Unveiling the mechanism of activation of the vraSRT system of Staphylococcus aureus by β-lactam antibiotics using photoactive compounds

Abstract

Staphylococcus aureus is the leading cause of nosocomial and community-acquired infections. The vraSRT system acts as a sentinel that can rapidly sense cell wall peptidoglycan damage and coordinate a response that leads to resistance to β-lactam and glycopeptide antibiotics. VraS is a membrane histidin-kinase and VraR a cytoplasmatic response regulator. However, the rol of VraT, another membrane protein, is yet unknown but essential for the survival of the bacteria. We still do not understand how VraS is activated in response to cell wall-active antibiotics. The interaction between VraS , VraT and dierent ampicillin-derived photo-anity probes was studied. Using a S. aureus reporter strain, which has a shuttle vector that allows expression of GFP under the control of the vraSRT operator region, we conrmed that the ampicillin photoprobes eectively activate the vraSRT system. The photo-anity probes were used for covalent labeling of VraS and VraT in E. coli BL21 Star DE3 spheroplasts. An interaction with VraS was evidenced by a shift in the electrophoretic mobility of the protein. MALDI-TOF/TOF analysis of the puried VraS-photoprobe complexes did not allow the identication of the site of crosslinking. We hypothesized that β-lactams could interact with the extracellular loop of VraS, a peptide not detected by MALDI-TOF/TOF. Hence, we introduced photoactive phenylalanine residues in that loop of VraS and evaluated labeling with the uorescent penicillin Bocillin-FL. No uorescent VraS was detected which indicated no direct interaction of the antibiotic with this loop. VraT has an extracellular C-terminal domain, as determined in a Proteinase K susceptibility assay, which does not interact directly with the ampicillin photoprobes. In conclusion, VraS interacts directly with β-lactams but its extracellular loop is not involved in the recognition. VraT participation in activation of the system is not as a receptor of the antibiotic.