Staphylococcus aureus, a Gram positive bacteria and a leading cause of hospital acquired infections, has quickly acquired resistance to many antibiotics. Despite being highly researched, all vaccines to date have failed. Adhesins on the surfaces of bacterial pathogens often target glycosaminoglycans which decorate the surfaces of mammalian cells. Heparin is a glycosaminoglycan used widely to mimic the highly sulphated regions of surface proteoglycans. Heparin-binding proteins produced by pathogenic bacteria are important factors that aid in adherence, colonisation and invasion of epithelial cells and were characterised as part of a larger study to better understand the S. aureus’ pathogenic mechanisms.
The surfacome of S. aureus (SH1000) was characterised using two methodologies: (1)surface proteins were tagged with biotin and captured by streptavidin affinity chromatography and (2)by cell surface protein trypsin shaving. 2D SDS-PAGE was utilised to separate protein isoforms and proteolytic cleavage products from whole cell lysates and surface biotinylated proteins. Unfractionated whole cell lysates were used to identify proteins that bound heparin in vitro via heparin-agarose affinity chromatography. Dimethyl labelling and LC-MS/MS were used to identify neo-N-termini. Proteins were identified by LC-MS/MS.
Using a combination of approaches we show that Ef-Tu was identified on the surface of S. aureus, both as an intact protein, and as cleaved fragments. We mapped the precise site of cleavage by characterising tryptic peptides that were dimethyl-labelled on the N-terminus, and by mapping fragments separated by 2D-SDS-PAGE. Some of these cleavage events process Ef-Tu into its sub-domains, supporting the hypothesis that processing increases functional proteome diversity. Both intact Ef-Tu and several cleavage fragments of Ef-Tu bound to heparin-agarose and were eluted by NaCl concentrations significantly above physiological levels. 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 as adhesins.