Vancomycin does not affect the enzymatic activities of purified VanSA

Abstract

VanS is a membrane-bound sensor histidine kinase responsible for sensing vancomycin and activating transcription of vancomycin-resistance genes. In the presence of vancomycin, VanS phosphorylates the transcription factor VanR, converting it to its transcriptionally active form. In the absence of vancomycin, VanS dephosphorylates VanR, thereby maintaining it in a transcriptionally inactive state. To date, the mechanistic details of how vancomycin modulates VanS activity have remained elusive. We have therefore studied these details in an in vitro system, using the full-length VanS and VanR proteins responsible for type-A vancomycin resistance in enterococci. Both detergent- and amphipol-solubilized VanSA display all the enzymatic activities expected for a sensor histidine kinase, with amphipol reconstitution providing a marked boost in overall activity relative to detergent solubilization. A putative constitutively activated VanSA mutant (T168K) was constructed and purified, and was found to exhibit the expected reduction in phosphatase activity, providing confidence that detergent-solubilized VanSA behaves in a physiologically relevant manner. In both detergent and amphipol solutions, VanSA’s enzymatic activities were found to be insensitive to vancomycin, even at levels many times higher than the antibiotic’s minimum inhibitory concentration. This result argues against direct activation of VanSA via formation of a binary antibiotic-kinase complex, suggesting instead that either additional factors are required to form a functional signaling complex, or that activation does not require direct interaction with the antibiotic.

Molecular mechanisms of vancomycin resistance

“…Another study further supported this model demonstrating that vancomycin induced significant thermostability and conformational changes in purified VanS A, while other potential signals such as the peptidoglycan components Ala‐ d ‐γ‐Glu‐Lys‐ d ‐Ala‐ d ‐Ala, N ‐acetylmuramic acid or d ‐Ala‐ d ‐Ala had no such effect . However, other studies contested this model, reporting the lack of a VanS A ‐vancomycin complex even at high concentrations of vancomycin and suggesting that the enzymatic activities of detergent or amphipol‐solubilized VanS A are not altered in the presence of vancomycin . A possible explanation of the contradictory results of these latter studies can be due to the VanS A solubilization using detergents, which are known to affect the function of sensor kinase membrane proteins .…”

“…In the absence of the signal, VanS catalyzes dephosphorylation of VanR suggesting that this protein also possesses phosphatase activity . Direct interaction between VanS A and VanR A supposedly through the cytosolic domain of VanS A has been reported …”

“…52 However, other studies contested this model, reporting the lack of a VanS A -vancomycin complex even at high concentrations of vancomycin and suggesting that the enzymatic activities of detergent or amphipol-solubilized VanS A are not altered in the presence of vancomycin. 50 A possible explanation of the contradictory results of these latter studies can be due to the VanS A solubilization using detergents, which are known to affect the function of sensor kinase membrane proteins. 50,53 Furthermore, the in vitro experimental setup in these studies also lacks the target of vancomycinthe D-Ala-D-Ala group of lipid II, and thus do not necessarily reflect the natural environment of VanS wherein the protein could form a proposed VanS Alipid II-vancomycin complex.…”

“…50 A possible explanation of the contradictory results of these latter studies can be due to the VanS A solubilization using detergents, which are known to affect the function of sensor kinase membrane proteins. 50,53 Furthermore, the in vitro experimental setup in these studies also lacks the target of vancomycinthe D-Ala-D-Ala group of lipid II, and thus do not necessarily reflect the natural environment of VanS wherein the protein could form a proposed VanS Alipid II-vancomycin complex. 54 In an attempt to minimize the effect of detergent, the S. coelicolor VanS (VanSSc) was recombinantly expressed and purified from S. coelicolor and E. coli membranes and shown to bind a photo-labeled vancomycin derivative.…”

“…18,49 Direct interaction between VanS A and VanR A supposedly through the cytosolic domain of VanS A has been reported. 50 The earliest model of VanS A activity was that it senses the presence of vancomycin through a direct interaction between this molecule at the protein's extracellular face. Recognition of vancomycin would trigger VanS A dimerization, autophosphorylation, and transfer of the phosphoryl group to VanR A .…”