CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway

Objectives Epithelial colonisation is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilise several host factors to associate with cells, including α5βl integrin and heparan sulphate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process while integrins are only critical for post-adhesion entry into human epithelial cells. Methods and Results Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion whereas blockade of β1 integrins did not affect adhesion but did reduce the subsequent internalisation of adhered bacteria. CD9 disruption or deletion increased β1 integrin-mediated internalisation, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalisation. Conclusions CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organisation to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogues could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. ### Competing Interest Statement PNM, RI and BC are co-inventors on patent WO2021175809A1, related to the peptides used in this study. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.