The binding of streptococci to human platelets is a postulated central mechanism in the pathogenesis of infective endocarditis. Bacterium-platelet binding may be important both for the initial attachment of blood-borne organisms to the valve surface, and for the subsequent formation of macroscopic vegetations. Our previous research has shown that platelet binding by Streptococcus mitis strain SF100 is mediated in part by two cell wall-associated proteins (PblA and PblB) that are encoded by a temperate bacteriophage (SM1). Expression of both proteins on the bacterial surface is required for maximum levels of platelet binding by SF100. The overall goal of this proposal is to delineate further the mechanisms by which these proteins contribute to platelet binding, and to determine the role of PblA and PblB mediated binding in the pathogenesis of endocarditis. We will first purify PblA and PblB, and examine the binding properties of each protein with human platelets in vitro, to determine whether either or both proteins can bind human platelets directly in vitro. Formal binding analysis will be done to determine whether binding resembles a receptor-ligand interaction. Purified PblA and PblB will also be used to identify their respective platelet binding sites, by far western blotting and by immunoprecipitation or affinity chromatography. Platelet membrane proteins bound by either PblA or PblB will then be identified by several methods as needed (e.g., N-terminal sequencing, or mass spectroscopy). We will also assess the mechanisms for the export of PblA and PblB to the bacterial surface, and whether these proteins form platelet-binding complexes with other phage structural proteins. To address the role of these adhesins in the pathogenesis of endocarditis, we will compare the virulence of SF100 and selected mutants in a rabbit model of endocardial infection. By characterizing streptococcal adhesins for platelets, this research will further define the role of platelet binding in the pathogenesis of endocarditis. In addition, it may identify novel targets for new preventative or therapeutic strategies.
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