INFECTION AND IMMUNITY, Oct. 1980, p. 140-146 Vol. 30, No. 1 0019-9567/80/10-0140/07$02.00/0

Purification and Immunological Characterization of a Rhamnose- Antigen from Streptococcus mutans 6715-T2 (Serotype g) AKRAPORN PRAKOBPHOL AND ROSEMARY LINZER* Department of Oral Biology, State University ofNew York at Buffalo, Buffalo, New York 14226

The serotype antigens of Streptococcus mutans have been described as cell wall-associated . In this study, an additional wall antigen was purified and characterized from S. mutans strain 6715-T2, a mutant of 6715 (serotype g). Strain 6715-T2 lost the serotype antigen during animal passage. Rhamnose-containing fractions were solubilized from bac- terial cells by extraction with 5% trichloroacetic acid at 40C for 18 h and with 0.01 N HOl at 100C for 20 min. Extracts were combined and purified on columns of diethylaminoethyl-Sephadex A-25 and Sephadex G-100. The purified sample contained 59% rhamnose, 31% glucose, 2.2% protein, and 0.24% phosphorus. The purified rhamnose-glucose polysaccharide (RGP/6715-T2) reacted strongly with antisera to whole cells of 6715-T2. Agar gel diffusion and comparative immuno- electrophoresis studies revealed that RGP/6715-T2 was serologically distinct from the serotype g and d polysaccharide antigens. These techniques also indicated immunological identity between RGP/6715-T2 and RGP/B13, a rhamnose-glu- cose polymer previously isolated from S. mutans B13, a serotype d strain. Antigen immunologically identical to RGP/6715-T2 was detected both in Rantz-Randall extracts from whole cells of S. mutans strains 6715, OMZ-65, and 6715-PT and in extracts from cells of 6715-T2 and C307, two mutant serotype g strains that lacked the serotype g antigen. In addition to peptidoglycan, strains of Strep- (This work has been presented in part previ- tococcus mutans serotypes a, d, and g possess ously [A. Prakobphol and R. Linzer, Abstr. rhamnose, glucose, and as their major Annu. Meet. Am. Soc. Microbiol. 1980, E111, p. wall (11). However, the wall-as- 69].) sociated serotype a, d, and g antigens are galac- tose-glucose polymers and do not contain rham- MATERIALS AND MERHODS nose (17). A rhamnose-glucose polysaccharide Bacterial strains and growth conditions. S. mu- (RGP) from S. mutans strain B13 (RGP/B13) tans strains 6715, 6715-T2 (mutant), OMZ-65, and B13 serotype d was recently purified and character- have been maintained in this department for several ized (25). The RGP/B13 was found to be chem- years. Cultures of S. mutans strains 6715-PT and ically and immunologically distinct from the se- 6715(C307) were kindly provided by Suzanne M. Mich- rotype antigen. In view of the similarities in cell alek. Cells were grown in Todd-Hewitt broth (THB) wall patterns (11) and deoxyribo- or its dialysate (Difco Laboratories, Detroit, Mich.). carbohydrate Both media were supplemented with 1.8% glucose, nucleic acid base contents (4) among strains 0.8% NaHCO3, 0.3% NaCl, and 0.15% K2HPO4. Cul- belonging to serotypes d and g, the existence of tures were grown at 37°C for 18 h, harvested by a rhamnose polymer in the cell walls of serotype centrifugation, washed twice with distilled water, and g strains seemed probable. lyophilized. Mutants of serotype g strains that apparently Purification of RGP from S. mutans strain lack the serotype polysaccharide antigen have 6715-T2. Lyophilized cells (10 g) of S. mutans 6715-T2 been reported in the literature (12, 13). The were suspended in 5% trichloroacetic acid (200 ml) and present study has used both mutant and parent stirred