Genomic approach to identifying the putative target of and mechanisms of resistance to mefloquine in mycobacteria

The emergence of mycobacterial resistance to multiple antimicrobials emphasizes the need for new compounds. The antimycobacterial activity of mefloquine has been recently described. Mycobacterium avium, Mycobacterium smegmatis, and Mycobacterium tuberculosis are susceptible to mefloquine in vitro, and activity was evidenced in vivo against M. avium. Attempts to obtain resistant mutants by both in vitro and in vivo selection have failed. To identify mycobacterial genes regulated in response to mefloquine, we employed DNA microarray and green fluorescent protein (GFP) promoter library techniques. Following mefloquine treatment, RNA was harvested from M. tuberculosis H37Rv, labeled with 32 P, and hybridized against a DNA array. Exposure to 4 x MIC resulted in a significant stress response, while exposure to a subinhibitory concentration of mefloquine triggered the expression of genes coding for enzymes involved in fatty acid synthesis, the metabolic pathway, and transport across the membrane and other proteins of unknown function. Evaluation of gene expression using an M. avium GFP promoter library exposed to subinhibitory concentrations of mefloquine revealed more than threefold upregulation of 24 genes. To complement the microarray results, we constructed an M. avium genomic library under the control of a strong sigma-70 (G13) promoter in M. smegmatis. Resistant clones were selected in 32 mu g/ml of mefloquine (wild-type M. avium, M. tuberculosis, and M. smegmatis are inhibited by 8 mu g/ml), and the M. avium genes associated with M. smegmatis resistant to mefloquine were sequenced. Two groups of genes were identified: one affecting membrane transport and one gene that apparently is involved in regulation of cellular replication.