Vol. 129, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Jan. 1977, p. 351-357 Copyright C 1977 American Society for Microbiology
Biochemical Study of the Relationship of Extracellular Glucan to Adherence and Cariogenicity in Streptococcus mutans and an Extracellular Polysaccharide Mutant M. C. JOHNSON, J. J. BOZZOLA,' I. L. SHECHMEISTER,* AND
I.
L. SHKLAIR
Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901,* and Dental Research Institute, U.S. Naval Training Center, Great Lakes, Illinois 60085 Received for publication 18 August 1976
A mutant of Streptococcus mutans, GS-5, which differed in extracellular polysaccharide (EPS) produced from sucrose, was used to study the role of EPS in the production of dental caries. The mutant proved to be identical to the parent strain in sugar fermentation, growth rate, and serotype. Strain GS-5 synthesized an EPS, which in electron micrographs appeared to be of fibrillar structure, whereas the mutant produced no fibrillar material but only a globular EPS. Analysis of the EPS revealed that about 30% of the glucose units in the GS5 polymer carried (1-3)-like bonds either as branch points or as part of the linear backbone and that the mutant material contained only about 3% of these linkages. When grown in sucrose broth, the proportion of the mutant culture adherent to the glass vessel was dramatically less than that of the parent strain. Caries scores produced in conventional rats by the mutant were significantly lower than those obtained with the parent strain. Since the only difference discovered between strain GS-5 and the mutant was the inability of the mutant to synthesize either a fibrillar EPS or an EPS with more than about 3% (1-3)like linkages, it was concluded that the fibrillar EPS of strain GS-5 contained about 30% (1-3)-like linkages and was necessary for adherence of the bacteria to surfaces and for production of dental caries in test animals.
The development of dental caries is due to an interaction of the oral microflora, especially Streptococcus mutans, the diet, particularly sucrose, and the dental surface (8, 11, 28). S. mutants produces multiple forms of the enzyme glucosyltransferase (EC 2.4.1.5). These enzymes synthesize glucose polymers from sucrose, which adhere to the dental surfaces and lead to the development of cariogenic dental plaque (11, 13-15, 18). The glucans were originally thought to be a-(1-6) dextrans (15, 19), but later work showed them to contain a high percentage of a-(1-3) linkages (16, 26), and this type of linkage was considered to be responsible for the insolubility and "stickiness" of the polysaccharides (16). Mutants of S. mutans that demonstrated a decreased cariogenicity and defective glucan synthesis were reported by de Stoppelaar et al. (6) and Tanzer et al. (35). Although the specific nature of the alteration in polysaccharide synthesis was not determined, a decreased production of water-insoluble polysaccharide and a lessened ability to adhere to hard surfaces were I Present address: Department of Microbiology, Medical College of Pennyslvania, Philadelphia, PA 19129.
reported (9). A morphological study of a similar mutant derived from S. mutans GS-5, and designated GS-511, was reported by our laboratory (22). The primary difference between the parent strain and the mutant was the absence of an intercellular fibrillar polysaccharide in specimens of the mutant, and it was suggested that the fibrillar polymer was necessary for adherence of S. mutans to hard surfaces. The present investigation is an extended characterization of S. mutans GS-5 and GS-511. The formation of a fibrillar intercellular polysaccharide meshwork, the proportion of (1-3)-like links in the extracellular polysaccharide, and the adherence to hard surfaces were studied in both of these organisms. These properties were found to be directly related to the cariogenic potential of the bacteria in test animals. MATERIALS AND METHODS Culture and culture conditions. A culture of S. mutans GS-5 was obtained from R. J. Gibbons (Forsyth Dental Center, Boston, Mass.). The procedures employed for the isolation of mutant GS-511 from GS-5 and for physiological characterization of these organisms were presented earlier (22). The growth media varied according to the nature of the experi351
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ment. The bacteria were maintained at -60°C in Todd-Hewitt broth (BBL) and cultured once in this medium before use. Growth rates of the organisms were determined in Todd-Hewitt broth using a 5% (vol/vol) inoculum in the usual manner. Counts were determined by placing 0.025-ml samples of 10fold serial dilutions onto mitis salivarius agar (Difco) with microdroppers. For the study of adherence to glass, the bacteria were grown in a clean sucrose broth that consisted of the following ingredients (per liter): Trypticase (BBL), 20 g; sucrose, 40 g; NaCl, 8 g; KCI, 0.5 g; Na2HPO4, 0.5 g; K2CO3, 1.0 g; and 5.0 g of yeast extract (Difco). For production and isolation of enzyme, the organisms were grown in a dialyzed broth, free of macromolecules for easier enzyme purification as described by Carlsson et al. (3). All cultures in this study were incubated at 35°C in an atmosphere of 90 to 95% N2 and 10 to 5% CO2. Serological studies. Antibodies were prepared by intramuscular injections of whole cells into eight New Zealand albino rabbits. The first inoculation consisted of 2 ml of viable cells (3 x 1010 cells/ml) emulsified with an equal volume of complete Freund adjuvant (Cappel) administered into each thigh. This was followed 3 weeks later by an injection of 1 ml of the same concentration of whole cells emulsified in incomplete Freund adjuvant. The rabbits were bled 4 weeks after the second injection and the sera were pooled. The type-specific antigens were extracted from whole cells with 5% (vol/vol) trichloroacetic acid as described by Slade (34). These antigens were dissolved in physiological saline to a final concentration of 2% (vol/vol) and used in all serological procedures. Immunodiffusion experiments were performed in petri plates (50 by 12 mm) filled with 0.85% Ionagar (Oxoid) in barbitone acetate buffer (Oxoid) at pH 8.6. The same medium on glass slides (1 by 3 inch [ca. 2.5 by 7.5 cm]) was used for immunoelectrophoresis. Wells were cut and filled with the antigen and subjected to electrophoresis at 6 V per cm for 1 h. The troughs were then filled with antisera, and the slides were stored at 4°C in a humid chamber for 1 week (5). Cellular adherence to glass. The relative adherence of the two bacterial strains to glass when grown in sucrose broth was determined according to the method described by Olson et al. (30). The organisms were inoculated into 2 ml of sucrose broth and incubated at a 300 angle for 36 h. The broth containing the nonadherent cells was decanted and collected, the culture tubes were washed twice with 2 ml of distilled water, and the washings were combined with the broth. To collect the adherent cells, the tubes were washed vigorously three times each with 2 ml of 0.01 N NaOH and the sides were scraped with wooden applicator sticks. The optical densities (ODs) of the cell suspensions were determined using a spectrophotometer (Beckman DB) at 540 nm. Hydroxylapatite column chromatography. The cells were cultured according to the procedure described by Carlsson et al. (3) in 5 liters of dialyzed broth until acid production ceased, when they were separated from the culture fluid by centrifugation at 10,400 x g for 15 min at 4°C. The supernatant fluid, which contained the enzymes, was diluted
