The mutans family of oral streptococci possesses antigens cross- reactive with human cardiac and skeletal muscle. This requires both caution in the development of anti-caries vaccines and intensive investigations of the immunochemistry of the cell surfaces of these microorganisms. There presently exists some confusion as to the exact nature of heart cross-reactive antigens (HRA); most reports link this property to wall-associated proteins (Spa A or P1), while others contest these findings. Recent work, however, pinpoints a 62,000 dalton (62KD) membrane-bound moiety possessing the properties of HRA. Attempts will be made to clone the 62KD membrane antigen into Escherichia coli in order to prepare purified protein and to obtain plasmids containing the relevant genetic material. Purified 62KD antigen will be analyzed as to its N-terminal amino acid sequence and its amino acid content. Because this antigen is reactive with monoclonal antibodies (mAbs) to Streptococcus pyogenes that also react with human myosin, the recombinant plasmid will be analyzed to determine the nucleotide sequence of inserted DNA in order to search for regions of homology with this eukaryotic protein. Also, cDNA probes for large regions of the myosin heavy chain will be employed in hybridization studies. If homology with myosin is found, synthetic peptides spanning these regions will be prepared as potential inhibitors in serologic assays and as immunogens to induce cross-reactive antibodies in rabbits. Inserts from the recombinant plasmid will be labeled to serve as DNA probes for the presence of this gene in chromosomal DNA for other streptococci. Simultaneously, the gene for P1 (S. mutans Ingbritt) will be cloned into E. coli to allow preparation of large amounts of purified protein. Anti-P1 immunoglobulins, affinity purified to remove other antibodies, will be used in inhibition assays to confirm the biologic role(s) of the protein in either potentiating adherence or allowing sucrose-induced cellular aggregation. The functional domains on P1 responsible for each activity will be determined either by BAL 31 exonuclease digestion of the P1 gene to obtain progressively shorter DNA inserts capable upon subcloning of yielding P1 fragments or direct cleavage of P1 by enzymes and/or cyanogen bromide. Reactive P1 fragments will be purified for N- terminal sequencing or, in the case of subclones, nucleotide sequencing would determine the relevant genetic message. Anti-P1 immunoglobulins against both cloned and native antigens will be reacted with detergent and aqueous extracts of human heart or with tissue sections by the immunogold technique. Rheumatoid factor will be removed by appropriate affinity purification. P1 also will be tested for direct binding to human heart homogenates or tissues. If positive reactions are obtained, reactive P1 epitopes (or binding sites) will be characterized as described above for functional domains. cDNA probes to detect genetic determinants for functional domains and/or epitopes will be synthesized for hybridization with DNA from streptococci.
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