Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System.

Kanchiyaphat Ariyachaokun,Anna D Grabowska,Olivier Neyrolles,Claude Gutierrez

doi:10.3390/toxins12050329

Abstract

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

Highlights

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is a strictly human-adapted obligate pathogen of major public health importance (WHO, Global Tuberculosis report, 2019)
In order to express the MbcT toxin in M. smegmatis, we used an expression vector, pGMCS-TetR-P1-mbcT, that can stably integrate into the mycobacterial chromosome, within the glyV
Upon transformation into a Mtb strain deleted for the endogenous mbcAT operon, pGMCS-TetR-P1-mbcT conferred anhydro-tetracycline (Atc)-dependent depletion of the essential metabolite NAD+, resulting in bacterial cell death [1]

Summary

Introduction

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is a strictly human-adapted obligate pathogen of major public health importance (WHO, Global Tuberculosis report, 2019). Toxins 2020, 12, 329 bactericidal toxin/antitoxin (TA) systems as new therapeutic tools [1]. TA systems are genetic modules encoding an endogenous protein toxin, which targets an essential process in the bacterial cell, and a toxin-neutralizing antidote, the so-called antitoxin [2,3,4]. The precise mechanisms remain to be characterized for most TA systems, it has been proposed that when cells are facing environmental stresses, the less stable antitoxin is degraded, leading to an increased production of toxin, resulting in growth arrest of the bacterial cells [5,6]. TA systems are classified into six families, depending on the nature of the antitoxin, i.e., ribonucleic acid (RNA) or protein, and its mechanism of toxin neutralization. The recently characterized MbcTA system encodes an NAD+

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Conclusion