J. BACTERIOL.
with 2 liters of distilled water and a slurry of about 250 ml of hydroxylapatite (Bio-Gel HT, Bio-Rad) was added. After continuous stirring for 2 h, the slurry was permitted to sediment overnight. It was then packed into a 2.5-cm-diameter column and washed with 0.01 M phosphate buffer at pH 6.8, until the OD280 of the eluant was near 0. The column was then eluted stepwise with phosphate buffers at pH 6.8, ranging in concentrations up to 0.5 M. The eluant from the column was continuously monitored at OD280 using an ultraviolet analyzer (Isco model UA-2). Glucosyltransferase activity was measured by determining the amount of reducing sugar released by the reaction of the enzymes with sucrose (3), and expressed as dextransucrase units (DSU) (21). Periodate oxidation of polysaccharides. The polysaccharides used in this study were synthesized by enzyme fractions eluted from hydroxylapatite. These enzymes were first dialyzed against 0.05 M phosphate buffer at pH 6.0; then 500 to 1,000 DSU and a few drops of toluene were added to 1 liter of 5% sucrose in the same buffer. The mixture was incubated at 37°C for 24 h. The resulting polysaccharides were precipitated with an equal volume of ethanol, redissolved in distilled water, and reprecipitated in three cycles and, finally, freeze-dried (29, 36). Approximately 40 mg of the polysaccharides was dissolved in 20 ml of distilled water and oxidized with 200 mg of sodium periodate in the dark at room temperature (26), and the amount of reacting periodate was measured spectrophotometrically (23). In the case of the glucan obtained from the GS-5 0.4 M enzyme fraction, periodate consumption became constant after 48 h of reaction; all other polysaccharides were oxidized after 24 to 36 h. The polysaccharides were separated from excess periodate by centrifugation and washed three times with distilled water. The supernatant fluids were tested for soluble carbohydrate with the anthrone reagent (33), and none was detected. The oxidized polysaccharides were resuspended in 5 ml of distilled water and reduced with sodium borohydride (1 mg per mg of polysaccharide) at room temperature for 4 h. A second addition of the same amount of sodium borohydride was then allowed to react overnight, and the excess sodium borohydride was decomposed with 1 to 2 ml of 1 N HCI. The glucans were hydrolyzed with 20 ml of 4 N HCI at 100°C for 2 h and neutralized batchwise with an excess of Dowex 2-4X (bicarbonate) resin (26), and samples were analyzed for glucose content, as a measure of unoxidized units indicating (1-3)-like bonds, with D-glucose oxidase (Glucostat reagent, Worthington). Since this assay is specific for glucose, no further effort was made to ensure that the hydrolysis product contained glucose. Evaluation of caries-producing ability. S. mutans GS-5 and the mutant GS-511 were tested in conventional Osborn-Mendle rats for their ability to produce dental caries. Two separate trials were performed. In the first experiment, two groups of 5 rats each were used, whereas the second experiment consisted of one group of 7 rats and one of 10 rats. Grampositive oral microflora was suppressed before the trials by adding 0.2% polycillin to the drinking wa-
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S. MUTANS EXTRACELLULAR POLYSACCHARIDE MUTANT
353
ter 2 days before birth of the test rats and continuing the treatment until the young were weaned (24). The test bacteria, made resistant to 1 mg of streptomycin per ml (8), were implanted immediately after weaning according to the method of Konig and Guggenheim (24), and 35 days later the animals were sacrificed and the teeth were scored by Samuel Rosen (Ohio State University) for incidence of caries.
RESULTS Physiological characteristics. As previously reported (22), strains GS-5 and GS-511 were identical in fermentation of sugars, reaction on blood agar, hydrolysis of arginine, and growth on mitis salivarius agar containing 40% sucrose. Growth rate studies revealed identical growth curves for both bacterial strains. Serological studies. Since the parent strain, S. mutans GS-5, belongs to Bratthal's serological group "c" (2), it was anticipated that this antigen would also be present on the mutant GS-511. Immunodiffusion of the trichloroacetic acid extracts of each strain formed a single line of precipitate with the homologous antiserum and a single precipitin line of identity with the heterologous antiserum (Fig. la). Immunoelectrophoresis of these extracts against GS-5 antiserum revealed a single arc of precipitation for each antigen with the same degree of mobility toward the cathode (Fig. lb). Cellular adherence to glass. When grown in the presence of sucrose, the two organisms showed striking differences in adherence to glass (Fig. 2). The sucrose broth samples conFIG. 1. (a) Immunodiffusion of trichloroacetic taining suspensions of nonadherent GS-511 acid extracts from GS-5 (on right) and GS-511 (on cells were about 17.3 times more dense than left) against GS-5 antisera (center well) shows a GS-5 samples. Resuspension of adherent GS-5 single precipitin line of identity. (b) Immunoelectroorganisms gave 6.51 times higher OD readings phoresis of trichloroacetic acid extracts from GS-5 well) and GS-511 (bottom well) against anti-GSthan the resuspended adherent GS-511 cul- 5(topserum (center trough). Both antigens revealed tures. It is evident that the majority of the GS-5 identical precipitin patterns with the same degree of cells were firmly attached to the surface of the mobility toward the cathode. culture vessel, whereas most of the GS-511 cells remained suspended in the culture medium. Hydroxylapatite chromatography. Repre- resented a 17-fold reduction in volume and a sentative results of the purification of extracel- recovery of about 25.6% of the activity. lular glucosyltransferase activity are shown in Periodate oxidation of polysaccharides. Table 1. Hydroxylapatite chromatography of The polysaccharides used for linkage determithe GS-5 culture fluid gave one pool of activity nation were synthesized by the enzymes from that eluted with 0.2 M phosphate buffer at pH the 0.2 and 0.4 M pools obtained from hydroxyl6.8 and another pool of activity that came off apatite. The average percentages of (1-3)-like with a 0.4 M phosphate buffer at the same pH. links present in these glucans are shown in The 0.2 M and the 0.4 M pools contained 960 Table 2. The enzymes from the 0.2 M pool of and 1,840 DSU, respectively. Together, the GS-5 produced polysaccharide which, in each of pools represented an 11-fold reduction in vol- the six different samples, contained no detectume and a recovery of about 28% of the activity. able periodate-resistant glucose units, whereas Purification of the GS-511 enzymes gave a 0.2 that synthesized by the 0.4 M pool possessed M pool with 2,520 DSU and a 0.4 M pool con- 30.70 ± 4.03% of these units, which represents taining 550 DSU. These two enzyme pools rep- 68.22 ± 8.95 ,umol of glucose intact after treat-
354
J. BACTERIOL.
JOHNSON ET AL.
ment with periodate. The glucose polymers
synthesized by the 0.2 M pool of GS-511 glucosyltransferases contained no a-(1-3)-linked glucose units. Only 3.48 + 1.40% of the polysaccharide produced by the enzymes from the 0.4 M fraction of GS-511 was (1-3)-like linked, determined from 7.73 3.11 ,umol ofunoxidized glucose. Evaluation of dental caries-producing ability. The two bacterial strains were tested for cariogenic potential in conventional rats and the average dental caries scores are given in Tables 3 and 4. In the first experiment, GS-5 produced an average dental caries score of 60.2 + 15.6, whereas the average dental caries score for GS-511 was 32.6 9.24. Comparison of these two means by the t test (32) showed them to be significantly different at the 0.005 level. In the second test, the respective scores for GS-5 and
GS-511 were 32 + 12.4 and 14.1 + 9.11, and comparison of these two means gave a t value of 3.38, which is statistically significant. Thus, mutant GS-511 demonstrated significantly less cariogenic potential than the parent strain GS5.
