Abstract
ABSTRACTGenetics-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.
Highlights
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has progressively developed resistance to the most effective first- and second-line antituberculosis drugs [1]
In order to identify novel mechanisms of resistance mediated at the level of transcription, we subjected drug-resistant and drugsusceptible strains of M. tuberculosis to comparative transcriptomics using RNA sequencing (Table 1)
We confirmed by our dual-color promoter assay and quantitative RT-PCR that this mutation leads to the reduced transcription of ethA, which encodes a monooxygenase that activates the prodrug ethionamide [22, 23]
Summary
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has progressively developed resistance to the most effective first- and second-line antituberculosis drugs [1]. Current molecular genetics-based tests, such as the GeneXpert MTB/RIF and GenoType MTBDRplus assays, have accelerated the clinical detection of known mutations causing RIF and/or isoniazid resistance [4, 5]. These and other genetic tests detect only MDR-TB and a limited number of mutations associated with resistance to second-line drugs [6]. Our current understanding of drug resistance in M. tuberculosis has developed through studying resistant mutants isolated in vitro and the accumulation of mutations in resistant clinical isolates [12] These studies have identified various genetic mechanisms of resistance, including target modification, loss of the enzymatic function required to activate prodrugs, and altered drug efflux [13, 14]
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