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Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant, persistent state. Activation of bipartite toxin-antitoxin modules is one of the mechanisms known to trigger such a state with low metabolic activity. M. tuberculosis harbors a large number of TA systems, mostly located within discernible genomic islands. We have investigated both the parDE TA systems of M. tuberculosis H37Rv and found that only the parDE2 locus (but not parDE1) encodes functional toxin (ParE2) and antitoxin (ParD2) proteins. The parDE2 locus was transcriptionally active from growth phase till late stationary phase in M. tuberculosis. A functional promoter located upstream of parD2 GTG start-site was identified by 5´-RACE and lacZ reporter assay. The ParD2 protein transcriptionally auto-regulated the parDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. We scrutinized the toxicity of ParE2 in recA1 and recA+ E. coli backgrounds to study the role of SOS response as a survival strategy and examine different phenotypes generated due to ParE2 expression. Ectopic expression of parE2 affected growth and viability of both the E. coli strains to different degrees. Live-dead staining (SG-I/Propidium Iodide) revealed that in a short span of 4h, the toxin killed ~54% of the recA1 strain compared to ~27% cells of the recA+ cells. In both cases, the majority of the live cells ~99.99% were viable but non-culturable (VBNCs) and only 0.01% of the population formed colonies under standard culture conditions. The results suggested that though more cells survived the toxic effect in the recA+ strain, VBNC formation was not solely dependent on SOS regulation. The toxic activity of ParE2, crucially dependent on C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin ParD2, both in vivo and in vitro. Under in vitro conditions, ParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. The ParE2 toxicity mediated DNA damage, triggered the activation of SOS response, leading to inhibition of cell division and formation of multi-nucleoid, long filamentous cells. ParE2 expression in E. coli resulted in a massive increase in sulA and tisB transcript levels, and loss of membrane potential in the recA+ strain, but and not in the recA1 (mutant) strain. This indicates the SOS-dependence of these phenotypes. Severe morphological aberrations observed under the electron-microscope in the parE2-expressing recA1 E. coli strain were largely mitigated when the toxin was expressed in the recA+ strain. Cells of the latter strain were extensively filamentous with smooth and intact membranes compared to the mutant recA1 cells that were wrinkled and corrugated with hyper-hydrated periplasmic spaces and large electron-lucent ‘vacuoles.’ This is an indication that a functional SOS- response is involved in the recovery of ParE2-intoxicated E. coli cells. As the effect of the toxin waned with time (perhaps due to proteolytic turnover), the E. coli cells resumed division and recovered their colony-forming ability. The ParE2-expressing recA+ E. coli strain produced significantly higher number of persisters than the recA1 strain, upon exposure to different antibiotics. Introduction of the parE2 gene alone into M. smegmatis (a surrogate host for Mtb) did not result in any transformants despite repeated attempts. However, an M. smegmatis strain containing the complete parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. This loss in colony-forming ability of a major proportion of the ParE2-expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. ParE2- triggered VBNC formation and persistence are hallmarks of dormancy, potentially relevant in tuberculosis. Our study has thus laid the foundation for future investigations to explore the physiological significance of parDE2 operon in Mtb persistence and dormancy.
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