±
DISCUSSION The reaction ofS. mutans with sucrose yields extracellular polysaccharide, which enables these bacteria to establish adherent cariogenic TABLE 2. Average percentage ofperiodate-resistant glucose units or links in polysaccharides produced by enzyme fractions eluted from hydroxylapatite
±
Enzyme Avg % (1-
rgaOrga-
GS-5
GS-511 | ADHERENT E
0.2 0.4
None 30.70
6 6
4.03
0.2 0.4
None 3.48
5 5
1.40
devia-
linkages tson
TABLE 3. Statistical treatment of caries scores obtained in conventional rats: first experiment
7
No. of test Avg caries Standard score deviation animals
Organism
F .6-
U)
z
No. of
.8
0
LO)
Standard
3)-like
fraction (M)
.5-
60.2 5 GS-5 32.6 GS-511 5 a Critical 99.5t8 is 3.335.
0
15.60 9.24
t value
3 40a
0.03
TABLE 4. Statistical treatment of caries scores obtained in conventional rats: second experiment Orga- No. of test Avg caries Standard t value GS-5
GS-511
FIG. 2. Relative absorbencies at 540 nm of cell suspensions of adherent and nonadherent cell populations of GS-5 and GS-511 grown in sucrose broth.
nism
animals
score
GS-5 GS-511
7 10
32.7 14.1
a
deviation
12.4 9.11
3.38a
Critical 99.5t15 is 2.947.
TABLE 1. Purification of glucosyltransferases from S. mutans GS-5 and GS-511
a
b
Source
Volume
Enzyme
Total units
% Yield
GS-5 Culture fluid Adsorbed to HAa 0.2 M fraction (40_70)b 0.4 M fraction (85-108)
5,000 240 184
2.0
10,000 4,340 960 1,840
100 43.5 9.6 18.4
12,500 4,150 2,520 550
100 34.5 21.0 4.6
GS-511 Culture fluid Adsorbed to HA 0.2 M fraction (21-39) 0.4 M fraction (71-82) HA, Hydroxylapatite. Fraction numbers.
4.0 10.0
5,000
2.5
180 110
14.0 5.0
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plaques on smooth dental surfaces in the highly fluid and abrasive environment of the oral cavity (28). This adherence of S. mutans is one of the possible processes that leads to dental caries. The mechanism of adherence has been studied by several investigators (11, 14, 17, 27, 31). The first step appears to be the reversible attachment of the bacteria or the capsular polysaccharide to the glycoprotein pellicle that coats the teeth (31). Sucrose is then necessary for the synthesis of certain insoluble or "sticky" polysaccharides, which form the framework of the dental plaque, and for the irreversible attachment of the bacteria to the teeth (13, 17). The insolubility or "stickiness" of the polysaccharides was reported to be related to their content of a-(1-3)-linked glucose units (16). The nature of the S. mutans enzymes that synthesize these polysaccharides has been studied (4, 10, 18, 27), and the number of different enzyme forms found associated with a specific strain varied from two (4, 10, 27) to six (18). It has been suggested that the variety of linkages found in the extracellular glucans resulted either from enzymes of different specificity (18) or that a single enzyme carries out the synthesis of different linkages (7). The present study demonstrated that S. mutans GS-5 produced at least two glucosyltransferases, or two groups of these enzymes, which synthesized glucans of different types. A mutant, S. mutans GS-511, was obtained that was altered in the synthesis of at least one of these types of extracellular glucans, and that proved to be identical to the parent strain in all other physiological and serological characteristics studied. Therefore, morphological and biochemical comparisons of the extracellular glucans were made in an attempt to elucidate their role in the etiology of dental caries. Transmission electron micrographs of sections of GS-5 (22) revealed two distinct types of polysaccharides, similar to those described by Guggenheim and Schroeder (19): (i) globular material in close association with the cell surface was found only in young cultures and then only at the periphery of the microcolonies, and (ii) fibrillar substances that were present in the intercellular spaces of both the young and old cultures. The only extracellular polysaccharide observed in both 9- and 24-h cultures of GS-511 was the globular material (22). The disappearance of the globular material around older GS-5 cells may have been due to its conversion into fibrillar glucan by the action of at least one additional enzyme. That is, one enzyme, or set of enzymes, may synthesize the globular material, which is then used as a receptor by a
355
second enzyme or set of enzymes to form the fibrillar polysaccharide. Recently, Kuramitsu and Ingersoll (25) provided evidence for such a synthetic sequence. Using antibodies to soluble and insoluble glucan-synthesizing enzymes, they demonstrated partial inhibition of cellular adherence with the former and almost complete inhibition with the latter types of antibodies. The possibility of this being due to mixing of activities was ruled out and it was suggested that synthesis of both a(1-3) and a-(1-6) glucans was necessary for adherence. A structure for S. mutans polysaccharide suggested by Long and Edwards (26), which consisted of an a-(1-6)-linked linear molecule with a-(1-3)-linked branches, would be compatible with this possibility. However, such a branched polysaccharide would not be expected to form fibrils. To form fibrils, several polysaccharide molecules must aggregate "side by side" and the side branches might interfere with this union. Glycol units that lack adjacent hydroxyl groups such as nonterminal units joined by (13) bonds or units involved in branching at both C2 and C4 are not affected by periodate oxidation (20). Thus, each mole of glucose liberated following hydrolysis of the oxidized and reduced polysaccharide is indicative of one of these two bonding possibilities. Since previous work has indicated the bonding in S. mutans polysaccharide to be either (1-6) or (1-3) bonding (16, 26) and the latter linkage was related to adherence (16), all periodate-resistant glucose units in this study were considered to represent units with (1-3)-like bonding. No distinction was made as to whether these linkages were involved in a branch point or a part of the backbone of the polymer. The percentages of periodate-resistant glucose units in polysaccharides synthesized by GS-5 and GS-511 glucosyltransferases, which eluted with 0.4 M phosphate buffer at pH 6.8, differed dramatically. This difference was considered to be a reflection of reality and not the result of differences in enzyme preparation, since it was repeated several times, and examination of polysaccharide isolated from bacterial culture yields similar results. The enzymes from GS-5 produced polysaccharides with about 30% periodate-resistant linkages, whereas those obtained from GS-511 contained only about 3% of these linkages. This indicates that the fibrillar polysaccharide observed in sections of GS-5 may have consisted of about 30% a-(13)-linked glucose units, and that it was synthesized by glucosyltransferases that eluted from hydroxylapatite with 0.4 M phosphate buffer.
