The histidine kinase VraS orchestrates the cell-wall stress response in Staphylococcus aureus via its direct interaction with glycopeptides and β-lactams

Multidrug-resistant Staphylococcus aureus is a major global health threat, with the VraTSR three-component system playing a key role in sensing and conferring resistance to cell-wall active antibiotics, particularly vancomycin. VraTSR comprises the membrane histidine kinase VraS, the cytoplasmic response regulator VraR, and the uncharacterized membrane protein VraT, which regulate the cell wall stress stimulon. However, the molecular signals sensed by VraTSR remain unknown. To elucidate the activation mechanism of this regulatory system, we investigated interactions with {beta}-lactams and glycopeptides. Using a transcriptional reporter strain, we confirmed VraTSR activation by {beta}-lactams, glycopeptides, a vancomycin-derived photoprobe (VPP), and the previously unreported activators A47934 and moenomycin A. Photo-crosslinking assays with VPP and full-length VraS expressed in membranes revealed a direct interaction with vancomycin, which was further confirmed in purified VraS reconstituted in liposomes. VPP binding was concentration-dependent, saturable, and displaced by vancomycin. Saturation transfer difference (STD) Nuclear Magnetic Resonance (NMR) experiments confirmed vancomycin binding to VraS and demonstrated ampicillin interaction, highlighting the involvement of aryl protons from both antibiotics. These findings establish VraS as a receptor for vancomycin and ampicillin. In contrast, assays with membrane vesicles expressing only VraT or co-expressing VraS/VraT did not show covalent adduct formation between VraT and VPP. While VraTs exact role remains unclear, its participation in antibiotic sensing or signal transduction cannot yet be excluded. These results demonstrate that vancomycin and ampicillin directly activate VraS, providing critical insights into the activation of the cell wall stress stimulon and the mechanisms underlying antibiotic resistance. Disrupting VraTSR signaling is a promising strategy to combat multidrug resistance in S. aureus, and we provide invaluable in vitro platforms for identifying potential VraS inhibitors. Author SummaryMultidrug-resistant Staphylococcus aureus poses a major global health threat due to its resistance to cell-wall active antibiotics. Our study focuses on the VraTSR three-component system, a key regulator of the cell wall stress response in S. aureus, whose activation signals have remained unknown. We demonstrate that VraS, the membrane histidine kinase of the system, acts as a direct receptor for vancomycin and ampicillin--two structurally distinct antibiotics. These findings uncover the activation mechanism of VraTSR and position VraS as a central player in antibiotic sensing and resistance. By identifying VraS as a direct antibiotic receptor, we provide a promising target for developing inhibitors to disrupt VraTSR signaling and restore antibiotic efficacy. Additionally, the in vitro platforms we established enable the identification and testing of potential VraS inhibitors. This study highlights the importance of understanding bacterial stress-response pathways to combat antibiotic resistance, offering critical insights for developing new therapeutic strategies against multidrug-resistant S. aureus, a growing global health challenge.