An antigen possessing the attributes of an adhesin has been identified in Streptococcus sanguis G9B. Cell surface components were extracted from G9B and a spontaneously occurring nonadherent mutant of G9B, strain Adh-, with a 2 mM barbital buffer, pH 8.6. The extract of G9B but not of Adh-absorbed more than 80% of the adhesion-inhibitory activity of anti-G9B immunoglobulin G (IgG). Immunoblots revealed 80-and 52-kilodalton (kDa) antigens present in the G9B extract but not in the Adhextract. Absorption of anti-G9B IgG with Adh-and G9B barbital extracts showed a correlation between the loss of the 80-and 52-kDa antibodies and the loss of adhesion-inhibitory activity. An antibody prepared against the 80-kDa antigen excised from sodium dodecyl sulfate-polyacrylamide gels recognized the 80-and 52-kDa antigens and another antigen of 62 kDa but did not inhibit adhesion. However, an antibody eluted from an electroblot containing the native protein from which the 80-kDa and related antigens were derived (the 80-kDa antigen complex) inhibited adhesion to the same extent as anti-G9B IgG. Periodate oxidation of the G9B barbital extract modified the 80-kDa antigen complex and resulted in the loss of 40% of its absorbing activity. The barbital extract also contained an endogenous enzyme responsible for producing the 62-and 52-kDa antigens from the 80-kDa protein and which, under optimal conditions, degraded the antigen completely, resulting in the loss of antibody-absorbing activity. The 80-kDa antigen complex has a molecular mass of more than 200 kDa in native polyacrylamide gels and a pl of 4.1 to 4.8. These observations suggest that the adhesin antigen in S. sanguis G9B is a large glycoprotein from which an 80-kDa antigen complex is derived.

Saliva-binding molecules of Streptococcus sanguis and their receptors were investigated. Streptococcal cell surfaces were extracted with a barbital buffer and examined immunochemically. Strains G9B and Blackburn, which adhere specifically to saliva-coated hydroxyapatite via immunologically related adhesins, possess 80-, 62-, and 52-kilodalton (kDa), and 52-, 42-, and 29-kDa polypeptides, respectively, which correlate with adhesion to saliva-coated hydroxyapatite. Nonadherent strains Adhand M-5 lack these antigens. In an immunoblot overlay, the putative adhesins bound to a 73-kDa receptor present in submandibular saliva but not in parotid saliva. G9B also contains a 160-kDa surface protein which bound to an unidentified receptor in both submandibular and parotid saliva samples. Blackburn barbital-extracted components bound to 78and 70-kDa receptors in parotid saliva. These bacterial-salivary interactions may be important in the regulation of oral ecology. * Corresponding author. MATERIALS AND METHODSBacteria and culture conditions. S. sanguis G9B and M-5 are reference strains originally isolated from dental plaque. Strain Blackburn (NCTC 10231) was obtained from R. M. Cole, National Institutes of Health, Bethesda, Md. Strain Adh-is a poorly adhering (see below) spontaneous mutant of G9B isolated from a chemostat culture (manuscript in preparation). After initial isolation, the strains were maintained at -70°C and subcultured only infrequently. The strains were characterized physiologically by the criteria of Carlsson (7) and Facklam (16) and fit the Facklam biotype I category. On the basis of the data of Rosan (32), strains G9B and Adhare serotype 1, whereas M-5 and Blackburn are serotype 2. All of these strains have approximately the same saliva-mediated aggregating activity (35).Cells were grown overnight at 37°C in Trypticase peptone broth (BBL Microbiology Systems, Div. Becton Dickinson and Co., Cockeysville, Md.) supplemented with 5 g of yeast extract (BBL) and 0.5% glucose as a carbon source.Saliva. Stimulated parotid saliva and submandibular saliva samples were used. Parotid saliva was collected in Curby cups from 15 individuals and pooled. Submandibular saliva was collected from one individual, previously found to have high adherence-promoting activity, by a plastic mouthpiece collector custom fit for the subject (5). The salivas were clarified by centrifugation (44,000 x g, 15 min) and either aliquoted and stored at -20°C or freeze-dried and stored at +40C.Adhesion assay. Bacterial adhesion to saliva-coated hydroxyapatite (SHA), an in vitro model of the tooth surface, and inhibition of adhesion by antibody were measured by the methods of Rosan et al. (34).Barbital extraction of bacteria. Cells were harvested and washed three times in H20 by centrifugation at 10,000 x g for 10 min at 40C. The pellet was suspended at 5 mg/ml in cold barbital buffer (2 mM sodium barbital, pH 8.6) and vortexed for 30 min at 40C. The cells were harvested by centrifugation (10,000 x g, 10 min, 4°C), and the supernatant was coll...

A genomic library of Streptococcus sanguis, strain G9B, was constructed and expressed in Escherichia coli using a lambda gt11 expression vector. The amplified library was probed with polyclonal anti-G9B IgG and 13 antigen-positive clones were isolated. A lysate of one clone, designated PP39, absorbed the adhesion-inhibitory activity of anti-G9B IgG. This clone contained an insert of approximately 2000 bp and expressed unique 200 and 53 kDa proteins that reacted with monospecific anti-adhesin antibody. The 200 kDa protein also reacted with anti-beta-galactosidase IgG, indicating that it is a fusion protein of which 84 kDa represents the streptococcal adhesin. The 84 and 53 kDa proteins are similar in size to the major polypeptides in a streptococcal antigen complex which is associated with the adhesion of G9B to saliva-coated hydroxyapatite. The 53 kDa fragment may result from post-translational cleavage of the recombinant polypeptide.

A spontaneous non‐adherent mutant (Adh−) of Streptococcus sanguis, strain G9B has been isolated from a chemostat culture employing a chemically defined medium (FMC) as a nutritional source. The Adh− mutant showed five‐fold less adhesion to saliva‐coated hydroxyapatite (SHA) compared with parent G9B. No difference in adhesion to uncoated hydroxyapatite (HA) was found between G9B and Adh−. The mutant had the same colonial, cellular morphology, and biochemical properties as the parent. A comparison of the antigens found in mutanolysin (M‐1, N‐acetylmuramidase) extracts of G9B and Adh− using crossed rocket immunoelectrophoresis (CIE), indicated that a surface antigen, designated A, reacted strongly in G9B but only weakly in Adh−. The adhesion of G9B but not Adh− to SHA was directly correlated with dilution rates in a chemostat. Adhesion to HA was not affected by the dilution rate. There was also a direct correlation between the concentration of the A antigen with adhesion of G9B to SHA. The concentration of A in Adh− was low at all dilution rates. Antibody to G9B specifically inhibited adhesion of G9B to SHA but antibody to Adh− had no effect on adhesion of either strain to SHA. Neither antibody affected adhesion to HA. A partially purified A antigen absorbed adhesion inhibitory activity of G9B IgG. Trypsin did not digest the A antigen but resulted in the extraction of the antigen from the surface causing the loss of the cells ability to adhere to SHA. Galactose oxidase treated G9B cells also showed a reduced adhesion to SHA but glucose oxidase had no effect. Periodate oxidation of G9B resulted in a loss of adhesion to SHA. The A antigen was found in purified cell walls and was labelled with 125I, indicating a surface location. The A antigen also reacted with affinity purified antibody against the G9B adhesin antigen. Immunoblots of the A antigen following SDS‐PAGE indicated it contained 80, 62, and 52 kDa polypeptides. These results suggest that the A antigen is a glycoprotein which is the native form of the adhesin responsible for the attachment of G9B to salivary pellicle.

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