Vol. 14, No. 1

INFECTION AND IMMUNITY, JUlY 1976, p. 203-211 Copyright © 1976 American Society for Microbiology

Printed in U.S.A.

Chemical and Immunological Properties of the Type f Polysaccharide Antigen of Streptococcus mutans SHIGEYUKI HAMADA,' KAMAL GILL, AND HUTTON D. SLADE* Department of Microbiology-Immunology, Northwestern Medical and Dental Schools, Chicago, Illinois 60611

Received for publication 4 February 1976

The type-specific cell wall polysaccharide antigen was extracted, purified, and characterized from type fStreptococcus mutans strains 0MZ175 and MT557. The antigen was extracted from lyophilized cells with 5% trichloroacetic acid at 85 C for 15 min or saline at 120 C for 30 min. The trichloroacetic acid antigen was chromatographically separated into three antigenic fractions on a diethylaminoethyl-Sephadex A-25 column. Antigen 1 (Ag1P), which was specific for type f antiserum, was further purified by passing through carboxymethyl-Sephadex C25 and Sephadex G-200 columns. It was a polysaccharide composed of 49% rhamnose and 47% glucose. No reaction was obtained with anti-polyglycerophosphate (PGP) serum. Antigen 2 was reactive with both type f and PGP antisera and contained significant amounts of protein and phosphorus. Antigen 3 was reactive only with PGP antiserum and had no type specificity. The polysaccharide antigen gave a single precipitin band against type-specific antiserum on immunodiffusion and immunoelectrophoresis. The presence of a--1,6-glucosidic linkages was indicated by a 90% inhibition of the precipitin reaction by isomaltose and a-methyl-D-glucopyranoside, adsorption to and release from a concanavalin A-Sepharose column, and reaction with an S. mutans (type e) glucan antiserum. This antiserum was used to show that the type f polysaccharide antigen did not contain free glucan. An analysis of the antigen released from the antigen-glucan antiserum complex showed the presence of rhamnose and glucose. This released antigen also reacted with an f antiserum, which did not react with commercial dextran. The results show that the type f polysaccharide antigen is the first of those S. mutans type-specific polysaccharides identified to be immunologically related to an S. mutans glucan. adherence of a, d, and e S. mutans cells to a glass surface (10, 26, 28). Inhibition of adherence by antibody to the S. mutans glucan and treatment of the cells with trypsin indicates that other surface components also participate in the adherence process (28). In view of these results, it was of interest to determine the chemical composition of the S. Lancefield group E antiserum (2, 9, 29). More mutans serotype f antigen, the relation of its recently, additional strains of S. mutans have structure to immunological specificity, and the relation of its properties to the other S. mutans been designated as serotypes f and g (29). The a and d antigens have been described as polysaccharide antigens. galactose-glucose polymers (13, 20, 25). In conMATERIALS ANI) METHODS trast, the b and c polymers are composed of rhamnose-galactose and rhamnose-glucose, reStreptococcal strains. Strains OMZ175 and SEll spectively (18, 27, 36). These polymers were of serotype f S. mutans (29) were supplied by B. shown by comparative immunoelectrophoresis Perch, Statens Seruminstitut, Copenhagen, Dento be the same as those present in the extracts mark. S. mutans strain MT557 (type f) and MT123 of S. mutans used to identify the a, b, c, and d (type e) were isolated from a Japanese child (9). The organisms were grown in Todd-Hewitt broth fortiserotypes (18, 20, 25, 27). Antibody against the a, d, and e polysaccha- fied with salts and glucose for 18 h at 37 C, harand lyophilized as previously described (25). ride antigens has been shown to inhibit the vested, Antisera. Strain SEll antiserum was provided by

Studies on the serotype-specific antigens a, b, c, and d of Streptococcus mutans have identified these substances as polysaccharides located in the cell wall of the microorganism (13, 18, 20, 25, 27, 36). The immunological differentiation of these groups (1, 2) agreed with studies on their genetic homology (5). A few strains designated type e were shown to cross-react with

Visiting Investigator from the Department of Microbiology, Osaka University Dental School, Osaka, Japan.

