The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or IIH) has been cloned into Escherichia coli (

The ability to adhere to salivary agglutinin-coated hydroxyapatite beads and to aggregate in the presence of fluid-phase salivary agglutinin was tested by using 25 isolates of mutans streptococci representing eight serotypes. Both adherence and aggregation activity correlated with expression of the Mr-185,000 cell surface antigen P1 on Streptococcus mutans serotype c, e, and f strains. In addition, it was shown that the P1 molecule itself served as the adhesin of S. mutans serotype c, since adherence was significantly inhibited by the presence of recombinant-specified Mr-150,000 P1. The ability of S. sobrinus strains to adhere or aggregate did not correlate with expression of the P1 cross-reactive antigen SpaA. There was also evidence for interaction with salivary agglutinin, as manifested by aggregation but not adherence of S. rattus serotype b, which does not express a P1 cross-reactive antigen. To understand the interaction of Pl with salivary agglutinin at the molecular level, a panel of 11 anti-Pt monoclonal antibodies was tested for inhibitory activity in adherence and aggregation inhibition assays. Overlapping, but not identical, subsets of monoclonal antibodies were found to inhibit adherence and aggregation, indicating that the interactions of P1 with salivary agglutinin which mediate these two phenomena are different. The localization of functional domains of P1 which may mediate the aggregation and adherence reactions is discussed.

DNA encoding the alanine-rich region (A-region) of the cell surface adhesin, P1, from Streptococcus mutans was subcloned and expressed as a fusion protein with the maltose-binding protein (MBP) of Escherichia coli. The A-region fusion protein was shown to competitively inhibit both adherence of S. mutans to salivary agglutinin-coated hydroxyapatite and fluid-phase agglutinin-mediated aggregation of this organism. MBP alone or an MBP-paramyosin fusion protein was not inhibitory. Proteolytic cleavage of the fusion protein into its component moieties, MBP and A-region, resulted in breakdown of the A-region into three main fragments. Western immunoblot analysis of calcium-dependent agglutinin binding to this preparation revealed binding specificity for a 28-kDa fragment. Thus, the A-region of P1 is an important domain which interacts directly with salivary agglutinin, and this interaction interferes with both the aggregation and the adherence mechanisms in vitro.

Eleven monoclonal antibodies (MAbs) specific for P1, the major protein surface antigen of Streptococcus mutans serotype c, were characterized by Western blot (immunoblot) analysis and by radioimmunoassay using whole bacterial cells. The approximate binding domains of the MAbs were determined by using full-length and truncated P1 polypeptides. The accessibility of these binding sites on the surfaces of intact bacteria was determined by radioimmunoassay. The ability of each MAb to cross-react with related proteins from strains of S. mutans serotypes e and f, S. sanguis, and S. sobrinus serotype g is also reported. ponents at the molecular level (7a). These MAbs will also prove useful for the analysis of functional similarities and differences among the immunologically related adhesin molecules. In addition, their cross-reactivity profile will be valuable in epidemiological studies, as it will allow certain 4425 on July 4, 2020 by guest http://iai.asm.org/ Downloaded from

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