at 4°C for 18 h. The cells were recovered by strains in an attempt to purify a rhamnose pol- centrifugation at 6,000 X g for 10 min and extracted in 0.01 N HCO (200 ml) at 100°C for 20 min. The trichlo- ymer from serotype g. The rhamnose polymer roacetic acid sample was extracted twice with an equal that was isolated was immunologically charac- volume of cold ether to remove the acid. Both the terized with respect to its relationship to the trichloroactic acid and the HCl extracts were neutral- RGP/B13 and the serotype g and d polysaccha- ized with 1 N NaOH, dialyzed against distilled water, ride antigens. and lyophilized. 140 VOL. 30, 1980 ANTIGENS OF S. MUTANS 141 The samples were purified separately on columns were assayed by the phenol-sulfuric acid method (6). of diethylaminoethyl (DEAE)-Sephadex A-25 (Phar- Rhamnose was measured by the methylpentose assay macia Fine Chemicals, Inc., Piscataway, N.J.). Column of Gibbons (9), substituting thioglycolic acid for cys- fractions were monitored for nucleotides and proteins teine-hydrochloride. Phosphorus was determined by at 260 nm and assayed for carbohydrates (6). Void- the method of Bartlett (1), and protein was measured volume fractions containing carbohydrates were by the method of Lowry et al. (21). Quantitation of pooled and lyophilized. These samples were combined specific was performed by using a Varian gas and further purified on a column of Sephadex G-100 chromatograph model 2740 as previously described (Pharmacia). Fractions composing the major carbo- (25). hydrate peak were pooled, dialyzed against distilled water, and lyophilized. RESULTS Preparation of additional antigens. Serotype d and g polysaccharide antigens were prepared from Preparation of a rhamnose polysaccha- cells ofS. mutans strain B13 and OMZ-65, respectively ride from S. mutans 6715-T2. Cells of S. mu- (18). RGP/B13 was purified from cell walls of S. tans strain 6715-T2 were extracted with 5% tri- mutans strain B13 as previously described (25). Crude chloroacetic acid at 4°C for 18 h. The neutralized extracts from whole cells of strains 6715, 6715-T2, B13, and dialyzed extract contained 70 mg of carbo- OMZ-65, 6715-PT, and 6715(C307) were prepared by hydrate with 14% rhamnose. Galactose, a autoclaving the cells in saline (20 mg/ml) at 120'C for found in serotype g polysaccharide antigen, was 30 min. not detected in the extract. The cells were fur- Preparation of antisera. Antisera to cells of S. mutans strains 6715-T2, OMZ-65, and B13 were pre- ther extracted with 0.01 N HCO at 100°C for 20 pared by using New Zealand white rabbits. Cells were min. This procedure yielded an additional 240 suspended in phosphate-buffered saline containing mg of carbohydrate. 0.5% Formalin for 16 h at room temperature (RT). The dialyzed and concentrated acid extracts After washing, the Formalin-killed cells were sus- were fractionated on columns of DEAE-Sepha- pended in phosphate-buffered saline containing 0.01% dex A-25 (Fig. 1). The major carbohydrate peak Formalin at a final cell concentration of 10 mg/ml. was recovered at the void volume for both sam- Rabbits received a total of nine intravenous injections ples. After dialysis, the two void-volume samples during a 3-week period. Doses increased from 0.2 to were combined and further purified on a column 2.0 ml. Test bleedings were begun at 4 weeks, and titers were assayed by using the capillary ofSephadex G-100. The fractions comprising the precipitin reaction with the respective autoclaved cell major carbohydrate peak were pooled as