Role of Streptococcal-Platelet Binding in Endocarditis

链球菌-血小板结合在心内膜炎中的作用

• 批准号: 6766006
• 负责人: PAUL M. SULLAM
• 金额: $ 33万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766006
• 关键词: Streptococcus; Streptococcus lactis; adhesin; bacteria infection mechanism; bacterial cytopathogenic effect; bacterial endocarditis; bacterial genetics; binding sites; cell membrane; disease /disorder model; flow cytometry; gene expression; genetic mapping; host organism interaction; human subject; laboratory rabbit; molecular cloning; operon; patient oriented research; platelets; protein purification; protein structure function; receptor; virulence; western blottings

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.

在感染性心内膜炎的发病机制中，链球菌与人血小板的结合是一个假定的中心机制。细菌-血小板结合对于血液传播的生物体最初附着在瓣膜表面以及随后的宏观植物的形成都可能是重要的。该项目的目标是进一步确定戈尔顿链球菌直接结合血小板的分子基础，以及结合在心内膜炎发病机制中的作用。我们在体外已经确定了戈登链球菌M99株的两个基因座，它们介导了该菌与人血小板的直接结合。第一个基因座(GSPA)编码一个117 kDa的细胞壁相关蛋白，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)，也可能是一种血小板结合蛋白。此外，该操纵子还含有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调节与血小板结合方面的作用。我们的第一个目标是确认GSPA或GSPB的表达与血小板结合有关，从而更广泛地表征这些基因座。这项工作将包括GspA和gspB突变体的互补性研究，GspA或GspB在乳酸乳球菌中的表达及其对该微生物与血小板结合的影响，以及gspB-secY2A2操纵子的额外定位。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和选定的突变体的毒力。通过表征链球菌黏附素对血小板的作用，这项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，它还可能为新的预防或治疗策略确定新的靶点。