Mycobacterium leprae DNA gyrase: expression, purification, inhibition by quinolones and functional analysis of two mutant enzymes
Abstract number: 1733_389
Matrat S., Petrella S., Cambau E., Sougakoff W., Jarlier V., Aubry A.
Objectives:M. leprae is still uncultivable in vitro and evaluation of antibiotic activity against this bacteria relies mainly on the mouse foot pad model that requires 8 to 12 months experiments. It is therefore important to establish an in vitro approach allowing to test rapidly the efficiency of quinolones against wild type and quinolone resistant mutants of M. leprae. Mutations in the DNA gyrase (the sole target of quinolones in M. leprae) was previously described. The aim of the present study was to purify the DNA gyrase of M. leprae in order (i) to test the ability of quinolones to inhibit this enzyme and (ii) to study the implication of gyrase mutations in the resistance of M. leprae clinical isolates to quinolones.
Methods: We separately overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits as His-Tagged proteins using pET plasmids harbouring the gyrA and gyrB genes. Two mutant enzymes (A91V in GyrA and D205N in GyrB, corresponding to amino acids GyrA 83 and GyrB 183 in E. coli numbering system), representing mutations found in M. leprae clinical isolates (Cambau et al., Lancet, 1997; Kim et al., FEMS, 2003), were introduced by site directed mutagenesis. Wild type and mutant subunits were purified by nickel chelate chromatography. The drug concentrations that induced 25% of DNA cleavage (CC25) were measured for 3 quinolones (MXF, OFX and LVX).
Results: The soluble 97.5-kDa GyrA and 74.5-kDa GyrB wild type and mutant subunits obtained after purification were mixed to reconstitute enzymes showing a DNA supercoiling activity. The CC25s of the GyrB mutant D205N were similar to those found with the wild type subunit. In contrast, the CC25s of OFX, LVX and MXF for the GyrA mutant A91V were 4-, 6- and 20-fold higher than the wild type ones respectively.
Conclusion: Our results show that mutation D205N in GyrB is not involved in acquired resistance of M. leprae to quinolones whereas A91V in GyrA is involved in quinolone resistance of M. leprae. In addition, we showed that drug-induced DNA cleavage assays from purified wild type and mutant M. leprae DNA gyrases represent a safe, quick and useful screening test for identifying quinolones with potential activity against M. leprae and related multidrug resistant strains.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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