Untangling topoisomerase inhibition by ParE toxins of Mycobacterium tuberculosis; a biochemical, structural, and phylogenetic approach

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of TB, remains a 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 (FQ) antibiotics indicates the necessity to develop both novel therapeutics and our understanding of Mtb growth during infection. ParDE type toxin-antitoxin systems may lie at this intersection, with ParE toxins targeting gyrase as responsive elements to both host-associated and drug-induced stress during infection. Here we present biochemical and biophysical analyses exploring the ParE1 and ParE2 poisoning of Mtb gyrase, trapping cleavage complexes potentially via a contrasting mechanism to FQ antibiotics. We also propose a novel mechanism of post-translational ParE1 toxin release and system activation via complex remodelling, a potential first in the field. We show the ParE2 toxin to be closely related to the RelE toxin family via phylogenetic analyses and as part of a collaboration with the Genevaux group (Toulouse, France) present the crystal structure of the Mtb RelBE1 complex. We highlight differences in the RelE1 toxin which may contribute to alternate and novel mechanisms of toxicity employed by Mtb RelE toxins, all three of which are upregulated during infection. Altogether, this study combines a range of techniques to better our understanding of three TA systems important to the adaptability of Mtb. We lay the foundations for future work focussing on the molecular basis of Mtb ParE and RelE toxicity through biochemical, biophysical, and structural studies.