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Antimicrobial Agents and Chemotherapy, January 2006, p. 104-112, Vol. 50, No. 1
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.1.104-112.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Novel Gyrase Mutations in Quinolone-Resistant and -Hypersusceptible Clinical Isolates of Mycobacterium tuberculosis: Functional Analysis of Mutant Enzymes

Alexandra Aubry,1,2* Nicolas Veziris,2 Emmanuelle Cambau,2,{dagger} Chantal Truffot-Pernot,2 Vincent Jarlier,2 and L. Mark Fisher1*

Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St. George's, University of London, London SW17 0RE, United Kingdom,1 Laboratoire de Bactériologie, Faculté de Médecine Pierre et Marie Curie, Université Paris VI, Paris, France2

Received 13 July 2005/ Returned for modification 6 September 2005/ Accepted 27 October 2005


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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.


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INTRODUCTION
 
The increasing incidence of multidrug-resistant tuberculosis (MDR-TB), which involves bacilli resistant to both isoniazid and rifampin, the two main anti-TB drugs, is a worldwide problem (33). Effective second-line drugs are needed for the treatment of MDR-TB (2). Fluoroquinolones are active against Mycobacterium tuberculosis and are the first new antimycobacterial drugs to be available since the discovery of rifampin (4, 16). Indeed, fluoroquinolones are nowadays part of the treatment recommended for MDR-TB (2, 10). Among the fluoroquinolones, those with the greatest activity against tuberculosis are the newer 8-methoxy derivatives, such as moxifloxacin (MOX) and gatifloxacin (GAT). These two agents exhibit bactericidal activities against M. tuberculosis that are significantly greater than those of ofloxacin (OFX) and levofloxacin (LVX) (20, 29).

The increasing use of quinolones in the community, especially to treat respiratory infectious diseases, can lead to decreased quinolone susceptibility in M. tuberculosis (16, 17). This raises interest in the mechanisms of quinolone action and resistance in this organism. Quinolones inhibit the bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, which are two related ATP-dependent enzymes that act by a double-stranded DNA break (14) and cooperate to facilitate DNA replication and chromosome segregation at cell division (23). Gyrase is unique in catalyzing the ATP-dependent introduction of negative supercoils into closed circular DNA. The enzyme consists of two proteins, GyrA and GyrB, encoded by the gyrA and gyrB genes, which form the catalytically active GyrA2GyrB2 complex (6, 9, 23). Surprisingly, there is no evidence of the topoisomerase IV parC and parE gene homologs in the genome of M. tuberculosis (8). It appears that DNA gyrase is the sole topoisomerase target for quinolones in M. tuberculosis.

The toxicity of quinolones on the bacterial cell is thought to involve the formation of a topoisomerase-drug-DNA ternary complex (13-15) that cellular processes convert into a lethal lesion (30, 31). Most mutations conferring bacterial resistance to quinolones occur in a short discrete segment termed the quinolone resistance-determining region (QRDR) of the DNA gyrase gyrA gene (and analogously in the topoisomerase IV parC gene), and more rarely in gyrB (or topoisomerase IV parE) (26). Laboratory studies on M. tuberculosis have revealed that single missense mutations in gyrA are associated with low-level quinolone resistance and that bacteria with high-level resistance generally have two missense mutations in gyrA or one mutation in gyrA and one in gyrB (22). Spontaneous quinolone resistance mutations, in laboratory-selected mutants, mostly affect Ala90 changed to Val and Asp94 mutated to Gly, His, Asn, Tyr, or Ala in GyrA and more rarely in GyrB at the Asp472 position (4, 22). The residues altered in M. tuberculosis GyrA are equivalent to Ser83 and Asp87 in Escherichia coli GyrA and to Asp426 in E. coli GyrB. It would appear these residues play a role in drug binding and resistance to quinolones in M. tuberculosis (4, 5, 22, 28, 36). However, there have been few studies at the enzyme level examining the effects of M. tuberculosis GyrA and GyrB mutations on quinolone susceptibility.

