to extend the molecule, other introduce branchpoints. S t i l l others may be responsible for modifying otherwise complete dextran. Depending on the proportions and activities of the different enzymes an i n f i n i t e variety of shapes, sizes and molecular weights resultSr(24,&0,51,72,122). Guggenheim and Schroeder (51) demonstrated the presence of a-1,4 and a-1,3 branchpoints using periodate oxidation and they also observed three morphological types of dextran i n osmium stained c e l l preparations which appeared to differ i n the degree of branching. S_. sanguis dextran has a large proportion of a-1,6 linkages and a much smaller but significant number of a-1,4 and a-1,3 linkages indicating a linear molecule with a large number of branches (120,142). S_. mutans dextran, on the other hand, is much more highly branched (99) as indicated by a much lower level of a-1,6 linkages and many more a-1,4 and a-1,3 branchpoints. The source of commerical dextran used in many of the studies mentioned in this thesis was Leucoriostoc mesenteroides (118). The molecular weight of the commercial dextran was assessed on the basis of light scattering and gel f i l t r a t i o n . The dextran molecular weights are really weight average molecular weights with a preponderance of molecules of the 3 size indicated but also measurable amounts of dextran of larger and smaller molecular weights.. As was. noted the dextrans. produced by the streptococci also vary widely i n molecular weight but those streptococcal dextrans used in the studies mentioned in this thesis were not characterized with respect to this property. The Leucbiibstoc dextran is a linear molecule with a high percentage of a-1,6 linkages and only a few, short side chains (140). S_. mutans which has been implicated in the formation of multisurface caries does u t i l i z e i t s dextran to bind to tooth surfaces (92) and also to mediate aggregation between bacterial cells (42,84,85,127). Some workers believe that the traits of adhesion to a solid surface and aggregation of cells are two distinct and separate functions, but both require the presence of dextransucrases-and exogenously supplied sucrose. Freedman and Tanzer (32) state that they have isolated mutants of SI mutans that aggregate in the same way as normal cells but do not produce much plaque on wires suspended in sucrose containing growth medium. They feel that aggregation may not necessarily be a glucan associated tr a i t since the only change they could detect was in the consistency of the dextran produced by the mutant. Bearing in mind that interbacterial aggregation i s a specific phenomenon the results could be explained by a change in the proportion of polysaccharide producing enzymes. S_. mutans normally produces a low level of levan (14,48,154), a polyfructan, from sucrose and a high level of dextran. If this proportion was reversed there would s t i l l be enough dextran to allow aggregation, since 3 molecules of dextran per c e l l is enough, but not enough to permit adherence. Alternatively, the proportions may not change but polysaccharide structure may be altered i n some way which affects adherence as would be expected i f the dextran was i n i t i a l l y soluble and only gradually modified into a form capable of binding to bacterial c e l l s . J. D. deStoppelaar (20) isolated mutants that were unable to adhere to solid surfaces and he was able to show that l i t t l e c e l l bound, al k a l i soluble glucan could be isolated from the mutant cells as compared to the parent strain. The mutant was s t i l l capable of producing large amounts of soluble polysaccharide but this material did not bind to the ce l l s . In this organism even addition of exogenous dextran isolated from the parent strain failed to cause aggregation. This indicates that there was a change in the c e l l surface which precluded binding of dextran. The mutant isolated by Freedman and Tanzer would appear to be very different from that of deStoppelaar. It would also seem that aggregation is indeed a glucan mediated tr a i t and a highly specific one requiring c e l l surface receptors on the S, mutans. 4 Specific attachment of bacteria to various surfaces of the human body, other than the oral cavity, has also been well documented, Neisseria have been shown to attach to epithelial cells of the genitourinary tract (102,152). Pathogenic Escherichia c o l i (22,74,153) and Vibrio chblerae (102) specifically bind to intestinal epithelial c e l l s . These are by no means the only examples (2,35,93,134,143). It might be noted that the a b i l i t y to remain in one site due to adherence is not an indication of pathogenicity but i t is a prerequisite for colonization by any bacteria (4) and thus aids i n building up a population of specific bacteria. Caries and periodontal disease result only when bacteria adhere to the oral surfaces involved. Much of the work that has been done with regard to determining the conditions required for the establishment of certain organisms and their subsequent role i n oral pathology is open to criticism. The studies vary i n the animal strains used, in the consistency and composition of the diets fed the animals, in the methods of assessing disease progression and also i n the measures used to control the total oral flora (7,10,12,30,31,34, 71,76-78,80,82,88-90,106,116,146,155). It i s possible to say that under the very special conditions of the experiments the bacteria under study caused a determinable level of oral disease. However, i n reality there are so many other factors involved that no one organism can be blamed as a major disease agent (62,151). It is impossible to l i s t a l l the factors which may affect the a b i l i t y of an organism to cause disease in the oral cavity so a few of the major factors w i l l have to suffice. Phosphate and trace elements appear to reduce caries i n general (8,9,53,56,87,100,108,109,121,129), possibly by intercalating with the hydroxylapatite l a t t i c e structure and thus giving i t more resistance to attack by acids. The levels of exogenously supplied sugars vary and so the levels of acid can vary. The oral flora varies from animal to animal (28,117,145) in the same species, between species, with diet (155), with age (115) and with bacterial concentration (66). In fact, a different bacterial population can quite often be seen at the same site sampled at different times (9,131,146). Since the environmental factors responsible for bacterial growth and persistence change continuously the bacterial population i s also continuously variable. The animal systems are thus a r t i f i c i a l and may have l i t t l e application to real l i f e let alone the human situation (6). 