STREPTOCOCCAL PLATELET BINDING AND ENDOCARDITIS

链球菌血小板结合和心内膜炎

• 批准号: 2673029
• 负责人: PAUL M. SULLAM
• 金额: $ 23.47万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2673029
• 关键词: Streptococcus sanguis; bacterial endocarditis; bacterial genetics; bacterial proteins; cell adhesion; cell adhesion molecules; human tissue; laboratory rabbit; molecular cloning; nucleic acid sequence; platelets; protein structure function; tissue /cell culture; transposon /insertion element; virulence

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.

描述（改编自申请人摘要）：直接绑定 链球菌至人血小板是一种假定的中心机制 心内膜炎的发病机理。 细菌 - 血小板结合可能至关重要 为了附着血传播生物， 随后形成感染的宏观植被。 目的 该项目是为了定义直接结合的分子基础 对人血小板的链球菌链球菌，并确定 在心内膜炎的发病机理中结合。 通过转座子 诱变，sanguis菌株M99的四个同源突变体 被生成，在体外最少结合血小板。 这些突变体会 为拟议的研究提供基础。 推定的链球菌基因 （“ spl〜）使用编码人体血小板的配体，使用 从低结合突变体得出的探针来筛选一个基因组文库 菌株M99，然后进行克隆和测序。 SPL基因产品将 然后通过克隆到PET矢量表达系统中纯化。 一次 孤立的，假定配体在体外与洗涤的人血小板结合 将检查使用SCATCHARD分析来确定绑定是否类似 受体 - 配体相互作用。 配体介导的结合在 心内膜炎的发病机理将在动物模型中通过 比较父母M99与同基因突变体的相对毒力 在SPL基因座中包含TN916DELTAE插入。 通过定义 在分子水平上结合链球菌 - 链路链血动板的机制，这 工作将为确定血小板在 心内膜炎的发病机理。 反过来，这可能为 制定新颖的诊断和治疗策略。