Platelet-interactive domains of Streptococcus gordonii surface protein PadA

Abstract

BackgroundThat the oral commensal bacterium Streptococcus gordonii causes infective endocarditis is due partly to its ability to bind and activate platelets. PadA was recently identified as a platelet-interactive protein on S gordonii, but little is known about its functional domains. Furthermore, platelet-interactive studies have often been done under static conditions, but it is increasingly evident that shear forces in vivo can affect these interactions. This study aimed to determine the roles of potential platelet-recognition aminoacid residue motifs AGD and RGT of PadA in platelet adhesion and activation, and to assess such interactions under static or shear conditions. MethodsAlanine substitutions of AGD/RGT motifs within the N-terminal region of 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 conditions (p-nitrophenol phosphate assay) compared with flow conditions (parallel flow perfusion chamber). Platelet activation (dense granule secretion) by rPadA proteins was measured by luciferase assay. FindingsAlanine-substituted rPadA proteins supported static platelet adhesion at concentrations comparable with wild-type rPadA. None of the rPadA proteins was able to bind platelets under flow. However, when platelets were bound under static conditions, all rPadA proteins maintained this attachment at shear forces of 50 s−1. Wild-type rPadA induced platelet activation—eg, spreading, dense granule release. These measures of activation were significantly impaired upon deletion of both AGD and RGT motifs (p<0·05). InterpretationThese data suggest that PadA acts with other S gordonii surface proteins to mediate firm platelet adhesion under flow. The motifs AGD and RGT, found also within fibrinogen, have a significant role in PadA-mediated platelet activation. Since PadA-like proteins are found across a range of microorganisms, determination of the molecular basis of PadA-platelet interactions could enable such mechanisms to be targeted in the management of infection-induced cardiovascular disease. FundingWellcome Trust.