5 Actinomyces viscosus i s a filamentous, gram^-positive organism found in human, rodent and canine dental plaque and i s most lik e l y present in the mouths of a wide variety of animals Cl,3,18,67,68,77,81,82,86,106,117,144,149). A. viscosus was f i r s t identified by Howell (67) in isolates taken from the plaque of hamsters with periodontal disease. Its description relied on the morphological and cultural characteristics of 169 isolates from 30 of 48 hamsters. Subsequent reports by Howell and Jordan (68) on i t s physiological and biochemical properties as well as by Jordan and Keyes (77) on attempts to induce experimental infections referred to A. viscosus as the "hamster organism." These workers believed that i t belonged in the Family Actino-mycetaceae but i t did not f i t in an existing genus since i t was catalase positive. A new genus, Odbritbmyces,was suggested with Odontomyces viscosus as the type species (69,80). Later the genus Actinomyces was redefined to include catalase positive organisms and so the "hamster organism" was f i n a l l y renamed Actinomyces viscosus (37). It varies in i t s biochemical properties from Actinomyces naeslundii, another oral Actiribmycete, only by the catalase reaction and so some workers feel that they are the same organism (29,38). The two organisms also cross-react antigenically but do not share completely identical surface antigens (38,141). Not much is known about the physiology of A. viscosus. The c e l l wall is also relatively ill-defined. It contains a typical gram-positive murein with some long chain fatty acids, a high level of 6-deoxytalose as well as mannose, rhamnose, galactose, glucose, alanine, glutamic acid, lysine and ornithine (135,147) but no diaminopimelic acid. There is an increasing amount of data indicating a role for A. viscosus in oral pathology (18); keeping i n mind the injunction noted previously with regard to the significance of results obtained from animal model systems. The organism can be implanted i n animals fed a wide variety of carbohydrates (80-82,114) unlike S_. mutans which requires sucrose to become established (5,8,10-13,15,17,34,40-42,50,79,84,92,94,99,112,123). Implantation of A. viscosus leads to the production of unique periodontal symptoms (52,76-78, 81,82,86,149). There has been a correlation between the rise in periodontal disease and a rise i n the numbers of A. viscosus after irradiation for the treatment of salivary gland carcinoma (106). There was no change in the numbers of A. riaesluridii or A. i s r a e l i i . This is one indication that A. viscosus and A. riaeslundii may be different i n more than just the catalase reaction. Finally, Hammond, et a l . (55) have detected a plasmid i n wild type A. viscosus which appears to be missing from some mutants that have been isolated. The 6 wild type organism was virulent while the mutants lacking the plasmid were avirulent. The plasmid appeared to be associated with the production of certain polymers on the bacterial c e l l surface, A, viscbsus may or may not be able to adhere directly to the tooth surface but i t can adhere to other bacteria. Gibbons and Nygaard (44) studied the relationships between a large variety of oral organisms and showed that A. viscbsus aggregated with Neisseria 17, Veillonella alcalescens, Leptbtrichia buccalis, Nbcardia A and jS, sanguis 11F3. A l l the organisms were grown in trypticase soy broth with no sucrose added which, as was made clear, i s required for glucan synthesis by oral organisms such as ^. sanguis and S^. mutans. Bacterial concentration was not considered important but this may i n fact be a v i t a l factor, (66). The concentration of organisms in plaque can be as high as 10 1 1 cells/ml. Furthermore, results outlined i n this thesis show that c e l l concentration can be important in interbacterial aggregation. There is also impressive but indirect evidence for an interaction between cocci and A. viscbsus. That i s the electron micrographs showing "corncob" formations, filamentous organisms with their surfaces completely covered with cocci (75,105,124). The modesof action of the adherence mechanisms of A. viscbsus have not been studied up un t i l now. In vitro studies of A. viscosus in our lab used dextran as a potential inhibitor of aggregation mediated by salivary glycoproteins. Dextran was found to actually enhance aggregation. This was very interesting since, as was mentioned, _S. sanguis and S^. mutans both produce large amounts of dextran from dietary sucrose. Thus, while A. viscosus produces levans (70,91,132)., ' i f / f e l l raffinose or sucrose, and other unidentified extracellular polysaccharide polymers and glycoproteins, i t appears that one molecule of significance i n i t s maintenance in the oral cavity may be dextran. The effect of dextran on aggregation led to a desire to investigate i t s relationship to A. viscosus and to determine how this polymer can mediate interactions with oral streptococci. Work has been done which indicates the interaction of A. viscosus with dextran is a specific phenomenon. Furthermore, work has been done which indicates that dextran is capable of mediating interactions involving both A. viscosus and oral streptococci such as S_, mutans and S. sanguis. MATERIALS AND METHODS A. ORGANISMS 1. Actinomyces viscosus (15987) ,v Actinomyces viscosus (.19246), Actinomyces  riaeslundii (19039) and Actinomyces i s r a e l i i (27027) were obtained from the American Type Culture Collection. They were grown in trypticase soy broth (TSB) or brain-heart infusion broth (BHI) i n static culture, aerobically at 37°C. Growth on solid medium required incubation in a 10% CO2 atmosphere. 2. Actinomyces viscosus strains were isolated from human dental plaque and identified according to the procedures of Georg, Pine and Gerencser (37). Freshly isolated strains grew in a compact pellet at the bottom of the culture vessel and, when shaken, swirled up in a large, viscous, stringy mass. Continual subculturing resulted i n a change i n which definite growth could be discerned throughout the culture medium. 3. Streptococcus mutans and Streptococcus sanguis strains were isolated from human carious lesions and human dental plaque respectively. Streptococcus  sanguis strains were isolated as described by Carlsson (11). Streptococcus  mutans strains were isolated as described by Gold, et a l . (47). A l l plates were incubated at 37°C in a 5-10% CO2 atmosphere. Cells for experimental procedures were grown in TSB, in static culture at 37°C. B. GROWTH STUDIES OF ACTINOMYCES VISCOSUS Growth in liquid culture was measured by total microscopic count, viable c e l l count, optical density and by the most probable number method. C. CELL STORAGE Stock cultures were stored at -70°C in growth medium supplemented with either s t e r i l e glycerol or dimethylsulfoxide to a f i n a l concentration of 10%. It was found that A. viscosus cells which were repeatedly subcultured gradually lost the a b i l i t y to aggregate. Because of this, cells were grown up from the stock cultures once a month. D. ISOLATION OF DEXTRAN 1. Dextrans of known molecular weight were purchased from Pharmacia (Canada). 2. Samples of dextran were isolated from various S_. sanguis and j5. mutans strains using a modification of the procedure described by Sidebotham and Weigel (140). 