Slowing down to get ahead: functional and structural characterisation of toxin-antitoxin systems from Mycobacterium tuberculosis

Toxin-antitoxin (TA) systems are ubiquitously found encoded on bacterial chromosomes and mobile genetic elements. They comprise a toxin protein, which interferes with an essential cellular process to inhibit growth, and an antitoxin, either sRNA or protein, which neutralises toxicity. TA modules are implicated in various roles, including plasmid maintenance, phage defence, and aiding pathogenicity. Mycobacterium tuberculosis boasts the greatest number of TA systems, with at least eighty identified thus far. The causative agent of tuberculosis, M. tuberculosis infects one third of the global population and was responsible for 1.4 million deaths in 2019 alone. Elucidating TA mechanisms that contribute to M. tuberculosis pathogenicity may help inform strategies to control future infections. This study has characterised a family of four TA systems identified in M. tuberculosis: MenA1-MenT1, MenA2-MenT2, MenA3-MenT3, and MenA4-MenT4. The MenT toxins are members of the widespread nucleotidyltransferase-like DUF1814 protein family. Functional tests showed that MenA3-MenT3 and MenA4-MenT4 inhibit growth in E. coli through a reversible mechanism. The X-ray crystallographic structures of the MenT1, MenT3, and MenT4 toxins were solved to 1.65 Å, 1.78 Å, and 1.23 Å, respectively. An additional MenT3 structure was solved to 1.59 Å which was phosphorylated at S78, an important residue for MenA3 antitoxicity. MenT1, MenT3 and MenT4 are bi-lobed globular proteins which feature a shared toxin fold and conserved active site. The crystal form of complexed MenA1:MenT1 was also solved to 1.44 Å, which shows MenA1 binding asymmetrically across two MenT1 protomers. Protein interaction studies indicated that the MenA-MenT family is comprised of multiple TA classes. Finally, biochemical assays demonstrated that MenT3 and MenT4 inhibit protein synthesis in vitro, implicating translation as the cellular target. This study’s characterisation of the MenA-MenT family expands our knowledge of M. tuberculosis TA systems, and helps reveal a novel mechanism by which a toxin inhibits bacterial growth.