Role of Streptococcal-Platelet Binding in Endocarditis

链球菌与人血小板的结合是感染性心内膜炎发病机制中的一个假定的中心机制。 细菌-血小板结合可能对血液传播的微生物最初附着在瓣膜表面以及随后形成肉眼可见的赘生物都很重要。 本项目的目标是进一步表征戈登链球菌直接血小板结合的分子基础，以及结合在心内膜炎发病机制中的作用。 我们已经确定了两个位点的S。gordonii菌株M99，其在体外介导该生物体与人血小板的直接结合。 第一个基因座（gspA）编码一个117 kDa的细胞壁相关蛋白，可以作为粘附素发挥作用。第二个基因座似乎是编码286 kDa细胞壁锚定蛋白（GspB）的操纵子（gspB-secY 2A 2），该蛋白也可能是血小板结合蛋白。 此外，该操纵子包含GspB输出所需的基因（secA 2和secY 2）。我们现在试图进一步研究这些基因及其产物在介导血小板结合中的作用。 我们的第一个目标是通过确认gspA或gspB的表达与血小板结合有关来更广泛地表征这些位点。 这项工作将包括gspA和gspB突变体的互补研究，GspA或GspB在乳酸乳球菌中的表达，并研究其对该生物体血小板结合的影响，以及gspB-secY 2A 2操纵子的额外作图。 然后，我们将纯化GspA和GspB，并检查每种蛋白质与体外人血小板的结合特性，从而确定结合是否类似于受体-配体相互作用。 纯化的GspA和GspB还将用于鉴定其各自的血小板结合位点。 为了评估这些粘附素在心内膜炎发病机制中的作用，我们将在兔心内膜炎感染模型中比较M99和选定突变体的毒力。 通过对链球菌与血小板粘附素的研究，本研究将进一步明确血小板结合在心内膜炎发病机制中的作用。 此外，它可以确定新的预防或治疗策略的新目标。

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. The goal of this project is to characterize further the molecular basis for direct platelet binding by Streptococcus gordonii, and the role of binding in the pathogenesis of endocarditis. We have identified two loci of S. gordonii strain M99 that mediate the direct binding of this organism to human platelets in vitro. The first locus (gspA) encodes a 117 kDa cell wall-associated protein that may function as an adhesin. The second locus appears to be an operon (gspB-secY2A2) encoding a 286 kDa cell wall anchored protein (GspB) that is also a likely platelet binding protein. In addition, this operon contains genes (secA2 and secY2) that are required for the export of GspB. We now seek to further examine the role of these genes and their products in mediating binding to platelets. Our first goal is to characterize more extensively these loci by confirming that expression of gspA or gspB is linked to platelet binding. This work will include complementation studies of gspA and gspB mutants, expression of GspA or GspB in Lactococcus lactis and studying its effects on platelet binding by that organism, and additional mapping of the gspB-secY2A2 operon. We will then purify GspA and GspB, and examine the binding properties of each protein with human platelets in vitro, thereby determining if binding resembles a receptor-ligand interaction. Purified GspA and GspB will also be used to identify their respective platelet binding sites. To assess the role of these adhesins in the pathogenesis of endocarditis, we will compare the virulence of M99 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.