In this paper, we have studied the susceptibility to quinolones of Mycobacterium tuberculosis DNA gyrase harboring known putative quinolone resistance mutations, i.e., A90V, D94G, D94H, and A90V plus D94G in GyrA and D472H in GyrB. Moreover, we have also examined the quinolone responses of gyrase complexes bearing T80A, A90G, and T80A plus A90V changes in GyrA and an N510D change in GyrB that we have recently encountered in some MDR-TB isolates. We established that the N510D GyrB mutation along with known GyrA mutations conferred resistance to a variety of quinolones. By contrast, the T80A and A90G alterations in GyrA were responsible for hypersusceptibility to quinolones in M. tuberculosis. These studies constitute the first detailed phenotypic examination of gyrase mutations and quinolone resistance and hypersusceptibility in M. tuberculosis.


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MATERIALS AND METHODS
 
Strains, plasmids, and reagents. The eight M. tuberculosis clinical isolates used in this study were obtained through the Centre National de Référence de la Résistance des Mycobactéries aux Antituberculeux, Laboratory of Bacteriology, Groupe Hospitalier Pitié-Salpêtrière, Paris, France, as was the quinolone-susceptible M. tuberculosis strain H37Rv. Strains were grown on Löwenstein-Jensen medium. Seven clinical isolates presented newly described mutations in their DNA gyrase genes: two strains carried a GyrA T80A mutation, four had the double mutation T80A plus A90G in GyrA, and one strain had a mutation resulting in a GyrB N510D change. One clinical isolate carrying the known GyrA A90V mutation was also studied.

Expression plasmids pATB and pBTB containing the respective wild-type (WT) gyrA and gyrB genes of M. tuberculosis have been described previously (1). Plasmids were transformed into E. coli BL21-CodonPlus({lambda}DE3)-RP cells (Stratagene) for protein expression.

Enoxacin (Sigma), ofloxacin and levofloxacin (Aventis), moxifloxacin (Bayer Pharma), and gatifloxacin (Grünenthal) were provided by the manufacturers. Supercoiled plasmid pBR322 DNA was purchased from New England Biolabs, and relaxed plasmid pBR322 DNA was from John Innes Enterprises, Ltd.

Determination of MICs. MICs were determined by the 1% standard proportion method on 7H11 agar supplemented with 10% oleic acid-albumin-dextrose-catalase (OSI) (21). MICs were defined as the lowest concentration of quinolone that inhibited more than 99% of the bacterial growth.

In vitro mutagenesis. Plasmids expressing mutant M. tuberculosis gyrA or gyrB genes were generated from pATB or pBTB using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primers for mutagenesis (Table 1) were synthesized by Sigma-Genosys, Ltd. After mutagenesis, plasmids were recovered, purified using the QIAGEN miniprep kit, and sequenced by Lark Technologies using previously described oligonucleotides (12).


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  		TABLE 1. Oligonucleotides used in site-directed mutagenesis of M. tuberculosis gyrA and gyrB genes

Overexpression and purification of wild-type and mutant gyrase proteins. Gyrase subunits were purified as previously described with minor modifications (1). First, E. coli BL21-CodonPlus-RP ({lambda}DE3) harboring mutant gyrA plasmids or mutant gyrB plasmids were cultured at 18°C and 28°C, respectively. Combined elution fractions from the nickel column were dialyzed overnight at 4°C against 2.5 liters of 50 mM Tris-HCl (pH 7.9) and then for 30 min against 1 liter of 50 mM Tris-HCl (pH 7.9) and 30% glycerol. In some preparations the concentration of imidazole used to elute the nickel column was increased from 1 to 3 M in the 4x elution buffer. Protein concentrations were estimated by the Bradford method (37).

Enzyme assays. DNA supercoiling and cleavage assays were carried out as described previously (1). DNA products were analyzed by electrophoresis in 1% agarose, stained with ethidium bromide, photographed, and quantified with an Alpha Innotech digital camera and associated software. To facilitate direct comparison, all incubations with wild-type and mutant enzymes were carried out and processed in parallel on the same day under identical conditions. All enzyme assays were done at least twice, with reproducible results.


