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

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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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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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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Role of the MexEF-OprN Efflux System in Low-Level Resistance of Pseudomonas aeruginosa to Ciprofloxacin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00101-11 ◽

2011 ◽

Vol 55 (12) ◽

pp. 5676-5684 ◽

Cited By ~ 48

Author(s):

Catherine Llanes ◽

Thilo Köhler ◽

Isabelle Patry ◽

Barbara Dehecq ◽

Christian van Delden ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Reference Strain ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Efflux System ◽

Content Type ◽

Clinical Strains ◽

Major Mechanism ◽

Level Resistance

ABSTRACTIn this study, we investigated the resistance mechanisms to fluoroquinolones of 85 non-cystic fibrosis strains ofPseudomonas aeruginosaexhibiting a reduced susceptibility to ciprofloxacin (MICs from 0.25 to 2 μg/ml). In addition to MexAB-OprM (31 of 85 isolates) and MexXY/OprM (39 of 85), the MexEF-OprN efflux pump (10 of 85) was found to be commonly upregulated in this population that is considered susceptible or of intermediate susceptibility to ciprofloxacin, according to current breakpoints. Analysis of the 10 MexEF-OprN overproducers (nfxCmutants) revealed the presence of various mutations in themexT(2 isolates),mexS(5 isolates), and/ormvaT(2 isolates) genes, the inactivation of which is known to increase the expression of themexEF-oprNoperon in reference strain PAO1-UW. However, these genes were intact in 3 of 10 of the clinical strains. Interestingly, ciprofloxacin at 2 μg/ml or 4 μg/ml preferentially selectednfxCmutants from wild-type clinical strains (n= 10 isolates) and from first-step mutants (n= 10) overexpressing Mex pumps, thus indicating that MexEF-OprN represents a major mechanism by whichP. aeruginosamay acquire higher resistance levels to fluoroquinolones. These data support the notion that thenfxCmutants may be more prevalent in the clinical setting than anticipated and strongly suggest the involvement of still unknown genes in the regulation of this efflux system.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01341-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6873-6881 ◽

Cited By ~ 13

Author(s):

Kathryn Winglee ◽

Shichun Lun ◽

Marco Pieroni ◽

Alan Kozikowski ◽

William Bishai

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Efflux Pump ◽

Transcriptional Regulator ◽

Cross Resistance ◽

Loss Of Function ◽

Strong Activity ◽

Content Type ◽

Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

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Resistance against Membrane-Inserting MmpL3 Inhibitor through Upregulation of MmpL5 in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01100-20 ◽

2020 ◽

Vol 64 (12) ◽

Author(s):

Ming Li ◽

Samuel Agyei Nyantakyi ◽

Mei-Lin Go ◽

Thomas Dick

Keyword(s):

Antibacterial Activity ◽

Mycobacterium Tuberculosis ◽

Mouse Model ◽

Mannich Base ◽

Efflux Pump ◽

Transcriptional Repressor ◽

Mannich Bases ◽

Mycolic Acid ◽

Low Level ◽

Level Resistance

ABSTRACT Spiroketal indolyl Mannich bases (SIMBs) present a novel class of membrane-inserting antimycobacterials with efficacy in a tuberculosis mouse model. SIMBs exert their antibacterial activity by two mechanisms. The indolyl Mannich base scaffold causes permeabilization of bacteria, and the spiroketal moiety contributes to inhibition of the mycolic acid transporter MmpL3. Here, we show that low-level resistance to SIMBs arises by mutations in the transcriptional repressor MmpR5, resulting in upregulation of the efflux pump MmpL5.

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Isoniazid Resistance in Mycobacterium tuberculosis Is a Heterogeneous Phenotype Composed of Overlapping MIC Distributions with Different Underlying Resistance Mechanisms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00092-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 11

Author(s):

Arash Ghodousi ◽

Elisa Tagliani ◽

Eranga Karunaratne ◽

Stefan Niemann ◽

Jennifer Perera ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Resistance Mechanisms ◽

Whole Genome ◽

Tube System ◽

Isoniazid Resistance ◽

Content Type ◽

Indicator Tube ◽

Promoter Mutations ◽

Level Resistance ◽

Growth Indicator

ABSTRACT MIC testing using the Bactec mycobacteria growth indicator tube system 960 of 70 phylogenetically diverse, isoniazid-resistant clinical strains of Mycobacterium tuberculosis revealed a complex pattern of overlapping MIC distributions. Whole-genome sequencing explained most of the levels of resistance observed. The MIC distribution of strains with only inhA promoter mutations was split by the current concentration endorsed by the Clinical and Laboratory Standards Institute to detect low-level resistance to isoniazid and is, consequently, likely not optimally set.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.11.4775-4777.2005 ◽

2005 ◽

Vol 49 (11) ◽

pp. 4775-4777 ◽

Cited By ~ 78

Author(s):

Maria R. Pasca ◽

Paola Guglierame ◽

Edda De Rossi ◽

Francesca Zara ◽

Giovanna Riccardi

Keyword(s):

Mycobacterium Tuberculosis ◽

High Resistance ◽

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Gene Encoding ◽

Resistance Level ◽

Carbonyl Cyanide ◽

Resistance Nodulation Division ◽

Efflux Pump Inhibitors ◽

Energy Dependent

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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In Vitro Study of Stepwise Acquisition of rv0678 and atpE Mutations Conferring Bedaquiline Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00292-19 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 4

Author(s):

Nabila Ismail ◽

Nazir A. Ismail ◽

Shaheed V. Omar ◽

Remco P. H. Peters

Keyword(s):

Mycobacterium Tuberculosis ◽

Solid Medium ◽

In Vitro Study ◽

Whole Genome ◽

Content Type ◽

Phenotypic Resistance ◽

Atcc Strain ◽

High Level ◽

Level Resistance

ABSTRACT Bedaquiline resistance within Mycobacterium tuberculosis may arise through efflux-based (rv0678) or target-based (atpE) pathway mutations. M. tuberculosis mutant populations from each of five sequential steps in a passaging approach, using a pyrazinamide-resistant ATCC strain, were subjected to MIC determinations and whole-genome sequencing. Exposure to increasing bedaquiline concentrations resulted in increasing phenotypic resistance (up to >2 μg/ml) through MIC determination on solid medium (Middlebrook 7H10). rv0678 mutations were dynamic, while atpE mutations were fixed, once occurring. We present the following hypothesis for in vitro emergence of bedaquiline resistance: rv0678 mutations may be the first transient step in low-level resistance acquisition, followed by high-level resistance due to fixed atpE mutations.

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