STREPTOCOCCAL PLATELET BINDING AND ENDOCARDITIS

描述（改编自申请人摘要）： 链球菌对人血小板的作用是一种假定的中枢机制， 心内膜炎的发病机制。 细菌-血小板结合可能是关键 用于将血液传播的生物体附着到瓣膜表面，以及用于 随后形成受感染的肉眼可见的赘生物。 的目的 这个项目是为了确定直接结合的分子基础， 血链球菌对人血小板的影响，并确定其作用 与心内膜炎的发病机制密切相关。 通过转座子 诱变，血链球菌菌株M99的四个同基因突变体具有 在体外最低限度地结合血小板。 这些变种人 为拟议的研究提供基础。 一种假定的链球菌基因 将使用以下方法鉴定编码人血小板配体的配体（“spl”）： 从低结合突变体衍生的探针筛选基因组文库， 菌株M99，然后进行克隆和测序。 spl基因产物将 然后通过克隆到pET载体表达系统中进行纯化。 一旦 分离的，假定配体与体外洗涤的人血小板的结合 将使用Scatchard分析进行检查，以确定结合是否类似于 受体-配体相互作用 配体介导的结合在细胞凋亡中的作用 心内膜炎的发病机制将在动物模型中解决， 比较亲本M99与同基因突变体的相对毒力 在spl基因座内含有Tn 916 deltaE插入。 通过定义 在分子水平上的链球菌-血小板结合机制， 这项工作将为确定血小板在糖尿病中的作用提供基础。 心内膜炎的发病机制。 反过来，这可能会为以下方面提供基础： 开发新的诊断和治疗策略。

DESCRIPTION (Adapted from applicants abstract): The direct binding of streptococci to human platelets is a postulated central mechanism in the pathogenesis of endocarditis. Bacterium-platelet binding may be critical for the attachment of blood-borne organisms to the valve surface, and for the subsequent formation of infected, macroscopic vegetations. The aim of this project is to define the molecular basis for the direct binding of Streptococcus sanguis to human platelets, and to determine the role of binding in the pathogenesis of endocarditis. By means of transposon mutagenesis, four isogenic mutants of Streptococcus sanguis strain M99 have been generated that bind platelets minimally in vitro. These mutants will provide a basis for the proposed research. A putative streptococcal gene ("spl~) encoding a ligand for human platelets will be identified, using probes derived from the low-binding mutants to screen a genomic library of strain M99, followed by cloning and sequencing. The spl gene product will then be purified, by cloning into a pET vector expression system. Once isolated, binding of the putative ligand to washed human platelets in vitro will be examined, using Scatchard analysis to determine if binding resembles a receptor-ligand interaction. The role of ligand mediated binding in the pathogenesis of endocarditis will be addressed in an animal model, by comparing the relative virulence of parental M99 with the isogenic mutant containing a Tn916deltaE insertion within the spl locus. By defining the mechanisms for streptococcal-platelet binding at the molecular level, this work will provide a basis for determining the role of platelets in the pathogenesis of endocarditis. In turn, this may provide a basis for developing novel diagnostic and therapeutic strategies.