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RESULTS
 
Characterization of quinolone-resistant and quinolone-hypersusceptible M. tuberculosis strains. As fluoroquinolones are now very important drugs for the treatment of MDR-TB, each multidrug-resistant clinical isolate submitted to the Centre National de Référence de la Résistance des Mycobactéries aux Antituberculeux is routinely tested for susceptibility to fluoroquinolones as recommended by the World Health Organization. The gyrA QRDR of MDR strains is also sequenced for mutations as a more rapid indicator of potential resistance to fluoroquinolones. In addition to strains bearing the previously described gyrA mutations encoding A90V (isolate A, this study), D94G, D94H, and A90V plus D94G alterations, six strains (strains C, D, E, F, G, and H) were identified with novel GyrA T80A or GyrA T80A plus A90G changes. One other strain (isolate B) lacking a gyrA mutation was resistant to ofloxacin and carried a gyrB QRDR change specifying a GyrB N510D mutation. These seven clinical strains bearing novel gyrase mutations were tested for their susceptibility to the quinolones recommended for the treatment of tuberculosis by the American Thoracic Society (2), i.e., moxifloxacin, gatifloxacin, levofloxacin, and ofloxacin Enoxacin, which is less active against M. tuberculosis, was also included in susceptibility testing. Quinolone-susceptible M. tuberculosis strain H37Rv was tested against the quinolones for comparison: four independent measurements gave the same MICs. For strains carrying mutations in gyrA, i.e., A90V, D94G, D94H, A90V plus D94G, and GyrA A90V and GyrB D472H, some MIC results were already available in the literature. Given the danger of manipulating such strains, which are mostly MDR, we did not determine their MICs (except for ofloxacin and the A90V mutation in GyrA) but used data available from the literature. Representative results are shown in Table 2.


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  		TABLE 2. Fluoroquinolone susceptibilities of M. tuberculosis H37Rv and clinical strains carrying mutations in gyrA and/or gyrB

The gatifloxacin, moxifloxacin, levofloxacin, ofloxacin, and enoxacin MICs for the wild-type M. tuberculosis strain H37Rv were 0.25, 0.5, 0.5, 1, and 8 µg/ml, respectively, values that were essentially identical to those reported previously (1, 20). The MICs for strain A with the GyrA A90V mutation were four- to eightfold higher than those for the WT for gatifloxacin, moxifloxacin, levofloxacin, ofloxacin, and enoxacin. Quinolone MICs for strains G and H carrying the T80A mutation were two- to eightfold higher than those for the WT (Table 2) but substantially lower than for strains bearing the single mutations A90V, D94G, and D94H, which are frequently associated with quinolone resistance. For the four strains carrying the double mutation T80A plus A90G (strains C, D, E, and F), the MICs are presented in Table 2. When comparing MIC values of those strains with those of the WT, 13 out of 20 are lower for the double mutant (1 to 2 dilutions), 5 are equal, and 2 are higher (1 dilution). Therefore, even if the difference is small, several of these strains were hypersusceptible to quinolones. For example, strain F exhibited a two- to fourfold reduction in MICs for all quinolones compared to strain H37Rv. The MICs for strain B with the GyrB N510D mutation were eightfold higher than those for the WT for gatifloxacin, moxifloxacin, levofloxacin, ofloxacin, and enoxacin. These results suggested that the GyrA T80A and GyrB N510D changes may confer resistance, whereas the GyrA T80A plus A90G combination may be responsible for hypersusceptibility.

Purification of DNA gyrase proteins engineered with mutations found in clinical isolates. Susceptibility testing can establish an association between a particular mutation and MICs, but it does not prove a phenotype. To examine directly the effects on quinolone susceptibility of the M. tuberculosis GyrA T80A, A90G, and GyrB N510D changes, as well as the commonly found GyrB D472H and GyrA A90V, D94G, and D94H alterations, we engineered plasmid clones that overexpress the mutant subunits in E. coli. We previously described plasmids pATB and pBTB that allow the respective IPTG-inducible expression of M. tuberculosis gyrA or gyrB genes from strain H37Rv (1). The presence of a histidine tag at the C-terminal end of GyrA and at the N-terminal end of GyrB allows rapid recovery of highly purified active subunits. Accordingly, we used site-directed mutagenesis on plasmids pATB and pBTB to introduce the desired mutations and purified the corresponding mutant proteins by nickel chelate column chromatography.