indi- extract. When high titers of antisera were obtained, the animals were exsanguinated by cardiac puncture. Antiserum to extracted cell walls of S. mutans B13 was prepared by a protocol similar to that previously described (25). Serological assays. Agar gel diffusion and com- parative immunoelectrophoresis were performed in gels containing 1.2% agarose (SeaKem agarose, Marine Colloids, Inc., Rockland, Maine) in 0.05 M sodium barbital-acetate buffer, pH 8.6. Immunoelectrophore- sis was performed at a constant voltage of 150 V for 45 min For the quantitative precipitin assay, 6-pl portions of antiserum were incubated with increasing concen- trations of antigen in saline containing 0.001% Mer- thiolate (final volume, 100 ,l) for 1 h at RT and EfMi (& overnight at 40C. Precipitates were collected by cen- FIG. 1. Chromatography of trichloroacetic acid trifugation in a Beckman Microfuge B (Beckman In- extract from S. mutans 6715-T2 cells on a column of struments, Inc., Fullerton, Calif.) and washed twice DEAE-Sephadex A-25. A sample obtained by extrac- with saline (1 ml each). The washed precipitates were tion of 6715-T2 cells with cold trichloroacetic acid dissolved in 1 ml of 2% Na2CO3 in 0.1 NaOH and was purified on a column of DEAE-Sephadex A-25 quantitated by the method of Lowry et aL (21). In (1.5 by 30 cm). The column was rinsed with 0.01 M inhibition assays, antiserum and inhibitor were incu- tris(hydroxymethyl)aminomethane (Tris)-hydrochlo- bated for 15 min at RT before the addition of antigen. ride, pH 8.2 (equilibration buffer). The arrow indi- In cross-precipitin assays, the initial supernatant after cates beginning of the development of the column centrifugation of the overnight reaction was reacted with a linear gradient between 0.05 and 1.0 M NaCl with the second antigen for 1 h at RT and overnight in the equilibration buffer. Samples (10 p1) of each at 40C. Antigen-antibody precipitates were collected fraction were used for assaying total sugars (optical and processed as described. All precipitin assays were density at 490 nm [OD490]; 0). Ultraviolet absorption done in triplicate. was measured at 260 nm (ODsw; A). Fractions at the Quantitative chemical analysis. Total sugars void volume were pooled as indicated. 142 PRAKOBPHOL AND LINZER INFECT. IMMUN. claved extracts from cells of strains 6715 and OMZ-65, but absent in extracts from cells of strain 6715-T2 (Fig. 3b). The precipitation pat-

I.-~ terns (Fig. 3c) indicated that the RGP/6715-T2 gLS and the serotype g antigen were serologically distinct. o0 A second mutant of serotype g, S. mutans 6715(C307), was reported to lack detectable se- 0.2 rotype g antigen as determined by agar diffusion and absorption assays (12). Gas-liquid chroma- 50 100 50 200 250 tography studies in this laboratory failed to de- Efflun (ml) tect galactose in whole-cell hydrolysates from FIG. 2. Chromatography of combined samples either strain 6715-T2 or 6715(C307), thus con- from DEAE-Sephadex A-25 columns on a column of firming the absence of the serotype polysaccha- Sephadex G-100. Void-volume samples obtained from ride. In agar gel diffusion, autoclaved extracts of purification of trichloroacetic acid and HCl extracts strains 6715-PT and 6715(C307) contained an on DEAE-Sephadex columns were combined and fil- tered on a column of Sephadex G-100. The column antigen that appeared immunologically identical was eluted with 0.01 M Tris-hydrochloride buffer, pH to the purified RGP/6715-T2 (Fig. 4). The parent 8.2. Fraction were assayed for total sugars (OD45o; strain 6715-PT also contained a component that 0) and ultraviolet absorption at 260 nm (A). Carbo- was serologically identical to the serotype g an- hydrate fractions were pooled as indicated. tigen, whereas this antigen was lacking in the extract from mutant strain 6715(C307). TABLE 1. Chemical composition of the purified S. mutans strain polysaccharide from 6715-T2 A.n' 7...