Lost in Translation: Characterisation of Translation-targeting Bacterial Toxin-Antitoxin Systems from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), represents the single deadliest human pathogen globally, accounting for over 1.25 million deaths per annum. Believed to contribute to its alarming success rate as an infectious agent are the remarkably high number of toxin-antitoxin (TA) systems found within the M. tuberculosis genome, with over 90 identified to date. TA systems encode a toxic effector molecule responsible for targeting an essential cellular process required for growth, such as DNA replication or protein synthesis, and an antitoxin responsible for neutralising toxicity in the absence of specific activating triggers. TA systems have been implicated in a variety of biological roles, including evasion of environmental stress and promoting virulence, both of which are key phenotypic traits of M. tuberculosis. Understanding the biological functions and targets of these systems might therefore reveal new strategies in the ongoing battle against M. tuberculosis. The research presented herein details structural, functional, biochemical, and biophysical characterisation of a widespread family of DUF1814 nucleotidyltransferase (NTase) toxins and cognate antitoxins from two unrelated human pathogens. The MenA-MenT TA systems from M. tuberculosis encode functional MenT1, MenT3, and MenT4 toxins, each of which block translation through modification of tRNA 3ʹ-ends, and cognate MenA antitoxins. Streptococcus agalactiae encodes a homologous AbiEi-AbiEii TA system that also encodes a DUF1814 family NTase toxin (AbiEii) and cognate antitoxin (AbiEi), though the biological target(s) and mechanism of toxin neutralisation are unknown. Biochemical studies revealed that, like MenT3, toxins MenT1 and AbiEii are phosphorylated at conserved active site residues when expressed in the presence of their cognate antitoxins. The X-ray crystallographic structure of phosphorylated MenT1 was solved to a resolution of 2.80 Å, revealing reduced positive charge and steric occlusion of the active site blocks toxin activity. Biophysical and functional assays showed that cognate antitoxins are likely not protein kinases, but instead function as regulators of phosphorylation activity by inducing toxin auto-phosphorylation. The AbiEi:AbiEii TA complex was also solved to a resolution of 2.20 Å, supporting the proposed function of antitoxins as inducers of toxin auto-phosphorylation. Protein interaction studies revealed perplexing diversity amongst the MenA-MenT classes in that MenA4 does not bind to or phosphorylate MenT4. Finally, biochemical assays and RNA sequencing revealed both the mode of action and likely target of AbiEii, whilst proof-of-concept hit compound screens highlighted the therapeutic potential of MenT toxins as novel drug targets. Collectively, this study advances our understanding of TA system functionality and reveals a novel mechanism of toxin neutralisation in a widespread family of NTases.