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JOHNSON ET AL.
The enzymes used in this investigation were collected after acid production had ceased and the bacteria were in stationary growth phase. A study of the proportions of linkages formed in earlier cultures, which may provide information regarding the sequence of polysaccharide synthesis, is currently in progress. The ability of GS-5 to adhere to glass when grown in sucrose broth was 6.5 times greater than that of GS-511, as determined by relative densities of suspensions of adherent cell populations. It can therefore be assumed that the globular polysaccharide produced by GS-511 was not adequate for attachment of S. mutans to hard surfaces. Finally, GS-511 demonstrated a significantly lower dental caries-inducing potential than did GS-5, when tested in conventional rats. Since the only discovered difference between the parent and mutant strains was the type of extracellular glucan synthesized, it may be assumed that the production of some specific polysaccharide is a critical factor in determining cariogenic potential. Hence, cariogenic GS-5 produced fibrillar polysaccharide consisting of about 30% (1-3)-likelinked glucose units, which immobilized the cells, and a globular polysaccharide closely associated with the cell surface, which disappeared from around older cells. Mutant GS-511, which differed from parent strain GS-5 only in that it failed to synthesize the fibrillar glucan, was nonadhering and possessed a markedly lower cariogenic potential. This indicates that the fibrillar glucan is a significant factor in the cause of dental caries. ACKNOWLEDGMENTS We wish to express our appreciation to Tom Stephens for the serology and to Mike Okuji for assistance with preparing the enzymes. This investigation was supported by training grant DE00144 from the National Institute of Dental Research. M.C.J. and J.J.B. were predoctoral trainees on this same training grant. LITERATURE CITED 1. Bozzola, J. J., M. C. Johnson, and I. L. Shechmeister. 1973. In situ multiple sampling of attached bacteria for scanning and transmission electron microscopy. Stain Technol. 48:317-325. 2. Bratthall, D. 1970. Demonstration of five serologic groups of streptococcal strains resembling Streptococcus mutans. Odontol. Revy 21:143-152. 3. Carlsson, J., E. Newbrun, and B. Krasse. 1969. Purification and properties of dextransucrase from Streptococcus sanguis. Arch. Oral Biol. 14:469-478. 4. Chludzinski, M., G. R. Germaine, and C. F. Schachtele. 1974. Purification and properties of dextransucrase from Streptococcus mutans. J. Bacteriol. 118:17. 5. Crowle, A. J. 1973. Immunoelectrophoreses, p. 305-352. In Immunoelectrophoresis. Academic Press Inc., New York. 6. de Stoppelaar, J. D., K. G. Konig, A. J. Plassachaert,
J. BACTERIOL. and J. S. van der Hovers. 1971. Decreased cariogenicity of a mutant of Streptococcus mutans. Arch. Oral Biol. 16:971-975. 7. Ebert, K. H., and M. Brosche. 1967. Origin of branches in native dextrans. Biopolymers 5:423-430. 8. Fitzgerald, R. J., and P. Keyes. 1960. Demonstration of the etiological role of streptococci in experimental caries in the hamster. J. Am. Dent. Assoc. 61:6-61. 9. Freedman, M. L., and J. M. Tanzer. 1974. Dissociation of plaque formation from glucan-induced agglutination in mutants of Streptococcus mutans. Infect. Immun. 10:189-196. 10. Fukui, K., Y. Fukui, and T. Moriyama. 1974. Purification and properties of dextransucrase and invertase from S. mutans. J. Bacteriol. 118:796-804. 11. Gibbons, R. J. 1968. Formation and significance of bacterial polysaccharides in caries etiology. Caries Res. 2:164-171. 12. Gibbons, R. J., K. Berman, P. Knoettmer, and B. Kapsimalis. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule-forming streptococci of human origin. Arch. Oral Biol. 11:549-560. 13. Gibbons, R. J., P. F. Depaola, D. M. Spinell, and Z. Skobe. 1974. Interdental localization of Streptococcus mutans as related to dental caries experience. Infect. Immun. 9:481-488. 14. Gibbons, R. J., and R. J. Fitzgerald. 1969. Dextraninduced agglutination of Streptococcus mutans and its potential role in the formation of microbial dental plaque. J. Bacteriol. 98: 341-346. 15. Gibbons, R. J., and M. Nygaard. 1968. Synthesis of insoluble dextran and its significance in the formation of gelatinous deposits by plaque-forming streptococci. Arch. Oral Biol. 13:1249-1262. 16. Guggenheim, B. 1970. Enzymatic hydrolysis of water insoluble glucan produced by glucosyltransferases from a strain of Streptococcus mutans. Helv. Odontol. Acta 14(V):89-108. 17. Guggenheim, B. 1970. Extracellular polysaccharides and microbial plaque. Int. Dent. J. 20:657-678. 18. Guggenheim, B., and E. Newbrun. 1969. Extracellular glucosyltransferase activity of an HS strain of Streptococcus mutans. Helv. Odontol. Acta 13:84-97. 19. Guggenheim, B., and H. E. Schroeder. 1967. Biochemical and morphological aspects of extracellular polysaccharides produced by cariogenic streptococci. Helv. Odontol. Acta 11:131-152. 20. Hay, G. W., B. A. Lewis, and F. Smith. 1965. Periodate oxidation of polysaccharides: general procedures, p. 357-360. In R. L. Whistler and J. N. BeMiller (ed.), Methods in carbohydrate chemistry, vol. 5. Academic Press Inc., New York. 21. Hehre, E. J. 1955. Polysaccharide synthesis from disaccharides. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 22. Johnson, M. C., J. J. Bozzola, and I. L. Shechmeister. 1974. Morphological study of Streptococcus mutans and two extracellular polysaccharide mutants. J. Bacteriol. 118:304-311. 23. Khym, J. X. 1972. Direct spectrophotometric determination of iodate following periodate oxidation of aglycol groups, p. 87-93. In R. L. Whistler and J. N. BeMiller (ed.), Methods in carbohydrate chemistry. Academic Press Inc., New York. 24. Konig, K. G., and B. Guggenheim. 1968. Implantation of antibiotic resistant bacteria and the production of dental caries in rats, p. 217-252. In P. A. Staple (ed.), Advances in oral biology, vol. 3. Academic Press Inc., New York. 25. Kuramitsu, H. K., and L. Ingersoll. 1976. Differential inhibition of Streptococcus mutans in vitro adherence by anti-glucosyltransferase antibodies. Infect. Immun. 13:1775-1777.