$ Component Composition (mg/100 mg) * ,~. 4, G -' G ^ ^ .: . Rhamnose 59 Glucose ...... 31 Phosphorus 0.24 * * 4, ''' Protein ...... 2.2 * * ',S' cated in Fig. 2, and the chemical composition of the sample is shown in Table 1. The purified polysaccharide contained 59% rhamnose, 31% glucose, and 2.2% protein. This rhamnose-glu- cose polymer will be referred to as RGP/6715- * 4 , . T2. Immunological distinction between RGP/6715-T2 and serotype g polysaccha- * : . 4 : ride. Antisera produced against whole cells of S. mutans 6715-T2 were examined for the presence of to the purified RGP/6715-T2. Whole-cell antiserum reacted strongly with the - 'i ..Y -. purified RGP/6715-T2 (Fig. 3a). This reaction * was not affected by preincubation ofthe purified antigen with protease. In agar diffusion against *... - autoclaved extracts from serotype g cells of

:': ( *0 strains 6715, 6715-T2, and OMZ-65, the anti- .. 6715-T2 serum detected one antigen which was * /. immunologically identical to the purified RGP/ 6715-T2 polysaccharide. The anti-6715-T2 serum did not react with purified serotype g antigen prepared from OMZ-65 cells. FIG. 3. Comparison ofRGP/6715-T2 and serotype Antiserum prepared to whole cells of S. mu- g antigen in agargel diffusion. Outer wells contained (i) purified RGP/6715-T2 and serotype g antigen and tans OMZ-65 reacted strongly with the serotype (ii) autoclaved extracts of strains 6715-T2, 6715, and g polysaccharide but did not cross-react with the OMZ-65, as indicated. The center wells contained purified RGP/6715-T2 (Fig. 3b). The serotype g antiserum: anti-6715-T2 (a); anti-OMZ-65 (b); and antigen was shown to be present in the auto- anti-6715-T2 plus anti-OMZ-65 (c). VOL. 30, 1980 ANTIGENS OF S. MUTANS 143 antibodies (antiserum to B13 cells) was placed in the upper trough and purified RGP/6715-T2 and serotype g antigen were placed in the lower trough. Antigen distinct from RGP/B13 or RGP/6715-T2 but identical to serotype g antigen was detected only in the OMZ-65 extract. In Fig. 6c, the continuous band below the OMZ-65 an- tigen arc is due to the reaction of the d antigen from the lower trough with specific anti-d anti- R G P/6 7i5 -T2 67!5-C3X07ext RP /67 5 -T2 bodies. This partial cross-reaction between se- rotype d and g antigens has been described in the literature (10, 24). Therefore, RGP/6715-T2 Anti -OMZ65 Anti-6715-T2 appears to be immunologically identical to RGP/B13, and these RGPs are immunologically * * distinct from the serotype g and d antigens.

g Ag 6715 -PT ext. 9 Ag Immunological identity between RGP/6715- T2 and RGP/B13 was also examined by a quan- titative cross-precipitin assay (Fig. 7). Increasing concentrations of purified RGP/B13 antigen FIG. 4. Examination of extracts from S. mutans were reacted with equal portions of anti-6715-T2 6715(C307) and 6715-PT in agar gel diffusion. Auto- 1 After claved cell extracts of strains 6715(C307) and 6715- serum at RT for h and 4VC overnight. PT as well as purified RGP/6715-T2 and serotype g removal of antigen-antibody precipitate by cen- antigen were examined, using anti-OMZ-65 serum on trifugation, the supernatant containing excess the left and anti-6715-T2 serum on the right. antibody