Unveiling the mechanism of activation of the VraSRT system of Staphylococcus aureus by vancomycin and peptidoglycan fragments

ANTINORI, M.A.; LLARRULL, L.I.; WRIGHT, G.

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

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 b-lactam and glycopeptide antibiotics. VraS is a membrane histidin-kinase and VraR a cytoplasmatic response regulator. However, the role of VraT, another membrane protein, is yet unknown but essential for manifestation of resistance. We still do not understand how VraS is activated in response to cell wall-active antibiotics. To evaluate whether the system is activated by direct interaction of glycopeptides or of cell-wall derived fragments with either VraS or VraT, a vancomycin-derived photoprobe (VPP) and peptidoglycan fragments were used.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 corroborated that b-lactam and glycopeptide antibiotics activate the VraSRT system. Peptidoglycan from S. aureus ATCC29213 was obtained and subjected to cleavage with Mutanolysin, Lysostaphin or both enzymes to generate different peptidoglycan fragments. Working with the reporter strain, we demonstrated that these fragments do not activate the system.A vancomycin-derived photoprobe (VPP), with a benzophenone photoaffinity label and a biotin linker, was used to evaluate if there was direct interaction of the antibiotic with VraS and/or VraT. VPP activated the VraSRT system and we evidenced formation of a VraSVPP adduct by Western blot. MS/MS analysis of the purified VraS and VraS-VPP complex did not allow identification of the site of crosslinking.In conclusion, the VraSRT system was not activated by peptidoglycan fragments. VraS interacted directly with vancomycin, but the exact site of interaction could not be determined. VraT participation in activation of the system is not as a receptor of the antibiotic.