Role of sialoglycan binding in the pathogenesis of streptococcal endocarditis

项目总结 口腔链球菌是感染性心内膜炎(IE)的主要病因。这些物种中有几个表达Siglec- 例如，富含丝氨酸重复序列(SRR)的粘附素可以将各种O-连接的唾液酸聚糖结合在人糖蛋白上，并 细胞。戈登链球菌M99 SRR粘附素GspB基因的表达可增强猪的致病性 IE的动物模型。这种增强的致病性被认为是由于GspB与 血小板受体GPIB上的三糖唾液酸T抗原(STA，一种核心1唾液酸聚糖)，导致 血流中的细菌附着在心脏瓣膜表面并形成植被。同基因 优先结合核心2唾液酸聚糖(例如唾液酸乳糖胺)的表达GspB变异体的M99突变体 与WT株相比，毒力较弱。这些发现表明，在类型上的差异 唾液酸聚糖结合(“选择性”)影响生物体启动和繁殖心内膜的能力 感染，一些相互作用增强致病作用，而另一些相互作用减少疾病。我们 假设在体内，血液中的链球菌与血小板或心脏表面的STAT结合 有助于引发感染，这种结合因高剪切流产生的血流动力学效应而增强。 相反，与血细胞上的核心2唾液酸聚糖结合可能导致细菌清除增加，从而 减弱毒力。瓣膜上细菌与核心1和核心2唾液酸聚糖的后续相互作用 可能通过对血小板活化、凝血或内皮愈合的不同影响来影响疾病的进展。 为了进一步探讨唾液酸结合选择性在IE发病机制中的作用，我们将通过Flow进行比较 上述同基因菌株在体外与血小板、红细胞和白细胞结合。我们还将研究是否 这些菌株与不同gpib唾液酸聚糖含量不同的血小板结合，以及是否与sta结合还是与core 2结合。 结构会影响血小板的激活。此外，我们将评估选择性如何影响依恋 细菌到受损的心脏表面，如用排列着固定血小板的微流体室模拟的那样， 血管性血友病因子、胶原或活化的人血管内皮细胞及其对血流动力学的影响 约束力上的力。我们还将通过评估菌株来探索选择性对植被形成的影响。 触发血小板收缩能力的差异和对血小板依赖的内皮愈合的影响。我们还将 比较这些菌株在体内产生的植物与毒力有关的关键特征，包括共同 用血小板定位和生物被膜生产。 这些研究将提供新的见解，以了解唾液酸聚糖的选择性和血流动力学力量如何影响 心内膜感染的发生和传播。通过定义这些事件的机制，本研究 将重新定义我们对链球菌IE发病机制的关键步骤的理解。这些概念和 这些发现将广泛适用于其他血管内病原体，并将为 微生物与宿主多糖的相互作用。

PROJECT SUMMARY The oral streptococci are a major cause of infective endocarditis (IE). Several of these species express Siglec- like, serine-rich repeat (SRR) adhesins that bind a variety of O-linked sialoglycans on human glycoproteins and cells. Expression of the SRR adhesin GspB of Streptococcus gordonii M99 results in increased virulence in animal models of IE. This enhanced pathogenicity is thought to be due the interaction of GspB with the trisaccharide sialyl-T antigen (sTa, a core 1 sialoglycan) on the platelet receptor GPIb, leading to increased attachment of bacteria in the blood stream to cardiac valve surfaces and vegetation formation. Isogenic mutants of M99 expressing GspB variants that preferentially bind core 2 sialoglycans,(e.g., sialyl-lactosamine) are less virulent, as compared with the WT strain. These findings indicate that differences in the type of sialoglycan bound ("selectivity") affect the ability of organisms to both initiate and propagate endocardial infection, with some interactions enhancing pathogenesis, while other interactions reducing disease. We hypothesize that in vivo, the binding of streptococci in the bloodstream to sTa on platelets or cardiac surfaces helps initiate infection, and that this binding is enhanced by hemodynamic effects created by high shear flow. In contrast, binding to core 2 sialoglycans on blood cells may lead to increased bacterial clearance, thereby attenuating virulence. Subsequent interactions of bacteria on valves with core 1 versus core 2 sialoglycans may affect disease progression via different effects on platelet activation, clotting, or endothelial healing. To further address the roles of sialoglycan binding selectivity in the pathogenesis of IE, we will compare by flow the above isogenic strains for their binding to platelets, RBCs and WBCs in vitro. We will also examine whether these strains bind platelets that differ in GPIb sialoglycan content, and whether binding to sTa versus core 2 structures affects platelet activation. In addition, we will assess how selectivity impacts the attachment of bacteria to damaged cardiac surfaces, as modeled by microfluidic chambers lined with immobilized platelets, von Willebrand Factor, collagen or activated human vascular endothelial cells, and the effect of hemodynamic forces on binding. We will also explore the impact of selectivity on vegetation formation by assessing strain differences in triggering platelet contractility and effects on platelet-dependent endothelial healing. We will also compare vegetations produced in vivo by these strains for key features linked to virulence, including co- localization with platelet and biofilm production. These studies will provide novel insights as to how sialoglycan selectivity and hemodynamic forces affect the initiation and propagation of endocardial infection. By defining the mechanisms for these events, this research will redefine our understanding of the key steps in the pathogenesis of streptococcal IE. These concepts and findings will be broadly applicable to other endovascular pathogens, and will provide novel insights into microbial interactions with host glycans.