Staphylococcus aureus is a leading cause of nosocomial-acquired infections and has rapidly acquired resistance to multiple antibiotics. Pathogenic and commensal bacteria present multifunctional surface adhesins that bind to a wide range of host molecules, particularly those associated with the extracellular matrix. Glycosaminoglycans are components of the extracellular matrix (ECM) that are targeted by bacterial adhesins. Heparin is a glycosaminoglycan that mimics highly sulphated regions of proteoglycans and it has been used as a bait to identify bacterial virulence factors that facilitate adherence, colonisation and invasion of target host cells.  

To characterise the surfacome of S. aureus we tagged surface proteins with biotin and identified them by streptavidin affinity chromatography and LC-MS/MS. A complimentary set of surface proteins were identified by shaving S. aureus with trypsin. Dimethyl labelling of neo-N-termini followed by LC-MS/MS was used to identify cleavage start sites. Unfractionated native WCL were used to identify heparin-binding proteins via heparin-agarose chromatography. We applied this technique to gain insights into the repertoire of S. aureus surface proteins that have evolved the ability to bind highly sulphated glycosaminoglycans and correlated the identities of these proteins with those from our surfacome studies.

318 proteins were identified on the surface of SH1000. Notably, Ef-Tu was identified as an intact protein and as cleaved fragments and both the intact molecule and the cleavage fragments bound heparin. Dimethyl-labelling studies of neo-N-termini enabled us to determine precise sites of cleavage within Ef-Tu. For the first time, these data suggest that Ef-Tu and processed fragments of Ef-Tu can perform alternate functions on the surface of S. aureus. Our studies indicate that cleavage events target a region of the molecule that separates the major subdomains within Ef-Tu, suggesting that processing may function to increase functional diversity.