8 One ml of a 24 hour TSB culture of the streptococcus was used to inoculate 100 mis of TSB containing 5% sucrose. This culture was incubated at 37°C for three days. Sodium hydroxide was then added to give a f i n a l concentration of 0.04 M and the culture was stirred at room temperature for twenty hours. The culture was then centrifuged at 1750 xg for fifteen minutes and the supernatant and pellet were treated as described below. SUPERNATANT The pH was adjusted to 4.0 Using glacial acetic acid. Cold ethanol (4°C) was added to give a f i n a l concentration of 40%. Ice cold ethanol was used throughout the isolation procedure. The supernatant was stored at 4°C overnight. It was then centrifuged^at 1750 xg for fifteen minutes. SUPERNATANT ~~ FRACTION I ^ Ethanol was added to give a fi n a l concentration of 70%. The solution was stored at 4°C overnight and then i t was centrifuged at 1750 xg for fifteen minutes. SUPERNATANT FRACTION II Discard. Purify as for Fraction I. The dextran in the pellet was purified by repeated reprecipitation with ethanol and deproteinized by the method of Sevaq, et a l . (125) PELLET Sodium hydroxide was added to a f i n a l concentration of 0.2 M. The ce l l s were dispersed and stirred at room temperature for one hour. The suspension was centrifuged at 12,000 xg for fifteen minutes to remove the c e l l s . Ethanol was added to a f i n a l concentration of 40%. This solution was stored at 4 C overnight and then centrifuged at 1750 xg for fifteen minutes. I r 1 SUPERNATANT FRACTION III Ethanol was added to a f i n a l Purify as for Fraction I. concentration of 70% and the solution was stored at 4 C overnight. It was then centrifuged at 1750 xg for fifteen minutes. , >- , SUPERNATANT FRACTION IV Discard. Purify as for Fraction I. Table 1 Contaminating glycosidase activity i n commercial dextranase PNP Substrate Temperature <°C) pH Buffer ymoles of substrate hydrolyzed per minute by 0.5 mg of dextranase units of enzyme per mg of dextranase g-D-galactopyranoside 30 4.6 0.05 M Na citrate 2.35 x 10 _ 3 2.12 X IO 2 a-D-galactopyranoside 30 4.6 0.05 M Na citrate 3.66 x 10" 3 1.36 X 10 2 8-D-glucoside 25 4.8 0.10 M Na acetate 3.30 x 10" 3 1.51 X 10 2 a-L-fucoside 25 6.0 0.05 M Na citrate 7.70 x 10~6 6.50 X 10 4 a-D-glucoside 25 4.8 0.10 M Na acetate 1.15 x 10" 3 4.35 X 10 2 N-acetyl^S-D-galactosaminide 30 4.6 0.05 M Na acetate 3.90 x 10" 3 1.28 X 10 2 N-acetyl-8-D-glucosaminide 30 4.6 0.05 M Na acetate 8.00 x 10" 4 6.25 X 10* B-glucuronide 37 5.0 0.20 M Na acetate 9.00 x 10~5 5.60 X 10 3 a-D-mannoside 25 4.5 0.05 M Na citrate 1.70 x IO"* 2.93 X 10 3 1 unit = the amount of enzyme needed to hydrolyze 1 Umole of substrate per minute under the conditions cited. ^iq3 3. lkC labelled dextran was isolated from streptococcal cultures grown in TSB containing 5% sucrose and further supplemented with 100 y l of sucrose [glucose- 1 "COO] (20 Uc/ml). 4. Analysis of the isolated dextrans a. Total carbohydrate was measured by the anthrone method as described by Scott and Melvin (137) or by the phenol^sulphuric acid method of Dubois, et.al (23) using dextran T2000 as the standard. b. Ketohexose (fructose) was determined by the cysteine-sulphuric acid method of Dische and Devi (21). c. Protein was measured by the Lowry method using BSA as the standard (107). d. The purity of the dextran samples was checked by thin layer chromatography. Dextran samples were dissolved in d i s t i l l e d water to a fi n a l concentration of 2.5 mg/ml. 100 yl/ml of sample was mixed with 100 u l of 4N HC1 and sealed in a glass ampoule. The mixture was hydrolyzed in a boiling water bath for one hour. The hydrolyzed samples were then freeze-dried in the presence of NaOH pellets. The residue was resuspended i n 100 y l of d i s t i l l e d water and 10 y l was chromatographed on a cellulose thin layer chromatography sheet (Polygram Cel 300; Macherey-Nagel and Co., Germany) i n n-butanol:acetic acidrwater (3:1:1). The chromatogram was developed using an aniline-diphenylamine spray followed by heating at 100°C for one to two minutes. Dextran T2000, glucose and fructose were used as standards. E. ANALYTICAL PROCEDURES 1. Glucose was measured by the glucostat assay (Sigma Chemical Co., Technical Bulletin 510). 2, Reducing sugar was measured by the Park-Johnson ferricyanide method (148). F. ENZYMES 1. Dextranase (Sigma, grade I) This enzyme, isolated from a Periicillium sp., is more accurately called endo-a-l,6-glucan 6-glucanhydrolase. It has an activity of 50 units per mg at pH 7.2, 20°C and using dextran 10 as a substrate. Contaminating enzymes such as proteases and glycosidases were present in the dextranase preparation. The casein assay as described by Rick (130) was performed using trypsin as: the control protease. Contaminating protease was present to a level of 0.14%, Many glycosidase act i v i t i e s were detected. The substrates and assay conditpns. are li s t e d in Table 1; a l l of the substrates tested were hydrolzyed to some degree by the dextranase preparation. The assays were run under optimum conditions, as. des.crtb.ed in Methods in Enzymology, Volume VIII and so the activities under our experimental conditions would most like l y be less. 2. Proteases The act i v i t i e s l i s t e d for these enzymes are optimal activities and so such are probably higher than those present under experimental conditions. a. Trypsin (Sigma, type XI) 7500-9000 BAEE units per mg (DCC treated to remove a-chymotrypsin activity) b. a-chymotrypsin (Sigma, type III) 25-35 BAEE units per mg c. Subtilisin-BPN' (Sigma, type:VII) 6- 9 units per mg d. . Subtilopeptidase-A (Sigma, type VIII) 7- 15 units per mg e. Proriase (Sigma, type V) 0.7-1.0 units per mg 3. Neuraminidase 0.5-1.5 units per mg 4. Hyaluronidase 3000-15,000 NF units per mg G. AGGREGATION ASSAY INVOLVING ACTINOMYCES VISCOSUS The simple, visual assay used to investigate the aggregation phenomenon consisted of mixing 80 u l of tris(hydroxymethyl)aminomethane buffer ( t r i s , 0.01 M, pH 7.2), 10 u l of washed cells (10 1 0 cells/ml of buffer) and 10 u l of test compound. The total reaction volume was always 100 ul unless otherwise noted. The mixture was gently agitated for ten minutes at room temperature and observed for the degree of aggregation using a stereomicroscope (lOx). Controls withouttldextran T2000 and with 500 ug/ml of dextran T2000 were included in every assay. Aggregation was assessed on a scale of 0, ±, +1 - +4 as determined by a comparison with the controls. The control lacking dextran should be 0, the control with dextran should be +4. This procedure was adhered to unless a test mixture showed greater aggregation than the dextran control. In this situation the tube showing maximum aggregation was scored +4 and the dextran control was scored accordingly. 12. Due to the vari a b i l i t y in the degree of aggregation from day to day the results of each experiment are internally consistent but in absolute terms a +4 on one day may not equal a +4 on another day. H. INTERBACTERIAL AGGREGATION In the studies on interbacterial aggregation i t was sometimes necessary to confirm that both the A. viscosus and streptococcus were involved in the aggregation process. Therefore interbacterial aggregation was measured by visual assessment as described previously and by counting the number of organisms remaining in suspension after aggregates had settled to the bottom of the reaction tube. The reaction mixtures were prepared as described below. A. viscosus 1.0 - 1.0 1.0 1.0 S. sanguis - 1.0 - 1.0 1.0 1.0 Tris buffer 1.1 1.1 1.0 - 0.1 1.0 Dextran T2000 - - 0.1 0.1 - 0.1 ml The reaction mixtures were incubated at room temperature with gentle agitation every fifteen minutes for one hour. Fifteen minutes after the fi n a l agitation a sample was removed from the surface of the reaction mixture. '' By this time the aggregates had settled to the bottom of the tube. The sample was then diluted i n exponential steps using s t e r i l e buffer and then plated out on Mitis-Salivarius agar and brain-heart infusion agar. Differential plate counts revealed any decrease i n colony forming units (cfu) of either A. viscosus or the streptococcus. 