Septicemia due to Staphylococcus aureus is often the consequence of a local infection (e.g. from catheters) which has gained access to the bloodstream. Once the bacteria is in the bloodstream, patients are at risk of developing endocarditis and other metastatic complications. One intriguing aspect in the pathogenesis of S. aureus infection is the ability of the microorganism to adhere to catheters and subsequently to the endothelium during transient bacteremia. Recent studies from this laboratory have suggested that S.aureus colonizes catheters and the endothelium by using a fibrinogen binding protein (FBP) to mediate adhesion to fibrinogen bound on respective surfaces. Our data have also shown that inflammatory mediators such as tumor necrosis factor can significantly augment the adherence of S.aureus to cultured human endothelial cells by promoting fibrinogen binding to the endothelium. To test the hypothesis that FBP is an important adhesin to both catheters and endothelium and to elucidate the mechanism of adherence, the structural gene for FBP in a selected strain will be expressed and sequenced. The cloned FBP will be purified and used to raise polyclonal and monoclonal antibodies. These antibodies mill be used to map out the fibrinogen binding domain of the adhesin (i.e. FBP). In addition, they will be tested in adherence assays with catheters and endothelium in both normal and cytokine-stimulated conditions. Further analysis of the FBP will be achieved by the construction of FBP deletion mutants lacking the fibrinogen binding phenotype by allele replacement. The mutants will be tested in staphylococcal adherence assays with catheters and cytokine-stimulated endothelium. The information generated from in vitro adherence studies will be applied to an in-vivo rabbit model of experimental endocarditis to determine if FBP plays a major role in mediating S.aureus adherence to heart valves during staphylococcal bacteremia. These proposed studies will enable us to determine if FBP is an important mediator of staphylococcal adherence to catheters and endothelium in both in vitro and in vivo settings. Since bacterial adherence always precedes tissue invasion, this knowledge would allow one to gain insights into devising strategies towards prevention of staphylococcal adherence to catheters, heart valves and other intravascular targets.
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