was reacted with 2 jg of RGP/6715-T2. Figure 7 shows that 2 tig of RGP/B13 was suf- Immunological relationship of RGP/ ficient to completely eliminate the reaction be- 6715-T2 to RGP/B13 and serotype d anti- tween anti-6715-T2 serum and RGP/6715-T2. gens. Cells of serotype d strains of S. mutans Thus, the two antigens were reacting with the had previously been shown to contain an RGP same population of antibodies. (RGP/B13) in addition to the serotype polysac- Hapten inhibition assays. Determinants on charide (25). Because of the close genetic rela- the RGP/6715-T2 and RGP/B13 molecules were tionship between the serotype d and g strains examined by using hapten inhibition of the (4), the RGP/B13 and serotype d antigens pu- quantitative precipitin assay. In a preliminary rified from S. mutans strain B13 were compared study, (glucose-a-1,4-glucose) was found with the antigen purified from 6715-T2. RGP/ to be a highly effective inhibitor of the reaction 6715-T2 and RGP/B13 appeared serologically between anti-6715-T2 serum and the RGP anti- identical when reacted with antiserum prepared gens. The inhibitory effect was concentration to 6715-T2 cells or when reacted with antiserum dependent; 5 pmol of maltose yielded 97% inhi- prepared to extracted walls from B13 (Fig. 5). bition of the reaction with RGP/6715-T2 (Fig. 8) This antiserum had previously been shown to be and 96% inhibition of the reaction with RGP/ specific for RGP/B13 (25). B13 (Table 2). The identity between RGP/6715-T2 and To compare the inhibitory potential ofvarious RGP/B13 and the distinction between these two haptens, samples of anti-6715-T2 serum were polysaccharides and the serotype d or g antigens incubated with 5 pmol of the respective saccha- were also examined by comparative immuno- electrophoresis. In Figure 6a, purified RGP/B13 I.. - An" - B13 / exl. walls and extracts from OMZ-65 and 6715-T2 cells * were placed in the wells and electrophoresed. * c_ After electrophoresis, anti-6715-T2 serum was placed in the upper trough and purified RGP/ 0 was lower The * * S 6715-T2 placed in the trough. Ar'671~-~ RC-P/Bi3 patterns of antigen-antibody precipitation showed the identity between RGP/6715-T2 and FIG. 5. Agar gel diffusion of RGP/6715-T2 versus RGP/B13 and the presence of an antigen iden- RGP/B13. Purified RGP/B13 and purified RGP/ tical to the purified RGPs in the OMZ-65 and 6715-T2 were reacted with anti-B13/extracted (ext.) 6715-T2 cell extracts. In Fig. 6b, a combination wall serum and anti-6715-T2 serum. Precipitation of anti-6715-T2 serum and an antiserum contain- patterns indicated complete immunological identity ing specific anti-d and cross-reacting anti-d-g between the antigens. 144 PRAKOBPHOL AND LINZER INFECT. IMMUN. +4- polymers (14, 18) and have been shown to be immunologically partially cross-reactive (10, 24). A second wall polysaccharide was recently pu- rified from serotype d strains and shown to be chemically and serologically distinct from the serotype d antigen (25). In the present report, a rhamnose-glucose polymer (RGP/6715-T2) was purified from S. mutans strains 6715-T2. Strain E:.;,.e,- 6715-T2 is a mutant of strain 6715 and lacks the serotype g antigen. The RGP/6715-T2 appeared both chemically and immunologically identical to the RGP/B13 (Fig. 5). Immunological identity was confirmed by comparative immunoelectro- phoresis (Fig. 6) and a quantitative precipitin f b An -- -r X X assay (Fig. 7). The newly isolated RGP/6715-T2 did not cross-react immunologically with the serotype g or d antigens (Fig. 3 and 6).

