Molecular Investigation of Resistance to the Antituberculous Drug Ethionamide in Multidrug-Resistant Clinical Isolates ofMycobacterium tuberculosis

ABSTRACTEthionamide (ETH) needs to be activated by the mono-oxygenase EthA, which is regulated by EthR, in order to be active againstMycobacterium tuberculosis. The activated drug targets the enzyme InhA, which is involved in cell wall biosynthesis. Resistance to ETH has been reported to result from various mechanisms, including mutations altering EthA/EthR, InhA and its promoter, the NADH dehydrogenase encoded byndh, and the MshA enzyme, involved in mycothiol biosynthesis. We searched for such mutations in 87 clinical isolates: 47 ETH-resistant (ETHr) isolates, 24 ETH-susceptible (ETHs) isolates, and 16 isolates susceptible to ETH but displaying an intermediate proportion of resistant cells (ETHSip; defined as ≥1% but <10% resistant cells). In 81% (38/47) of the ETHrisolates, we found mutations inethA,ethR, orinhAor its promoter, which mostly corresponded to new alterations inethAandethR. The 9 ETHrisolates without a mutation in these three genes (9/47, 19%) had no mutation inndh, and a single isolate had a mutation inmshA. Of the 16 ETHSipisolates, 7 had a mutation inethA, 8 had no detectable mutation, and 1 had a mutation inmshA. Finally, of the 24 ETHsisolates, 23 had no mutation in the studied genes and 1 displayed a yet unknown mutation in theinhApromoter. Globally, the mechanism of resistance to ETH remained unknown for 19% of the ETHrisolates, highlighting the complexity of the mechanisms of ETH resistance inM. tuberculosis.

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Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

mBio ◽

10.1128/mbio.01767-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 17

Author(s):

Shichun Lun ◽

David Miranda ◽

Andre Kubler ◽

Haidan Guo ◽

Mariama C. Maiga ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Synthetic Lethality ◽

Multidrug Resistant ◽

Cell Wall Biosynthesis ◽

Drug Resistant ◽

Content Type ◽

Innate Resistance ◽

Extensively Drug Resistant

ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. IMPORTANCE The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of β-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to β-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with β-lactams in the mouse model.

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RNA expression analysis of efflux pump genes in clinical isolates of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis

10.26226/morressier.56d5ba2cd462b80296c94d42 ◽

2016 ◽

Author(s):

Hee Joo Lee

Keyword(s):

Mycobacterium Tuberculosis ◽

Expression Analysis ◽

Efflux Pump ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Rna Expression ◽

Drug Resistant ◽

Extensively Drug Resistant

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Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dku161 ◽

2014 ◽

Vol 69 (9) ◽

pp. 2369-2375 ◽

Cited By ~ 32

Author(s):

Tomasz Jagielski ◽

Zofia Bakuła ◽

Katarzyna Roeske ◽

Michał Kamiński ◽

Agnieszka Napiórkowska ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Isoniazid Resistance ◽

Detection Of Mutations

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Label-Free Comparative Proteomics of Differentially Expressed Mycobacterium tuberculosis Protein in Rifampicin-Related Drug-Resistant Strains

Pathogens ◽

10.3390/pathogens10050607 ◽

2021 ◽

Vol 10 (5) ◽

pp. 607

Author(s):

Nadeem Ullah ◽

Ling Hao ◽

Jo-Lewis Banga Ndzouboukou ◽

Shiyun Chen ◽

Yaqi Wu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Control Strategies ◽

Multidrug Resistant ◽

Differentially Expressed ◽

Effective Control ◽

Drug Resistant ◽

Differentially Expressed Proteins ◽

Label Free ◽

Candidate Target

Rifampicin (RIF) is one of the most important first-line anti-tuberculosis (TB) drugs, and more than 90% of RIF-resistant (RR) Mycobacterium tuberculosis clinical isolates belong to multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. In order to identify specific candidate target proteins as diagnostic markers or drug targets, differential protein expression between drug-sensitive (DS) and drug-resistant (DR) strains remains to be investigated. In the present study, a label-free, quantitative proteomics technique was performed to compare the proteome of DS, RR, MDR, and XDR clinical strains. We found iniC, Rv2141c, folB, and Rv2561 were up-regulated in both RR and MDR strains, while fadE9, espB, espL, esxK, and Rv3175 were down-regulated in the three DR strains when compared to the DS strain. In addition, lprF, mce2R, mce2B, and Rv2627c were specifically expressed in the three DR strains, and 41 proteins were not detected in the DS strain. Functional category showed that these differentially expressed proteins were mainly involved in the cell wall and cell processes. When compared to the RR strain, Rv2272, smtB, lpqB, icd1, and folK were up-regulated, while esxK, PPE19, Rv1534, rpmI, ureA, tpx, mpt64, frr, Rv3678c, esxB, esxA, and espL were down-regulated in both MDR and XDR strains. Additionally, nrp, PPE3, mntH, Rv1188, Rv1473, nadB, PPE36, and sseA were specifically expressed in both MDR and XDR strains, whereas 292 proteins were not identified when compared to the RR strain. When compared between MDR and XDR strains, 52 proteins were up-regulated, while 45 proteins were down-regulated in the XDR strain. 316 proteins were especially expressed in the XDR strain, while 92 proteins were especially detected in the MDR strain. Protein interaction networks further revealed the mechanism of their involvement in virulence and drug resistance. Therefore, these differentially expressed proteins are of great significance for exploring effective control strategies of DR-TB.

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