The vancomycin-sensing mechanisms of VanS proteins in vancomycin-resistant enterococci

Elizabeth Josephine D'Lauro

doi:10.17918/00011531

Vancomycin-resistant Enterococci (VRE) are one of the leading causes of Enterococcal infections worldwide. Because vancomycin has been used as a last-resort antibiotic, resistance towards this drug leaves few other treatment options available. It is imperative to understand the mechanism of resistance to develop new ways to fight VRE. Vancomycin binds and sequesters Lipid II, an intermediate in cell-wall synthesis, resulting in cell death. VRE evade cell death via remodeling of Lipid II to decrease vancomycin's binding affinity. This resistance phenotype is regulated by a two-component system, VanSR. VanS is a membrane-bound histidine kinase that senses vancomycin and, in response, phosphorylates VanR. VanR is a transcription factor that, when phosphorylated, directs transcription of several Lipid II-remodeling genes, activating resistance. However, the mechanistic and structural details behind VanS sensing and signal transduction remain unknown. Furthermore, various VRE strains potentially have different mechanisms of activation. With our focus on two of the most clinically prevalent VRE strains, type-A and type-B, we investigated two different methods of antibiotic sensing, indirect and direct. In the case of an indirect mechanism, we questioned whether Lipid II plays a role in modulating the activities of VanS. We reconstituted VanS into nanodiscs, a membrane-like environment, and incorporated Lipid II. We then measured the autophosphorylation and dephosphorylation activities of VanS from type-A (VanSA) and from type-B (VanSB) VRE. We demonstrated that autophosphorylation does not seem to be affected in the presence of Lipid II. However, preliminary data for dephosphorylation of VanSA suggests that Lipid II is potentially stimulating this activity. We also studied a direct sensing mechanism for VanSB. We have previously shown that vancomycin binds directly to the protein. To identify the exact site where vancomycin binds, we used multiple molecular docking programs to predict and dock vancomycin onto the surface of VanS. We then tested these predicted sites in vitro using mutagenesis to see if we can disrupt the protein and ligand binding interaction. Next, we tested these mutants' autophosphorylation activity using in vitro activity assays. With these experiments, we have identified a putative binding site of the antibiotic. Together, these studies will elucidate how VRE are able to sense vancomycin and activate resistance at the molecular level.

The vancomycin-sensing mechanisms of VanS proteins in vancomycin-resistant enterococci

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The vancomycin-sensing mechanisms of VanS proteins in vancomycin-resistant enterococci

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