13 RESULTS A. DEXTRAN INDUCED AGGREGATION OF ACTINOMYCES VISCOSUS 1. Effect of dextran concentratton The degree of aggregation was shown to be dependent on the dextran concentration as can be seen i n Figure 1. On occasion.visible aggregation has occurred with as l i t t l e as 5 ng/ml of dextran T2000 (3 molecules of dextran per bacterial c e l l ) . 2. Effect of dextran molecular Weight The effect of dextran molecular weight on aggregation i s shown i n Table 2. Aggregation was at a maximum using dextran with a molecular weight of 2.0 x 106 daltons (dextran T2000). No activity could be detected using dextran having a molecular weight less than 1.1 x 105 D. It should be noted that the molecular weights represent an average value as determined by light scattering and gel f i l t r a t i o n . What i n fact might be occurring i s a titration of dextran T2000 i n the various lower molecular weight preparations. Aggregation caused by streptococcal dextran was generally more intense than that produced by Leuconostoc dextran. This may be due to the fact that such streptococcal dextrans have molecular weights i n the range of 10 7-10 8 D. 3. Ion Requirements The role of ions in aggregation was determined by replacing the t r i s buffer with varying concentrations of divalent and monovalent cations in d i s t i l l e d water. The assay was performed as described using 500-yg/ml of dextran T2000. The results are shown in Table 3. It was shown that divalent cations were more efficient at maintaining the a b i l i t y to +2 aggregate. Mg was able to sustain a +1 level ot aggregation at 6.1 x 10 M while Na + maintained +1 aggregation at 1.8 x 10 3 M. There was no requirement for a specific ion since many cations support aggregation (113) and aggregation occurs i n t r i s buffer. 4. Cell Modification a. Enzymatic treatment In order to study the nature of the receptors on the c e l l surface A. viscosus was treated with a number of hydrolytic enzymes. Washed cells were resuspended in'.;tris buff er at a concentration, of 10 1 0 cells/ml. A 14 Figure 1. E f f e c t of dextran concentrat ion on dextran induced aggregation of Actinomyces v i s c o s u s A g g r e g a t i o n 16 Table 2 Effect of dextran molecular weight on aggregation of Actinomyces viscosus Dextran Molecular Weight Dextran Concentration (ug/ml) (x 103) 5000 500 50 5 0.5 0.05 10 0 0 0 0 0 0 20 0 0 0 0 0 0 70 + + 0 0 0 0 110 +1 + 0 0 0 0 150 +2 +1 + 0 0 0 250 +2 +1 +1 +1 0 0 500 +3 +2 +1 +1 0 0 2000 +4 +4 +3 +2 +1 o 17 Table 3 Ion requirements for dextran induced aggregation of Actinomyces viscosus NaCl (M) Aggregation MgCl 2 00 Aggregation 2.0 x 10 - 3 +2 8.0 X 10"" +3 1.8 x 10 - 3 +1 7.6 X 10"* +2 1.7 x 10 - 3 0 7.3 X 10"* +2 1.5 x 10 - 3 0 7.0 X 10"* +2 6.7 X 10~* +1 6.4 X 10"* +1 6.1 X 10"* +1 6.0 X i o " * 0 There was no aggregation in the absence of added dextran. 18 100 y l volume of cells was mixed with 100 y l of enzyme solution or with buffer. The mixtures were incubated at room temperature and at timed intervals samples were removed and placed on ice. Each sample was centrifuged at 4°C and the cells were washed twice with buffer and fina l l y resuspended i n 100 y l of buffer. The samples were then tested for their a b i l i t y to aggregate i n the presence of dextran T2000. As can be seen in Table 4, treatment with the various proteases reduced or eliminated aggregation. Dextranase rapidly eliminated aggregation while neuraminidase and hyaluronidase had no effect. As was discussed previously (Table 1) the "dextranase" was actually a mixture of dextranase, a number of other glycosidases and protease. It was important to determine which enzyme was involved in eliminating the aggregating a b i l i t y . The protease was not considered because other proteases at much higher concentrations were not as effective as the "dextranase." Low molecular weight dextran was added to the reaction mixture to compete with the bacterial c e l l surfaces for enzyme. Low molecular weight dextran (1.0 x 10* D) was chosen because i t neither causes aggregation nor inhibits aggregation. Washed cells were resuspended to a f i n a l concentration of 2.0 x 10 1 0 cells/ml. The cells were treated with dextranase alone, dextranase plus LMW dextran, LMW dextran alone or they were untreated. The contents of each tube were thoroughly mixed and then incubated at room temperature for fifteen minutes. The contents of each tube were centrifuged and washed twice before resuspending i n 1J0 ml of buffer. The cells were then assayed for dextran induced aggregating activity. Table 5 shows that the LMW dextran protected the cells from the enzymatic activity. In order to provide stronger evidence that the dextranase was the active component i n the enzyme mixture cells were incubated with "dextranase" i n the presence of other glycoside substrates; each at a concentration of 450 yg/ml. The cells were collected as usual and treated with dextranase alone, dextranase plus glycoside substrates, glycoside substrates alone or the cells were l e f t untreated. The aggregating activity was eliminated regardless of whether the glycosides were present or not as can be seen in Table 5. There may be an undetected glycosidase activity strong enough to give the results seen but the evidence points to dextranase as the enzyme responsible for eliminating aggregating activity. 19 Table 4 Effect of treatment with various hydrolyztic enzymes on dextran induced aggregation of Actinomyces viscosus Enzymatic Time of Treatment (min) Treatment (units) 0 5 10 20 30 40 50 60 Cells alone 0 0 0 0 0 0 0 0 Cells + dextran +4 +4 +4 +4 +4 +4 +4 +4 a-chymotrypsin(0.3) +4 +4 +4 +4 +4 +3 +2 +1 Subtilisin BPN*(0.75) +4 +4 +4 +3 +2 0 0 0 Neuraminidase(0.1) +4 +4 +4 +4 +4 +4 +4 +4 Trypsin(80) +4 +4 +2 +1 0 0 0 0 Hyaluronidase(90) +4 +4 +4 +4 +4 +4 +4 +4 Pronase(0.4) +4 +4 +3 +2 +2 +1 0 0 Subtilopeptidase(0.1) +4 +4 +2 0 0 0 0 0 Dextranse(2.5) +4 +4 +2 +1 0 0 0 0 20 Table 5 Inhibition of dextranase activity Treatment Dextran T2000 Concentration (yg/ml) 0 5000 500 50 5 0.5 0.05 Dextranase 0 +1 +1 0 0 0 0 Dextranase + LMW Dextran 0 +3 +4 +4 +2 +1 0 LMW Dextran 0 +3 +4 +4 +3 +1 0 Dextranase + Glycosides 0 +1 o: 0 0 0 0 Glycosides 0 +4 +4 +2 +1 0 0 None 0 +3 +4 +4 +3 +1 0 21 There was a possibility that the dextranase was functioning by attaching to the surface of A. viscosus cells and degrading the exogenously supplied dextran. This possibility was investigated by assaying for dextranase activity bound to the c e l l surface. 