RGP/ 6715-T2 ( 2,g) mom ~0.4 H CS_ R' g 03 \; > )

id 0.2 - p !1.11 IIIHill MI.I., 0.I FIG. 6. Comparative immunoelectrophore8sW of RGP/6715-T2, RGP/B13, and serotype antige?ns. Pu-. 20 &O rifled RGP/B13 and autoclaved extracts from OMZ-10 O0 65 and 6715-T2 cells were placed in the welUls and RGP/ 8 13 (49 electrophoresed. Anti-6715-T2 serum (a), anti-65715-T2 FIG. 7. Quantitative cross-precipitin reaction with serum combined with antiserum containing anti-d RGP/B13 and RGP/6715-T2. Increasing concentra- plus anti-d-g antibodies (b), and antiserum coWntain- tions of RGP/B13 were reacted with anti-6715-T2 ing anti-dplus anti-d-g antibodies (c) wereplaiced in serum (6 ul) for 1 h at RT and 40C overnight (0). the upper troughs. Purified RGP/6715-T2 (a), RGPI After centrifugation to remove antigen-antibody com- 6715-T2 combined with serotype g antigen (b), and Plexes, the supernatants were reacted with 2 pg of serotype d antigen (c) were placed in lower tyroughs RGP/6715-T2 (0). for comparison with the electrophoresed antigo,ens. ride for 15 min before the addition of the3 RGP antigen. The inhibitory effects of the v,arious haptens are summarized in Table 2. In ad[dition to maltose, (glucose-a-1,6-gliucose) was a highly effective inhibitor, 93 and 8i8% for RGP/6715-T2 and RGP/B13 respectivelyr. Sac- zI0- charides with ,f-1-linked glucose moleculess were w U significantly less inhibitory. No significantt inhi- w bition was found when rhamnose was ussed as 9. the inhibitor. This suggested that the deltermi- nants on the RGP antigens reacting with anti- 2 4 6 8 10 6715-T2 serum were mainly terminal a-i-lgluco- MALTOSE (JLMOLES) syl residues. FIG. 8. Inhibitory effect of maltose in the quanti- tativeprecipitin assay. Anti-6715-T2 serum (6 Id) was DISCUSSION incubated with increasing concentrations ofmaltose The at RT for 15 min before the additon of2 pg ofRGPI serotype d and g antigens of S. mlutans 6715-T2. Antigen-antibody complexes were quanti- have been identified as galactose-glucosfa wall tated as described in Materials and Methods. VOL. 30, 1980 ANTIGENS OF S. MUTANS 145 TABLE 2. Inhibition of the precipitin reaction necessary for insoluble synthesis and cell between anti-6715-T2 serum and RGP/6715-T2 or accumulation (19, 23). However, the role of the RGPB13a serotype polysaccharides in the cariogenicity of % Inhibition' S. mutans is unclear. Hirasawa et al. (12) have Inhibitor RGP/ RGP/ described a mutant of S. mutans 6715, desig- 6715-T2 B13 nated C307, that apparently did not produce the serotype g antigen but did retain a high degree a-l-Methylglucopyranoside ...... 40 38 of cariogenicity [M. Hirasawa, T. A. Brown, H. ,8-1-Methylglucopyranoside ...... 23 20 Kiyono, T. Shiota, R. R. Arnold, J. L. Babb, and Maltose (glucose-a-1,4-glucose) ... 97 96 Isomaltose (glucose-a-1,6-glucose) 93 88 J. R. McGhee, J. Dent. Res. 58(Special Issue (glucose-,8-1,4-glucose) 37 40 A):378, 1979]. Although lacking the serotype g Gentiobiose (glucose-,8-1,6-glucose) 45 32 antigen, mutant C307 was shown to possess an L-Rhamnose ...... 16 6 antigen that was immunologically identical to a Anti-6715-T2 serum (6 A1) was incubated with 5 the RGP/6715-T2 antigen (Fig. 4). As a surface lumol of the inhibitor for 15 min at RT before the component, the RGP may have an effect on the addition of purified RGP/6715-T2 or RGP/B13 (2 ug). binding of glucosyltransferase and the caries ac- b Mean of triplicate assays. tivity demonstrated by the C307 mutant. There- fore, the RGP antigens may be of interest for Rhamnose-containing polysaccharides are future protection studies. common to the walls of many streptococci. The serotype antigens of S. mutans serotypes c, e, ACKNOWLEDGMENTS and f are