Description of compensatory gyrA mutations restoring fluoroquinolone susceptibility in Mycobacterium tuberculosis.

OBJECTIVES: Resistance to fluoroquinolones (FQs) in Mycobacterium tuberculosis (Mtb) is mainly due to mutations in DNA gyrase (GyrA2B2), with the most common substitutions located at positions 90 and 94 in GyrA. Two clinical MDR Mtb (MDR-TB) strains harbouring an A90E or D94N substitution in GyrA were found to be surprisingly susceptible to FQs (ofloxacin MIC ≤2 mg/L). We studied the impact of the additional GyrA substitutions found in these strains (T80A and T80A + A90G, respectively) on FQ susceptibility. METHODS: Mutants of interest were generated by site-specific mutagenesis of GyrA alleles. WT and mutant TB DNA gyrase subunits were overexpressed in Escherichia coli and purified, and the in vitro susceptibility to FQs of their DNA supercoiling reaction was studied. RESULTS: IC50s of mutant gyrase complexes bearing GyrA D94N and A90E were 3- to 36-fold higher than WT IC50s, whereas IC50s of gyrase bearing T80A + A90G + D94N and T80A + A90E were close to the WT IC50s. CONCLUSIONS: We demonstrated that substitutions T80A and A90G restore FQ susceptibility when associated with a substitution implicated in high-level FQ resistance. Line probe assay misclassification of MDR-TB strains as pre-XDR or XDR can be corrected by sequence analysis of gyrA.

Mycobacterium tuberculosis DNA gyrase possesses two functional GyrA-boxes.

In contrast with most bacteria which possess two type II topoisomerases (topoisomerase IV and DNA gyrase), Mycobacterium tuberculosis possesses only one, DNA gyrase, which is functionally a hybrid enzyme. Functional differences between the two type IIA topoisomerases are thought to be specified by a CTD (C-terminal DNA-binding domain), which controls DNA recognition. To explore the molecular mechanism responsible for the hybrid functions of the M. tuberculosis DNA gyrase, we conducted a series of sequence analyses and structural and biochemical experiments with the isolated GyrA CTD and the holoenzyme. Although the CTD displayed a global structure similar to that of bona fide GyrA and ParC paralogues, it harbours a second key motif similar in all respects to that of the conserved GyrA-box sequence motif. Biochemical assays showed that the GyrA-box is responsible for DNA supercoiling, whereas the second GyrA-box-l (GyrA-box-like motif) is responsible for the enhanced decatenation activity, suggesting that the mechanistic originality of M. tuberculosis DNA gyrase depends largely on the particular DNA path around the CTD allowed for by the presence of GyrA-box-l. The results of the present study also provide, through phylogenetic exploration of the entire Corynebacterineae suborder, a new and broader insight into the functional diversity of bacterial type IIA topoisomerases.