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

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.

Identification of a Toxin–Antitoxin System That Contributes to Persister Formation by Reducing NAD in Pseudomonas aeruginosa

“…Therefore, reducing the intracellular level of NAD + by ResA might rewire the cell metabolism and interfere with the redox homeostasis, which might contribute to increased antibiotic tolerance. A recent study demonstrated that overexpression of a M. tuberculosis toxin MbcT in Mycobacterium smegmatis depleted the intracellular NAD+ and lead to cell death [ 51 ]. We suspect that ResA might display obvious growth inhibitory effects under certain environmental stresses such as carbon or amino acid starvation.…”

Nucleotidyltransferase toxin MenT targets and extends the aminoacyl acceptor ends of serine tRNAs in vivo to control Mycobacterium tuberculosis growth

“…M. tuberculosis encodes an unusually high number of TA systems, over 86, representing close to 4% of its genome (Akarsu et al , 2019; Sala et al , 2014). This includes multiple homologs from conserved TA families that have been shown to be induced under relevant stress conditions, including hypoxia, macrophage engulfment, or drug exposure (Ramage et al , 2009; Keren et al , 2011; Ariyachaokun et al , 2020). Although their contribution to M. tuberculosis physiology and virulence is currently unknown, it has been proposed that activated toxins could modulate M. tuberculosis growth under certain conditions, thereby contributing to survival in the human host (Keren et al , 2011; Barth et al , 2021; Sala et al , 2014).…”

Toxin-mediated depletion of nicotinamide dinucleotides drives persister formation in a human pathogen

“…Some members have recently been implicated in solvent tolerance, plasmid stability or stress-mediated growth inhibition 12,14,[20][21][22] . E.g., the MbcA-MbcT system of M. tuberculosis is significantly upregulated in a variety of stress conditions, including persister cells 23 , hypoxic stress 24 , starvation 25 , and in human macrophages 26,27 . Similarly, the natR-natT module of P. aeruginosa is induced under oxidative stress conditions, exposure to antibiotics and under host-like conditions [28][29][30] and natT mutants show reduced survival during antibiotic treatment and in macrophages 30 .…”