Mechanisms for ligand binding by serine-rich adhesins of Gram-positive pathogens

描述(申请人提供)：富含丝氨酸重复序列(SRR)糖蛋白是在许多革兰氏阳性细菌中发现的粘附素大家族。这些表面成分是广泛人类感染的重要毒力决定因素。GspB是戈登链球菌的SRR粘附素，通过与血小板受体GPIb上的三糖唾液酸T抗原(Sta)相互作用而介导与人血小板的结合。这种结合似乎在感染性心内膜炎的发病机制中很重要，因为GspB结合区的突变会导致体外血小板结合减少，并在这种疾病的动物模型中降低毒力。GspB结合的三个特性对于链球菌靶向心内膜可能是非常重要的：亲和力、选择性和FOW增强。首先，GspB与其血小板受体的结合是一种高亲和力相互作用(KD 2.4 x 10-8M)。其次，GspB有一个非常有选择性的结合光谱，其中sta是它的主要配体。第三，GspB介导的细菌与STA的结合可以通过类似于血管内系统内的流体剪切流水平来增强。结合起来，这三种结合特性可能针对血液传播的链球菌与固定在心内膜损伤部位的血小板(从而启动感染)。GspB介导的结合也可能有助于随后形成含有细菌和血小板的肉眼心内膜损伤(赘生物)。这个项目试图定义GspB结合亲和力，选择性， 和流动增强，以及这些特性对毒力的相对重要性。目标1将研究GspB结合域的分子结构如何赋予亲和力和选择性。我们将确定与sta及相关化合物共结晶的GspB结合区的结构，选择关键结构域和残基进行靶向突变，并研究这些突变对结合亲和力和选择性的影响。两个结合性质不同的GspB同源物(HSA和SRPA)也将被评估。目标2将决定 GspB的结构特征，有助于流动增强结合，以及结合是否通过形成捕获键发生。我们将具体研究GspB的富含丝氨酸的重复结构域在流动增强结合中的作用。目的3将评估配基亲和力、选择性和血流增强结合在感染性心内膜炎发病机制中的作用。戈登链球菌M99株的同基因变种，其配体结合特性不同，将使用这种疾病的共同感染模型来比较其相对毒力。该项目将为这种新型细菌粘附素结合碳水化合物的结构基础以及链球菌与人类血小板结合的机制提供重要的见解。此外，这些研究可能为以SRR糖蛋白结合为靶点的感染性心内膜炎的新疗法提供基础。

DESCRIPTION (provided by applicant): The serine-rich repeat (SRR) glycoproteins are a large family of adhesins found in many Gram-positive bacteria. These surface components are important virulence determinants for a broad range of human infections. GspB is an SRR adhesin of Streptococcus gordonii that mediates binding to human platelets through its interaction with the trisaccharide sialyl-T antigen (sTa) on the platelet receptor GPIb. This binding appears to be important for the pathogenesis of infective endocarditis, since mutagenesis of the GspB binding region results in decreased platelet binding in vitro, and reduced virulence in an animal model of this disease. Three properties of GspB binding may be highly important for the targeting of streptococci to the endocardium: affinity, selectivity, and fow enhancement. First, the binding of GspB to its platelet receptor is a high affinity interaction (KD 2.4 x 10-8 M). Second, GspB has a very selective binding spectrum, with sTa being its principal ligand. Third, GspB-mediated binding by bacteria to sTa is enhanced by levels of fluidic shear flow similar to those within the endovascular system. In combination, these three binding properties may target blood-borne streptococci to platelets immobilized at sites of endocardial injury (thereby initiating infection). GspB-mediated binding may also contribute to the subsequent formation of macroscopic endocardial lesions (vegetations) containing bacteria and platelets. This project seeks to define the molecular basis for GspB binding affinity, selectivity, and flow enhancement, and the relative importance of these properties for virulence. Aim 1 will examine how the molecular architecture of the GspB binding domain confers affinity and selectivity. We will determine the structure of the GspB binding region cocrystallized with sTa and related compounds, select key domains and residues for targeted mutagenesis, and examine the impact of these mutations on binding affinity and selectivity. Two GspB homologs (Hsa and SrpA) that differ in their binding properties will also be evaluated. Aim 2 will determine the structural features of GspB that contribute to flow-enhanced binding, and whether binding occurs via the formation of catch bonds. We will specifically examine the contribution of the serine-rich repeat domains of GspB in flow-enhanced binding. Aim 3 will assess the impact of ligand affinity, selectivity and flow-enhanced binding on the pathogenesis of infective endocarditis. Isogenic variants of S. gordonii strain M99 that differ in their ligand binding properties will be compared for relative virulence, using a well-established co-infection model of this disease. This project will provide significant insights into the structural basis for carbohydrate binding by this novel group of bacterial adhesins, as well as the mechanisms for streptococcal binding to human platelets. In addition, these studies could provide a basis for novel therapies for infective endocarditis that target SRR glycoprotein binding.