STREPTOCOCCAL PLATELET BINDING AND ENDOCARDITIS

描述(改编自申请者摘要)：直接约束 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 用于将血液传播的微生物附着到阀门表面，以及用于 随后形成受感染的肉眼可见的植物。的目的是 这个项目是为了定义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 结合在心内膜炎发病机制中的作用。通过转座子的方式 血链球菌M99菌株四个等基因突变株的诱变 在体外最小限度地结合了血小板。这些变种人会 为拟议的研究提供基础。一种可能的链球菌基因 (“SPL~”)编码人类血小板的配体将被识别，使用 从低结合突变体中衍生的探针用于筛选沙门氏菌基因组文库 菌株M99，然后进行克隆和测序。SPL基因产物将 然后通过克隆入pET载体表达系统进行纯化。一次 体外分离与洗涤的人血小板结合的假定配体 将进行检查，使用Scatchard分析来确定绑定是否类似 受体与配体的相互作用。配体介导的结合在细胞周期调控中的作用 心内膜炎的发病机制将在动物模型中进行研究，通过 亲本M99与同基因突变株的相对毒力比较 在SPL基因座内含有Tn916deltaE插入。通过定义 在分子水平上链球菌与血小板结合的机制，这 这项工作将为确定血小板在心脏疾病中的作用提供依据 心内膜炎的发病机制。反过来，这可能会为 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.