Mechanism of signal transduction of VraSR: A mediator of antibiotic resistance in Staphylococcus aureus

Staphylococcus aureus is a pathogen of greatest concern due to the emergence of antibiotic resistant strains. It has been discovered that S. aureus response to antibiotics that target cell-wall biosynthesis such as beta-lactams and vancomycin is coordinated by a two-component regulatory system named VraSR. This study provides for the first time insights on the mechanism of signal transduction of VraSR system. We have shown that the cytoplasmic portion of VraS catalyzes an ATP-dependent autophosphorylation and subsequently donates the high energy phosphoryl group to its response regulator VraR in a rapid phosphotransfer reaction. The acceptor of the migrating phosphoryl group is Asp 55 located within regulatory domain of VraR. We have demonstrated that the event of phosphorylation triggers conformational changes in VraR that result in formation of VraR phosphorylated dimers which were established as biologically active species. Most importantly, VraR utilizes a different strategy to regulate the output response of the VraSR signaling pathway. The effector domain prevents dimerization in unphosphorylated VraR by holding the N-terminal domain in a conformation that is capable of catalyzing rapid phosphotransfer from VraS phosphate. VraS on the other hand tightly controls the phosphorylation state of VraR by its kinase and phosphatase activities and in this way facilitates rapid switch on and off of the response of S. aureus to the cell wall damage. Moreover, we have provided first evidence for the transcriptional activity of VraR by investigating its interaction with vraSR operon control region. We have shown that VraR binds to three distinct sites each with unique features. VraR-binding to the most conserved site is phosphorylation independent and dimerization of the protein was proposed to be induced upon binding to DNA. However, phosphorylation is required for VraR-binding to the secondary site that overlaps with the---35 region of the promoter. By using independent DNA-binding assays we have demonstrated that phosphorylated VraR is involved in recruitment of RNAP at the vraSR promoter. Under conditions of cell wall stress activated VraR is able to stimulate high level of transcription of the genes from vraSR operon.