The overall aim of this research project is to study the molecular mechanisms of the adherence of bacteria to mammalian cells. For this purpose the lipoteichoic acid-mediated attachment of group A streptococci and the type 1 fimbriae-mediated attachment of Escherichia coli will be studied in detail as paradigms of the process of adhesion and colonization. Specific efforts will be directed toward: (1) defining the structural domain(s) of the fibronectin molecule on oropharyngeal epithelial cells that recognize group A streptococci and its lipoteichoic acid, (2) defining the orientation and the anchorage of the lipoteichoic acid-protein complex on the surface of intact streptococci, (3) defining primary tertiary and quaternary structural determinants of type I fimbriae of E. coli that mediate the binding to mannose-containing receptors on eukaryotic cells; monoclonal antibodies will be employed to define the structural elements necessary for binding, and (4) determining the protective activity of monoclonal antibodies directed against primary, tertiary and quaternary structural epitopes of type I fimbriae in a mouse model of E. coli-induced pyelonephritis. Human plasma fibronectin will be purified by affiniity chromatography over gelatin and arginine Sepharose columns. The molecule will be cleaved with various proteolytic enzymes and the fragments examined for binding to streptococci and LTA employing radio immunochemical techniques in combination with electroimmunoblotting. The formation of complexes between M protein and LTA will be studied by immunoelectrophoresis, radiolabeling techniques and by preparing lipoteichoic acid substituted to varying degrees with alanine residues. Native and synthetic subpeptides of M protein will be used to determine the minimum structure required for complex formation. Type I fimbriase will be purified from E coli and used to prepare sets of monoclonal antibodies directed against various structural epitopes. The reactivities of the antibodies with dissociated and reassembled fimbriae will be studied in vitro and in vivo. Mice will be injected intraperitoneally with monoclonal antibodies and challenged intravesically with type I fimriated E. coli 24 hrs later. Colonization by the organisms will be evaluated by culturing samples of the kidney and bladder 5 days after challenge. The proposed studies should provide information needed to develop new approaches to the prevention of serious infectious diseases arising from bacterial colonization of susceptible mucosal surfaces.
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