Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida.
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 inFrancisella novicidaclones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that thein vitroFQ 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 inF. novicidaand pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition inF. novicidais 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-ΔK488 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 inFrancisella.
TIMC-IMAG UMR 5525, UGA CNRS, Grenoble, France.|Centre Hospitalier Universitaire Grenoble-Alpes, Centre National de Référence des Francisella, CS 10217, Grenoble, France.
Yvan
Y
Caspar
TIMC-IMAG UMR 5525, UGA CNRS, Grenoble, France.|Centre Hospitalier Universitaire Grenoble-Alpes, Centre National de Référence des Francisella, CS 10217, Grenoble, France.
Claire
C
Siebert
Centre Hospitalier Universitaire Grenoble-Alpes, Centre National de Référence des Francisella, CS 10217, Grenoble, France.
Vivien
V
Sutera
TIMC-IMAG UMR 5525, UGA CNRS, Grenoble, France.|Université de Caen Normandie, EA4655 U2RM (Unité de Recherche Risques Microbiens), Caen, France.
Corinne
C
Villers
Sorbonne Université, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, Team 13, INSERM U1135, Paris, France.|AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France.
Alexandra
A
Aubry
Institut Pasteur, Paris, France.|CNRS, UMR 3528, Paris, France.|Université Paris Diderot, Sorbonne Paris Cité, Paris, France.
Claudine
C
Mayer
TIMC-IMAG UMR 5525, UGA CNRS, Grenoble, France.|Centre Hospitalier Universitaire Grenoble-Alpes, Centre National de Référence des Francisella, CS 10217, Grenoble, France.
Max
M
Maurin
TIMC-IMAG UMR 5525, UGA CNRS, Grenoble, France patricia.renesto@univ-grenoble-alpes.fr.
Patricia
P
Renesto
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D003001:Cloning, Molecular; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D027101:DNA Topoisomerase IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D024841:Fluoroquinolones / Q000737:chemistry / Q000494:pharmacology; D005603:Francisella / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones; D011994:Recombinant Proteins; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase
DNA gyrase; Francisella; fluoroquinolones