B. Perch. For preparation of the antiserum, cells were cultured in Todd-Hewitt broth (29). The antise203

204

HAMADA, GILL, AND SLADE

rum against strain OMZ175 was obtained from N. Masuda, Osaka University Dental School, Osaka, Japan. Cells grown in Trypticase soy broth (BBL) were used as vaccines. Antisera were prepared in rabbits by intravenous injections of the antigens described above (9). Antiserum specific for polyglycerophosphate (PGP; glycerol teichoic acid) of group A Streptococcus pyogenes was kindly provided by T. Myoda of the DuPont Institute. One lot of antiserum obtained from a rabbit immunized with S. mutans strain Ingbritt (serotype c) whole cells was found to have anti-PGP activity without showing type c specificity and was used as an anti-PGP serum. Antiserum specific for glucans was obtained by immunizing a rabbit with cells of S. mutans strain MT123 (type e) grown in Todd-Hewitt broth containing 1% sucrose. The antiserum, however, contained no type e-specific antibody and was used as an antiglucan serum. Extraction of antigens from whole cells of strains OMZ175 and MT557. Lyophilized cells of strains OMZ175 were extracted by the following methods: (i) autoclaving the cells in saline (20 mg/ ml) at 120 C for 15 min (30); (ii) holding the cells in water (20 mg/ml) at 100 C for 10 min, 20 min, or 1 h; (iii) holding the cells in 0.01 N HCl at 100 C for 10 or 20 min; or (iv) treating the cells in 5% trichloroacetic acid at 4 C overnight (24). The suspensions were centrifuged, and the supernatants were tested for the presence of antigenic substances by the capillary precipitin test. Lyophilized cells of strain MT557 were extracted by autoclaving the cells in saline (20 mg/ml) at 120 C for 30 min. The centrifuged supernatant, which contained crude antigen, was lyophilized after extensive dialysis against water. Purification of type f antigen. A 20-g portion of lyophilized whole cells of strain OMZ175 was extracted with 400 ml of 5% trichloroacetic acid at 85 C for 15 min with stirring. The suspension was centrifuged, and the precipitated cells were extracted again under the same conditions. The supernatants were combined and extensively dialyzed against distilled water (ca. 50 liters). After removing the insoluble materials by centrifugation, the antigen extract was evaporated to 800 ml and added to 3.2 liters of acetone. The precipitate that formed after overnight incubation at -20 C was dissolved in distilled water (800 ml), and the pH was adjusted to 7.2 with 3 N NaOH. The water-insoluble material was again removed by centrifugation, the supernatant was precipitated with 4 volumes of cold acetone, and the precipitate was dried from ether. The crude antigen was purified by using column chromatography with diethylaminoethyl-Sephadex A-25, carboxySephadex C-25, Sephadex G-75, and Sephadex G-200 as previously described (18). Lyophilized cells of strain MT557 were extracted by autoclaving in saline (20 mg/ml) at 120 C for 30 min. The centrifuged supernatant, which contained crude antigen, was lyophilized after extensive dialysis against water. The type f antigen of strain MT557 was purified by passing through a diethylaminoethyl-Sephadex

INFECT. IMMUN.A-25 column under the game conditions as for the OMZ175 antigen (18). The immunologically active material appeared in the void volume of the tris (hydroxymethyl) aminoethane (Tris)hydrochloride (pH 7.4) buffer. No further purification was carried out. Extracts (30) of OMZ175 cell walls were found by precipitin test to contain the f antigen. Serological procedures. Capillary precipitin tests were performed as previously reported (35). Immunodiffusion was carried out in 0.7% Ionagar (Oxford) in saline, and immunoelectrophoresis was carried out in barbital-HCl buffer (pH 8.2; ionic strength, 0.05). Whole cell agglutination was carried out as previously described (10). Chemical analyses. Total hexose, rhamnose, protein, phosphorus, and sugars were measured as described previously (20). Sugars were also determined quantitatively by gas-liquid chromatography as previously described (24). Characterization of antigen. Quantitative precipitin reactions and their inhibition by haptens were carried out as described previously (31). Purified antigen 1P was treated with dextranase (Sigma), which was active against a-1,6-glucosidic linkages, and filtered on a Sephadex G-75 column (1.5 by 55 cm) after heat inactivation of the enzyme (100 C for 2 min). The column was eluted with water, and the two sugar peaks obtained were analyzed by gas-liquid chromatography (24). The purified antigen was also charged on a concanavalin A-Sepharose (Pharmacia) column (0.9 by 19.5 cm) and eluted with 0.01 M Tris-hydrochloride containing 1 M NaCl and 10-3 M MnCl2 (pH 7.2), followed by a stepwise elution with 0.05 M glucose in the same buffer (17). Serological purification and specificity of antigen. Samples of antigen 1P (0.5 mg) were combined with anti-OMZ175 and antiglucan sera (0.2 ml). The volume was made up to 1 ml with saline. The reaction mixtures were incubated at 37 C for 1 h and 5 C overnight. The precipitated antigen-antibody complexes were centrifuged in a Beckman minifuge and washed three times with saline. The precipitates were dissolved in 0.4 ml of 0.1 N HCI and incubated at 25 C for 10 min. Equal volumes of 5% trichloroacetic acid were added, and incubation was continued at 25 C for 10 min and at 5 C for 10 min. The precipitated antibody proteins were removed by centrifugation. The supernatants were extracted three times with equal volumes of ether, neutralized, and lyophilized (19, 22). The dry samples were dissolved in 0.05 ml of distilled water and tested for precipitin reaction against anti-OMZ175, antiglucan, and anti-SEll sera and analyzed for sugars by gas-liquid chromatography.

RESULTS The specificity of type f antiserum was confirmed by whole cell agglutination and/or capillary precipitin tests. Table 1 shows the results of whole cell agglutination using various serotypes of S. mutans and 0MZ175 whole cell antiserum. Unadsorbed 0MZ175 serum signifi-

VOL. 14, 1976

POLYSACCHARIDE TYPE f ANTIGENS OF S. MUTANS

TABLE 1. Whole cell agglutination of S. mutans by type f antiserum" Strain (serotype)

Ant-OM175Anti-OMZ175 serum srm adsorbed with MT703 cells

HS6 (a)