Our studies with purified fimbrial proteins have demonstrated an interaction with salivary-coated hydroxyapatite (sHAP) that involves the carboxyl third of the major 43 kDa fimbrillin subunit. In contrast, immunodominant regions appear to reside in the N-terminus of the fimbrillin subunit. The principal goal of this study is to further define the structure-function relationship of fimbriae and fimbrial-associated adhesins of P. gingivalis. Molecular genetic approaches to studying the fimbriae will be used to evaluate the genes specifying the P. gingivalis fimbrial-associated adhesins. DNA sequences adjacent to the 5' and 3' ends of the fimbrillin gene will be examined to determine whether the gene encoding for fimbrillin is part of a larger polycistonic unit that also contains other fimbrial-associated components. In order to further define the sHAP binding domains of the fimbrillin monomer, we propose to introduce site-directed mutations in the binding sites of fimbrillin domains identified in our previous studies. The mutant genes will be expressed as fusion proteins, purified, and tested in the in vitro system for adherence to sHAP or salivary components in solution. We will also pursue the direct approach of insertional inactivation of fimbrial gene and complementation studies. Mutant genes will be inserted into shuttle vectors which will be used to complement fimA gene deficient mutants generated in our laboratory. We will expand our studies to other strains of P. gingivalis by comparing the variable and constant epitopes of the 43 kDa fimbrillin subunit from several strains. Completion of these studies will provide, at the molecular level, an understanding of the antigenic heterogeneity observed among the strains. Synthetic peptides of the variable regions will be used to identify their role in attachment to salivary-coated HAP as well as in immune reactivity of fimbriae. Thus, the studies outlined in this subproject could provide important information needed for a rational approach for immunization to modulate colonization of the oral cavity.
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