Characterization of an adhesion antigen of Streptococcus sanguis G9B

Abstract

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.

Isolation and characterization of a non‐adherent mutant of Streptococcus sanguis G9B

“…All of the studies reported here used the same pools of antisera and the IgG derived from these pools. Mouse anti-80-kDa serum was prepared using essentially the same protocol described previously for rabbit anti-80-kDa serum (24,25), In brief barbital extracts of G9B were subjected to SDS-PAGE (see below) and the 80-kDa band was eut out of the gel, macerated in 0.15 NaCI and mixed with an equal volume (ca. 0.2 ml) of Freund's complete adjuvant.…”

“…The second dimension used 6,7 ml agar'ose and was run at 2.4 V/cm for 18 h on a glass plate cooled to 4 C. The gels wer'e washed in saline, dried, stained with Coornassie Brilliant Blue (CBB) and destained prior to photography (3), Quantitation of the antigen was performed by estimating the height of the rockets (22). In some studies an intermediate gel containing an affinity purified antibody against the 80 kDa antigen complex (25) was inserted between the first and second dimension.…”

“…3A. The antigens (and antibodies) wer"e extracted with 0.05 M Tris-HCl, pH8.6, containing 2% SDS, lO'/o glycerol, 5% 2-rnercaptoethanol and 0.002% brornophenol blue at 100°C for 3 min and SDS-PAGE gels run as described previously (24,25). The pr-oteins were tr-ansferr^ed to nitrocellulose (0.45 /«nm pore size, Schleicher & Sehuell, Keane, NH) by electrophoretie blotting (47) in 20 mM Tris buffer, containing 1.125% glycine and 20"/o rnethanol; transfer was at 115 tnA for 18 h at 4 C, The nitr'ocellulose was washed with phosphate-buffered saline (PBS) containing 0.1% Tween-20.…”

“…5C) shows that some antigens have been extracted by trypsjn treatment but the antigenic activity of the A antigen does not appear to have been affected. Subsequent studies of a trypsin treated extract containing the A antigen showed that it still absorbed anti-adhesion aetivity (25). Several other antigens also do not appear to be trypsin sensitive.…”

“…Affinity purified antibody to the 80 kDa adhesin complex whieh inhibited adhesion to SHA (25) was plaeed in an intermediate gel in CIE absorbed the A antigen from the M-1 extract (Fig. 6B) compared to control serum (Fig.…”