The Chromosomal parDE2 Toxin–Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization

Abstract

Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant persistent state. Activation of 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 the parDE2 operon of M. tuberculosis H37Rv encoding MParE2 toxin and MParD2 antitoxin 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 MParD2 protein transcriptionally regulated the PparDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. In Escherichia coli, ectopic expression of MParE2 inhibited growth in early stages, with a drastic reduction in colony forming units. Live-dead analysis revealed that the reduction was not due to cell death alone but due to formation of viable but non-culturable cells (VBNCs) also. The toxic activity of the protein, identified in the C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin MParD2, both in vivo and in vitro. MParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. Introduction of parE2 gene in the heterologous M. smegmatis host prevented growth and colony formation by the transformed cells. An M. smegmatis strain containing the parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. Loss in colony-forming ability of a major part of the MParE2 expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. Our study has laid the foundation for future investigations to explore the physiological significance of parDE2 operon in mycobacterial pathogenesis.

Relationship between the Viable but Nonculturable State and Antibiotic Persister Cells

“…Demidenok and colleagues showed that overexpression of the VapB antitoxin prevented cells from entering the VBNC state, while overexpression of the VapC toxin induced the VBNC state (108). Similarly, overexpression of the MParE2 toxin led to a decline in culturability due to entry into the VBNC state (109). Recently, we reported that VBNC cells of Vibrio vulnificus have significantly elevated expression of several homologues of the type II toxins of RelE and HipA compared to log-phase and stationary-phase cultures (26).…”

Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity

“…This ParE toxin was later demonstrated to inhibit E. coli DNA gyrase in vitro, and its expression in cells recapitulated a filamentous phenotype consistent with the identified in vitro inhibition (Jiang, Pogliano, Helinski, & Konieczny, ). Other reports have corroborated in vitro ParE‐mediated gyrase inhibition for chromosomally encoded toxins from Pseudomonas aeruginosa (Pa), Vibrio cholera (Vc), and Mycobacterium tuberculosis (Mt; Gupta et al, ; Muthuramalingam, White, Murphy, Ames, & Bourne, ; Yuan et al, ). The filamentous phenotype arising from ParE toxin exposure is consistent with DNA gyrase inhibition, which is stalled during the catalytic cycle, resulting in fragmented DNA that can trigger the SOS and other responses (Kreuzer, ; Reece & Maxwell, ; Williams & Schumacher, ).…”

“…The filamentous phenotype arising from ParE toxin exposure is consistent with DNA gyrase inhibition, which is stalled during the catalytic cycle, resulting in fragmented DNA that can trigger the SOS and other responses (Kreuzer, ; Reece & Maxwell, ; Williams & Schumacher, ). While there are many triggers for SOS, the filamentous phenotype has been observed upon overexpression of multiple ParE toxins for Pa, Vc, Mt, and Caulobacter crescentus (Cc; Fiebig, Castro Rojas, Siegal‐Gaskins, & Crosson, ; Gupta et al, ; Muthuramalingam et al, ; Yuan et al, ), as well as with other gyrase‐inhibiting proteins (CcdB and Fic toxins, SmbC; Dao‐Thi et al, ; De Jonge et al, ; Harms et al, ; Nakanishi, Oshida, Matsushita, Imajoh‐Ohmi, & Ohnuki, ; Sprenger et al, ; Van Melderen, Bernard, & Couturier, ) and compounds (quinolones, novobiocin; Handel, Hoeksema, Freijo Mata, Brul, & Kuile, ; Torres‐Barcelo, Kojadinovic, Moxon, & MacLean, ). Interestingly, a MazF toxin also induces filamentation and results in persister cells, but only when ciprofloxacin has also been administered to the culture (Cho, Carr, Whitworth, Johnson, & Wilson, ).…”

“…However, in the ParE toxin subfamily, sequence identity is only around 12%, while conservation is stronger with a range of 30%–80% (e.g., see Figure ). Reports vary on which portion of the large DNA gyrase heterotetramer that the ParE toxins target (Gupta et al, ; Sterckx et al, ; Yuan et al, ). Therefore, despite the high prevalence of ParE toxin sequences in bacterial genomes, they remain one of the few toxins to have an unknown molecular mechanism for interaction with their target.…”

Adjuvant Potential of Poly-α- l -Glutamine from the Cell Wall of Mycobacterium tuberculosis

“…For protein expression and purification, all of the Escherichia coli recombinant strains were cultured in Luria-Bertani (LB) medium (Difco) under standard conditions. M. tuberculosis cultures were grown as described earlier by Gupta et al (54). All of the cultures were grown at 37°C with shaking at 180 rpm.…”