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Genetic changes associated with glycopeptide resistance in Staphylococcus aureus: predominance of amino acid substitutions in YvqF/VraSR.

ABSTRACT: To further understand the mechanism of intermediate-level glycopeptide resistance, resulting from multiple endogenous mutations, in both laboratory-derived and clinically isolated Staphylococcus aureus.Laboratory-derived S. aureus strains were generated under selection using a variety of cell-wall-active antibiotics. Complete sequences of 27 genes, including 17 two-component histidine kinase sensors, were then compared with those of their susceptible parent strain. Further genetic analysis was performed on 125 clinical S. aureus isolates and 42 geographically diverse isolates of vancomycin-intermediate S. aureus (VISA).Selective pressure using imipenem resulted in single point mutations leading to amino acid substitutions in two genes: vraS, encoding a two-component histidine kinase sensor; and SA1702 (also called yvqF, located immediately upstream of vraS), encoding a conserved hypothetical protein. The accumulation of the mutation in two distinct proteins-MsrR, a peptide methionine sulphoxide reductase regulator, and TcaA, a teicoplanin-resistance-associated protein-correlated with further increases in the glycopeptide MIC. The prevalence of YvqF/VraSR mutants among 125 clinical isolates along with the corresponding teicoplanin MICs was as follows: 0% (0/39), < or =1 mg/L; 48.6% (17/35), 2 mg/L; 72.7% (24/33), 4 mg/L; 93.8% (15/16), 8 mg/L; and 100% (2/2), 16 mg/L. Genetic analysis of 42 VISA isolates also identified the predominant amino acid substitutions in YvqF/VraS: 9 isolates (21.4%) revealed mutations in YvqF, followed by 7 isolates with mutations in VraS (16.7%).Our findings provide novel insights into the high prevalence and genetic diversity of YvqF/VraSR mutants among clinical S. aureus isolates with reduced susceptibility to teicoplanin.

SUBMITTER: Kato Y

PROVIDER: S-EPMC2800785 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Genetic changes associated with glycopeptide resistance in Staphylococcus aureus: predominance of amino acid substitutions in YvqF/VraSR.

Kato Yoshihisa Y Suzuki Takahisa T Ida Takashi T Maebashi Kazunori K

The Journal of antimicrobial chemotherapy 20100101 1

<h4>Objectives</h4>To further understand the mechanism of intermediate-level glycopeptide resistance, resulting from multiple endogenous mutations, in both laboratory-derived and clinically isolated Staphylococcus aureus.<h4>Methods</h4>Laboratory-derived S. aureus strains were generated under selection using a variety of cell-wall-active antibiotics. Complete sequences of 27 genes, including 17 two-component histidine kinase sensors, were then compared with those of their susceptible parent str ...[more]

PMID: 19889788

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Project description:Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants.IMPORTANCE Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.

Project description:Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to approximately 128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.

Project description:BACKGROUND: To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. RESULTS: In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. CONCLUSION: Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.

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Genetic changes associated with glycopeptide resistance in Staphylococcus aureus: predominance of amino acid substitutions in YvqF/VraSR.

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Genetic changes associated with glycopeptide resistance in Staphylococcus aureus: predominance of amino acid substitutions in YvqF/VraSR.

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