Molecular Characterization of Mycobacterium ulcerans DNA Gyrase and Identification of Mutations Reducing Susceptibility to Quinolones In Vitro

Abstract

Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans. Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase. However, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in M. ulcerans remains unknown. This study investigated the detailed molecular mechanism of M. ulcerans DNA gyrase and the contribution of FQ resistance in vitro using recombinant proteins from the M. ulcerans subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC₅₀ of FQs against Ala91Val and Asp95Gly mutants of M. ulcerans shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type (WT) enzyme. Similarly, the quinolone concentrations required to induce 25% of the maximum DNA cleavage (CC₂₅) was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of the WT/mutant M. ulcerans DNA gyrase and FQ side chains were assessed by molecular docking studies. This was the first elaborative study demonstrating the contribution of mutations in M. ulcerans DNA GyrA subunit to FQ resistance in vitro.

Keywords: Buruli ulcer disease; DNA gyrase; Mycobacterium ulcerans; fluoroquinolone resistance; molecular docking study; supercoiling assay.

FIG 1

Purity of recombinant M. ulcerans DNA gyrase subunits. (A) Black and blue circles represent the FQ binding domain and the location of the N terminus his₆-tag, respectively. (B) Five to twenty percent SDS-PAGE (ATTO, Tokyo, Japan), of WT and mutant M. ulcerans DNA gyrase subunits from shinshuense and Agy99 strains. Approximately 3 μM each subunit was loaded into each well. Lanes 1 and 2: WT M. ulcerans shinshuense GyrA and GyrB subunit, respectively; lanes 3 and 4: WT M. ulcerans Agy99 GyrA and GyrB subunits, respectively. Lane M, size markers (kDa, Bio-Rad Lab. Inc., Japan); lanes 5 and 6: Ala91Val and Asp95Gly M. ulcerans shinshuense GyrA mutants, respectively; lanes 7 and 8: Ala91Val and Asp95Gly M. ulcerans Agy99 GyrA mutants, respectively.

FIG 2

ATP-dependent DNA supercoiling activities of WT M. ulcerans DNA gyrases. Three μM of GyrA and GyrB (M. ulcerans shinshuense), and 3 μM GyrA and GyrB (M. ulcerans Agy99) in the presence or absence of ATP. Lanes 1 and 5: GyrA and GyrB from shinshuense and Agy99 strains, respectively; lanes 2 and 6: absence of GyrA subunit; lanes 3 and 7: absence of GyrB subunit; lane 6: absence of ATP. R* and S* denote relaxed and supercoiled pBR322 DNA, respectively.

FIG 3

Concentration-dependent DNA gyrase supercoiling assays. Assays were performed using a fixed concentration (3 μM) of GyrA (left panel) with various concentrations of GyrB (1, 2, 3, 6, 12, and 24 μM) or fixed GyrB with variable GyrA concentrations (right panel). (A) WT M. ulcerans shinshuense; (B) WT Agy99; (C) Ala91Val and Asp95Gly M. ulcerans shinshuense mutants; (D) Ala91Val and Asp95Gly M. ulcerans Agy99 mutants. number 1 and 7, 1 μM; number 2 and 8, 2 μM; number 3 and 9, 3 μM; number 4 and 10, 6 μM; number 5 and 11, 12 μM; number 6 and 12, 24 μM, respectively. The optimum levels of DNA gyrase subunits are denoted by white arrows.

FIG 4

Temperature-dependent M. ulcerans shinshuense and Agy99 DNA gyrases supercoiling activities. Assays were performed at 20°C, 25°C, 30°C, 37°C, 42°C, and 50°C using 3 μM GyrA and GyrB for WT M. ulcerans shinshuense (●), and 3 μM WT M. ulcerans Agy99 GyrA and GyrB (○). Electrophoresis results are shown below the graph. Assays were performed in triplicate. S represents supercoiled pBR322 DNA.

FIG 5

Inhibition of WT and mutant M. ulcerans DNA gyrase supercoiling activities by FQs. Optimum concentrations of DNA gyrase subunits in the presence or absence of the indicated amounts (μg/mL) of (A) CIP, (B) MOX, and (C) LVX. Inhibition of WT/mutant M. ulcerans shinshuense and Agy99 DNA gyrase was shown in the left and right panels, respectively. R denotes relaxed pBR322 DNA.

FIG 6

CIP-mediated DNA cleavage complex by WT and mutants M. ulcerans shinshuense DNA gyrases. Supercoiled pBR322 DNA (0.1 μg) was incubated with each WT and mutants M. ulcerans DNA gyrases in the presence of increasing LVX concentrations indicated (0 to 320 μg/mL). After the addition of each 3 μL of 2% SDS and 1 mg/mL proteinase K for 30 min at 37°C, and then the reactions were stopped, and the mixture samples were analyzed by electrophoresis in 1% agarose gels. S and L denote supercoiled and linearized pBR322 DNA, respectively.

FIG 7

CIP binding mode in WT and mutant M. ulcerans shinshuense DNA gyrase A subunit. Molecular docking studies of WT and mutant M. ulcerans DNA gyrase with FQs were performed using the MOE software. The binding modes of CIP (A) and MOX (B) were shown within the catalytic site of M. ulcerans shinshuense DNA GyrA and the mutated amino acids (Ala91Val and Asp95Gly). Amino acids 90 to 96 were depicted in wheat color. The dotted red line indicates hydrogen bonding, and the distance between the amino acid residues and a side chain of FQs is indicated.