Cyclic nucleotide-binding proteins in mycobacteria

Abstract

Mycobacterium tuberculosis, one of the leading causes of death worldwide even today, employs the second messenger cAMP extensively for its pathogenicity and persistence. Intracellular and extracellular levels of cAMP in Mtb are severalfold higher than those of other commonly studied microbes. Mtb has sixteen putative adenylyl cyclases and ten cyclic nucleotide binding (CNB) domain-containing proteins. These CNB proteins mediate various activities like transcriptional regulation or acetylation, based on the other functional domains they contain, presumably via a cAMP-mediated crosstalk. An essential universal stress protein, Rv1636, which does not have a canonical CNB domain, acts as a cAMP sink and regulates cAMP signalling. This work encompasses a detailed overview of the CNB proteins in mycobacteria, their gene expression and their functions. The research delves into the evolution and functional aspects of the effector proteins. Mycobacteria were exposed to environmental perturbants, such as detergent or hypoxic, acidic stress conditions, to mimic the scenario encountered by the pathogen during infection. The study provides quantitative insights into cyclic nucleotide binding, gene expression, and cAMP levels under diverse conditions, laying the groundwork for predictive modelling of mycobacterial behaviour. Two previously uncharacterised proteins, Rv0073 and Rv2564, initially identified as putative glutamine transporters, were biochemically characterised. The allosteric crosstalk between the CNB domain and the ATPase domain of these proteins was examined. Knockout and complement strains for rv0073 were generated, and the biological roles of Rv0073 and Rv2564 were investigated. The mycobacterial Universal Stress Protein Rv1636 binds to cAMP and serves as a reservoir. A part of this study focused on determining the mechanism of secretion of Rv1636. Gene knock-down experiments using CRISPRi-dCas9 were performed to verify the essentiality of the USP. Using a molecular-docking approach, two potential natural compounds that target Rv1636 were identified, verified biochemically, and tested experimentally using mycobacterial cultures. Detailed analysis of the effect of one of the two compounds on Mtb establishes the relevance of USP as a promising drug target for combating tuberculosis. Overall, this study provides information about cAMP signalling in mycobacteria, biochemically delineating the cNMP-binding activities of the effector proteins, commenting on levels of cAMP and gene expression of the CNB proteins under different growth conditions and also focusing on the essentiality and drug targetability of the USP, Rv1636.