VraSR Two-Component Regulatory System Contributes to mprF -Mediated Decreased Susceptibility to Daptomycin in In Vivo -Selected Clinical Strains of Methicillin-Resistant Staphylococcus aureus

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistantStaphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients withS. aureus infections after treatment with DAP. Different point mutations were found in themprF gene in DAP-resistant (DR) strains. Investigation of themprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutatedmprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested thatmprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory systemvraSR . Inactivation ofvraSR resulted in increased DAP susceptibility, while complementation ofvraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact ofvraSR andmprF mutations in the cell wall. Moreover, overexpression ofvraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations inmprF ,vraSR contributes to DAP resistance in the present group of clinical strains.