Inducible auto-phosphorylation regulates a widespread family of nucleotidyltransferase toxins.

Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA1 induces auto-phosphorylation of MenT1 by repositioning the MenT1 phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA3 is similarly able to induce auto-phosphorylation of cognate toxin MenT3. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.

Toxin release by conditional remodelling of ParDE1 from Mycobacterium tuberculosis leads to gyrase inhibition.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a growing threat to global health, with recent efforts towards its eradication being reversed in the wake of the COVID-19 pandemic. Increasing resistance to gyrase-targeting second-line fluoroquinolone antibiotics indicates the necessity to develop both novel therapeutics and our understanding of M. tuberculosis growth during infection. ParDE toxin-antitoxin systems also target gyrase and are regulated in response to both host-associated and drug-induced stress during infection. Here, we present microbiological, biochemical, structural, and biophysical analyses exploring the ParDE1 and ParDE2 systems of M. tuberculosis H37Rv. The structures reveal conserved modes of toxin-antitoxin recognition, with complex-specific interactions. ParDE1 forms a novel heterohexameric ParDE complex, supported by antitoxin chains taking on two distinct folds. Curiously, ParDE1 exists in solution as a dynamic equilibrium between heterotetrameric and heterohexameric complexes. Conditional remodelling into higher order complexes can be thermally driven in vitro. Remodelling induces toxin release, tracked through concomitant inhibition and poisoning of gyrase activity. Our work aids our understanding of gyrase inhibition, allowing wider exploration of toxin-antitoxin systems as inspiration for potential therapeutic agents.

MenT nucleotidyltransferase toxins extend tRNA acceptor stems and can be inhibited by asymmetrical antitoxin binding.

Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, has a genome encoding a remarkably high number of toxin-antitoxin systems of largely unknown function. We have recently shown that the M. tuberculosis genome encodes four of a widespread, MenAT family of nucleotidyltransferase toxin-antitoxin systems. In this study we characterize MenAT1, using tRNA sequencing to demonstrate MenT1 tRNA modification activity. MenT1 activity is blocked by MenA1, a short protein antitoxin unrelated to the MenA3 kinase. X-ray crystallographic analysis shows blockage of the conserved MenT fold by asymmetric binding of MenA1 across two MenT1 protomers, forming a heterotrimeric toxin-antitoxin complex. Finally, we also demonstrate tRNA modification by toxin MenT4, indicating conserved activity across the MenT family. Our study highlights variation in tRNA target preferences by MenT toxins, selective use of nucleotide substrates, and diverse modes of MenA antitoxin activity.

Toxin-antitoxin systems as mediators of phage defence and the implications for abortive infection.

Bacteria have evolved a broad range of defence mechanisms to protect against infection by their viral parasites, bacteriophages (phages). Toxin-antitoxin (TA) systems are small loci found throughout bacteria and archaea that in some cases provide phage defence. The recent explosion in phage defence system discovery has identified multiple novel TA systems with antiphage activity. Due to inherent toxicity, TA systems are thought to mediate abortive infection (Abi), wherein the host cell dies in response to phage infection, removing the phage, and protecting clonal siblings. Recent studies, however, have uncovered molecular mechanisms by which TA systems are activated by phages, how they mediate toxicity, and how phages escape the defences. These new models reveal dazzling complexity in phage-host interactions and provide further evidence that TA systems do not in all cases inherently perform classic Abi, suggesting an evolved conceptual definition is required.