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)的致病机制尚不清楚，限制了 制定预防和治疗策略。然而，细菌与血小板的结合被认为是 感染的关键一步。口腔链球菌和中型链球菌属绿色链球菌，它们是 IE的常见原因，但很少被研究。末端唾液酸是许多绿色分子的主要血小板受体。 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介导的 属于粘附素的富丝氨酸重复蛋白(SRRP)家族。这一范式因识别一种 口腔沙门氏菌中新的唾液酸结合蛋白ASAA。唾液酸介导SRRPs和ASAA的结合 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包含两个推定的 类Siglec域。第一个Siglec-like结构域包含一个唾液酸结合基序；然而，第二个 Siglec-like结构域是第一个完全缺乏这个基序的结构域。一种由两个符号组成的多肽- 相似和独特的结构域直接与血小板上的唾液酸结合。然而，每个类Siglec域的角色 结合的特定蛋白质也是未知的。在四种引起IE的物种中发现了ASAA同源基因，没有 先前描述的粘连机制。总而言之，这些数据导致了这样的假设：ASAA和 同源基因是一个新的唾液酸结合粘附素家族的成员，它通过结合宿主而促进IE 组件通过两个类似Siglec的域。三个目标将检验这一假设。目标1：确定 绑定IE相关主机组件的两个ASAA Siglec结构域。定义的突变体和重组 表达的蛋白将被用来分解两个不同的Siglec样结构域对血小板的贡献 并鉴定每个结构域结合的特定蛋白。此外，IE相关主机的范围 ASAA结合的成分以及两个Siglec结构域在这些相互作用中的作用将是 已经成立了。目的2：确定ASAA是否是唾液酸结合粘附素家族的成员。这个 ASAA同源基因在血小板结合中的作用将被研究，重点是S.mitis。目标3：确定 使用3D人体微血管模型研究ASAA，特别是唾液酸结合对IE的贡献。一次切割- 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结构域在植被的建立上。此外，实验将确定 这种黏附机制是否可以成为治疗的靶点。此应用程序具有重要意义，因为它侧重于 以前没有描述过结合血小板机制的细菌，这是发展的关键一步 亚急性IE。该提议的创新方面包括定义新的类Siglec结构域的功能和 利用尖端的3D微血管模型。这项研究还可能确定一个新的唾液酸家族- 由多个IE引起的物种产生的结合粘附素，可能与SRRP结合相同的受体 使这些互动成为一个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.