Co-Expression of DevR and DevRN-Aph Proteins Is Associated with Hypoxic Adaptation Defect and Virulence Attenuation of Mycobacterium tuberculosis

BACKGROUND: The DevR response regulator is implicated in both hypoxic adaptation and virulence of Mycobacterium tuberculosis (M. tb). DevR regulon genes are powerfully induced in vivo implicating them in bacterial adaptation to host control strategies. A better understanding of DevR function will illumine the way for new strategies to control and treat tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Towards this objective, we used a combination of genetic, microbiological, biochemical, cell biological tools and a guinea pig virulence assay to compare the hypoxic adaptation and virulence properties of two novel M. tb strains, namely, a devR disruption mutant, Mut1, that expresses C-terminal truncated N-terminal domain of DevR (DevR(NTD)) as a fusion protein with AphI (DevR(N)-Kan), and its complemented strain, Comp1, that expresses intact DevR along with DevR(N)-Kan. Comp1 bacteria exhibit a defect in DevR-mediated phosphosignalling, hypoxic induction of HspX and also hypoxic survival. In addition, we find that Comp1 is attenuated in virulence in guinea pigs and shows decreased infectivity of THP-1 cells. While Mut1 bacilli are also defective in hypoxic adaptation and early growth in spleen, they exhibit an overall virulence comparable to that of wild-type bacteria. CONCLUSIONS/SIGNIFICANCE: The hypoxic defect of Comp1 is associated to a defect in DevR expression level. The demonstrated repression of DevR function by DevR(N)-Kan suggests that such a knockdown approach could be useful for evaluating the activity of DevRS and other two-component signaling pathways. Further investigation is necessary to elucidate the mechanism underlying Comp1 attenuation

Appropriate DevR (DosR)-Mediated Signaling Determines Transcriptional Response, Hypoxic Viability and Virulence of Mycobacterium tuberculosis

Background: The DevR(DosR) regulon is implicated in hypoxic adaptation and virulence of Mycobacterium tuberculosis. The present study was designed to decipher the impact of perturbation in DevR-mediated signaling on these properties. Methodology/Principal Findings: M. tb complemented (Comp) strains expressing different levels of DevR were constructed in Mut1 * background (expressing DevR N-terminal domain in fusion with AphI (DevRN-Kan) and in Mut2DdevR background (deletion mutant). They were compared for their hypoxia adaptation and virulence properties. Diverse phenotypes were noted; basal level expression (,5.362.3 mM) when induced to levels equivalent to WT levels (,25.869.3 mM) was associated with robust DevR regulon induction and hypoxic adaptation (Comp 9 * and 10*), whereas low-level expression (detectable at transcript level) as in Comp 11 * and Comp15 was associated with an adaptation defect. Intermediate-level expression (,3.361.2 mM) partially restored hypoxic adaptation functions in Comp2, but not in Comp1 * bacteria that coexpressed DevRN-Kan. Comp * strains in Mut1 * background also exhibited diverse virulence phenotypes; high/very low-level DevR expression was associated with virulence whereas intermediate-level expression was associated with low virulence. Transcription profiling and gene expression analysis revealed up-regulation of the phosphate starvation response (PSR) in Mut1 * and Comp11 * bacteria, but not in WT/Mut2DdevR/other Comp strains, indicating a plasticity in expression pathways that is determined by the magnitude of signaling perturbation through DevRN-Kan

Definition of blindness under National Programme for Control of Blindness: Do we need to revise it?

A review appropriateness of the current definition of blindness under National Programme for Control of Blindness (NPCB), Government of India. Online search of peer-reviewed scientific published literature and guidelines using PubMed, the World Health Organization (WHO) IRIS, and Google Scholar with keywords, namely blindness and visual impairment, along with offline examination of reports of national and international organizations, as well as their cross-references was done until December 2016, to identify relevant documents on the definition of blindness. The evidence for the historical and currently adopted definition of blindness under the NPCB, the WHO, and other countries was reviewed. Differences in the NPCB and WHO definitions were analyzed to assess the impact on the epidemiological status of blindness and visual impairment in India. The differences in the criteria for blindness under the NPCB and the WHO definitions cause an overestimation of the prevalence of blindness in India. These variations are also associated with an over-representation of refractive errors as a cause of blindness and an under-representation of other causes under the NPCB definition. The targets for achieving elimination of blindness also become much more difficult to achieve under the NPCB definition. Ignoring differences in definitions when comparing the global and Indian prevalence of blindness will cause erroneous interpretations. We recommend that the appropriate modifications should be made in the NPCB definition of blindness to make it consistent with the WHO definition

Importance of the α10 helix for DevR activation: A road map for screening inhibitors against DevR-mediated dormancy of Mycobacterium tuberculosis

