mmpL7 Gene of Mycobacterium tuberculosis Is Responsible for Isoniazid Efflux in Mycobacterium smegmatis

ABSTRACT The Mycobacterium tuberculosis mmpL7 gene, encoding a hypothetical resistance nodulation division transporter, confers a high resistance level to isoniazid when overexpressed in Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine and CCCP (carbonyl cyanide m-chlorophenylhydrazone). Energy-dependent efflux of isoniazid from M. smegmatis cells expressing the mmpL7 gene was observed.

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Rv2686c-Rv2687c-Rv2688c, an ABC Fluoroquinolone Efflux Pump in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.8.3175-3178.2004 ◽

2004 ◽

Vol 48 (8) ◽

pp. 3175-3178 ◽

Cited By ~ 108

Author(s):

Maria Rosalia Pasca ◽

Paola Guglierame ◽

Fabio Arcesi ◽

Marco Bellinzoni ◽

Edda De Rossi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Abc Transporter ◽

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Resistance Level ◽

Carbonyl Cyanide ◽

Efflux Pump Inhibitors ◽

Energy Dependent

ABSTRACT The Mycobacterium tuberculosis Rv2686c-Rv2687c-Rv2688c operon, encoding an ABC transporter, conferred resistance to ciprofloxacin and, to a lesser extent, norfloxacin, moxifloxacin, and sparfloxacin to Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide m-chlorophenylhydrazone, and verapamil. Energy-dependent efflux of ciprofloxacin from M. smegmatis cells containing the Rv2686c-Rv2687c-Rv2688c operon was observed.

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MmpL3 Is the Cellular Target of the Antitubercular Pyrrole Derivative BM212

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05270-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 324-331 ◽

Cited By ~ 141

Author(s):

Valentina La Rosa ◽

Giovanna Poce ◽

Julio Ortiz Canseco ◽

Silvia Buroni ◽

Maria Rosalia Pasca ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Protein A ◽

Multidrug Resistant ◽

Whole Genome ◽

Content Type ◽

Genomic Libraries ◽

Cellular Target ◽

Efflux Pump Inhibitors

ABSTRACTThe 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates andMycobacterium tuberculosisresiding within macrophages as well as againstMycobacterium aviumand other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. SpontaneousMycobacterium smegmatis,Mycobacterium bovisBCG, andM. tuberculosisH37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in themmpL3gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterialmembraneprotein,large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanidem-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [14C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

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Characterization of P55, a Multidrug Efflux Pump inMycobacterium bovis and Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.3.800-804.2001 ◽

2001 ◽

Vol 45 (3) ◽

pp. 800-804 ◽

Cited By ~ 82

Author(s):

Pedro E. A. Silva ◽

Fabiana Bigi ◽

Marı́a de la Paz Santangelo ◽

Maria Isabel Romano ◽

Carlos Martı́n ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Tetracycline Resistance ◽

Drug Efflux ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Reading Frame ◽

Carbonyl Cyanide

ABSTRACT The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of Mycobacterium tuberculosisH37Rv, annotated as a probable drug efflux protein. Genes similar toP55 were present in all species of the M. tuberculosis complex and other mycobacteria such asMycobacterium leprae and Mycobacterium avium. By Western blotting, P55 was located in the membrane fraction ofM. bovis. When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatiscells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.

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Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

Journal of Bacteriology ◽

10.1128/jb.182.19.5479-5485.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5479-5485 ◽

Cited By ~ 34

Author(s):

Helena I. M. Boshoff ◽

Valerie Mizrahi

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Intracellular Localization ◽

Wild Type ◽

Gene Encoding ◽

Allelic Exchange ◽

Mutant Strains ◽

Established Role ◽

Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

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Optimized Nile Red Efflux Assay of AcrAB-TolC Multidrug Efflux System Shows Competition between Substrates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00620-10 ◽

2010 ◽

Vol 54 (9) ◽

pp. 3770-3775 ◽

Cited By ~ 83

Author(s):

Jürgen A. Bohnert ◽

Brian Karamian ◽

Hiroshi Nikaido

Keyword(s):

Antimicrobial Agents ◽

Efflux Pump ◽

Nile Red ◽

Proton Conductor ◽

Multidrug Efflux ◽

Pump System ◽

Tetraphenylphosphonium Chloride ◽

Carbonyl Cyanide ◽

Efflux Pump Inhibitors ◽

Multidrug Efflux System

ABSTRACT AcrAB-TolC is the major constitutively expressed efflux pump system that provides resistance to a variety of antimicrobial agents and dyes in Escherichia coli. However, no systematically optimized real-time dye efflux assay has been published for the measurement of its activity and for detection of possible competition between substrates. Here, we report on the development of such an assay using a lipophilic dye, Nile Red. Energy-depleted cells were loaded with the dye in the presence of low (10 μM or less) concentrations of the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The CCCP was then removed, and efflux was triggered by energization with glucose. Various known efflux pump inhibitors and antimicrobials were checked for the ability to slow down Nile Red efflux, presumably through competition. Besides the known inhibitors Phe-Arg-β-naphthylamide and 1-naphthyl-methylpiperazine, several tetracyclic compounds (doxorubicin, minocycline, chlortetracycline, doxycycline, and tetracycline) and tetraphenylphosphonium chloride were found to interfere with dye efflux. This inhibition could not be explained by the depletion of proton motive force. None of the other tested antimicrobials, including macrolides, fluoroquinolones, and β-lactams, had any impact on Nile Red efflux, even at concentrations of up to 1 mM.

