Site-specific mutation of Staphylococcus aureus VraS reveals a crucial role for the VraR-VraS sensor in the emergence of glycopeptide resistance

Article Article scientifique Article Site-specific mutation of Staphylococcus aureus VraS reveals a crucial role for the VraR-VraS sensor in the emergence of glycopeptide resistance eng 2011 author Galbusera Elena author Renzoni Adriana Maria 134435 author Andrey Diego Olivier 0000-0003-3247-9274 542856 author Monod Antoinette 22547 author Barras Christine 26040 author Tortora Paolo author Polissi Alessandra 27026 author Kelley William 28396 1008-1020 10.1128/AAC.00720-10 21173175 0066-4804 An initial response of Staphylococcus aureus to encounter with cell wall-active antibiotics occurs by transmembrane signaling systems that orchestrate changes in gene expression to promote survival. Histidine kinase two-component sensor-response regulators such as VraRS contribute to this response. In this study, we examined VraS membrane sensor phosphotransfer signal transduction and explored the genetic consequences of disrupting signaling by engineering a site-specific vraS chromosomal mutation. We have used in vitro autophosphorylation assay with purified VraS[64-347] lacking its transmembrane anchor region and tested site-specific kinase domain histidine mutants. We identified VraS H156 as the probable site of autophosphorylation and show phosphotransfer in vitro using purified VraR. Genetic studies show that the vraS(H156A) mutation in three strain backgrounds (ISP794, Newman, and COL) fails to generate detectable first-step reduced susceptibility teicoplanin mutants and severely reduces first-step vancomycin mutants. The emergence of low-level glycopeptide resistance in strain ISP794, derived from strain 8325 (ΔrsbU), did not require a functional σ(B), but rsbU restoration could enhance the emergence frequency supporting a role for this alternative sigma factor in promoting glycopeptide resistance. Transcriptional analysis of vraS(H156A) strains revealed a pronounced reduction but not complete abrogation of the vraRS operon after exposure to cell wall-active antibiotics, suggesting that additional factors independent of VraS-driven phosphotransfer, or σ(B), exist for this promoter. Collectively, our results reveal important details of the VraRS signaling system and predict that pharmacologic blockade of the VraS sensor kinase will have profound effects on blocking emergence of cell wall-active antibiotic resistance in S. aureus. Anti-Bacterial Agents/pharmacology Bacterial Proteins/genetics/metabolism Blotting, Northern DNA-Binding Proteins/genetics/metabolism Glycopeptides/pharmacology Microbial Sensitivity Tests Mutation Polymerase Chain Reaction Staphylococcus aureus/drug effects/genetics/metabolism Dewey 616 54 Département de microbiologie et médecine moléculaire 21B false 63 Acquisition et expression de facteurs de virulence chez Staphylococcus aureus 86 19 Glycopeptide résistance 28 Antimicrobial agents and chemotherapy 55 3 020c6129-b114-496e-a69d-a09f51814789 Article (Published version) Restricted 2011_Galbusera_Site-Specific_Antimicrobial.pdf 1010016 application/pdf restrictedAccess Faculté de médecine / Section de médecine fondamentale / Département de microbiologie et médecine moléculaire

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Site-specific mutation of Staphylococcus aureus VraS reveals a crucial role for the VraR-VraS sensor in the emergence of glycopeptide resistance	Antimicrobial agents and chemotherapy			2011	733	0

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