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26. Long, L. W., and J. R. Edwards. 1972. Detailed structure of a dextran from a cariogenic bacterium. Carbohydr. Res. 24:216-217. 27. Mukasa, H., and H. D. Slade. 1974. Mechanism of adherence of Streptococcus mutans to smooth surfaces. III. Purification and properties of enzyme complex responsible for adherence. Infect. Immun. 10:11351145. 28. Newbrun, E. 1967. Sucrose, the arch criminal of dental caries. Odontol. Revy 18:373-386. 29. Newbrun, E. 1972. Extracellular polysaccharides synthesized by glucosyltransferases of oral streptococci: composition and susceptibility to hydrolysis. Caries Res. 6:132-147. 30. Olson, G. D., A. S. Bleiweiss, and P. A. Small. 1972. Adherence inhibition of Streptococcus mutans: an assay reflecting a possible role of antibody in dental
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caries prophylaxis. Infect. Immun. 5:419-427. 31. Rolla, G. 1971. Adsorption of dextran to saliva-treated hydroxyapatite. Arch. Oral Biol. 16:527-533. 32. Roscoe, J. T. 1966. Fundamental research statistics for the behavioral sciences. Holt, Rinehart and Winston,
New York. 33. Scott, T. A., and E. H. Melvin. 1953. Determination of dextran with anthrone. Anal. Chem. 25:1656-1716. 34. Slade, H. D. 1965. Extraction of cell wall polysaccharide antigen from streptococci. J. Bacteriol. 90:667-671. 35. Tanzer, J. M., M. L. Freedman, R. J. Fitzgerald, and R. H. Larson. 1974. Diminished virulence of glucan synthesis-defective mutants of Streptococcus mutans. Infect. Immun. 10:197-235. 36. Wood, J. M., and P. Critchley. 1966. The extracellular polysaccharide produced from sucrose by cariogenic streptococcus. Arch. Oral Biol. 11:1039-1042.
JOURNAL OF BACTERIOLOGY, Jan. 1977, p. 351-357 Copyright C 1977 American Society for Microbiology
Biochemical Study of the Relationship of Extracellular Glucan to Adherence and Cariogenicity in Streptococcus mutans and an Extracellular Polysaccharide Mutant M. C. JOHNSON, J. J. BOZZOLA,' I. L. SHECHMEISTER,* AND
I.
L. SHKLAIR
Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901,* and Dental Research Institute, U.S. Naval Training Center, Great Lakes, Illinois 60085 Received for publication 18 August 1976
A mutant of Streptococcus mutans, GS-5, which differed in extracellular polysaccharide (EPS) produced from sucrose, was used to study the role of EPS in the production of dental caries. The mutant proved to be identical to the parent strain in sugar fermentation, growth rate, and serotype. Strain GS-5 synthesized an EPS, which in electron micrographs appeared to be of fibrillar structure, whereas the mutant produced no fibrillar material but only a globular EPS. Analysis of the EPS revealed that about 30% of the glucose units in the GS5 polymer carried (1-3)-like bonds either as branch points or as part of the linear backbone and that the mutant material contained only about 3% of these linkages. When grown in sucrose broth, the proportion of the mutant culture adherent to the glass vessel was dramatically less than that of the parent strain. Caries scores produced in conventional rats by the mutant were significantly lower than those obtained with the parent strain. Since the only difference discovered between strain GS-5 and the mutant was the inability of the mutant to synthesize either a fibrillar EPS or an EPS with more than about 3% (1-3)like linkages, it was concluded that the fibrillar EPS of strain GS-5 contained about 30% (1-3)-like linkages and was necessary for adherence of the bacteria to surfaces and for production of dental caries in test animals.
The development of dental caries is due to an interaction of the oral microflora, especially Streptococcus mutans, the diet, particularly sucrose, and the dental surface (8, 11, 28). S. mutants produces multiple forms of the enzyme glucosyltransferase (EC 2.4.1.5). These enzymes synthesize glucose polymers from sucrose, which adhere to the dental surfaces and lead to the development of cariogenic dental plaque (11, 13-15, 18). The glucans were originally thought to be a-(1-6) dextrans (15, 19), but later work showed them to contain a high percentage of a-(1-3) linkages (16, 26), and this type of linkage was considered to be responsible for the insolubility and "stickiness" of the polysaccharides (16). Mutants of S. mutans that demonstrated a decreased cariogenicity and defective glucan synthesis were reported by de Stoppelaar et al. (6) and Tanzer et al. (35). Although the specific nature of the alteration in polysaccharide synthesis was not determined, a decreased production of water-insoluble polysaccharide and a lessened ability to adhere to hard surfaces were I Present address: Department of Microbiology, Medical College of Pennyslvania, Philadelphia, PA 19129.
reported (9). A morphological study of a similar mutant derived from S. mutans GS-5, and designated GS-511, was reported by our laboratory (22). The primary difference between the parent strain and the mutant was the absence of an intercellular fibrillar polysaccharide in specimens of the mutant, and it was suggested that the fibrillar polymer was necessary for adherence of S. mutans to hard surfaces. The present investigation is an extended characterization of S. mutans GS-5 and GS-511. The formation of a fibrillar intercellular polysaccharide meshwork, the proportion of (1-3)-like links in the extracellular polysaccharide, and the adherence to hard surfaces were studied in both of these organisms. These properties were found to be directly related to the cariogenic potential of the bacteria in test animals. MATERIALS AND METHODS Culture and culture conditions. A culture of S. mutans GS-5 was obtained from R. J. Gibbons (Forsyth Dental Center, Boston, Mass.). The procedures employed for the isolation of mutant GS-511 from GS-5 and for physiological characterization of these organisms were presented earlier (22). The growth media varied according to the nature of the experi351