Seven different mutant GyrA proteins and two mutant GyrB proteins were obtained at high purity (>90%) in milligram amounts (Fig. 1) and free of contaminating host topoisomerase activity. Initially, we followed the same purification protocol as used previously for wild-type subunits (1). However, better yields and specific activities were obtained by increasing the level of imidazole in the 4x column elution buffer from 1 to 3 M.



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  		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.

Activities of mutant M. tuberculosis DNA gyrase complexes. The expressed gyrase subunits were stable, and their activities were determined in a DNA supercoiling assay in the presence of excess complementary wild-type subunit (Fig. 2). With the exception of the GyrB D472H protein, which was inactive, all the mutant proteins reconstituted DNA supercoiling activity. Moreover, the specific activities of the mutant GyrA and GyrB proteins were comparable to those measured for the wild-type proteins. Thus, the specific activities were 8.9 x 103 and 5 x 103 U/mg for the wild-type GyrA and GyrB proteins, respectively; 6.5 x 103, 2.9 x 103, 1.2 x 103, 8.3 x 103, 7.4 x 103, 5.3 x 103, and 4.2 x 103 U/mg for the GyrA proteins with T80A, A90G, T80A plus A90G, A90V, D94G, D94H, and A90V plus D94G mutations, respectively; and 5 x 103 U/mg for the GyrB N510D mutant protein.



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  		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.

Effects of mutations on quinolone inhibition of DNA gyrase. The 50% inhibitory concentrations (IC50s) for a range of quinolones were determined for the WT enzyme and eight mutant proteins using a DNA supercoiling assay (Table 3). Figure 3 show representative data obtained for each mutant gyrase using moxifloxacin, one of the most potent quinolones tested. In reviewing the data, it is helpful to bear in mind that A90 and D94 residues of M. tuberculosis GyrA are equivalent to S83 and D87 residues of E. coli GyrA, whose mutation is associated with quinolone resistance. Complexes bearing A90V, D94G, and D94H mutations in GyrA were resistant to moxifloxacin inhibition with IC50s of 35, 50 and 90 µg/ml, respectively (Fig. 3a) compared with an IC50 of 2 µg/ml for the wild type (Fig. 3b). Moreover, the combination of A90V and D94G mutations in GyrA resulted in a IC50 of >160 µg/ml. The same pattern of resistance was seen for other quinolones, with gatifloxaxin and moxifloxacin showing similar inhibitory potentials and levofloxacin and ofloxacin being much less effective against both wild-type and mutant enzymes (Table 3).


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  		TABLE 3. Fluoroquinolone activities against mutant M. tuberculosis DNA gyrase



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  		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.

It is interesting that gyrase bearing the novel N510D mutation in GyrB was also highly resistant to inhibition by moxifloxacin with an IC50 of 35 µg/ml (Fig. 3a), a 17-fold increase over that for the wild-type enzyme. This mutation conferred similar increases in resistance to gatifloxacin, levofloxacin, and ofloxacin (Table 3). The N510 residue in M. tuberculosis GyrB is equivalent to residue 464 in E. coli GyrB. Therefore, it lies outside the classical QRDR of E. coli GyrB defined by resistance hot spots D426 and K447 (35).

In regard to the T80A and A90G mutations in GyrA, gyrase complexes bearing either mutation alone exhibited moxifloxacin IC50s similar to those of the wild-type enzyme, i.e., 1 and 2 µg/ml, respectively (Fig. 3b). However, gyrase bearing both mutations had a moxifloxacin IC50 of 0.5 µg/ml, which is fourfold lower than that of the wild-type enzyme (Fig. 3b and Table 3). Two- to 14-fold-higher sensitivities of the complexes of GyrA T80A and GyrA A90G were also seen for other quinolones, including ofloxacin, which was initially used to identify the hypersusceptibility of strains bearing those particular changes (Table 3). It appears that the T80A and A90G mutations individually cause weak hypersusceptibility to quinolones and that the combination of mutations gives rise to higher susceptibility.

