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

Abstract

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Cadependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR 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 of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR 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 in mprF, vraSR contributes to DAP resistance in the present group of clinical strains. Staphylococcus aureus is the most common Gram-positive pathogen among skin and soft tissue infections (2). Methicillin resistance in S. aureus is mediated by the acquisition of a penicillin-binding protein (PBP), PBP 2a, which has decreased affinity for ␤-lactam antibiotics but can continue to cross-link the cell wall once the native PBPs (i.e., PBPs 1 to 4) have been inactivated (23). A distinctive feature for most methicillin-resistant S. aureus (MRSA) strains is the heterogeneous expression of resistance, characterized by a small proportion (Յ0.1%) of the population expressing a high level of homogeneous resistance while most of the other isolates in the population express resistance to 10 g/ml (12, 15, 43). Daptomycin (DAP) is a cyclic anionic lipopeptide antibiotic that is produced by Streptomyces roseosporus (3) and is approved for treatment of skin and skin structure infections as well as treatment of bacteremia and right-side endocarditis caused by MRSA (1). The mechanism of action involves disruption of cytoplasmic membrane function, resulting in depolarization and cell death due to disruption of critical metabolic functions, such as protein, DNA, and RNA synthesis (2).The incidence of DAP resistance in clinical isolates is very low, and resistant strains display small increases in MIC (2). The exact m...

Molecular Bases Determining Daptomycin Resistance-Mediated Resensitization to β-Lactams (Seesaw Effect) in Methicillin-Resistant Staphylococcus aureus

“…Decreased susceptibility to DAP in S. aureus has been reported to lead to clinical failures in patients with MRSA deep-site infections, such as endocarditis and abscesses (28)(29)(30). Previously, we identified two major factors that mutually cooperate in the acquisition of DAP resistance; one is related to the cell membrane (mrpF mutations), and the second affects cell wall factors (VraSR) (6). Moreover, we observed that the DAP r phenotype was accompanied by increased susceptibility to OXA, the so-called seesaw effect.…”

“…Sequencing of all PCR amplification products was performed at the Nucleic Acid Research Facility at Genewiz (South Plainfield, NJ). Analysis of the mprF sequence in wild-type strains and mutants was performed by using mprF-specific primers as previously described (6). Consensus sequences were assembled from both orientations with Lasergene (v12) software (DNAStar, Madison, WI).…”

“…First, we recently demonstrated that PrsA, a lipoprotein acting as a posttranslocational chaperone, is involved in ␤-lactam resistance by affecting the amounts of PBP 2a in the CM (26); in addition, prsA expression is regulated by the two-component system VraSR (14). Second, we have shown that acquisition of DAP r involves the upregulation of genes controlling CW synthesis and turnover, including vraSR (6). Unpublished transcriptome RNA sequencing (RNA-Seq) results suggest that the vraSR and prsA genes in the DAP r CB1634 strain are upregulated compared to their level of regulation in the DAP s CB1631 strain, suggesting a link between the mprFL826F mutation present in CB1634 and changes in the expression of both the vraSR and prsA genes.…”

“…To address this, we analyzed PBP 2a protein levels using membrane protein extracts from DAP r CB1634, CB1634 ΔmprF, and CB1634 ΔmprF complemented with either wild-type (WT) mprF or a previously isolated mprF mutant with an L-to-F amino acid change at position 826 (mprFL826F) that is associated with decreased susceptibility to DAP (6). As depicted in Fig.…”

Resistencia a antibióticos de última línea en cocos Gram positivos: la era posterior a la vancomicina

“…Otro sistema regulador de dos componentes relacionado con el desarrollo de la resistencia a la daptomicina es el vraSR (también relacionado con el desarrollo de S. aureus de sensibilidad intermedia a la vancomicina), el cual se ha encontrado sobreexpresado en cepas resistentes a la daptomicina. Aún más, la inactivación de estos genes en dichas cepas revierte la sensibilidad a este antibiótico (101). Por último, los genes vraF (proteína transportadora de tipo ABC) y vraG (proteína transportadora de tipo permeasa) también han sido asociados con el desarrollo de resistencia a la daptomicina en S. aureus (102).…”