Functional Characterization of PknI-Rv2159c Interaction in Redox Homeostasis of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis adapts to stress conditions by responding to the signals from its external environment. M. tuberculosis genome encodes 11 eukaryotic like serine/threonine protein kinases (STPK) and their importance in regulating the physiology and virulence of the bacteria are being explored. Previous study from our lab identified the M. tuberculosis STPK, PknI interacts with two peroxidase proteins such as Rv2159c and Rv0148. In this study, we have characterized the biological function behind the PknI-Rv2159c interaction in M. tuberculosis. Point mutation of Ala-Gly-Trp motif identified that only Ala49 and Gly50 amino acids of Rv2159c are responsible for interaction and there is no phosphorylation involved in the PknI-Rv2159c interaction. Rv2159c is a member from the carboxymuconolactone decarboxylase family with peroxidase activity. Enzymatic assays with catalytic site point mutants showed that Cys84 of Rv2159c was responsible for its alkylhydroperoxidase activity. Interestingly, interaction with PknI increased its peroxidase activity by several folds. Gene knockdown of Rv2159c in M. tuberculosis showed increased sensitivity to peroxides such as cumene hydroperoxide and hydrogen peroxide. Proteomic analysis of differentially expressing Rv2159c strains by 2D gel electrophoresis and mass spectrometry revealed the differential abundance of 21 proteins. The total absence of oxidoreductase, GuaB1 suggests the essential role of Rv2159c in redox maintenance. Our findings provide new insights on signaling mechanisms of PknI in maintaining the redox homeostasis during oxidative stresses.

An Overview on the Potential Antimycobacterial Agents Targeting Serine/Threonine Protein Kinases from Mycobacterium tuberculosis

“…PknE, PknH, PknI and PknK, toghether with PknG, have important roles in Mtb intracellular survival [21,[79][80][81][82]. For instance, PknE has been demonstrated to promote the suppression of apoptosis, as shown by the use of a pknEdeleted Mtb strain [79,83], and to be involved in adaptive responses [84] and HIV co-infection [85]; PknH is implicated in Mtb dormancy [86] and in the regulation of cell wall biosynthesis [87], while PknI plays a role in the redox homeostasis during oxidative stress [88]. Moreover, advances in integrated proteomic approaches allowed to assign novel biological functions to the different STPKs.…”

Lineage-Specific Proteomic Signatures in the Mycobacterium tuberculosis Complex Reveal Differential Abundance of Proteins Involved in Virulence, DNA Repair, CRISPR-Cas, Bioenergetics and Lipid Metabolism

“…MshD involved in mycothiol (MSH) biosynthesis has a similar function to glutathione ( Newton et al, 2008 ). Mutations in mshD resulted in MSH production defect ( Newton et al, 2005 ; Venkatesan et al, 2016 ). An ahpC mutant Mtb showed decreased survival in macrophages ( Master et al, 2002 ).…”

Role of a Putative Alkylhydroperoxidase Rv2159c in the Oxidative Stress Response and Virulence of Mycobacterium tuberculosis

“…Rv2159c has also been reported to interact with Rv0148, a putative short chain dehydrogenase involved in intermediary metabolism, homeostasis, virulence, and drug resistance of M. tuberculosis [18,19]. Gene knockdown studies on Rv2159c report reduced in vitro survival of the bacteria and a higher level of sensitivity to oxidative stress, compared to the wild type strain [14]. These findings suggest that Rv2159c has a potential role in the virulence of M. tuberculosis.…”

“…In addition, presence of cysteine residues at the catalytic site (Cys-Pro-Try-Cys) of Rv2159c contributes to peroxidase activity. Enzymatic assays of the catalytic site indicate that Cys84 is a key residue for alkylhydroperoxidase activity of Rv2159c [14]. A protein-BLAST analysis revealed that Rv2159c shares amino acid sequence identity with similar proteins in other mycobacterial species, including M. angelicum (100% identity) and M. riyadhense (79.4% identity).…”