Characterization of the mechanism of activation of the vraSRT system of Staphylococcus aureus using antiobiotic-derived photoprobes

BELLUZO, B.; MÉNDEZ, L.; LLARRULL, L.I.; ANTINORI, M.; TESTERO, S.A.

Buenos Aires

Congreso; Joint Meeting of Bioscience Societies; 2017

Abstract: Staphylococcus aureus is the leading cause of both nosocomial and community-acquired infections. In S. aureus the vraSRT three-component system acts as a sentinel that can rapidly sense cell wall peptidoglycan damage and coordinate a response that leads to resistance to beta-lactam and glycopeptide antibiotics. VraS and VraR encode a histidin-kinase and a response regulator, respectively. However, the rol of VraT is yet unknown. We still do not understand how VraS is activated in response to cell wall-active antibiotics.The interaction between VraS (the possible sensor), VraT and different photoprobes derived from ampicillin was studied. The photoaffinity probes were used for covalent labelling of VraS and VraT in E. coli spheroplasts, and the interaction was evidenced by an electrophoretic mobility shift in the case of VraS, although no interaction was seen with VraT. We used a S. aureus reporter strain to confirm that the ampicillin-derived photoprobes effectively activate the vraSRT system. The VraS-photoprobe complexes were purified and analyzed by MALDI-TOF/TOF. In this study we also addressed the topology of VraT in membranes. Using a Proteinase K susceptibility assay we determined that the C-terminal domain of VraT has extra-cytoplasmatic location.