Prophage-Encoded Binding of S. mitis to Human Platelets

描述（由申请方提供）：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种假定的中心机制。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。 我们以前的研究表明，缓症链球菌SF 100菌株的血小板结合部分是由温和噬菌体（SM 1）编码的两种细胞壁相关蛋白（PblA和PblB）介导的。 这两种蛋白质在细菌表面的表达是SF 100最大水平的血小板结合所必需的。 本提案的总体目标是进一步阐明这些蛋白质促进血小板结合的机制，并确定PblA和PblB介导的结合在心内膜炎发病机制中的作用。我们将首先纯化PblA和PblB，并在体外检测每种蛋白与人血小板的结合特性，以确定是否一种或两种蛋白可以在体外直接结合人血小板。将进行正式的结合分析，以确定结合是否类似于受体-配体相互作用。纯化的PblA和PblB也将用于通过远蛋白质印迹和通过免疫沉淀或亲和层析来鉴定它们各自的血小板结合位点。然后根据需要通过几种方法鉴定与Pb 1A或Pb 1B结合的血小板膜蛋白（例如，N-末端测序或质谱）。我们还将评估PblA和PblB出口到细菌表面的机制，以及这些蛋白质是否与其他噬菌体结构蛋白形成血小板结合复合物。为了阐明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SF 100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。

DESCRIPTION (provided by applicant): 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.