Platelet interactive domains of Streptococcus gordonii surface protein PadA

Oral commensal bacterium Streptococcus gordonii is a causative agent of cardiac disease infective endocarditis. These pathogenic capabilities are due, in part, to the ability of S. gordonii to bind and activate platelets. PadA was recently identified as a platelet-interactive protein of S. gordonii, but little is known regarding its functional domains. Furthermore, such studies have been performed under static conditions, but it is increasingly evident that shear forces in vivo may influence these interactions. Objectives: This study aimed to determine the roles of potential platelet-recognition amino acid residue motifs AGD and RGT of PadA in platelet adhesion and activation, and to assess such interactions under static or shear conditions. Methods: Alanine substitutions of AGD/RGT motifs in PadA were generated by site-directed mutagenesis, and recombinant forms of these proteins (rPadA) were expressed and purified.  Platelet adhesion to rPadA fragments was measured under static (p-nitrophenol phosphate assay) versus flow (parallel flow perfusion chamber) conditions.  Platelet activation (dense granule secretion) by rPadA proteins was measured by luciferase assay. Results: Alanine-substituted rPadA proteins supported static platelet adhesion at levels comparable to wild-type. No rPadA protein was able to bind platelets under flow but, once platelets were bound, all rPadA proteins maintained this attachment at shear forces of 50 s^-1. Wild-type rPadA induced platelet activation. This was significantly impaired upon deletion of both AGD and RGT motifs. Conclusion: These data suggest that PadA works in concert with other S. gordonii surface proteins to mediate firm platelet adhesion under flow. Furthermore, PadA motifs AGD and RGT have been shown to play a significant role in platelet activation.  PadA-like proteins are found in a number of microorganisms. Thus, determining the molecular basis of PadA-platelet interactions may enable such mechanisms to be targeted in the management of infection-induced cardiovascular disease. Supported by Wellcome Trust WT097285MA awarded to JH.