Role of Streptococcal-Platelet Binding in Endocarditis

链球菌与人血小板的结合被认为是感染性心内膜炎发病机制的中心机制。细菌-血小板结合对于血液传播的生物最初附着在瓣膜表面和随后形成宏观植被都可能是重要的。本项目的目的是进一步表征戈氏链球菌直接结合血小板的分子基础，以及这种结合在心内膜炎发病机制中的作用。我们已经鉴定出两个基因座的戈氏链球菌菌株M99介导这种有机体直接结合人血小板在体外。第一个位点（gspA）编码一个117 kDa的细胞壁相关蛋白，该蛋白可能具有粘附素的功能。第二个位点似乎是一个操纵子（GspB - secy2a2），编码286 kDa的细胞壁锚定蛋白（GspB），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（secA2和secY2）。我们现在寻求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基因座。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，研究其对血小板结合的影响，以及gspB- secy2a2操纵子的附加定位。然后，我们将纯化GspA和GspB，并在体外检测每种蛋白与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也将用于鉴定它们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，我们将在兔心内膜感染模型中比较M99和选定突变体的毒力。通过对血小板链球菌黏附素的表征，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，它可能为新的预防或治疗策略确定新的靶点。

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.