Recognition of a Streptococcus sanguis Platelet-interactive Protein

Interaction between oral streptococci and human platelets is thought to be central to the colonisation of vegetations in the pathogenesis of infective endocarditis. We have previously shown that platelet GPIb acts as an adhesion receptor for Streptococcus sanguis but the bacterial adhesin involved has not been reported. Objective: to identify the bacterial molecule that interacts with platelet GPIb. Methods: S.sanguis 133-79 cells were collected at different phases of growth and digested with mutanolysin (250 units/ml) for 1h at 37°C. A portion of each extract was labelled with N-hydroxysuccinimide-biotin. Soluble GPIb was prepared from platelets by intrinsic calpain cleavage followed by wheatgerm agglutinin affinity chromatography, and sepharose CL-6B chromatography. Western blots of GPIb were probed with the biotin-labelled S.sanguis extracts, the GPIb-band was then excised, boiled in SDS-sample buffer to split the GPIb-and interactive protein and again Western blotted. Blots were probed with avidin-conjugated horseradish peroxidase. A portion of unlabelled S.sanguis extract was passed through a GPIb-affinity matrix and eluted with 0.1M glycine-HCl pH 2.5. The eluate was examined by SDS-PAGE. Results: The ligand blotting experiments revealed a GPIb-interactive protein of 58kDa in extracts of stationary phase cells only, while a similar sized protein (60.2kDa) was recovered from the GPIb affinity matrix. In both cases the yield of protein was low and it did not appear to stain with silver or Coomassie blue. Conclusion: A 59kDa protein or fragment of a protein from S.sanguis binds directly to GPIb. This protein may act as the major adhesin for this organism to platelets.