The VanS sensor histidine kinase from type-B VRE recognizes vancomycin directly.

V ancomycin- r esistant e nterococci (VRE) are among the most common causes of nosocomial infections and have been prioritized as targets for new therapeutic development. Many genetically distinct types of VRE have been identified; however, they all share a common 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 or its effects; it then transduces this signal to the VanR transcription factor, thereby alerting the organism to the presence of the antibiotic. 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 a purified VanRS system from one of the most clinically prevalent forms of VRE, type B. We show that in a native-like membrane environment, 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.

Significance statement: When v ancomycin- r esistant e nterococci (VRE) sense the presence of vancomycin, they remodel their cell walls to block antibiotic binding. This resistance phenotype is controlled by the VanS protein, a histidine kinase that senses the antibiotic or its effects and signals for transcription of resistance genes. However, the mechanism by which VanS detects the antibiotic has remained unclear, with no consensus emerging as to whether the protein interacts directly with vancomycin, or instead detects some downstream consequence of vancomycin's action. Here, we show that for one of the most clinically relevant types of VRE, type B, VanS is activated by direct binding of the antibiotic. Such mechanistic insights will likely prove useful in circumventing vancomycin resistance.