Vancomycin-resistant enterococci (VRE) are among the most common causes of nosocomial infections, which can be challenging to treat. VRE have acquired a suite of resistance genes that function together to confer resistance to vancomycin. Expression of the resistance phenotype is controlled by the VanRS two-component system. This system senses the presence of the antibiotic, and responds by initiating transcription of resistance genes. VanS is a transmembrane sensor histidine kinase, and plays a fundamental role in antibiotic resistance by detecting vancomycin and then transducing this signal to VanR. Despite the critical role played by VanS, fundamental questions remain about its function, and in particular about how it senses vancomycin. Here, we focus on purified VanRS systems from the two most clinically prevalent forms of VRE, types A and B. We show that in a native-like membrane environment, the enzymatic activities of type-A VanS are insensitive to vancomycin, suggesting that the protein functions by an indirect mechanism that detects a downstream consequence of antibiotic activity. In contrast, the autokinase activity of type-B VanS is strongly stimulated by vancomycin. We additionally demonstrate that this effect is mediated by a direct physical interaction between the antibiotic and the type-B VanS protein, and localize the interacting region to the protein's periplasmic domain. This represents the first time that a direct sensing mechanism has been confirmed for any VanS protein.
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.
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.
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