AbstractBackground The ability of the oral bacterium Streptococcus gordonii to bind platelets and extracellular matrix (ECM) contributes to its virulence in infective endocarditis. Surface protein PadA has recently been found to be crucial for platelet activation. The hypothesis is that PadA is dependent upon another surface protein (Hsa) for S gordonii to activate platelets and adhere to ECM. We aimed to determine the respective roles of Hsa and PadA in platelet adhesion, and ascertain PadA function in ECM binding. Methods S gordonii DL1 ΔpadA and ΔpadAΔhsa knockout mutants were generated by allelic replacement. Mutants were complemented using PadA or Hsa expression plasmids under the control of a nisin-inducible promoter. PadA expression by knockout and knockin strains was confirmed by western immunoblot of cell-wall protein extracts. Platelet adhesion to bacteria was measured under static conditions in a p-nitrophenol assay. Bacterial adhesion to ECM proteins was determined by crystal violet assay. Findings Static platelet adhesion by S gordonii ΔpadA mutant was reduced by 30% compared with wild-type. ΔpadAΔhsa was more than 80% reduced in binding platelets. Expression of padA in ΔpadAΔhsa failed to restore any platelet adhesion, whereas expression of hsa in ΔpadAΔhsa mutant restored binding to 70% of wild-type levels. The ΔpadA mutant cells were reduced in binding cellular fibronectin by 25% and vitronectin by 60%. Deletion of hsa abrogated vitronectin binding. Complementation of ΔpadAΔhsa with either hsa or padA alone did not restore vitronectin binding. Interpretation PadA requires the presence of Hsa to interact with platelets. PadA has a minor role in binding cellular fibronectin alongside other surface adhesins. In vitronectin binding, Hsa requires the presence of functional PadA for efficient binding. These results suggest that the S gordonii surface-anchored proteins Hsa and PadA work in concert to mediate processes relevant to host colonisation and pathogenesis. Funding Wellcome Trust (grant WT097285MA awarded to JH).
Bibliographical noteArticle type: Conference Poster abstract
Special Issue: Spring Meeting for Clinician Scientists in Training 2017