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ment. The bacteria were maintained at -60°C in Todd-Hewitt broth (BBL) and cultured once in this medium before use. Growth rates of the organisms were determined in Todd-Hewitt broth using a 5% (vol/vol) inoculum in the usual manner. Counts were determined by placing 0.025-ml samples of 10fold serial dilutions onto mitis salivarius agar (Difco) with microdroppers. For the study of adherence to glass, the bacteria were grown in a clean sucrose broth that consisted of the following ingredients (per liter): Trypticase (BBL), 20 g; sucrose, 40 g; NaCl, 8 g; KCI, 0.5 g; Na2HPO4, 0.5 g; K2CO3, 1.0 g; and 5.0 g of yeast extract (Difco). For production and isolation of enzyme, the organisms were grown in a dialyzed broth, free of macromolecules for easier enzyme purification as described by Carlsson et al. (3). All cultures in this study were incubated at 35°C in an atmosphere of 90 to 95% N2 and 10 to 5% CO2. Serological studies. Antibodies were prepared by intramuscular injections of whole cells into eight New Zealand albino rabbits. The first inoculation consisted of 2 ml of viable cells (3 x 1010 cells/ml) emulsified with an equal volume of complete Freund adjuvant (Cappel) administered into each thigh. This was followed 3 weeks later by an injection of 1 ml of the same concentration of whole cells emulsified in incomplete Freund adjuvant. The rabbits were bled 4 weeks after the second injection and the sera were pooled. The type-specific antigens were extracted from whole cells with 5% (vol/vol) trichloroacetic acid as described by Slade (34). These antigens were dissolved in physiological saline to a final concentration of 2% (vol/vol) and used in all serological procedures. Immunodiffusion experiments were performed in petri plates (50 by 12 mm) filled with 0.85% Ionagar (Oxoid) in barbitone acetate buffer (Oxoid) at pH 8.6. The same medium on glass slides (1 by 3 inch [ca. 2.5 by 7.5 cm]) was used for immunoelectrophoresis. Wells were cut and filled with the antigen and subjected to electrophoresis at 6 V per cm for 1 h. The troughs were then filled with antisera, and the slides were stored at 4°C in a humid chamber for 1 week (5). Cellular adherence to glass. The relative adherence of the two bacterial strains to glass when grown in sucrose broth was determined according to the method described by Olson et al. (30). The organisms were inoculated into 2 ml of sucrose broth and incubated at a 300 angle for 36 h. The broth containing the nonadherent cells was decanted and collected, the culture tubes were washed twice with 2 ml of distilled water, and the washings were combined with the broth. To collect the adherent cells, the tubes were washed vigorously three times each with 2 ml of 0.01 N NaOH and the sides were scraped with wooden applicator sticks. The optical densities (ODs) of the cell suspensions were determined using a spectrophotometer (Beckman DB) at 540 nm. Hydroxylapatite column chromatography. The cells were cultured according to the procedure described by Carlsson et al. (3) in 5 liters of dialyzed broth until acid production ceased, when they were separated from the culture fluid by centrifugation at 10,400 x g for 15 min at 4°C. The supernatant fluid, which contained the enzymes, was diluted
J. BACTERIOL.
with 2 liters of distilled water and a slurry of about 250 ml of hydroxylapatite (Bio-Gel HT, Bio-Rad) was added. After continuous stirring for 2 h, the slurry was permitted to sediment overnight. It was then packed into a 2.5-cm-diameter column and washed with 0.01 M phosphate buffer at pH 6.8, until the OD280 of the eluant was near 0. The column was then eluted stepwise with phosphate buffers at pH 6.8, ranging in concentrations up to 0.5 M. The eluant from the column was continuously monitored at OD280 using an ultraviolet analyzer (Isco model UA-2). Glucosyltransferase activity was measured by determining the amount of reducing sugar released by the reaction of the enzymes with sucrose (3), and expressed as dextransucrase units (DSU) (21). Periodate oxidation of polysaccharides. The polysaccharides used in this study were synthesized by enzyme fractions eluted from hydroxylapatite. These enzymes were first dialyzed against 0.05 M phosphate buffer at pH 6.0; then 500 to 1,000 DSU and a few drops of toluene were added to 1 liter of 5% sucrose in the same buffer. The mixture was incubated at 37°C for 24 h. The resulting polysaccharides were precipitated with an equal volume of ethanol, redissolved in distilled water, and reprecipitated in three cycles and, finally, freeze-dried (29, 36). Approximately 40 mg of the polysaccharides was dissolved in 20 ml of distilled water and oxidized with 200 mg of sodium periodate in the dark at room temperature (26), and the amount of reacting periodate was measured spectrophotometrically (23). In the case of the glucan obtained from the GS-5 0.4 M enzyme fraction, periodate consumption became constant after 48 h of reaction; all other polysaccharides were oxidized after 24 to 36 h. The polysaccharides were separated from excess periodate by centrifugation and washed three times with distilled water. The supernatant fluids were tested for soluble carbohydrate with the anthrone reagent (33), and none was detected. The oxidized polysaccharides were resuspended in 5 ml of distilled water and reduced with sodium borohydride (1 mg per mg of polysaccharide) at room temperature for 4 h. A second addition of the same amount of sodium borohydride was then allowed to react overnight, and the excess sodium borohydride was decomposed with 1 to 2 ml of 1 N HCI. The glucans were hydrolyzed with 20 ml of 4 N HCI at 100°C for 2 h and neutralized batchwise with an excess of Dowex 2-4X (bicarbonate) resin (26), and samples were analyzed for glucose content, as a measure of unoxidized units indicating (1-3)-like bonds, with D-glucose oxidase (Glucostat reagent, Worthington). Since this assay is specific for glucose, no further effort was made to ensure that the hydrolysis product contained glucose. Evaluation of caries-producing ability. S. mutans GS-5 and the mutant GS-511 were tested in conventional Osborn-Mendle rats for their ability to produce dental caries. Two separate trials were performed. In the first experiment, two groups of 5 rats each were used, whereas the second experiment consisted of one group of 7 rats and one of 10 rats. Grampositive oral microflora was suppressed before the trials by adding 0.2% polycillin to the drinking wa-
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353
ter 2 days before birth of the test rats and continuing the treatment until the young were weaned (24). The test bacteria, made resistant to 1 mg of streptomycin per ml (8), were implanted immediately after weaning according to the method of Konig and Guggenheim (24), and 35 days later the animals were sacrificed and the teeth were scored by Samuel Rosen (Ohio State University) for incidence of caries.