rhamnose-glucose polymers; the sero- This work was supported by Public Health Service grants type antigen of S. mutans serotype b is a rham- DE 05017 and DE 07034 from the National Institute of Dental Research. nose-galactose polymer (reviewed in reference We thank Lynette M. Guindon and Thomasine C. Bodkin 17). Among the ,8-hemolytic streptococci, group for their excellent technical assistance and Hector A. Velasco antigens A, B, C, E, F, G, L, and R and type for his photographic work. antigens ofgroups D and F all contain rhamnose (2, 3, 5, 15, 22, 26-29). Although rhamnose is a LITERATURE CITED major constituent, the antigenic specificity usu- 1. Bartlett, G. IL 1959. Phosphorus assay in column chro- ally resides in the side chains of matography. J. Biol. Chem. 234:466-468. In 2. Bleiweis, A. S., and R. M. Krause. 1965. The cell walls rhamnose. the present report, the antigenic ofgroup D streptococci. I. The immunochemistry of the determinants of RGP/6715-T2 and RGP/B13 type I carbohydrate. J. Exp. Med. 122:237-249. appeared to be nonreducing a-i-linked glucose 3. Coligan, J.E., W. C. Schnute, Jr., and T. J. Kindt. units (Table 2 and Fig. 8). The anti-whole-cell 1975. Immunochemical and chemical studies on strep- tococcal group-specific carbohydrates. J. Immunol. 144: serum was highly specific for the a-linkage; mal- 1654-1658. tose and isomaltose inhibited the precipitin re- 4. Coykendall, A. L. 1977. Proposal to elevate the subspe- action with the RGP antigens between 88 and cies of Streptococcus mutans to species status, based on 97%. The 1)-linked cellobiose and gentibiose only their molecular composition. Int. J. Syst. Bacteriol. 27: inhibited between 32 and 45%. In contrast, in- 26-30. 5. Curtis, S. N., and R. M. Krause. 1964. Immunochemical hibition ofthe precipitin reaction between RGP/ studies on the specific carbohydrate of group G strep- B13 and an antiserum prepared to acid-ex- tococci. J. Exp. Med. 119:997-1004. tracted cell walls demonstrated two populations 6. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, not Both a- and and F. Smith. 1956. Colorimetric method for determi- of antibodies (data shown). fB- nation of sugars and related substances. Anal. Chem. linked nonreducing glucose units inhibited the 28:350-356. reaction about equally, and the inhibition was 7. Fitzgerald, R. J., H. V. Jordan, and H. R. Stanley. found to be somewhat additive. This suggested 1960. Experimental caries and gingival pathologic units were changes in the gnotobiotic rat. J. Dent. Res. 39:923- that 1)-linked glucose exposed during 935. the preparation and acid extraction of the walls. 8. Fitzgerald, R. J., and P. H. Keyes. 1960. Demonstration Neither the anti-whole-cell nor the cell wall of the etiologic role of streptococci in experimental serum was significantly inhibited by rhamnose, caries in the hamster. J. Am. Dent. Assoc. 61:9-19. that rhamnose the 9. Gibbons, M. N. 1955. The determination of methylpen- suggesting may comprise toses. Analyst 80:268-276. backbone of the RGP antigen. 10. Hamada, S., N. Masuda, and S. Kotani. 1978. Demon- S. mutans is usually found associated with stration of serotype d and g specificities of Streptococ- dental caries in humans (16, 20) and has been cus mutans by immunodiffusion. Arch. Oral Biol. 23: shown to induce caries in animals 495-499. gnotobiotic 11. Hardie, J. M., and G. H. Bowden. 1974. Cell wall and (7, 8). Antibodies to the wall-associated serotype serological studies on Streptococcus mutans. Caries antigens have been shown to block the binding Res. 8:301-316. to the cell of glucosyltransferase, an enzyme 12. Hirasawa, M., H. Kiyono, T. Shiota, S. M. 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