Potencies of different quinolones in stabilizing the cleavable complex of gyrase reconstituted with wild-type or mutant subunits. To compare cleavable-complex stabilization achieved with the clinically approved antitubercular fluoroquinolones and to examine the effects of GyrA and GyrB mutations, supercoiled pBR322 was incubated with gyrase in the absence or presence of drugs at a wide range of concentrations, and DNA breakage was induced by the subsequent addition of sodium dodecyl sulfate (SDS). Following incubation with proteinase K (to remove covalently bound GyrA protein), DNA samples were analyzed by agarose gel electrophoresis. The drug concentration that caused 25% linearization of the input DNA (CC25) was determined at least twice in independent experiments with similar results (Table 3). Figure 4 shows the results of a representative cleavage experiment in which supercoiled pBR322 was incubated with equal amounts of wild-type or mutant M. tuberculosis enzyme in the absence or presence of moxifloxacin. Because the wild-type gyrase showed a marked DNA-relaxing activity, we included 1 mM ATP which blocked DNA relaxation in our assays (1). Drug stabilization of the cleavable complex is expected to generate linear DNA on denaturation. For wild-type gyrase (Fig. 4a), inclusion of moxifloxacin or gatifloxacin produced a dose-dependent increase in linear DNA product, with CC25 values of 1 to 2 µg/ml for both drugs.



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  		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.

In contrast to wild-type enzyme, mutant GyrA gyrases containing A90V, D94G, D94H, and A90V plus D94G changes associated with quinolone resistance each exhibited a low level of DNA cleavage activity for moxifloxacin with CC25 values of 4, 4 to 8, 8, and ≥64 µg/ml, respectively (Fig. 4b and Table 3). Broadly similar results were found using gatifloxacin, but levofloxacin and ofloxacin were less active against the wild type and the mutant enzyme (Table 3). It is clear that A90V, D94G, and D94H mutations in GyrA reduce DNA cleavage activity some four- to eightfold, and the double A90V D94G mutation reduces DNA cleavage activity by some 32- to 64-fold. Surprisingly, for the GyrB N510D mutant, the quinolone CC25s were similar to those of the WT, and some cleavage activity was observed without drug (Fig. 5).



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  		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.

For the newly described mutations found in clinical strains, we observed that the ability to induce a cleavable complex was two- to fourfold more efficient for moxifloxacin, gatifloxacin, levofloxacin, and ofloxacin for the GyrA T80A mutant than for the WT (Table 3 and Fig. 6). For the GyrA A90G and the GyrA with the T80A plus A90G double mutation, the CC25s were two- to eightfold lower than for the WT for the four fluoroquinolones tested (Table 3 and Fig. 6). Thus, the combination of T80A and A90G mutations in GyrA resulted in a DNA gyrase that was hypersensitive to drug action in the cleavage assay.



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  		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.


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DISCUSSION
 
We have characterized several multidrug-resistant clinical isolates of M. tuberculosis carrying novel mutations in their gyrA and/or gyrB genes 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 N510D mutation in gyrB was also associated with quinolone resistance. Although strains carrying the novel GyrA T80A alteration were slightly resistant to quinolones, surprisingly, several isolates bearing a novel combination of GyrA T80A plus A90G changes were hypersusceptible to ofloxacin and other quinolones. We examined the effects of these changes at the molecular level by expressing recombinant mutant GyrA and GyrB proteins and reconstituting gyrase activity. Mutant gyrase complexes bearing GyrA A90V, D94G, and D94H and GyrB N510D were resistant to quinolone inhibition, 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 changes were hypersusceptible to the action of many quinolones, an effect that was reinforced for complexes bearing both mutations. This is the first report of quinolone hypersusceptibility in M. tuberculosis and the first detailed enzymatic analysis of hypersusceptibility and resistance in this pathogen.

