MECHANISMS OF PLATELET BINDING BY VIRIDANS STREPTOCOCCI

据推测，血小板和细菌的结合是 心内膜炎的发病机制。然而，这两种机制都没有 细菌-血小板结合，也不是它在疾病过程中的作用 感染，已经被定义了。这项提议的目的是孤立和 鉴定绿色链球菌群的表面成分 调节与人血小板的结合。为了增加……的可能性 成功的配基纯化，概述了两种不同的策略， 可单独使用或组合使用，以恢复 莱兰德。作为第一种方法，我们将使用转座子突变来 产生血链球菌M99菌株的非结合变种。这个 然后通过免疫亲和力恢复链球菌的配体 层析；如果需要，将进行额外的纯化， 采用高效液相色谱(FPLC)和高效液相色谱法(HPLC法)。作为一种替代的配体纯化方法，a 描述了多步骤层析程序(离子交换、凝胶 渗透和反相色谱)，其中配体是 从整个细菌的变态溶血素提取液中回收。 一旦分离，结合特性(动力学，饱和性， 配体及其血小板受体的可逆性)将 评估过了。血小板受体的数量及其结合亲和力 对于配基将通过划痕分析来确定。以确认 完整的链球菌对血小板的黏附是由配体介导的， 我们将检测纯化的配体是否抑制血小板-链球菌 结合，用流式细胞仪测定。抑制研究将 也可以进行链球菌与免疫球蛋白的预培养 F(ab‘)2为配体所特有。以确定是否通过配体结合 对血小板聚集很重要，我们还研究了 纯化的配体和上述F(ab‘)2片段对血小板聚集的影响 被链球菌感染。 此外，血小板结合在心内膜炎中的作用将是 在动物模型中进行了评估。转座子的相对毒性- 将检测诱导的非结合突变体，以其能力来衡量 引发和延续心内膜感染。通过评估血小板- 细菌在细胞和分子水平上的结合，这项研究 应该有助于确定结合在糖尿病发病机制中的重要意义 心内膜炎。此外，这项工作还可以为定义 血小板的免疫功能，并用于检测 链球菌与其他细胞株，如内皮细胞和 单核细胞。

The binding of platelets and bacteria is a postulated central event in the pathogenesis of endocarditis. However, neither the mechanism for bacterial-platelet binding, nor its contribution to the course of infection, have been defined. The aim of this proposal is to isolate and characterize the surface component of viridans group streptococci that mediates binding to human platelets. To enhance the likelihood of successful ligand purification, two distinct strategies are outlined, which can be employed independently or in combination, to recover the ligand. As a first approach, we will employ transposon mutagenesis to produce a nonbinding variant of Streptococcus sanguis strain M99. The streptococcal ligand will then be recovered via immuno-affinity chromatography; if needed, additional purification will be performed, using FPLC and HPLC. As an alternative method for ligand purification, a multi-step chromatographic procedure is described (ion exchange, gel permeation, and reverse phase chromatography), in which the ligand is recovered from mutanolysin extracts of whole bacteria. Once isolated, the binding properties (kinetics, saturability, reversibility) of the ligand with its platelet receptor will be assessed. The number of platelet receptors, and their binding affinity for the ligand will be determined by Scratched analysis. To confirm that adherence of intact streptococci to platelets is mediated by the ligand, we will examined if the purified ligand inhibits platelet-streptococcal binding, as measured by a flow cytometric assay. Inhibition studies will also be performed, in which streptococci are preincubated with IgG F(ab')2 specific for the ligand. To determine if binding by the ligand is important for platelet aggregation, we also examine the effect of the purified ligand and the above F(ab')2 fragments on platelets aggregation by streptococci. In addition, the role of platelet binding in endocarditis will be evaluated in an animal model. The relative virulence of the transposon- induced nonbinding mutant will be examined, as measured by its ability to initiate and perpetuate endocardial infection. By assessing platelet- bacterial binding at the cellular and molecular level, this research should help define the significance of binding in the pathogenesis of endocarditis. Moreover, this work may provide a basis for defining the immunologic functions of platelets, and for examining the interaction of streptococci with other cell lines, such as endothelial cells and monocytes.