The dimerization interface in VraR is essential for induction of the cell wall stress response in Staphylococcus aureus: a potential druggable target

Ghazal Tajbakhsh,Dasantila Golemi-Kotra

doi:10.1186/s12866-019-1529-0

Abstract

BackgroundStaphylococcus aureus remains a medical challenge in the treatment of bacterial infections. It has acquired resistance to commonly used antibiotics, and to those considered to be the last weapons in treating staphylococcal infections, such as vancomycin. Studies have revealed that S. aureus is capable of mounting a rapid response to antibiotics that target cell wall peptidoglycan biosynthesis, such as β-lactams and vancomycin. The two-component system VraSR has been linked to the coordination of this response. VraS is a histidine kinase that undergoes autophosphorylation in the presence of signals elicited upon cell wall damage and it then transfers its phosphoryl group to VraR. VraR is a response regulator protein that functions as a transcription factor. Phosphorylation of VraR leads to its dimerization, which is required for optimum binding to its target promoters. Two-component systems have been targeted for the development of antibacterial agents. Deletion of the vraS or vraR gene has been shown to re-sensitize S. aureus to β-lactams and vancomycin.ResultsIn this study, we explored perturbation of the VraR phosphorylation-induced activation as a means to inhibit the VraSR-mediated signal transduction pathway. We show that dimerization of VraR is essential for the phosphorylation-induced activation of VraR. A single point mutation in the dimerization interface of VraR, in which Met13 was replaced by Ala, led to the inability of VraR to dimerize and to bind optimally to the target promoter. The consequences of these in vitro molecular deficiencies are equally dramatic in vivo. Complementation of a vraR deletion S. aureus strain with the vraRM13Ala mutant gene failed to induce the cell wall stress response.ConclusionsThis study highlights the potential of targeting the phosphorylation-induced dimerization of VraR to disrupt the S. aureus cell wall stress response and in turn to re-sensitize S. aureus to β-lactams and vancomycin.

Highlights

Staphylococcus aureus remains a medical challenge in the treatment of bacterial infections
Phosphorylation of VraRM13A Phosphorylation of Vancomycin-resistance-associated response regulator protein (VraR) and VraRM13A by a small molecule donor such as acetyl phosphate was assessed by Phos-tagTM Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE): the un-phosphorylated and phosphorylated protein species were separated by the SDS-PAGE as a result of the phosphorylated species mobility being slowed down by the Phostag reagent in the SDS-PAGE (Fig. 3)
The protein bands, stained with coomassie blue, were quantified by ImageJ (NIH). These experiments showed that after 45 min of incubation the VraRM13A variant underwent about 10% phosphorylation compared to 73% of phosphorylation measured for VraR

Summary

Introduction

Staphylococcus aureus remains a medical challenge in the treatment of bacterial infections. Studies have revealed that S. aureus is capable of mounting a rapid response to antibiotics that target cell wall peptidoglycan biosynthesis, such as β-lactams and vancomycin. VraS is a histidine kinase that undergoes autophosphorylation in the presence of signals elicited upon cell wall damage and it transfers its phosphoryl group to VraR. Two-component signaling systems (TCSs) are prevalent in bacteria [1, 2] They enable coupling of a diverse array of adaptive responses to environmental stimuli including antibiotic stress [1, 3]. Their absence in high eukaryotic systems makes them a prime target for development of novel antimicrobial agents [4]. About 60% of all identified RRs act as transcription factors [5]

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