1.0 ml of cells (10 1 0 cells/ml) was incubated with dextranase at room temperature for half an hour. The cells were then washed twice and resuspended i n 1.0 ml of buffer. LMW dextran was added and the mixture was incubated at room temperature for half an hour. No release of maltose or reducing sugar could be detected using either the glucostat assay or the Park-Johnson method. Thus dextranase was not attached to the cells at a level which could be detected by these assay systems. Some c e l l preparations aggregated i n the absence of exogenously added dextran. To determine i f this phenomenon was similar to dextran induced aggregation, a sample of such cells was treated with dextranase. Dextranase was added to 1.0 ml of cells (10 1 0 cells/ml) and self-aggregation was noted at various times. Table 6 shows that self-aggregation was reduced from +4 to +1 within 10 minutes. Self-aggregation never f e l l below the +1 level. If A. viscosus does indeed produce a dextranase susceptible polymer, and displays i t on i t s surface, hydrolytic products might be detected after enzyme treatment. 5.0 mis of strongly aggregating cells were treated with 2.5 units of dextranase for one hour at room temperature. The cells were removed by centrifugation and the supernatant was freeze-dried. The residue was resuspended to 1.0 ml in d i s t i l l e d water and duplicate glucostat,and Park-Johnson assays were performed. There was no difference between the supernatants of treated and untreated c e l l s . There may be too low a level of material released to be detected or the enzyme may cleave a carbohydrate chain but i t may not be released from the c e l l surface. b. Heating More information about the nature of the c e l l surface receptors can be gained by knowing their resistance to boiling. Washed cells were resuspended to a concentration of 10 1 0 cells/ml i n d i s t i l l e d water, i n t r i s buffer and i n 10 * M CaC l2. One ml samples of each suspension were placed i n a boiling water bath and at timed intervals samples were removed and assayed for their a b i l i t y to aggregate. Table 7 shows that the cells suspended i n water lost a l l of their a b i l i t y to aggregate after 10 minutes while the cells suspended in t r i s or 10 ** M CaC l2 s t i l l aggregated to a +1 level after being heated at 100°C for one hour. This indicates that the ionic component stabilizes the receptor and that the receptor could be a glycoprotein. 22 Table 6 Effect of dextranase treatment on self-aggregating Actinomyces viscosus Time Treated (min) Treated Not Treated 0 +4 +4 10 +1 +4 20 +1 +4 30 +1 +4 40 +1 +4 50 +1 +4 60 +1 +4 23 Table 7 Effect of heating on dextran induced aggregation of Actinomyces viscosus Heating Time Suspending Medium (min) Tris Distilled^H 20 10 _ l f M CaCl 2 0 +4 +4 +4 1 +4 +4 +4 2 +4 +4 +4 3 +4 +4 +4 4 +4 +3 +4 5 +4 +1 +4 10 +4 0 +4 20 +3 0 +3 30 +3 0 +1 6 0 + 1 0 +1 24 ' 5. Eh 3-mercaptoethanol, dithiothreitol and cysteine-HCl were a l l tested at levels up to 1% for their effect on aggregation. No significant differences between cells treated with reducing agent;or cells treated with t r i s buffer were seen. 6. pjl Since pH is an important consideration when discussing the role of an organism i n the oral cavity i t i s necessary to know i f pH has an effect on aggregation of A. viscosus. Cells were washed twice i n buffer of the indicated pH (Table 8) and were thenr: ^ resuspended to a concentration of 10 1 0 cells/ml i n the same buffer. As can be seen aggregation was unaffected between pH 5.0 and 8.5. Below pH 5.0 self-aggregation was beginning to occur while above pH 8.5 dextran induced aggregation no longer occurred. 7. Inhibitory Compounds The compounds listed in Table 9 were selected from a l i s t of a large number of compounds tested for their effect on aggregation. The concentrations list e d are the lowest at which there was a definite inhibition of aggregation. In no instance, at these concentrations, was there a visible precipitate formed between inhibitor and dextran T2000. Cellobiose and dextran 10 had no effect on aggregation. A concentration of dextran T2000 of 500 yg/ml was generally used for many studies as i t gave a definite positive but i t was near the shoulder of the dextran concentration ti t r a t i o n curve so any changes i n the level of aggregation should appear more clearly than i f the system was saturated with dextran. In the inhibition studies, however, the dextran concentration was always titrated prior to each experiment to determine the optimal level for detection of inhibition. 8. Aggregation caused by compounds other than dextran Some of the compounds tested for their effect on dextran induced aggregation were shown to cause aggregation by themselves (Table 10). Concanavalin A and phytohemagglutinin are plant lectins well known for their a b i l i t i e s to bind specific glycosides (5,25,49). Con A and EHA required up to one hour to induce aggregation to the levels noted; Con A had a slightly stronger aggregating effect and reacted more rapidly than PHA). Fibrinogen is a large molecular weight glycoprotein with a high carbohydrate content. Its 25 Table § Effect of pH on dextran induced aggregation of Actinomyces viscosus pH A. viscosus A. viscosus + dextran T2000 3.0 +3 +3 3.5 +2 +3 4.0 +1 +2 4.5 + +2 5.0 0 +3 5.5 0 +3 6.0 0 +3 6.5 0 +3 7.0 0 +4 7.5 0 +4 8.0 0 +2 8.5 0 +3 9.0 0 0 9.5 0 0 10.0 0 0 10.5 0 0 pH 3.0-7.0 pH 7.5-9.0 pH 9.5-10.5 citrate-phosphate buffer t r i s buffer carbonate-bicarbonate buffer 26 Table a Inhibition of dextran induced aggregation Compound Concentration (M) Aggregation Cellobiose 1.0 X 10"2 +4 Dextran 10 1.0 X 10 3 +4 Fetuin 1.0 X 10"9 +2 Fibrinogen 2.0 X 10" 8 +1 Bovine Submaxillary Mucin 5.0 X 10"9 +1 Concanavalin A 5.0 X 10 6 0 Phytohemagglutinin 4.0 X 10" 6 +2 EDTA 3.0 X 10" 5 +2 Urea 4.0 0 Sucrose 1.5 X 10" 5 +2 Trehalose 1.5 X 10"* +2 Melezitose 1.0 X 10"7 +1 Mannose 3.0 X 10 3 +2 Dextran T2000 2.5 X 10" 5 +4 None - 0 27 Table 10 Aggregation of Actinomyces viscosus by compounds other than dextran T2000 Compound Concentration (M) Aggregation S. sanguis dextran 0 .5 yg/mlt +4 S. mutans dextran 0 .5 ug/ml +4 Concanavalin A 5 .0 X 10 6 +3 Phytohemagglutinin 4 .0 X 10 6 +1 Fetuin 1 .0 X 10 6 0 Bovine Submaxillary mucin 5 .0 X 10" 7 0 Bovine serum albumin 7 .5 X 10 6 0 Fibrinogen 2 .0 X 10~8 +1 Cellobiose 1 .0 X 10"2 0 Dextran T2000 2 .5 X 10~5 +4 None - 0 f Assuming a molecular weight = 10 7-108, the concentration would be 5.0-50.0 x 10 8M. 28 effect may or may not be specific, although lOOx less was needed to cause aggregation as was required to inhibit dextran aggregation. 9. Aggregation by streptococcal dextrans S.* sanguis and S_. mutans dextrans generally caused much stronger aggregation than dextran T2000. The isolated streptococcal dextrans were a l l greater than 95% carbohydrate. Glucose was the only sugar component detected. No protein was detectable i n the preparations. As can be seen in Table 11 the streptococcal dextrans were a l l better than dextran T2000 in mediating aggregation. To determine i f dextran was attaching to the c e l l surface of A. viscosus, llfC labelled dextran (500 ug/ml) was mixed with cells resuspended to 10 1 0 cells/ml. The mixtures (Table 12) were incubated at room temperature for one hour and then the cells were washed with tris., buffer un t i l no xftc could be detected i n the supernatant. The cells were digested overnight at 37°C with NCS Tissue Solubilizer (Amersham/Searle). The ce l l s were then suspended in a toluene .'methanol :liquifluor (NEN) s c i n t i l l a t i o n f l u i d (600:400:42) and counted. Uptake of ll*C was i n the ranges shown in Table 12. A 10% uptake, assuming each molecule to be 2.0 x 10 6 D, indicates each c e l l had 150 molecules attached to i t s surface. Since the preparations are most l i k e l y a wide variety of molecular weights the uptake of high molecular weight molecules would be higher than 10%. 