Dataset Information

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida.

ABSTRACT: Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC50) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-?K488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella.

SUBMITTER: Caspar Y

PROVIDER: S-EPMC5365679 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Publications

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida.

Caspar Yvan Y Siebert Claire C Sutera Vivien V Villers Corinne C Aubry Alexandra A Mayer Claudine C Maurin Max M Renesto Patricia P

Antimicrobial agents and chemotherapy 20170324 4

Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in <i>Francisella novicida</i> clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the <i>in vitro</i> FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were comp ...[more]

PMID: 28167561

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Project description:Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of these strains for humans. This strategy could be extended to other closely-related bacterial species for inter-strain and inter-species analyses.

Dataset Information

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida.

Publications

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida.

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