Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis

Abstract

Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.

Fig 1

(A) Replacement of the M. tuberculosis mmr gene. An allelic exchange substrate (AES) for replacement of mmr (Rv3065) was generated by cloning 576-bp upstream and 996-bp downstream regions on either side of the hyg resistance cassette of pYUB854. The AES was obtained by PCR or restriction digestion of this segment; (B) integrative plasmid pCRS5 that contains a copy of mmr and that was used to complement the Mmr knockout mutant; (C) replicative plasmid pCVZ2 used to overexpress mmr in H37Rv.

Fig 2

Accumulation of ethidium bromide at 1 μg/ml by M. tuberculosis H37Rv, H37Rv MmrKO (H37Rv with the mmr gene inactivated), and H37Rv::pCVZ2 (H37Rv containing pCVZ2).

Fig 3

Effect of efflux inhibitors on the accumulation of ethidium bromide for M. tuberculosis H37Rv (A), H37Rv MmrKO (B), and H37Rv::pCVZ2 (C). Ethidium bromide and efflux inhibitors were used at 1/2 the MIC to not compromise cellular viability. CCCP, carbonyl cyanide m-chlorophenylhydrazone; CPZ, chlorpromazine; EPI, efflux pump inhibitor; VP, verapamil.