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

被引:160
作者
Aubry, A
Veziris, N
Cambau, E
Truffot-Pernot, C
Jarlier, V
Fisher, LM
机构
[1] Univ Paris 06, Fac Med Pierre & Marie Curie, Bacteriol Lab, F-75634 Paris 13, France
[2] Univ London, Mol Genet Grp, Mol & Metab Signalling Ctr, Div Basic Med Sci, London SW17 0RE, England
关键词
D O I
10.1128/AAC.50.1.104-112.2006
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Mutations in the DNA gyrase GyrA(2)GyrB(2) 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 [IC(50)s] 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 IC(50)s 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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页码:104 / 112
页数:9
相关论文
共 37 条
[1]  
[Anonymous], 2003, AM J RESP CRIT CARE, V167, P603
[2]   Mycobacterium tuberculosis DNA gyrase:: Interaction with quinolones and correlation with antimycobacterial drug activity [J].
Aubry, A ;
Pan, XS ;
Fisher, LM ;
Jarlier, V ;
Cambau, E .
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2004, 48 (04) :1281-1288
[3]   Fluoroquinolone susceptibility among Mycobacterium tuberculosis isolates from the United States and Canada [J].
Bozeman, L ;
Burman, W ;
Metchock, B ;
Welch, L ;
Weiner, M .
CLINICAL INFECTIOUS DISEASES, 2005, 40 (03) :386-391
[4]   Crystal structure of the breakage-reunion domain of DNA gyrase [J].
Cabral, JHM ;
Jackson, AP ;
Smith, CV ;
Shikotra, N ;
Maxwell, A ;
Liddington, RC .
NATURE, 1997, 388 (6645) :903-906
[5]   SELECTION OF A GYRA MUTANT OF MYCOBACTERIUM-TUBERCULOSIS RESISTANT TO FLUOROQUINOLONES DURING TREATMENT WITH OFLOXACIN [J].
CAMBAU, E ;
SOUGAKOFF, W ;
BESSON, M ;
TRUFFOTPERNOT, C ;
GROSSET, J ;
JARLIER, V .
JOURNAL OF INFECTIOUS DISEASES, 1994, 170 (02) :479-483
[6]   Resistance to quinolones in mycobacteria [J].
Cambau, E ;
Jarlier, V .
RESEARCH IN MICROBIOLOGY, 1996, 147 (1-2) :52-59
[7]   DNA topoisomerases: Structure, function, and mechanism [J].
Champoux, JJ .
ANNUAL REVIEW OF BIOCHEMISTRY, 2001, 70 :369-413
[8]   Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates [J].
Cheng, AFB ;
Yew, WW ;
Chan, EWC ;
Chin, ML ;
Hui, MMM ;
Chan, RCY .
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2004, 48 (02) :596-601
[9]   Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence [J].
Cole, ST ;
Brosch, R ;
Parkhill, J ;
Garnier, T ;
Churcher, C ;
Harris, D ;
Gordon, SV ;
Eiglmeier, K ;
Gas, S ;
Barry, CE ;
Tekaia, F ;
Badcock, K ;
Basham, D ;
Brown, D ;
Chillingworth, T ;
Connor, R ;
Davies, R ;
Devlin, K ;
Feltwell, T ;
Gentles, S ;
Hamlin, N ;
Holroyd, S ;
Hornby, T ;
Jagels, K ;
Krogh, A ;
McLean, J ;
Moule, S ;
Murphy, L ;
Oliver, K ;
Osborne, J ;
Quail, MA ;
Rajandream, MA ;
Rogers, J ;
Rutter, S ;
Seeger, K ;
Skelton, J ;
Squares, R ;
Squares, S ;
Sulston, JE ;
Taylor, K ;
Whitehead, S ;
Barrell, BG .
NATURE, 1998, 393 (6685) :537-+
[10]   Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases [J].
Corbett, KD ;
Berger, JM .
ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE, 2004, 33 :95-118