Ser/Thr Phosphorylation of <i>Mycobacterium tuberculosis</i> Type II Rel TA module by Protein Kinase K interferes with toxin neutralization: A novel mode of TA regulation

Abstract Toxin-Antitoxin (TA) modules represent genetic elements implicated in bacterial persistence and antibiotic tolerance. Remarkably, Mycobacterium tuberculosis encodes 90+ TA modules, the majority of which are Type II TA comprising of a toxin component and an antitoxin counterpart that neutralizes the toxin. Upon exposure to stress, the antitoxin is degraded, releasing the toxin which then acts to halt cellular growth. Given that TA modules dictate social behavior of a population, we hypothesize that their regulation must be exquisitely controlled to avoid superfluous growth inhibition and initiation of persistence. However, the regulation and coordination of TA modules is poorly understood. Herein, we describe for the first time, a novel regulatory mechanism for Type II TA modules involving post-translational modification (PTM). Using computational tools, we observed that over 85 % of the M. tuberculosis TA proteins possess potential Ser/Thr phosphosites highlighting them as putative substrates for M. tuberculosis Ser/Thr protein kinases (STPK). We demonstrate that members of the RelBE family are subjected to O- phosphorylation by PknK, a stress-responsive growth regulatory STPK. Mass spectrometry confirmed multiple sites of PknK-mediated phosphorylation in the RelJK TA module. To gain insights into the functional impact of this PTM, we conducted in vitro binding and phenotypic growth studies with the wild type and mutant RelJK proteins. Our findings indicate that phosphorylation of Thr77 residue in RelK toxin compromises its binding to the RelJ antitoxin. These results suggest a potential role for O- phosphorylation in influencing the interaction dynamics of the TA module components. Importance Bacterial pathogens rely on the phenomenon of persistence as a survival strategy to combat the adverse environmental conditions encountered during infection. As a stochastic process, the driving force(s) that potentiate the formation of persisters in a bacterial population are largely unclear. This study is a step towards the discovery of intricate regulatory mechanisms that coordinate a synchronized TA cellular program. We propose a model wherein the TA module is regulated post translationally, specifically via Ser/Thr phosphorylation disrupting the interaction between the toxin and antitoxin proteins as a mechanism to regulate TA function.