10. Culture age and growth medium The a b i l i t y of A. viscosus to aggregate i n the presence of dextran i s a function of the age of the culture from which the cells were harvested (Figure 2). A. viscosus was grown i n trypticase soy broth (TSB), brain-heart infusion broth (BHI), heart infusion broth (HI), nutrient broth (NB), thioglycollate broth (TB) and i n TSB containing 5% sucrose. Optical density at 660 nm and the a b i l i t y to aggregate was measured atidaily intervals. As can be seen the cells grown in both NB and BHI aggregated strongly while cells from TSB varied i n their aggregating a b i l i t y . Cells grown i n TB achieved the same c e l l density as those grown i n TSB or BHI but showed no aggregating a b i l i t y while cells grown in HI reached the same density as cells grown i n NB. HI grown cells showed a +3 level of aggregation for the f i r s t two days of growth and then lost a l l of their a b i l i t y to aggregate. Growth of cells in the presence of sucrose did not have an effect on their a b i l i t y to aggregate i n the presence of exogenously added dextran but i t did enhance self-aggregation. BHI appeared to be the 29 Table H Aggregation of Actinomyces viscosus by dextrans isolated from Streptococcus sanguis and Streptococcus mutans strains-Dextran Fraction Aggregation S. mutans 6 : Fraction I § +2 s. mutans 6 : Fraction II +3 s. mutans 6 : Fraction IV +3 s. mutans 4 : Fraction I +2 s. mutans 4 : Fraction II +2 s. mutans 4 : Fraction III +3 s. sanguis 3: Fraction I 0 s. sanguis 3: Fraction II +3 s. sanguis 9: Fraction I +4 s. sanguis 9: , Fraction t III +3 Dextran T2000 +2 None 0 §?Streptococcal dextrans had a f i n a l concentration + of 500 ng/ml t Dextran T2000 had a f i n a l concentration of 50 ug/ml 30 Table 12 Uptake of 1^C-dextran by Actinomyces viscosus Dextran Fraction Uptake (%) Aggregation S. mutans 4: Fraction I 3 12.7 +4 S. mutans 4: Fraction II 8.5 +4 £[. mutans 6: Fraction I 5.5 +4 S_. mutans 6: Fraction II 10.0 +4 S_. mutans 6: .Fraction III 4.6 0 Dextran T2000 - +2 None _ Q § Streptococcal dextrans had a f i n a l concentration of 500 ng/ml t Dextran T2000 had a f i n a l concentration of 50 yg/ml 31 Figure 2. E f f e c t of c u l t u r e age and growth medium on dextran induced aggregation of Actinomyces v iscosus 32 0 1 2 3 4 5 6 c o -4—> CD D) 0) _^ D> < O D 6 6 0 A g g r e g a t i o n 5 6 0 1 2 3 4 5 6 A g e ( d a y s ) 33 best choice of growth medium because of the strong, stable aggregation i t produced and the better c e l l yield. 11. Aggregation of other Actinomycetes In order to determine i f other Actinomycetes were capable of aggregation with dextran a number of strains of A. viscosus and other Actinomyces species were examined using the aggregation assay. As shown in Table 13 a l l were aggregated by dextran except the A. naeslundii which showed a consistently high level of self-aggregation. The results indicated that dextran induced aggregation was not limited to one strain of A. viscosus. B. INTERBACTERIAL AGGREGATION The previous experiments were aimed at gaining an insight into the characteristics of dextran mediated aggregation of A. viscosus. It was of interest to determine i f A. viscosus would interact with oral organisms known to synthesize dextrans. The majority of dextran i n the oral cavity is produced by S^. sanguis and S^. mutans. Because of this there is a strong likelihood of an interaction between either of these two organisms and A. viscbsus. It i s thus important to determine i f dextran can mediate aggregation between these organisms. 1. Interactions between streptococci and A. viscosus The visual assay indicated that aggregation was occurring but i t gave no indication i f both c e l l types were involved. To prove that both A. viscbsus and the streptococcus were coaggregating plate counts were made of the reaction mixtures to determine i f there was a decrease in the unaggregated populations of both c e l l types. When the cells were mixed in the proportions shown in Table 14 definite visual aggregation was seen and a decrease in cfu was detected as indicated. Microscopic examination of aggregates was further proof that both the streptococci and A. viscosus were involved in aggregate formation. 2. Sugars used by streptococci to mediate aggregation with A. viscosus In order to determine which sugars can be used to mediate aggregation, several strains of S_. mutans and S^. sanguis were grown in TSB and TSB supplemented with a sugar to a f i n a l concentration of 1%. Eleven different sugars were tested. The cells were harvested and washed three times in t r i s buffer and then tested for their a b i l i t y to coaggregate with A. viscosus. 34 Table 13 Aggregation of other Actinomycetes by dextran Organism Aggregation With Dextran Without Dextran A. viscosus ATCC 15987 +4 0 A. viscosus ATCC 19246 +4 0 A. viscosus 32 +4 +1 A. viscosus 33 +4 0 A. viscosus 41 +4 0 A. viscosus 59 +2 0 A. naeslundii ATCC 19039 +2 +2 A. i s r a e l i i ATCC 27037 +2 0 35 Table 14 Interbacterial aggregation of and Actinomyces viscosus sucrose grown Streptococcus sanguis Bacterial Mixture Cells/ml Decrease in cfu (%) Aggregation S. sanguis 109 0 0 A. viscosus 109 0 0 S. sanguis + A. viscosus 109 109 57 86 +4 S. sanguis + A. viscosus 109 10 8 15 84 +3 S. sanguis + A. viscosus 109 10 7 84 90 +3 S. sanguis + A. viscosus 109 10 6 0 0 0 36 To insure that there was no soluble dextran the streptococci were washed with buffer u n t i l the washings no longer aggregated A. viscosus. The A. viscosus and one of the streptococci were mixed so that there were 10 9 cells/ml of each i n the reaction mixture. Streptococci grown in TSB did not coaggregate with A. viscosus while cells grown i n the presence of sucrose gave consis-tently strong, positive aggregation for both S^. sanguis and _S. mutans. No other sugar supplement resulted i n such aggregation. S_. mutans, however, strongly self-aggregated when grown in the presence of sucrose. Because of the self-aggregation problem S^. sanguis was used almost exclusively for the experiments on interbacterial aggregation. 