RESULTS Physiological characteristics. As previously reported (22), strains GS-5 and GS-511 were identical in fermentation of sugars, reaction on blood agar, hydrolysis of arginine, and growth on mitis salivarius agar containing 40% sucrose. Growth rate studies revealed identical growth curves for both bacterial strains. Serological studies. Since the parent strain, S. mutans GS-5, belongs to Bratthal's serological group "c" (2), it was anticipated that this antigen would also be present on the mutant GS-511. Immunodiffusion of the trichloroacetic acid extracts of each strain formed a single line of precipitate with the homologous antiserum and a single precipitin line of identity with the heterologous antiserum (Fig. la). Immunoelectrophoresis of these extracts against GS-5 antiserum revealed a single arc of precipitation for each antigen with the same degree of mobility toward the cathode (Fig. lb). Cellular adherence to glass. When grown in the presence of sucrose, the two organisms showed striking differences in adherence to glass (Fig. 2). The sucrose broth samples conFIG. 1. (a) Immunodiffusion of trichloroacetic taining suspensions of nonadherent GS-511 acid extracts from GS-5 (on right) and GS-511 (on cells were about 17.3 times more dense than left) against GS-5 antisera (center well) shows a GS-5 samples. Resuspension of adherent GS-5 single precipitin line of identity. (b) Immunoelectroorganisms gave 6.51 times higher OD readings phoresis of trichloroacetic acid extracts from GS-5 well) and GS-511 (bottom well) against anti-GSthan the resuspended adherent GS-511 cul- 5(topserum (center trough). Both antigens revealed tures. It is evident that the majority of the GS-5 identical precipitin patterns with the same degree of cells were firmly attached to the surface of the mobility toward the cathode. culture vessel, whereas most of the GS-511 cells remained suspended in the culture medium. Hydroxylapatite chromatography. Repre- resented a 17-fold reduction in volume and a sentative results of the purification of extracel- recovery of about 25.6% of the activity. lular glucosyltransferase activity are shown in Periodate oxidation of polysaccharides. Table 1. Hydroxylapatite chromatography of The polysaccharides used for linkage determithe GS-5 culture fluid gave one pool of activity nation were synthesized by the enzymes from that eluted with 0.2 M phosphate buffer at pH the 0.2 and 0.4 M pools obtained from hydroxyl6.8 and another pool of activity that came off apatite. The average percentages of (1-3)-like with a 0.4 M phosphate buffer at the same pH. links present in these glucans are shown in The 0.2 M and the 0.4 M pools contained 960 Table 2. The enzymes from the 0.2 M pool of and 1,840 DSU, respectively. Together, the GS-5 produced polysaccharide which, in each of pools represented an 11-fold reduction in vol- the six different samples, contained no detectume and a recovery of about 28% of the activity. able periodate-resistant glucose units, whereas Purification of the GS-511 enzymes gave a 0.2 that synthesized by the 0.4 M pool possessed M pool with 2,520 DSU and a 0.4 M pool con- 30.70 ± 4.03% of these units, which represents taining 550 DSU. These two enzyme pools rep- 68.22 ± 8.95 ,umol of glucose intact after treat-
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JOHNSON ET AL.
ment with periodate. The glucose polymers
synthesized by the 0.2 M pool of GS-511 glucosyltransferases contained no a-(1-3)-linked glucose units. Only 3.48 + 1.40% of the polysaccharide produced by the enzymes from the 0.4 M fraction of GS-511 was (1-3)-like linked, determined from 7.73 3.11 ,umol ofunoxidized glucose. Evaluation of dental caries-producing ability. The two bacterial strains were tested for cariogenic potential in conventional rats and the average dental caries scores are given in Tables 3 and 4. In the first experiment, GS-5 produced an average dental caries score of 60.2 + 15.6, whereas the average dental caries score for GS-511 was 32.6 9.24. Comparison of these two means by the t test (32) showed them to be significantly different at the 0.005 level. In the second test, the respective scores for GS-5 and
GS-511 were 32 + 12.4 and 14.1 + 9.11, and comparison of these two means gave a t value of 3.38, which is statistically significant. Thus, mutant GS-511 demonstrated significantly less cariogenic potential than the parent strain GS5.
±
DISCUSSION The reaction ofS. mutans with sucrose yields extracellular polysaccharide, which enables these bacteria to establish adherent cariogenic TABLE 2. Average percentage ofperiodate-resistant glucose units or links in polysaccharides produced by enzyme fractions eluted from hydroxylapatite
±
Enzyme Avg % (1-
rgaOrga-
GS-5
GS-511 | ADHERENT E
0.2 0.4
None 30.70
6 6
4.03
0.2 0.4
None 3.48
5 5
1.40
devia-
linkages tson
TABLE 3. Statistical treatment of caries scores obtained in conventional rats: first experiment
7
No. of test Avg caries Standard score deviation animals
Organism
F .6-
U)
z
No. of
.8
0
LO)
Standard
3)-like
fraction (M)
.5-
60.2 5 GS-5 32.6 GS-511 5 a Critical 99.5t8 is 3.335.
0
15.60 9.24
t value
3 40a
0.03
TABLE 4. Statistical treatment of caries scores obtained in conventional rats: second experiment Orga- No. of test Avg caries Standard t value GS-5
GS-511
FIG. 2. Relative absorbencies at 540 nm of cell suspensions of adherent and nonadherent cell populations of GS-5 and GS-511 grown in sucrose broth.
nism
animals
score
GS-5 GS-511
7 10
32.7 14.1
a
deviation
12.4 9.11
3.38a
Critical 99.5t15 is 2.947.
TABLE 1. Purification of glucosyltransferases from S. mutans GS-5 and GS-511
a
b
Source
Volume
Enzyme
Total units
% Yield
GS-5 Culture fluid Adsorbed to HAa 0.2 M fraction (40_70)b 0.4 M fraction (85-108)
5,000 240 184
2.0
10,000 4,340 960 1,840
100 43.5 9.6 18.4
12,500 4,150 2,520 550
100 34.5 21.0 4.6
GS-511 Culture fluid Adsorbed to HA 0.2 M fraction (21-39) 0.4 M fraction (71-82) HA, Hydroxylapatite. Fraction numbers.
4.0 10.0
5,000
2.5
180 110
14.0 5.0
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plaques on smooth dental surfaces in the highly fluid and abrasive environment of the oral cavity (28). This adherence of S. mutans is one of the possible processes that leads to dental caries. The mechanism of adherence has been studied by several investigators (11, 14, 17, 27, 31). The first step appears to be the reversible attachment of the bacteria or the capsular polysaccharide to the glycoprotein pellicle that coats the teeth (31). Sucrose is then necessary for the synthesis of certain insoluble or "sticky" polysaccharides, which form the framework of the dental plaque, and for the irreversible attachment of the bacteria to the teeth (13, 17). The insolubility or "stickiness" of the polysaccharides was reported to be related to their content of a-(1-3)-linked glucose units (16). The nature of the S. mutans enzymes that synthesize these polysaccharides has been studied (4, 10, 18, 27), and the number of different enzyme forms found associated with a specific strain varied from two (4, 10, 27) to six (18). It has been suggested that the variety of linkages found in the extracellular glucans resulted either from enzymes of different specificity (18) or that a single enzyme carries out the synthesis of different linkages (7). The present study demonstrated that S. mutans GS-5 produced at least two glucosyltransferases, or two groups of these enzymes, which synthesized glucans of different types. A mutant, S. mutans GS-511, was obtained that was altered in the synthesis of at least one of these types of extracellular glucans, and that proved to be identical to the parent strain in all other physiological and serological characteristics studied. Therefore, morphological and biochemical comparisons of the extracellular glucans were made in an attempt to elucidate their role in the etiology of dental caries. Transmission electron micrographs of sections of GS-5 (22) revealed two distinct types of polysaccharides, similar to those described by Guggenheim and Schroeder (19): (i) globular material in close association with the cell surface was found only in young cultures and then only at the periphery of the microcolonies, and (ii) fibrillar substances that were present in the intercellular spaces of both the young and old cultures. The only extracellular polysaccharide observed in both 9- and 24-h cultures of GS-511 was the globular material (22). The disappearance of the globular material around older GS-5 cells may have been due to its conversion into fibrillar glucan by the action of at least one additional enzyme. That is, one enzyme, or set of enzymes, may synthesize the globular material, which is then used as a receptor by a
