Harnessing tuberculosis toxins to manipulate bacterial growth

Background. Mycobacterium tuberculosis is the biggest infectious disease killer worldwide, with 1.6 million deaths per annum and increasing rates of antimicrobial-resistant infections. Strategies to manage tuberculosis require fundamental research to drive innovation in therapeutics, but the World Health Organisation has highlighted the scarcity of new approaches.

An opportunity exists to address these points by harnessing "toxin-antitoxin" (TA) systems in bacteria that target essential steps such as translation, DNA replication and cell wall synthesis. The abundance of TA systems throughout bacteria, and M. tuberculosis in particular, suggests these systems represent a rich source of new biochemistry and antibacterial targets.

TA systems encode two components, a toxic protein that targets an essential cellular process, and an antagonistic antitoxin, which blocks toxin activity when cells are growing under favourable conditions. Although the processes that lead to toxin activation remain under debate, it has been proposed that under certain stress conditions, increased toxin transcription and synthesis may lead to activation.

This in turn reduces growth rate, which can provide a means to survive with minimal metabolic burden until favourable conditions return. TA systems are remarkably abundant in M. tuberculosis, which encodes more than 80 putative systems that are thought to contribute to the success of M. tuberculosis as a human pathogen. Many of the putative M. tuberculosis toxins tested thus far were shown to inhibit bacterial growth, and the highly toxic nature of some toxins suggests that their antibacterial mechanisms could be developed into antimicrobials.

Research Plan. In our recent work, we established and characterised the MenAT TA family of nucleotidyltransferases, which were proven to be active in killing tuberculosis. Toxin MenT3 targets and modifies all four mycobacterial serine tRNAs. The MenAT family also represents a new class of type VII TA systems, regulated through phosphorylation by the cognate MenA antitoxin.

These discoveries prompt two new routes for controlling the growth of M. tuberculosis; (i) modulating toxin activity and (ii) inhibiting amino-acyl charging of serine tRNAs. This project aims to explore these opportunities through a combination of biochemistry, structural biology and in silico methods encompassing protein dynamics modelling, docking studies and structure-based drug design.

Objective 1 - Toxin inhibition. Use our high resolution structures of toxin MenT3 and MenT4 with VirtualFlow, to screen and identify potential small molecule inhibitors. Hits will be screened for toxin inhibition through established biochemical assays, microbiology and X-ray crystallographic studies.

Objective 2 - Dysregulation of toxin-antitoxin interactions. Structural and biophysical studies will be performed to build models for the control of MenT toxins through phosphorylation by cognate MenA antitoxins.

Objective 3 - Target toxin targets. MenT3 modifies serine tRNAs, indicating the seryl-tRNA synthetase as a potential antibiotic target. Structural studies of the seryl-tRNA synthetase from M. tuberculosis will be combined with in silico screening methods to identify and characterise potential inhibitors.

Strategic vision. Studying the recently identified MenAT toxin-antitoxin family is timely and addresses an unmet research need, by characterising new potential antibiotic targets in a relevant human pathogen through a combination of biochemical and computational methodologies.