3. Effect of c e l l concentration on interbacterial aggregation Cell concentration appears to be an important factor i n bacterial colonization of oral surfaces and i t also appears to be a factor i n in vitro aggregation reactions. Table 14 shows that when the numbers of S_. sanguis were kept constant at 109 cells/ml only those cells grown in TSB containing sucrose reacted with A. viscosus to cause a decrease in cfu. Those cells grown in TSB alone showed no decrease i n cfu nor did the A. viscosus with which they were mixed. Controls containing only S^. sanguis or A. viscosus did not aggregate. If the numbers of A. viscosus were kept constant at 109 cells/ml in the reaction mixture and the numbers of streptococci were varied, vi s i b l e aggregation occurred but this was never reflected i n a lowering of cfu as the aggregates did not settle to the bottom of the reaction tube. Table 15 shows that a decrease in cfu could be detected i f the level of A. viscosus was raised to 10 1 0 cells/ml and the concentration of S_. sanguis was varied. When iS. sanguis grown i n TSB and resuspended to 10 1 0 cells/ml was mixed with A. viscosus resuspended to the same concentration there was a 30% decrease i n S_. sanguis cfu and a 35% decrease i n A. viscosus cfu. If the S_. sanguis concentration was lowered below 10 1 0 cells/ml no decrease in cfu could be detected. Sucrose grown j>. sanguis coaggregated with A. viscosus as shown in Table 15. The results indicated that interbacterial aggregation was occurring and possibly that there were more receptor sites on the surface of the S_. sanguis cells than on the surface of the A. viscosus cells since ten times as many A. viscosus cells were required to support detectable aggregation. 37 Table 15 ^  Interbacterial aggregation of Actinomyces viscosus and sucrose grown Streptococcus sanguis u (%) Aggregation 0 0 +4 +3 +2 0 Bacterial Mixture Cells/ml Decrease i n A. viscosus 10 1 0 0 S. sanguis 10 1 0 0 A. viscosus + 10 1 0 95 S,. sanguis 10 i 0 95 A. viscosus + 10 l u 79 S. sanguis 10 9 99 A. viscosus + 10!° 74 S. sanguis' 10 8 99 A. viscosus + 10 1 0 0 S. sanguis 10 7 0 38 4. Ion Requirements Since dextran induced aggregation of e i t h e r A. v iscbsus or o r a l s t r e p t o c o c c i requires the presence of ions i t was l i k e l y that i n t e r b a c t e r i a l aggregation mediated by dextran a lso had a s i m i l a r requirement. A . v i s c o s u s c e l l s and S_. sanguis c e l l s were washed i n the tes t s o l u t i o n s l i s t e d i n Table 16 and f i n a l l y resuspended to a concentra t ion of 1 0 1 0 c e l l s / m l . They were assayed f o r coaggregation and as can be seen an i o n i s r e q u i r e d f o r i n t e r b a c t e r i a l aggregat ion. 5. pH The pH of the o r a l environment i s an important parameter. Aggregation of A . v iscosus was shown to occur between pH 5.0 and 8.5 with no d e f i n i t e maximum. The range of pH f o r i l x n t e r b a c t e f i a l aggregation was i d e n t i c a l . 6. E f f e c t of c e l l age on i n t e r b a c t e r i a l aggregation C e l l surfaces change wi th the age of the c e l l s and c u l t u r a l condi t ions (63,113,126,128,135,147,150) and such changes may a f f e c t the a b i l i t y to aggregate. In one experiment the age of the _S_. sanguis c u l t u r e was kept constant at 18 hours while the age of the A. v iscosus c u l t u r e was v a r i e d from one to four days. C e l l s were washed arid resuspended to a concentrat ion of 1 0 1 0 c e l l s / m l i n b u f f e r . The age of the A . v iscosus c e l l s was not found to be a f a c t o r i n i n t e r b a c t e r i a l aggregat ion. In another experiment the age of the A . v i s c o s u s c e l l s was kept constant at four days w h i l e the age of the S^ . sanguis c u l t u r e was v a r i e d between 8 hours and 48 hours . There was no change i n the a b i l i t y to coaggregate although there appeared to be a s l i g h t increase i n the tendency to se l f -aggregate on the part of the S_. sanguis c e l l s at 48 hours . From the r e s u l t s so f a r i t seems that glucose grown S_. sanguis c e l l s do not produce a receptor while sucrose grown c e l l s do. I t should be p o s s i b l e to demonstrate an increase i n the a b i l i t y to coaggregate with an increased time of growth i n the presence of sucrose . To demonstrate t h i s , S_. sanguis was grown i n TSB alone overnight and then at var ious times sucrose was added to a f i n a l concentrat ion of 5%. I t was p o s s i b l e to show (Table 17) that the length of time the S^ . sanguis c e l l s were grown i n the presence of sucrose determined the l e v e l of coaggregation that was achieved when such c e l l s were mixed with A . v i s c o s u s . The maximum aggregating a b i l i t y was reached a f t e r twelve hours of growth i n the presence of sucrose . 39 Table 16 Ion requirement for interbacterial aggregaiton Reaction Mixture Suspending Medium A. viscosus ^. sanguis A. viscosus + J3. sanguis Tris 10"2 M, pH 7.2 0 +1 +4 Tris CaCl 10-1* M, pH 7.2 10"1* M, pH 7.2 0 +1 +3 0 +1 +3 D i s t i l l e d H20 0 0 +1 40 Table 17 Incubation of S_. sanguis with sucrose: Effect on interbacterial aggregation Reaction Mixture Time (hours) 0 0 4 8 12 A. viscosus 0 - - - -S_. sanguis o 5 t 0 0 0 0 A. JL-viscosus + sanguis t +1 +2 +3 +4 § Sucrose was never added. t Sucrose was added and the cells were immediately washed. 41 1. Effect of exogenously added dextran on interbacterial aggregation It i s advantageous to show in as many ways as possible that the molecule mediating interbacterial aggregation is dextran. In an experiment aimed at proving that dextran does play a role in aggregation, dextran T2000 ( 5 mg/ml) was mixed with washed cells of A. viscosus. The cells were incubated at room temperature for 30 minutes and then washed three times in t r i s buffer and resuspended to the original concentration of 10 1 0 cells/ml. S.. • sanguis cells grown in TSB (10 1 0 c e l l s / ml) were then mixed with an equal volume A. viscosus cells that had been treated with dextran. Table 18 shows that a decrease in cfu occurred. In the absence of dextran there was no decrease in cfu. The reverse experiment was also performed. S^. sanguis (10 1 0 cells/ml) was mixed with dextran T2000 (5 mg/ml), dextran 10 (5 mg/ml) or dextran isolated from a culture of the same organism (5 mg/ml). S_. mutans was treated in the same way. As can be seen in Table 18, dextran T2000 and streptococcal dextran both mediated interbacterial aggregation while dextran 10 did not. 8. Cell Modification Treatment of A. viscosus with dextranase and various proteases severely impaired or eliminated i t s a b i l i t y to aggregate with dextran T2000. If dextran i s the link between A. viscosus and the streptococci such treatment should have a similar effect on coaggregation. The enzymes li s t e d in Table 19 were incubated with A. viscosus cells (10 1 0 cells/ml) for one hour. The cells were then washed three times i n t r i s buffer and resuspended to the original concentration. A. viscosus and sanguis (grown in the presence of sucrose) were mixed in a 1:1 ratio. As can be seen in Table 19 a l l of the enzymes eliminated interbacterial aggregation. 42 Table 18. Interbacterial aggregation mediated by exogenously added dextran Reaction Mixture Decrease in cfu (%) Aggregation A. viscosus 0 0 A. viscosus + 99 +3 dextran S_. sanguis 0 0 A. viscosus + 0 S^. sanguis 0 0 A. viscosus + 99 dextran + +4 S_. sanguis 97 Table 19 43 Interbacterial aggregation mediated by exogenously added dextran Reaction Mixture Decrease i n cfu (%) Aggregation A. viscbsus 0 0 S_. sanguis 0 0 _S. sanguis + 0 0 Dextran 10 S^. sanguis + 0 +1 Dextran T2000 jS. sanguis + 0 +1 Sanguis dextran A. viscosus + 10 +1 S_. sanguis -A. viscosus + 0 0 S_. sanguis + Dextran 10 A. viscosus + 99 +2 _S_. sanguis + 97 Dextran T2000 A. viscosus + 99 +3 S_. sanguis + 99 Sanguis dextran S_. mutans 0 +1 S. mutans + 0 +1 Dextran 10 JS. mutans + 25 +2 Dextran T2000 .0 mutans + 98 +4 Mutans dextran A. viscosus + 33 +2 S_. mutans 50 A. viscosus + 0 +1 S_. mutans + 0 Dextran 10 A. viscosus + 99 +3 S.