CONICET | Buscador de Institutos y Recursos Humanos

Staphylococcus aureus is the leading cause of nosocomial andcommunity-acquired infections. The vraSRT system acts as a sentinel that canrapidly sense cell wall peptidoglycan damage and coordinate a response thatleads to resistance to â-lactam and glycopeptide antibiotics. VraS is amembrane histidin-kinase and VraR a cytoplasmatic response regulator. However,the rol of VraT, another membrane protein, is yet unknown but essential for thesurvival of the bacteria. We still do not understand how VraS is activated inresponse to cell wall-active antibiotics. The interaction between VraS, VraTand different ampicillin-derived photo-affinity probes was studied. Using a S.aureus reporter strain, which has a shuttle vector that allows expression ofGFP under the control of the vraSRT operator region, we confirmed that theampicillin photoprobes effectively activate the vraSRT system. Thephoto-affinity probes were used for covalent labeling of VraS and VraT in E.coli BL21 Star DE3 spheroplasts. An interaction with VraS was evidenced by ashift in the electrophoretic mobility of the protein. MALDI-TOF/TOF analysis ofthe purified VraS-photoprobe complexes did not allow the identification of thesite of crosslinking. We hypothesized that â-lactams could interact with theextracellular loop of VraS, a peptide not detected by MALDI-TOF/TOF. Hence, weintroduced photoactive phenylalanine residues in that loop of VraS andevaluated labeling with the fluorescent penicillin Bocillin-FL. No fluorescentVraS was detected which indicated no direct interaction of the antibiotic withthis loop. VraT has an extracellular C-terminal domain, as determined in aProteinase K susceptibility assay, which does not interact directly with theampicillin photoprobes. In conclusion, VraS interacts directly with â-lactamsbut its extracellular loop is not involved in the recognition. VraTparticipation in activation of the system is not as a receptor of theantibiotic.