Defining a novel mechanism of adhesion present in multiple infective endocarditis causing species

导致亚急性感染性心内膜炎（IE）的致病机制知之甚少，限制了对IE的研究。 制定预防和治疗策略。然而，细菌与血小板的结合被认为是 感染的关键步骤口腔链球菌和缓症链球菌是草绿色组链球菌， 常见的，但很少研究，IE的原因。末端唾液酸是许多草绿色体的主要血小板受体 群链球菌。直到最近，所有已知的链球菌-唾液酸相互作用都是由成员 富含丝氨酸的重复蛋白（SRRP）家族的粘附素。这一模式因确定了一个 新的唾液酸结合蛋白AsaA在S.口腔SRRP和AsaA的结合均由唾液酸介导 免疫球蛋白样凝集素（Siglec）样结构域。虽然大多数SRRP包含一个，但AsaA包含两个假定的 类Siglec结构域。第一个Siglec样结构域含有唾液酸结合基序;然而，第二个Siglec样结构域含有唾液酸结合基序。 Siglec样结构域是第一个被描述为完全缺乏这种基序的结构域。由两个Siglec组成的多肽， Like和Unique结构域直接结合血小板上的唾液酸。然而，每个类Siglec结构域的作用 结合的特异性蛋白质是未知的。AsaA直系同源物在四种IE致病物种中被鉴定， 先前描述的粘附机制。总之，这些数据导致了AsaA和 直系同源物是唾液酸结合粘附素新家族的成员，其通过结合宿主而促进IE 通过两个类似Siglec的域的组件。三个目标将检验这一假设。目标1：确定 两个AsaA Siglec样结构域结合IE相关宿主组分。确定的突变体和重组 表达的蛋白质将用于解析两个不同的Siglec样结构域对血小板聚集的贡献。 结合，并鉴定每个结构域结合的特异性蛋白质。此外，IE相关主机的范围 AsaA结合的组分和两个Siglec样结构域在这些相互作用中的作用将被 确立了习目的2：确定AsaA是否是唾液酸结合粘附素新家族的成员。的 AsaA直系同源物在血小板结合中的作用将被研究，重点是S.缓解。目标3：确定 AsaA的贡献，特别是唾液酸结合，IE使用3D人类微血管模型。一个切割- 边缘生理相关和易处理的3D微血管模型将用于评估唾液酸的作用， AsaA和Siglec-like结构域在植被建立中的作用。此外，实验将确定 这种粘附机制是否可以作为治疗靶点。该应用程序非常重要，因为它侧重于 细菌没有先前描述的结合血小板的机制，这是发展血小板的关键步骤。 亚急性IE该提议的创新方面包括定义新的Siglec样结构域的功能， 利用先进的3D微血管模型。这项研究还可能确定一个新的唾液酸家族- 由多种IE致病物种产生的结合粘附素，可能与SRRP结合相同的受体 使这些相互作用成为有吸引力的干预目标。

The pathogenic mechanisms leading to subacute infective endocarditis (IE) are poorly understood, limiting the development of preventative and therapeutic strategies. However, bacterial binding to platelets is accepted as a key step in infection. Streptococcus oralis and Streptococcus mitis are viridans group streptococci that are common, but rarely studied, causes of IE. Terminal sialic acid is the major platelet receptor for many viridans group streptococci. Until recently, all known streptococcal-sialic acid interactions were mediated by members of the serine-rich repeat protein (SRRP) family of adhesins. That paradigm was shifted by identification of a novel sialic acid-binding protein, AsaA, in S. oralis. Binding of both SRRPs and AsaA is mediated by sialic acid immunoglobulin-like lectin (Siglec)-like domains. While most SRRPs contain one, AsaA contains two putative Siglec-like domains. The first Siglec-like domain contains a sialic acid-binding motif; however, the second Siglec-like domain is the first described to completely lack this motif. A polypeptide consisting of the two Siglec- like and Unique domains directly binds sialic acid on platelets. However, the role of each Siglec-like domain and the specific proteins bound are unknown. AsaA orthologs were identified in four IE-causing species with no previously described mechanisms of adhesion. Together, these data led to the hypothesis that AsaA and orthologs are members of a novel family of sialic acid-binding adhesins, that contribute to IE by binding host components via two Siglec-like domains. Three aims will test this hypothesis. Aim 1: Determine the role of the two AsaA Siglec-like domains in binding IE-relevant host components. Defined mutants and recombinantly expressed proteins will be used to resolve the contribution of the two different Siglec-like domains to platelet binding and to identify the specific proteins bound by each domain. Furthermore, the range of IE-relevant host components bound by AsaA and the role of the two Siglec-like domains in these interactions will be established. Aim 2: Determine whether AsaA is a member of a novel family of sialic acid-binding adhesins. The role of AsaA orthologs in platelet binding will be investigated, with a focus on S. mitis. Aim 3: Determine the contribution of AsaA, and specifically sialic acid binding, to IE using a 3D human microvessel model. A cutting- edge physiologically relevant and tractable 3D microvessel model will be used to assess the role of sialic acid, AsaA, and the Siglec-like domains in establishment of a vegetation. Furthermore, experiments will ascertain whether this adhesion mechanism could be a therapeutic target. This application is significant as it focuses on bacteria with no previously described mechanisms of binding platelets, a critical step in development of subacute IE. Innovative aspects of this proposal include defining the function of a novel Siglec-like domain and utilizing a cutting-edge 3D microvessel model. This study will also likely identify a novel family of sialic acid- binding adhesins produced by multiple IE-causing species, which may bind the same receptor as SRRPs making these interactions an attractive target for intervention.