Previous work has focused on cataloguing mutations in quinolone-resistant isolates of M. tuberculosis. A mutation at GyrA position 94 (D94G or D94A) is most commonly followed by a mutation at position 90 (A90V) (3, 7, 27; C. Truffot-Pernot, unpublished data). Laboratory-selected mutants bearing such mutations have quinolone MICs that are up to eightfold higher than the wild-type strain (22). We found that recombinant gyrase complexes bearing GyrA A90V, D94G, or D94H were at least 3.5-fold and up to 30-fold more resistant than wild-type enzyme to inhibition by quinolones (Fig. 3a and Table 3) with additive resistance effects for gyrase carrying both GyrA A90V and D94G mutations. The rank order for catalytic inhibition of wild-type and mutant gyrase (and for cleavage complex formation) was moxifloxacin = gatifloxacin > levofloxacin > ofloxacin, i.e., similar to that observed for growth inhibition of the different bacterial mutants (Tables 2 and 3). The results of these studies with defined enzyme mutants establish unequivocally that the commonly encountered mutations in GyrA do confer resistance to quinolones in M. tuberculosis.

The one published study of quinolone inhibition of M. tuberculosis gyrase was limited to two mutant enzymes and measured just the effects of sitafloxacin, levofloxacin, ciprofloxacin, and sparfloxacin in DNA supercoiling assays (25). The 50% inhibitory concentrations (IC50s) determined for levofloxacin against gyrase reconstituted with wild-type GyrA, GyrA A90V, and GyrA A90V plus D94G were 13.9, >400, and >400 µg/ml, respectively, which compare favorably with the values of 16, 170, and >1,600 µg/ml, respectively, reported here (Table 3). However, our analysis is more extensive in terms of different quinolones and different mutants and extends to studies of DNA cleavage as well as supercoiling inhibition.

In addition to GyrA changes, the novel GyrB N510D mutation was also associated with fourfold increases in resistance to quinolones in some isolates (Table 2). Although GyrB mutations have not been implicated heretofore in clinical resistance, it has been shown that ciprofloxacin challenge of M. tuberculosis can select gyrB mutants (36). Moreover, analysis of 62 gyrB alleles revealed that 52 involved changes at residue 510: N to Y in 47 mutants, N to K in 2 mutants, N to D in 2 mutants, and N to T in 1 mutant. We have found that the GyrB N510D mutation confers 12- to 30-fold increases in the resistance of M. tuberculosis gyrase to quinolones, i.e., increases comparable to those seen for mutations at position 90 or 94 in GyrA (Table 3). Given these findings, it is curious that the N510D change had no effect on DNA cleavage (Fig. 5), suggesting it has novel consequences for quinolone interactions with the enzyme. Further work will be needed to investigate these differential effects.

Perhaps the most surprising observation was the identification of GyrA T80A plus A90G mutations with hypersusceptibility to quinolones both in vivo and in DNA supercoiling and cleavage assays in vitro (Tables 2 and 3 and Fig. 3 and 6). The T80A mutation resulted in a twofold sensitization of gyrase to quinolone inhibition and two- to eightfold reductions in the CC25s for moxifloxacin, gatifloxacin, levofloxacin, and ofloxacin measured in cleavage assays (Table 3). That the quinolone MICs of strains bearing this GyrA T80A change were equal to or slightly higher than that of the H37Rv strain may be due to the presence of another mutation(s) in another location, e.g., outside the QRDRs, or other unexplored mechanisms, such as efflux. The GyrA A90G mutation had little effect on IC50 values (except for a 4-fold reduction for enoxacin) but stimulated cleavage some 4- to 16-fold (Table 3). Interestingly, the combination of the two mutations potentiated the greatest susceptibility to inhibition of DNA supercoiling and cleavage stimulation (Table 3). These results add a new dimension in thinking about quinolone interactions with the GyrA QRDR in mycobacteria. It remains to be established whether tuberculosis arising from strains with this phenotype is particularly susceptible to treatment with moxifloxacin, gatifloxacin, ofloxacin, and other quinolone drugs.

