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 (IC 50 ) 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 in Francisella.

Francisella tularensis : FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation

“…Whether the hypothetical link between fupA/B mutations and ciprofloxacin susceptibility [5] is another specific feature of the fusion gene, or whether it is inherited from fupA and/or fupB ancestors is currently unknown. In agreement with the latter hypothesis, we observed that a highly FQ-resistant mutant of F. novicida (U112_Fno3) bearing a functionally-inert DNA gyrase mutation (GyrA_ΔE524, ΔS525) also had a nonsense mutation in FTN_0444 ( fupA ) [7]. Moreover, fupA was identified as one of seven mutated genes in a ciprofloxacin-resistant mutant of F. tularensis SCHU S4, alongside gyrA and parE [14].…”

“…The topic of antibiotic tolerance is complex, and the resistance of F. tularensis to FQ is definitely not solely a consequence of DNA gyrase mutations [5,7]. The work presented in this paper reveals a new pathway based on FupA/B or FupA alterations through which FQ could drive the emergence of drug resistance.…”

“…as in most Gram-negative bacteria, DNA gyrase is the primary target of FQ [2]. In a previous study [7], using genomic analysis combined with functional DNA supercoiling and cleavage assays, we showed that other mechanisms, independent of GyrA or GyrB mutations also contribute to FQ resistance in F. novicida . Based on careful analysis of both microarrays and next-generation sequencing data, a similar conclusion was drawn with the attenuated F. tularensis subsp.…”

Genetic Determinants of Antibiotic Resistance in Francisella

“…The specific regions altered in Gyr A that give Francisella enhanced resistance are residues 83 and 87, which are involved in the catalytic domain, 43, 523, and 524 which are involved with DNA binding ( Figure 2A ). Those altered in GyrB are catalytic residues 464–466, DNA binding residues 486, 487, and 747, as well as mutation in residues 570 and 86, which have unknown roles in protein functionality ( Sutera et al, 2014b ; Caspar et al, 2017 ) ( Figure 2B ).…”

Mutational switch-backs can accelerate evolution of Francisella to a combination of ciprofloxacin and doxycycline

“…Doxycycline is a second generation tetracycline that inhibits bacterial translation by reversible binding to the 30S ribosomal subunit (23,24). Ciprofloxacin is a quinolone that inhibits bacterial replication by binding to DNA gyrases and topoisomerase IV (25)(26)(27). The importance of the use of these two drugs in a mass casualty setting underscores the need to understand the efficacy of these drugs and the evolvability of resistance to this combination.…”