Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump

Abstract

The lfrA gene cloned from chromosomal DNA of quinolone-resistant Mycobacterium smegmatis mc 2 -552 conferred low-level resistance to fluoroquinolones when present on multicopy plasmids. Sequence analysis suggested that lfrA encodes a membrane efflux pump of the major facilitator family (H. E. Takiff, M. Cimino, M. C. Musso, T. Weisbrod, R. Martinez, M. B. Delgado, L. Salazar, B. R. Bloom, and W. R. Jacbos, Jr., Proc. Natl. Acad. Sci. USA 93: [362][363][364][365][366] 1996). In this work, we studied the role of LfrA in the accumulation of fluoroquinolones by M. smegmatis. The steady-state accumulation level of a hydrophilic quinolone, norfloxacin, by M. smegmatis harboring a plasmid carrying the lfrA gene was about 50% of that by the parent strain but was increased to the same level as that of the parent strain by addition of a proton conductor, carbonyl cyanide m-chorophenylhydrazone. Norfloxacin efflux mediated by LfrA was competed for strongly by ciprofloxacin but not by nalidixic acid. Furthermore, we showed that portions of norfloxacin accumulated by starved cells were pumped out upon reenergization of the cells, and the rates of this efflux showed evidence of saturation at higher intracellular concentrations of the drug. These results suggest that the LfrA polypeptide catalyzes the active efflux of several quinolones.The reemergence of tuberculosis worldwide has once again produced a major public health problem. Tuberculosis and other mycobacterial infections are often difficult to treat because mycobacteria are intrinsically resistant to most common antibiotics, apparently because of their extremely low cell wall fluidity and permeability (1,13,14,18,35). The situation is made worse by the dramatic increase in multidrug-resistant strains (6, 7). Fluoroquinolones inhibit bacterial DNA gyrase and are active against mycobacteria in vitro; thus, they seem to be promising for development as antituberculous agents. Unfortunately, fluoroquinolone-resistant clinical isolates of Mycobacterium tuberculosis have already appeared (3, 31, 33). Mutations conferring resistance to quinolones have been reported for several bacterial species (2), including the gram-negative bacteria Escherichia coli (5,10,22) and Pseudomonas aeruginosa (4, 16, 28) and the gram-positive bacterium Staphylococcus aureus (11,23,24,36). Two principal mechanisms have been described: (i) alteration of DNA gyrase and (ii) decreased drug accumulation in the cell as a result of either decreased influx or increased efflux. Alteration of DNA gyrase is usually the result of a mutation in the gyrA or, less frequently, the gyrB gene. The genes that encode the DNA gyrase of M. tuberculosis H37Rv have been cloned and sequenced, and mutations associated with quinolone resistance have been found in the gyrA gene from clinical isolates (3, 33).In gram-positive bacteria, it has been shown that active efflux pumps play an important role in the decreased accumulation of quinolones. An example is the norA gene product of S. aureus, which confers resistance to quin...

A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability

“…A previous study suggested that the major site of penetration for the hydrophobic molecules norfloxacin and chenodeoxycholate is the lipid domain of the cell wall (23). These results, together with increased resistance to more hydrophobic antibiotics, suggest that the cell wall permeability of the mutant to lipophilic molecules is decreased.…”

Molecular Properties of Bacterial Multidrug Transporters

“…NorA and Bmr confer higher levels of resistance to hydrophilic quinolones, such as norfloxacin and ciprofloxacin, than to hydrophobic quinolones, such as sparfloxacin, ofloxacin, and nalidixic acid (104,171,271,278). Similarly, norfloxacin transport by LfrA is inhibited by hydrophilic and not by hydrophobic quinolones (134). This specificity for hydrophilic quinolones was also observed for PmrA and NorM (63,163).…”

“…From the rapidly growing Mycobacterium smegmatis, which was used as a model genetic system for M. tuberculosis, the lfrA gene, which confers low-level fluoroquinolone resistance when present on a multicopy plasmid, was isolated. LfrA confers resistance to hydrophilic quinolones but not to the more hydrophobic quinolones (134). Hybridization analysis showed that LfrA homologs are present in M. tuberculosis and Mycobacterium avium (244).…”

Bioenergetics of the Staphylococcal Multidrug Export Protein QacA

“…Consistent with these assumptions was the observation that the amount of the fluorescent substrate in loaded cells was proportional to the concentration used. Similar kinetic studies of multidrug efflux systems using whole cells have been performed in P. aeruginosa (32) and Mycobacterium smegmatis (33).…”