The Histidine Kinase VraS Interacts with Vancomycin and Penicillins Through its Membrane‐Anchored N‐terminal Domain

Abstract Multidrug‐resistant Staphylococcus aureus ( S. aureus ) is a major global health threat, with the VraTSR three‐component system playing a key role in conferring resistance to cell‐wall active antibiotics, through regulation of the cell wall stress stimulon. The molecular signals sensed by VraTSR remain unknown. We investigated interactions of the membrane histidine kinase VraS with β‐lactams and glycopeptides. Photo‐crosslinking assays with a vancomycin‐derived and an ampicillin‐derived photoprobe revealed direct interaction of these two classes of antibiotics with full‐length VraS. Saturation transfer difference (STD) Nuclear Magnetic Resonance experiments confirmed vancomycin, ampicillin and penicillin G binding to VraS, with the involvement of aryl protons from the antibiotics. STD NMR assays with truncated versions of VraS demonstrated that ampicillin and vancomycin bind to the membrane‐anchored N‐terminal region of VraS. In contrast, assays with membrane vesicles expressing only VraT or co‐expressing VraS/VraT did not show covalent adduct formation between VraT and the vancomycin‐derived photoprobe. VraS p‐azido‐L‐phenylalanine mutants demonstrated participation of the extracellular loop of VraS in β‐lactam binding. These results demonstrate that vancomycin and penicillins directly interact with VraS, an interaction that could be involved in activation of the cell wall stress stimulon and the mechanisms underlying antibiotic resistance.