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The msaABCR operon regulates resistance in vancomycin-intermediate Staphylococcus aureus strains.

ABSTRACT: Vancomycin-intermediate Staphylococcus aureus (VISA) strains present an increasingly difficult problem in terms of public health. However, the molecular mechanism for this resistance is not yet understood. In this study, we define the role of the msaABCR operon in vancomycin resistance in three clinical VISA strains, i.e., Mu50, HIP6297, and LIM2. Deletion of the msaABCR operon resulted in significant decreases in the vancomycin MIC (from 6.25 to 1.56 ?g/ml) and significant reductions of cell wall thickness in strains Mu50 and HIP6297. Growth of the mutants in medium containing vancomycin at concentrations greater than 2 ?g/ml resulted in decreases in the growth rate, compared with the wild-type strains. Mutation of the msaABCR operon also reduced the binding capacity for vancomycin. We conclude that the msaABCR operon contributes to resistance to vancomycin and cell wall synthesis in S. aureus.

SUBMITTER: Samanta D

PROVIDER: S-EPMC4249426 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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The msaABCR operon regulates resistance in vancomycin-intermediate Staphylococcus aureus strains.

Samanta Dhritiman D Elasri Mohamed O MO

Antimicrobial agents and chemotherapy 20140825 11

Vancomycin-intermediate Staphylococcus aureus (VISA) strains present an increasingly difficult problem in terms of public health. However, the molecular mechanism for this resistance is not yet understood. In this study, we define the role of the msaABCR operon in vancomycin resistance in three clinical VISA strains, i.e., Mu50, HIP6297, and LIM2. Deletion of the msaABCR operon resulted in significant decreases in the vancomycin MIC (from 6.25 to 1.56 μg/ml) and significant reductions of cell wa ...[more]

PMID: 25155591

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Project description:Resistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin. IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

Project description:BackgroundPersister cells comprise a phenotypic variant that shows extreme antibiotic tolerance resulting in treatment failures of bacterial infections. While this phenomenon has posed a great threat in public health, mechanisms underlying their formation in Staphylococcus aureus remain largely unknown. Increasing evidences of the presence of persister cells in recalcitrant infections underscores the great urgency to unravel the mechanism by which these cells develop. Previously, we characterized msaABCR operon that plays roles in regulation of virulence, biofilm development and antibiotic resistance. We also characterized the function of MsaB protein and showed that MsaB is a putative transcription factor that binds target DNA in response to nutrients availability.ResultsIn this study, we compared the number of persister cell in wild type, msaABCR deletion mutant and the complemented strain in two backgrounds USA300 LAC and Mu50. Herein, we report that msaABCR deletion mutant forms significantly less number of persister cells relative to wild type after challenge with various antibiotics in planktonic and biofilm growth conditions. Complementation of the msaABCR operon restored wild type phenotype. Combined antibiotic therapy along with msaABCR deletion significantly improves the killing kinetics of stationary phase and biofilm S. aureus cells. Transcriptomics analysis showed that msaABCR regulates several metabolic genes, transcription factors, transporters and enzymes that may play role in persister cells formation, which we seek to define in the future.ConclusionsThis study presented a new regulator, msaABCR operon, that is involved in the persister cells formation, which is a poorly understood in S. aureus. Indeed, we showed that msaABCR deletion significantly reduces the persister cells formation in all growth phases tested. Although, we have not yet defined the mechanism, we have shown that msaABCR regulates several metabolic, transporters, and extracellular proteases genes that have been previously linked with persister cells formation in other bacterial systems. Taken together, this study showed that inactivation of the msaABCR operon enhances the effectiveness of antibiotics for the treatment of S. aureus infections, especially in context of persister cells.

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The msaABCR operon regulates resistance in vancomycin-intermediate Staphylococcus aureus strains.

Publications

The msaABCR operon regulates resistance in vancomycin-intermediate Staphylococcus aureus strains.

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