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 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 g/ml of mefloquine (wild-type M. avium, M. tuberculosis, and M. smegmatis are inhibited by 8 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. Mycobacterial diseases remain among the world’s leading infection problems. Tuberculosis is a major infectious disease with approximately 8 million new cases worldwide causing an estimated 2 million deaths annually (4). Pathogenic mycobacteria initiate long-term infection in the lungs by entering host macrophages and spreading rapidly. Although effective therapeutic regimens exist, the prevalence of Mycobacterium tuberculosis strains resistant to available antimicrobial agents and the emergence of multidrug resistance underscore the need for additional compounds targeted at new pathways. Mycobacterium avium, an environmental organism, is a major opportunistic pathogen that causes bacteremia and disseminated disease in patients in the advanced stages of AIDS. In non-AIDS patients, M. avium is associated with pulmonary infection (9). Although treatment with new macrolides and rifabutin became available in the last several years, complete killing of the bacteria is usually not achieved. There is an urgent need for new compounds to improve mycobacterial therapy. Based on broad screening of compounds, we found that mefloquine (a derivate of 4-quinoline methanol) is bactericidal against M. avium in vivo, and it is active against M. tuberculosis in vitro (3). Previously, we have shown that mefloquine is effective against M. avium isolates resistant to macrolides, quinolones, and rifamycins (3), which suggests a novel target in mycobacteria. Passages of M. avium in the presence of elevated levels of mefloquine, as well as the screening of mycobacterial transposon libraries, did not select for resistant colonies. Attempts to obtain resistant mutants by in vivo selection have also failed, suggesting that the target of mefloquine is either lethal or multiple. A number of studies with other bacteria have employed DNA microarrays to narrow down the possible target(s) for the drug. Therefore, to examine the effect of mefloquine on the level of gene expression in M. tuberculosis and M. avium ,w e applied two different techniques, i.e., the commercially available M. tuberculosis DNA microarray and, because the M. avium array has not yet been annotated, a green fluorescent protein (GFP) promoter library of M. avium genes. Some of the genes identified using both approaches for the two mycobacterial species were then overexpressed in M. avium, identifying mechanisms of resistance and a putative mefloquine target.