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second enzyme or set of enzymes to form the fibrillar polysaccharide. Recently, Kuramitsu and Ingersoll (25) provided evidence for such a synthetic sequence. Using antibodies to soluble and insoluble glucan-synthesizing enzymes, they demonstrated partial inhibition of cellular adherence with the former and almost complete inhibition with the latter types of antibodies. The possibility of this being due to mixing of activities was ruled out and it was suggested that synthesis of both a(1-3) and a-(1-6) glucans was necessary for adherence. A structure for S. mutans polysaccharide suggested by Long and Edwards (26), which consisted of an a-(1-6)-linked linear molecule with a-(1-3)-linked branches, would be compatible with this possibility. However, such a branched polysaccharide would not be expected to form fibrils. To form fibrils, several polysaccharide molecules must aggregate "side by side" and the side branches might interfere with this union. Glycol units that lack adjacent hydroxyl groups such as nonterminal units joined by (13) bonds or units involved in branching at both C2 and C4 are not affected by periodate oxidation (20). Thus, each mole of glucose liberated following hydrolysis of the oxidized and reduced polysaccharide is indicative of one of these two bonding possibilities. Since previous work has indicated the bonding in S. mutans polysaccharide to be either (1-6) or (1-3) bonding (16, 26) and the latter linkage was related to adherence (16), all periodate-resistant glucose units in this study were considered to represent units with (1-3)-like bonding. No distinction was made as to whether these linkages were involved in a branch point or a part of the backbone of the polymer. The percentages of periodate-resistant glucose units in polysaccharides synthesized by GS-5 and GS-511 glucosyltransferases, which eluted with 0.4 M phosphate buffer at pH 6.8, differed dramatically. This difference was considered to be a reflection of reality and not the result of differences in enzyme preparation, since it was repeated several times, and examination of polysaccharide isolated from bacterial culture yields similar results. The enzymes from GS-5 produced polysaccharides with about 30% periodate-resistant linkages, whereas those obtained from GS-511 contained only about 3% of these linkages. This indicates that the fibrillar polysaccharide observed in sections of GS-5 may have consisted of about 30% a-(13)-linked glucose units, and that it was synthesized by glucosyltransferases that eluted from hydroxylapatite with 0.4 M phosphate buffer.
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JOHNSON ET AL.
The enzymes used in this investigation were collected after acid production had ceased and the bacteria were in stationary growth phase. A study of the proportions of linkages formed in earlier cultures, which may provide information regarding the sequence of polysaccharide synthesis, is currently in progress. The ability of GS-5 to adhere to glass when grown in sucrose broth was 6.5 times greater than that of GS-511, as determined by relative densities of suspensions of adherent cell populations. It can therefore be assumed that the globular polysaccharide produced by GS-511 was not adequate for attachment of S. mutans to hard surfaces. Finally, GS-511 demonstrated a significantly lower dental caries-inducing potential than did GS-5, when tested in conventional rats. Since the only discovered difference between the parent and mutant strains was the type of extracellular glucan synthesized, it may be assumed that the production of some specific polysaccharide is a critical factor in determining cariogenic potential. Hence, cariogenic GS-5 produced fibrillar polysaccharide consisting of about 30% (1-3)-likelinked glucose units, which immobilized the cells, and a globular polysaccharide closely associated with the cell surface, which disappeared from around older cells. Mutant GS-511, which differed from parent strain GS-5 only in that it failed to synthesize the fibrillar glucan, was nonadhering and possessed a markedly lower cariogenic potential. This indicates that the fibrillar glucan is a significant factor in the cause of dental caries. ACKNOWLEDGMENTS We wish to express our appreciation to Tom Stephens for the serology and to Mike Okuji for assistance with preparing the enzymes. This investigation was supported by training grant DE00144 from the National Institute of Dental Research. M.C.J. and J.J.B. were predoctoral trainees on this same training grant. LITERATURE CITED 1. Bozzola, J. J., M. C. Johnson, and I. L. Shechmeister. 1973. In situ multiple sampling of attached bacteria for scanning and transmission electron microscopy. Stain Technol. 48:317-325. 2. Bratthall, D. 1970. Demonstration of five serologic groups of streptococcal strains resembling Streptococcus mutans. Odontol. Revy 21:143-152. 3. Carlsson, J., E. Newbrun, and B. Krasse. 1969. Purification and properties of dextransucrase from Streptococcus sanguis. Arch. Oral Biol. 14:469-478. 4. Chludzinski, M., G. R. Germaine, and C. F. Schachtele. 1974. Purification and properties of dextransucrase from Streptococcus mutans. J. Bacteriol. 118:17. 5. Crowle, A. J. 1973. Immunoelectrophoreses, p. 305-352. In Immunoelectrophoresis. Academic Press Inc., New York. 6. de Stoppelaar, J. D., K. G. Konig, A. J. Plassachaert,
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26. Long, L. W., and J. R. Edwards. 1972. Detailed structure of a dextran from a cariogenic bacterium. Carbohydr. Res. 24:216-217. 27. Mukasa, H., and H. D. Slade. 1974. Mechanism of adherence of Streptococcus mutans to smooth surfaces. III. Purification and properties of enzyme complex responsible for adherence. Infect. Immun. 10:11351145. 28. Newbrun, E. 1967. Sucrose, the arch criminal of dental caries. Odontol. Revy 18:373-386. 29. Newbrun, E. 1972. Extracellular polysaccharides synthesized by glucosyltransferases of oral streptococci: composition and susceptibility to hydrolysis. Caries Res. 6:132-147. 30. Olson, G. D., A. S. Bleiweiss, and P. A. Small. 1972. Adherence inhibition of Streptococcus mutans: an assay reflecting a possible role of antibody in dental
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caries prophylaxis. Infect. Immun. 5:419-427. 31. Rolla, G. 1971. Adsorption of dextran to saliva-treated hydroxyapatite. Arch. Oral Biol. 16:527-533. 32. Roscoe, J. T. 1966. Fundamental research statistics for the behavioral sciences. Holt, Rinehart and Winston,
New York. 33. Scott, T. A., and E. H. Melvin. 1953. Determination of dextran with anthrone. Anal. Chem. 25:1656-1716. 34. Slade, H. D. 1965. Extraction of cell wall polysaccharide antigen from streptococci. J. Bacteriol. 90:667-671. 35. Tanzer, J. M., M. L. Freedman, R. J. Fitzgerald, and R. H. Larson. 1974. Diminished virulence of glucan synthesis-defective mutants of Streptococcus mutans. Infect. Immun. 10:197-235. 36. Wood, J. M., and P. Critchley. 1966. The extracellular polysaccharide produced from sucrose by cariogenic streptococcus. Arch. Oral Biol. 11:1039-1042.