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Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00940-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 15

Author(s):

Yumeng Zhang ◽

Jia Zhang ◽

Peng Cui ◽

Ying Zhang ◽

Wenhong Zhang

Keyword(s):

Mycobacterium Tuberculosis ◽

Efflux Pump ◽

Binding Studies ◽

Content Type ◽

Clinical Strains ◽

Efflux Pump Inhibitors ◽

Level Resistance ◽

Pyrazinoic Acid ◽

Increased Susceptibility ◽

Efflux Proteins

ABSTRACT Pyrazinamide (PZA) is a critical drug used for the treatment of tuberculosis (TB). PZA is a prodrug that requires conversion to the active component pyrazinoic acid (POA) by pyrazinamidase (PZase) encoded by the pncA gene. Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1 mutations, clinical strains without these mutations are known to exist. While efflux of POA was demonstrated in Mycobacterium tuberculosis previously, the efflux proteins involved have not been identified. Here we performed POA binding studies with an M. tuberculosis proteome microarray and identified four efflux proteins (Rv0191, Rv3756c, Rv3008, and Rv1667c) that bind POA. Overexpression of the four efflux pump genes in M. tuberculosis caused low-level resistance to PZA and POA but not to other drugs. Furthermore, addition of efflux pump inhibitors such as reserpine, piperine, and verapamil caused increased susceptibility to PZA in M. tuberculosis strains overexpressing the efflux proteins Rv0191, Rv3756c, Rv3008, and Rv1667c. Our studies indicate that these four efflux proteins may be responsible for PZA/POA efflux and cause PZA resistance in M. tuberculosis. Future studies are needed to assess their roles in PZA resistance in clinical strains.

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The mycobacterial efflux pump EfpA can induce high drug tolerance to many anti-tuberculosis drugs, including moxifloxacin, in Mycobacterium smegmatis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00262-21 ◽

2021 ◽

Author(s):

Deepika Rai ◽

Sarika Mehra

Keyword(s):

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Drug Tolerance ◽

Inducible Promoter ◽

Major Facilitator Superfamily ◽

Expression Level ◽

Efflux Pump Inhibitor ◽

Gene Encoding ◽

Active Efflux ◽

Major Facilitator

Active efflux of drugs across the membrane is a major survival strategy of bacteria against many drugs. In this work, we characterize an efflux pump EfpA, from the major facilitator superfamily, that is highly conserved among both slow growing and fast-growing mycobacterium species and has been found to be upregulated in many clinical isolates of Mycobacterium tuberculosis . The gene encoding EfpA from Mycobacterium smegmatis was over-expressed under both constitutive and an inducible promoter. Expression of efpA gene under both the promoters resulted in greater than 32-fold increased drug tolerance of M. smegmatis cells to many first-line (rifampicin, isoniazid and streptomycin) and second-line (amikacin) anti-tuberculosis drugs. Notably, drug tolerance of M. smegmatis cells to moxifloxacin increased by more than 180-fold when efpA was over-expressed. The increase in minimum inhibitory concentration (MIC) correlated with the decreased uptake of drugs including norfloxacin, moxifloxacin and ethidium bromide and the high MIC could be reversed in the presence of an efflux pump inhibitor. A correlation was observed between the MIC of drugs and the efflux pump expression level, suggesting that the latter could be modulated by varying the expression level of the efflux pump. The expression of high levels of efpA did not impact the fitness of the cells when supplemented with glucose.The efpA gene is conserved across both pathogenic and non-pathogenic mycobacteria. The efpA gene from the Mycobacterium bovis BCG/ M. tuberculosis , which is 80% identical to efpA from M. smegmatis , also led to decreased antimicrobial efficacy to many drugs, although the fold-change was lower. When over-expressed in M. bovis BCG, an 8-fold higher drug tolerance to moxifloxacin was observed . This is the first report of an efflux pump from mycobacterium species that leads to higher drug tolerance to moxifloxacin, a promising new drug for the treatment of tuberculosis.

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Structural Plasticity and Distinct Drug-Binding Modes of LfrR, a Mycobacterial Efflux Pump Regulator

Journal of Bacteriology ◽

10.1128/jb.00631-09 ◽

2009 ◽

Vol 191 (24) ◽

pp. 7531-7537 ◽

Cited By ~ 26

Author(s):

Marco Bellinzoni ◽

Silvia Buroni ◽

Francis Schaeffer ◽

Giovanna Riccardi ◽

Edda De Rossi ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Drug Binding ◽

Binding Modes ◽

Transcriptional Repressors ◽

Gene Encoding ◽

Drug Binding Site ◽

Conformational Plasticity ◽

Local Unfolding ◽

Calorimetric Studies

ABSTRACT The TetR-like transcriptional repressor LfrR controls the expression of the gene encoding the Mycobacterium smegmatis efflux pump LfrA, which actively extrudes fluoroquinolones, cationic dyes, and anthracyclines from the cell and promotes intrinsic antibiotic resistance. The crystal structure of the apoprotein form of the repressor reveals a structurally asymmetric homodimer exhibiting local unfolding and a blocked drug-binding site, emphasizing the significant conformational plasticity of the protein necessary for DNA and multidrug recognition. Crystallographic and calorimetric studies of LfrR-drug complexes further confirm the intrinsic flexibility of the homodimer, which provides a dynamic mechanism to broaden multidrug binding specificity and may be a general property of transcriptional repressors regulating microbial efflux pump expression.

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Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02637-16 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 13

Author(s):

Jichan Jang ◽

Ryangyeo Kim ◽

Minjeong Woo ◽

Jinsun Jeong ◽

Da Eun Park ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Efflux Pump ◽

Ex Vivo ◽

Drug Candidate ◽

Content Type ◽

Tb Treatment ◽

Pump Activity ◽

Efflux Pump Inhibitors ◽

Combinatorial Strategy

ABSTRACT New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo, indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis.

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