Vancomycin's interactions with cellular targets drive its antimicrobial activity, and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycin's interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications, as compared to previous photoprobes. Using proteins fused to vancomycin's main cell-wall target, D-alanyl-D-alanine, we use mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying novel vancomycin-binding proteins.
The antibiotic vancomycin is used as a last resort to treat persistent infections caused by Gram‐positive pathogens. Vancomycin kills bacteria by binding a peptidoglycan precursor, thereby inhibiting cell‐wall biosynthesis. An alarming type of resistance to this antibiotic comes in the form of vancomycin‐resistant Enterococci(VRE). VRE have acquired genes that allow them to remodel the cell‐wall precursor and prevent vancomycin binding. Expression of these remodeling genes is under control of the VanSR two‐component system. VanS is a membrane‐bound sensor kinase that recognizes the vancomycin signal, and in response activates the transcription factor VanR, which activates expression of the remodeling genes. However, very little is known about how VanS senses the antibiotic. To date, nine different types of VRE have been discovered, with VanA and VanB types responsible for the vast majority of human infections. Since vancomycin can induce the expression of both VanA and VanB resistance genes, we hypothesize that the VanS proteins from these types are activated by directly binding to vancomycin. We used in vitro autokinase assays to show that vancomycin directly activates VanS from VanB VRE (VanSB), while having no direct effect on VanSA. We isolated the VanSB periplasmic sensor domain and used fluorescence anisotropy to show that it directly binds to a fluorescent vancomycin analog. Computational modeling predicts that the VanSB sensor domain adopts a PAS‐like fold, and HDX‐MS experiments supported this prediction and identified a potential vancomycin‐binding site. We also developed vancomycin photoprobes to confirm this binding site and to elucidate vancomycin’s orientation in the interaction. These results demonstrate how VanSBcan directly sense vancomycin in the environment to activate the resistance mechanism in VanB VRE, providing a promising therapeutic target to combat these dangerous pathogens.
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