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-Glucose 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 polysaccharides. In this study, an additional wall polysaccharide 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 carbohydrate 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 galactose as their major Annu. Meet. Am. Soc. Microbiol. 1980, E111, p. wall carbohydrates (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 sugars 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 sugar 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 antibody 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.