Whole-Transcriptome and -Genome Analysis of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates Identifies Downregulation of ethA as a Mechanism of Ethionamide Resistance

Antimicrob Agents Chemother. 2017 Nov 22;61(12):e01461-17. doi: 10.1128/AAC.01461-17. Print 2017 Dec.


Genetics-based drug susceptibility testing has improved the diagnosis of drug-resistant tuberculosis but is limited by our lack of knowledge of all resistance mechanisms. Next-generation sequencing has assisted in identifying the principal genetic mechanisms of resistance for many drugs, but a significant proportion of phenotypic drug resistance is unexplained genetically. Few studies have formally compared the transcriptomes of susceptible and resistant Mycobacterium tuberculosis strains. We carried out comparative whole-genome transcriptomics of extensively drug-resistant (XDR) clinical isolates using RNA sequencing (RNA-seq) to find novel transcription-mediated mechanisms of resistance. We identified a promoter mutation (t to c) at position -11 (t-11c) relative to the start codon of ethA that reduces the expression of a monooxygenase (EthA) that activates ethionamide. (In this article, nucleotide changes are lowercase and amino acid substitutions are uppercase.) Using a flow cytometry-based reporter assay, we show that the reduced transcription of ethA is not due to transcriptional repression by ethR Clinical strains harboring this mutation were resistant to ethionamide. Other ethA promoter mutations were identified in a global genomic survey of resistant M. tuberculosis strains. These results demonstrate a new mechanism of ethionamide resistance that can cause high-level resistance when it is combined with other ethionamide resistance-conferring mutations. Our study revealed many other genes which were highly up- or downregulated in XDR strains, including a toxin-antitoxin module (mazF5 mazE5) and tRNAs (leuX and thrU). This suggests that global transcriptional modifications could contribute to resistance or the maintenance of bacterial fitness have also occurred in XDR strains.

Keywords: RNA sequencing; RNA-seq; drug resistance; drug susceptibility testing; flow cytometry; fluorescent reporter; mymA; promoter; tuberculosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antitubercular Agents / pharmacology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Drug Resistance, Multiple, Bacterial / genetics*
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism
  • Ethionamide / pharmacology*
  • Gene Expression Regulation, Bacterial
  • Genome, Bacterial*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Isoniazid / pharmacology
  • Mutation
  • Mycobacterium tuberculosis / drug effects
  • Mycobacterium tuberculosis / enzymology
  • Mycobacterium tuberculosis / genetics*
  • Mycobacterium tuberculosis / isolation & purification
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Promoter Regions, Genetic
  • RNA, Bacterial / genetics*
  • RNA, Bacterial / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Transcriptome*
  • Tuberculosis, Multidrug-Resistant / drug therapy
  • Tuberculosis, Multidrug-Resistant / microbiology
  • Tuberculosis, Multidrug-Resistant / pathology


  • Antitubercular Agents
  • Bacterial Proteins
  • EthR protein, Mycobacterium tuberculosis
  • RNA, Bacterial
  • Repressor Proteins
  • Oxidoreductases
  • ethA protein, Mycobacterium tuberculosis
  • Endoribonucleases
  • MazF protein, Mycobacterium tuberculosis
  • Ethionamide
  • Isoniazid