-mutans + 90 Dextran T2000 _A. viscosus + 99 + ^ S. mutans + 99 Mutans dextran Table 20 Effect of enzymatic treatment of Actinbmcyes viscosus on interbacterial aggregation with Streptococcus sanguis Reaction Mixture Aggregation after enzyme treatment Trypsin a-chymotrypsin Subtilisin Subtilopeptidase Dextranase None A. viscosus 0 0 0 0 0 0 A. viscosus + Dextran T2000 0 0 0 0 0 *2 A. viscosus + S. sanguis 0 0 0 0 0 *4 45 DISCUSSION This thesis represents the f i r s t time a close look at the biochemical nature of the interaction of dextran with A ."'viscosus has been undertaken. The only other work showing a specific interaction with dextran have been the studies involving _S. mutans (42,84,85). It was f e l t that any interaction between cocci and filamentous organisms (75,105,124) such as A. viscosus was due to some other factor (44). The work described i n this thesis demonstrates for the f i r s t time that dextran mediated aggregation of A. viscosus exists and is a specific phenomenon. Aggregation can occur with as few as 3 molecules of high molecular weight dextran per bacterial c e l l . Various proteases were shown to eliminate the a b i l i t y to aggregate indicating that the receptor contains'protein. Treatment of the cells with dextranase also eliminates aggregating a b i l i t y indicating that the receptor i s possibly a glycoprotein containing a carbohydrate moiety with a-1,6 linked glucose units. The presence of a polysaccharide component i s further indicated by the fact that sugars can inhibit the aggregation reaction as can complex glycoproteins such as concanavalin A. Also, heating at 100°C in the presence of 10 11 M divalent cations reduces but does not eliminate aggregating a c t i v i t y . T h e a b i l i t y to aggregate is variable and may even disappear spontaneously indicating a change in the surface receptors or a loss of the receptors. Out of a l l the bacteria found i n the oral cavity only two, S^. mutans and A. viscosus, have been shown to aggregate in the presence of dextran. Not only does A. viscosus aggregate i n the presence of dextran but i t coaggregates in the presence of other organisms that are capable of producing dextran, such as S_. sanguis and S_. mutans. This interaction of two dissimilar organisms via dextran shares many features with aggregation involving only A. viscosus or only IS. mutans. Thus dextran may be a major factor involved i n causing aggregation of these bacteria on the enamel surfaces of the teeth. The importance of dextran as a molecule capable of mediating bacterial aggregation cannot be stressed enough. The interactions of dextran with other molecules i n the oral cavity are extremely complex and only super-f i c i a l l y understood. Soluble dextran which is free in the saliva can become bound to enamel surfaces or i t may interact with salivary glyco-proteins and precipitate onto the tooth surface. In either state i t is s t i l l free to bind to A. 'viscosus. In one case the cells are immediately attached to the tooth surface and i n the other case the aggregates formed 46 w i l l p r e c i p i t a t e and b i n d to the tooth s u r f a c e . Soluble dextran which has been bound to the surface of A . y i s c o s u s c e l l s may be presented i n a way that enables i t to a l so b i n d to other b a c t e r i a such as _S» mutans and S_. sanguis . S_. mutans may bind to the enamel surfaces of i t s own accord v i a i n s o l u b l e dextran attached to i t s s u r f a c e . Free f l o a t i n g S. mutans c e l l s that are coated w i t h dextran can b i n d e i t h e r S^ . sanguis or A . v iscosus c e l l s and p r e c i p i t a t e onto the tooth s u r f a c e . S_. sanguis a l s o produces i n s o l u b l e , c e l l bound dextran which does not appear to be as e f f i c i e n t i n causing s e l f - a g g r e g a t i o n but i t i s q u i t e capable of a l l o w i n g coaggregation w i t h jS. mutans or A, v iscosus and thus r e s u l t i n g i n the formation of la rge b a c t e r i a l aggregates. A simple model to e x p l a i n dextran induced aggregation of A . v iscosus which accounts f o r a l l the r e s u l t s to date can be suggested. The data suggests that, the receptors c o n s i s t of a g l y c o p r o t e i n , p o s s i b l y c o n t a i n i n g a d e x t r a n - l i k e moiety attached to the c e l l w a l l and with a p r o t e i n receptor at the f r e e end of t h i s carbohydrate moiety to which dextran attaches thus r e s u l t i n g i n aggregation. The sugar component would c o n s i s t p a r t i a l l y of glucose u n i t s l i n k e d with a-1,6 bonds. Maximum aggregation would occur when the c e l l surfaces are covered with the g l y c o p r o t e i n r e c e p t o r s . The v a r y i n g l e v e l s of aggregation could be due to a general lowering of the number of receptors or a decrease i n the number of competent c e l l s ; that i s , only a c e r t a i n percentage of the p o p u l a t i o n would have r e c e p t o r s . I f the g l y c o -p r o t e i n receptor was reduced to only the sugar component or was absent a l together no aggregation would take p l a c e . On the other hand i f there were both complete g l y c o p r o t e i n receptors and p a r t i a l receptors (only glucan) s e l f - a g g r e g a t i o n would takeplace . Since the dextranase recognized the "dextran" moiety on the c e l l s u r f a c e , the p r o t e i n receptors would a lso recognize the sugar component as dextran and b i n d to i t . Maximum s e l f -aggregation would l i k e l y occur when there were equal proport ions of the two c o n d i t i o n s ; e i t h e r equal proport ions on each c e l l or two d i f f e r e n t populat ions of c e l l s . Some unknown f a c t o r i s r e s p o n s i b l e f o r t h i s v a r i a b i l i t y . Hammond, et a l . (55) reported the presence of a plasmid i n A. v iscosus that i s apparently responsible f o r v i r u l e n c e , and may be r e l a t e d to the presence of a polysacchar ide on the surface of the c e l l . Further work has to be done on t h i s problem to completely e l u c i d a t e the phenomenon. Use of a h i g h l y p u r i f i e d . d e x t r a n a s e would put to r e s t any doubts on the quest ion of the s t r u c t u r e of the sugar component of 47 the r e c e p t o r . A c t u a l i s o l a t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of the receptor i s another n e c e s s a r y . s t e p , Examination of the r o l e , o f dextran i n aggregation could Be extended by preparing a r t i f i c i a l molecules containing glucose u n i t s l i n k e d together wi th an assortment of bonds and an assortment of molecular weights . In t h i s way i t would be p o s s i b l e to determine the p o r t i o n of the dextran molecule i n v o l v e d i n the i n t e r a c t i o n . E l e c t r o n microscopy could be used to examine the u l t r a s t r u c t u r e of the i n t e r a c t i n g species and molecules at the time of the i n t e r a c t i o n . The r o l e of the polymers produced by A . v iscbsus i n aggregation could a lso be examined. 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