Novel gyrase mutations in quinolone-resistant and -hypersusceptible clinical isolates of Mycobacterium tuberculosis: functional analysis of mutant enzymes

Abstract

Mutations in the DNA gyrase GyrA2GyrB2 complex are associated with resistance to quinolones in Mycobacterium tuberculosis. As fluoroquinolones are being used increasingly in the treatment of tuberculosis, we characterized several multidrug-resistant clinical isolates of M. tuberculosis carrying mutations in the genes encoding the GyrA or GyrB subunits associated with quinolone resistance or hypersusceptibility. In addition to the reported putative quinolone resistance mutations in GyrA, i.e., A90V, D94G, and D94H, we found that the GyrB N510D mutation was also associated with ofloxacin resistance. Surprisingly, several isolates bearing a novel combination of gyrA T80A and A90G changes were hypersusceptible to ofloxacin. M. tuberculosis GyrA and GyrB subunits (wild type [WT] and mutants) were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. Mutant proteins were produced similarly from engineered gyrA and gyrB alleles by mutagenesis. MICs, enzyme inhibition, and drug-induced DNA cleavage were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. Mutant gyrase complexes bearing GyrA A90V, D94G, and D94H and GyrB N510D were resistant to quinolone inhibition (MICs and 50% inhibitory concentrations [IC50s] at least 3.5-fold higher than the concentrations for the WT), and all, except the GyrB mutant, were less efficiently trapped as a quinolone cleavage complex. In marked contrast, gyrase complexes bearing GyrA T80A or A90G were hypersusceptible to the action of many quinolones, an effect that was reinforced for complexes bearing both mutations (MICs and IC50s up to 14-fold lower than the values for the WT). This is the first detailed enzymatic analysis of hypersusceptibility and resistance in M. tuberculosis.

FIG. 1.

SDS-polyacrylamide gel electrophoresis analysis of highly purified wild-type and mutant M. tuberculosis GyrA and GyrB proteins. The mutation carried by each recombinant protein is shown above the appropriate lane. Wild-type M. tuberculosis GyrA (WT-A) and GyrB (WT-B) are also shown. The His-tagged proteins were overexpressed in E. coli, purified by nickel resin chromatography, separated on an SDS-7.5% polyacrylamide gel, and stained with Coomassie brilliant blue. Lanes M, size protein markers with sizes shown on the left.

FIG. 2.

Supercoiling activity of M. tuberculosis GyrA and GyrB gyrase proteins. Relaxed pBR322 DNA (0.4 μg) was incubated with M. tuberculosis H37Rv GyrA (20 ng) and GyrB (20 ng) wild-type proteins or with mutant enzymes reconstituted with GyrA T80A, A90G, T80A plus A90G, A90V, D94G, D94H, or A90V plus D94G or GyrB D472H, N510D, respectively. Reactions were stopped, and the DNA was examined by electrophoresis in 1% agarose. Lane WT, wild-type; lane a, relaxed pBR322. N, R, and S denote nicked, relaxed, and supercoiled DNA, respectively.

FIG. 3.

Inhibitory activity of moxifloxacin against the supercoiling activity of M. tuberculosis DNA gyrase, wild-type (WT) and eight mutant proteins. Relaxed pBR322 DNA (0.4 μg) was incubated with gyrase activity (2 U) reconstituted from WT GyrA with WT GyrB and GyrB N510D, WT GyrB with GyrA with D94H, A90V plus D94G, A90V, and D94G (a) and from WT GyrB and WT GyrA and GyrA with T80A, A90G, and T80A plus A90G (b). Incubation was carried out in the presence of 1 mM ATP and in the absence or presence of the indicated amounts (in μg/ml) of moxifloxacin. Reactions were stopped, and the DNA was examined by electrophoresis in 1% agarose. N, R, and S denote nicked, relaxed, and supercoiled DNA, respectively.

FIG. 4.

Quinolone-mediated DNA cleavage by M. tuberculosis WT DNA gyrase and by mutant GyrA gyrase bearing A90V, D94G, D94H, or A90V plus D94G. In the presence of 1 mM ATP, supercoiled pBR3222 DNA (0.4 μg) was incubated with M. tuberculosis WT GyrB (0.24 μg) and WT GyrA (0.225 μg), gatifloxacin (GAT), and moxifloxacin (MOX) at the concentrations (μg/ml) indicated above the lanes (a) and with M. tuberculosis WT GyrB (0.24 μg) and GyrA bearing either A90V, D94G, D94H, or A90V plus D94G (0.225 μg) and moxifloxacin at the concentrations indicated (μg/ml) (b). After addition of SDS and proteinase K, DNA samples were analyzed by electrophoresis in 1% agarose. R, L, and S denote relaxed, linear, and supercoiled DNA, respectively. TL, pBR322 linearized by EcoRI; TS, supercoiled pBR322 used as the substrate in the assay.

FIG. 5.

Supercoiled pBR3222 DNA (0.4 μg) was incubated with M. tuberculosis WT GyrA (0.225 μg) and GyrB N510D (0.24 μg) in the presence of 1 mM ATP and four different fluoroquinolones: ofloxacin (OFX), levofloxacin (LVX), gatifloxacin (GAT), and moxifloxacin (MOX) at the concentrations indicated (μg/ml). After addition of SDS and proteinase K, DNA samples were analyzed by electrophoresis in 1% agarose. Lanes a, supercoiled pBR322. R, L, and S denote relaxed, linear, and supercoiled DNA, respectively.

FIG. 6.

Quinolone-mediated DNA cleavage by M. tuberculosis WT DNA gyrase and by mutant GyrA gyrase bearing T80A, A90G, or T80A plus A90G. Supercoiled pBR3222 DNA (0.4 μg) was incubated with M. tuberculosis WT GyrB (0.24 μg) and GyrB with either T80A, A90G, or T80A plus A90G and WT GyrA (0.225 μg) in the presence of 1 mM ATP and moxifloxacin at the concentrations (μg/ml) indicated above the lanes. After addition of SDS and proteinase K, DNA samples were analyzed by electrophoresis in 1% agarose. R, L, and S denote relaxed, linear, and supercoiled DNA, respectively. TL is the pBR322 linearized by EcoRI.