The antibiotic sensing mechanism of VanS from type-B vancomycin-resistant enterococci

Vancomycin-resistant Enterococci (VRE) are able to sense the presence of vancomycin and subsequently activate resistance against the antibiotic. This activation mechanism is regulated by VanS, a membrane-bound sensor histidine kinase. In the presence of vancomycin, VanS initiates a signaling cascade that leads to the expression of resistance genes in VRE. However, the molecular details of how VanS senses vancomycin remain a mystery. Vancomycin sensing could involve direct binding to the antibiotic, whether by VanS or adaptor proteins that transmit the signal to VanS. Thus, identification of vancomycin-binding proteins will unravel the mechanisms by which VRE sense the antibiotic. To this end, we created diazirine-based photoprobes that specifically label known vancomycin-binding proteins. We also developed an anti-vancomycin antibody, allowing for the direct detection of photolabeled protein without the need for a reporter tag. Using these photoprobes and solution-based assays, we demonstrated that vancomycin directly binds to the periplasmic sensor domain of VanS from type-B VRE (VanSB). Computational models and HDX-MS results indicate that the VanSB sensor adopts a PAS-like fold. An extensive buffer screen was implemented to find conditions that stabilize the VanSB sensor, allowing for structural studies by NMR. Together, this work demonstrates that VanSB directly binds to vancomycin to activate resistance in type-B VRE. Also, our novel photoprobes will be useful tools for elucidating vancomycin-sensing mechanisms in other pathogens.