EthA/R-Independent Killing of Mycobacterium tuberculosis by Ethionamide.

Michelle L T Ang,Vanessa Koh,Paola Florez De Sessions,Siti Z Zainul Rahim,Martin L Hibberd,Wenwei Lin,Kevin Pethe,Sylvie Alonso

doi:10.3389/fmicb.2017.00710

Abstract

Ethionamide (ETH) is part of the drug arsenal available to treat multi-drug resistant tuberculosis. The current paradigm of this pro-drug activation involves the mycobacterial enzyme EthA and the transcriptional repressor, EthR. However, several lines of evidence suggest the involvement of additional players. The ethA/R locus was deleted in Mycobacterium bovis BCG and three Mycobacterium tuberculosis (MTB) strains. While complete resistance to ETH was observed with BCG ethA/R KO, drug susceptibility and dose-dependent killing were retained in the ethA/R KO MTB mutants, suggesting the existence of an alternative pathway of ETH bio-activation in MTB. We further demonstrated that this alternative pathway is EthR-independent, whereby re-introduction of ethR in ethA/R KO MTB did not lead to increased resistance to ETH. Consistently, ethA KO MTB (with intact ethR expression) displayed similar ETH susceptibility profile as their ethA/R KO counterparts. To identify the alternative ETH bio-activator, spontaneous ETH-resistant mutants were obtained from ethA/R KO MTB and whole genome sequencing identified single nucleotide polymorphisms in mshA, involved in mycothiol biosynthesis and previously linked to ETH resistance. Deletion of mshA in ethA/R KO MTB led to complete ETH resistance, supporting that the role of MshA in ETH killing is EthA/R-independent. Furthermore mshA single KO MTB displayed levels of ETH resistance similar or greater than those obtained with ethA/R KO strains, supporting that mshA is as critical as ethA/R for ETH killing efficacy.

Highlights

One-third of the world population is presently infected with Mycobacterium tuberculosis (MTB), and this worldwide epidemic appears to be deteriorating
Concentration of drug required to inhibit 50% growth of mycobacteria compared to drug-free control (MIC50) was measured for the respective ethA/R KO mutant strains in order to determine their level of resistance to ETH and to the two other thiocarbamide-containing drugs ISO and TAC
Complete resistance to ETH was not seen with the MTB ethA/R KO mutants whereby a dose-dependent killing could still be observed as evidenced by the sigmoidal MIC curves obtained (Supplementary Figure S2)

Summary

Introduction

One-third of the world population is presently infected with Mycobacterium tuberculosis (MTB), and this worldwide epidemic appears to be deteriorating. Underlying this endemic is the emerging epidemic of multi-drug resistant (MDR-TB) and extensively drug resistant (XDR-TB) TB strains that have severely undermined control efforts (WHO, 2016). With dwindling treatment options for MDR and XDR-TB that are decades old, one of the pertinent key issues faced by the TB research community is the daunting challenge of synthesizing new anti-TB drugs with novel modes of action (Koul et al, 2011). The emergence of clinical resistance less than 3 years after BDQ introduction to medical use is likely to limit the impact of this new TB drug (Bloemberg et al, 2015). More appropriate clinical trials to properly evaluate the efficacy of anti-TB drugs used in MDR and XDR-TB patient groups are necessary along with the improvement in TB diagnostics for a wider coverage of drug susceptibility testing (Koul et al, 2011)

Methods

Results

Discussion

Conclusion