Much of our understanding of quinolone interactions with gyrase has come from studies of the E. coli enzyme. The E. coli GyrA QRDR comprises residues 67 to 106 that form a catabolite gene activator protein (CAP)-like helix-turn-helix motif thought to lie at the quinolone-DNA interface (24). Residues S83 and D87 (equivalent to A90 and D94 in M. tuberculosis GyrA) are commonly mutated in quinolone-resistant strains, and both residues lie in the {alpha}4 helix of the helix-turn-helix region. Mutation of S83 to alanine results in low-level drug resistance; alteration to bulky hydrophobic side chains, such as leucine, valine, phenylalanine, or tyrosine, confers high-level resistance (11, 34), most likely by reducing drug binding (32). Interestingly, the intrinsic resistance of many Mycobacterium species, such as M. avium, M. smegmatis, and M. tuberculosis, to quinolones is related to the presence of an alanine (rather than serine) in their GyrA proteins at the position analogous to S83 (18, 19). Substitution of A90 with valine leads to yet higher resistance to quinolones in M. tuberculosis (Table 3). Unlike quinolone-resistant E. coli wherein S83 is more commonly mutated than D87, the reverse applies in M. tuberculosis (5, 27, 28). Mutation of D94 in M. tuberculosis occurs more frequently than at A90 and leads to greater effects on MIC and increased resistance over A90 changes in DNA supercoiling and DNA cleavage. The molecular basis underlying these differences is not understood.

Quinolone hypersusceptibility of M. tuberculosis is also identified with mutations in the CAP-like domain of GyrA involving changing the key A90 residue to glycine or converting T80, located in the {alpha}3 helix, to alanine (Table 3). The hypersusceptibility to fluoroquinolones observed for the A90G mutant is in concordance with observations made in E. coli. Indeed, it has been shown that changing Ser83 to Gly in E. coli does not confer resistance, whereas changing Ser83 to Ala does (34). Both T80A and A90G substitutions reduce the size of the respective side chains and could introduce a measure of flexibility into protein folding in the CAP region. We speculate that these alterations, especially in combination, enhance quinolone binding to the gyrase complex, thereby accounting for the observed hypersusceptibility. Earlier studies have reported that hypersusceptibility to certain quinolones in E. coli stems from GyrB mutations at positions 426 and 447, which constitute part of the GyrB QRDR (26). It has been suggested that quinolone binding pockets in gyrase may lie at an interface between GyrA and GyrB subunits (11, 35). This proposal attractively accommodates the finding that mutations of GyrA and GyrB residues (such as N510D in M. tuberculosis) are implicated in resistance and hypersusceptibility to quinolones.


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ACKNOWLEDGMENTS
 
We thank Claudine Mayer and Xiao-Su Pan for helpful discussions and Aurelie Chauffour and Solenne Maucolin for M. tuberculosis MIC determinations.

This work was supported by grants from the Fondation pour la Recherche Médicale, Ministère de l'Education Nationale et de la Recherche (grant UPRES 1541), and Association Française Raoul Follereau. Work in the Fisher group was supported by the Biotechnology and Biological Sciences Research Council.


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FOOTNOTES
 
* Corresponding author. Mailing address for Alexandra Aubry: Faculté de Médecine Pierre et Marie Curie, Université Paris VI, 91, boulevard de l'Hôpital, 75634 Paris Cedex 13, France. Phone: 33 1 40 77 97 46. Fax: 33 1 45 82 75 77. E-mail: aubry{at}chups.jussieu.fr. Mailing address for L. Mark Fisher: Division of Basic Medical Sciences, St. George's, University of London, London SW17 0RE, United Kingdom. Phone: 44 208 725 5782. Fax: 44 208 725 2992. E-mail: lfisher{at}sgul.ac.uk. Back

{dagger} Present address: Laboratoire de Bactériologie-Virologie-Hygiène, Centre Hospitalo-Universitaire Henri Mondor, Assistance Publique-Hôpitaux de Paris, Faculté de Médecine de Créteil, Université Paris XII, Paris, France. Back


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Antimicrobial Agents and Chemotherapy, January 2006, p. 104-112, Vol. 50, No. 1
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.1.104-112.2006
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