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