Objective/Background: Bacterial persistence is the hallmark of tuberculosis (TB) and poses the biggest threat to the success of any antitubercular drug regimen. The DevR/DosR dormancy regulator of Mycobacterium tuberculosis belongs to the NarL subfamily of response regulators and is essential for M. tuberculosis persistence in macaque models of TB. The DevR/DosR crystal structure revealed a unique (αβ)4 topology instead of the classical (αβ)5 structure found in the receiver domain of other regulators in this subfamily. It was proposed that phosphorylation may culminate in the formation of a DNA-binding-competent dimeric species via α10–α10 helix interactions. Here, we deciphered the role of the α10 helix in activation of the DevR/DosR response regulator in M. tuberculosis. Methods: Wild-type (WT) and mutant DevR [α10-helix-deleted DevR (DevRΔα10)] proteins were cloned in suitable plasmids and expressed in Escherichia coli and M. tuberculosis strains. An in vitro phosphorylation assay was performed using acetyl phosphate, and the dimeric/oligomeric status of WT DevR and mutant proteins in the presence or absence of phosphorylation was assessed by glutaraldehyde-based in vitro cross-linking, followed by western blot analysis. Additionally, recombinant M. tuberculosis strains expressing WT and mutant DevR proteins were assessed for dormancy regulon gene expression under aerobic and hypoxic conditions by western blot analysis. An electrophoretic mobility shift assay was performed to assess the in vitro DNA-binding activity of DevR proteins to the target DNA, and biophysical characterization was performed using circular dichroism spectroscopy, fluorescence spectroscopy, and thermal shift assays. Results: Our results revealed that DevR structure and activity are modulated by phosphorylation-dependent α10 helix dimerization. In its hyperphosphorylated state, DevRΔα10 is defective in DNA binding and exhibits an open and less stable conformation. The combined results of in vitro cross-linking and genetic analysis established an essential role for the α10 helix in postphosphorylation dimerization of DevR and gene activation. The importance of the α10 helix for dormancy regulon induction in M. tuberculosis established the α10–α10 helix interaction as a novel target in the DevR-signaling pathway for developing inhibitors against DevR, a key regulator of hypoxia-triggered dormancy. Conclusion: This study established the importance of the α10 helix for DevR activation in M. tuberculosis and proposed a novel molecular tool to screen small-molecule inhibitors targeting dimerization of DevR in the absence (inactive state) or presence of phosphorylation (active state) to combat latent TB infection. This concept can be extended to screen inhibitors against response regulators where dimerization is crucial for their activation

DNA Aptamer Targets Mycobacterium tuberculosis DevR/DosR Response Regulator Function by Inhibiting Its Dimerization and DNA Binding Activity

Tuberculosis is recognized as one of the major public health threats worldwide. The DevR-DevS (DosR/DosS) two-component system is considered a novel drug target in Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, owing to its central role in bacterial adaptation and long-term persistence. An increase in DevR levels and the decreased permeability of the mycobacterial cell wall during hypoxia-associated dormancy pose formidable challenges to the development of anti-DevR compounds. Using an in vitro evolution approach of Systematic Evolution of Ligands by EXponential enrichment (SELEX), we developed a panel of single-stranded DNA aptamers that interacted with Mtb DevR protein in solid-phase binding assays. The best-performing aptamer, APT-6, forms a G-quadruplex structure and inhibits DevR-dependent transcription in Mycobacterium smegmatis. Mechanistic studies indicate that APT-6 functions by inhibiting the dimerization and DNA binding activity of DevR protein. In silico studies reveal that APT-6 interacts majorly with C-terminal domain residues that participate in DNA binding and formation of active dimer species of DevR. To the best of our knowledge, this is the first report of a DNA aptamer that inhibits the function of a cytosolic bacterial response regulator. By inhibiting the dimerization of DevR, APT-6 targets an essential step in the DevR activation mechanism, and therefore, it has the potential to universally block the expression of DevR-regulated genes for intercepting dormancy pathways in mycobacteria. These findings also pave the way for exploring aptamer-based approaches to design and develop potent inhibitors against intracellular proteins of various bacterial pathogens of global concern

Multifaceted remodeling by vitamin C boosts sensitivity of Mycobacterium tuberculosis subpopulations to combination treatment by anti-tubercular drugs

Bacterial dormancy is a major impediment to the eradication of tuberculosis (TB), because currently used drugs primarily target actively replicating bacteria. Therefore, decoding of the critical survival pathways in dormant tubercle bacilli is a research priority to formulate new approaches for killing these bacteria. Employing a network-based gene expression analysis approach, we demonstrate that redox active vitamin C (vit C) triggers a multifaceted and robust adaptation response in Mycobacterium tuberculosis (Mtb) involving similar to 67% of the genome. Vit C-adapted bacteria display well-described features of dormancy, including growth stasis and progression to a viable but non-culturable (VBNC) state, loss of acid-fastness and reduction in length, dissipation of reductive stress through triglyceride (TAG) accumulation, protective response to oxidative stress, and tolerance to first line TB drugs. VBNC bacteria are reactivatable upon removal of vit C and they recover drug susceptibility properties. Vit C synergizes with pyrazinamide, a unique TB drug with sterilizing activity, to kill dormant and replicating bacteria, negating any tolerance to rifampicin and isoniazid in combination treatment in both in-vitro and intracellular infection models. Finally, the vit C multi-stress redox models described here also offer a unique opportunity for concurrent screening of compounds/combinations active against heterogeneous subpopulations of Mtb. These findings suggest a novel strategy of vit C adjunctive therapy by modulating bacterial physiology for enhanced efficacy of combination chemotherapy with existing drugs, and also possible synergies to guide new therapeutic combinations towards accelerating TB treatment

Estimation of intracellular DevR_N-Kan and DevR concentration in <i>M. tb</i> under hypoxia.

<p>(<b>A</b>) A standard curve was generated for DevR_N-Kan protein as for DevR protein. (<b>B</b>) <i>M.tb</i> lysates prepared from hypoxic cultures (15 µg) were electrophoresed and analyzed by immunoblotting as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035847#pone-0035847-g003" target="_blank">Figure 3</a>. ‘−’ and ‘+’ indicate absence and presence of HspX expression. Inset, Ratio of normalized DevR_N-Kan and DevR proteins in various Comp strains. The values shown are the averages of two independent determinations.</p

Immunoblot analysis.

<p>Lysates of <i>M. tb</i> strains were electrophoresed and subjected to SDS-PAGE and immunoblot analysis using polyclonal antibodies against (<b>A</b>) DevR, (<b>B</b>) HspX and (<b>C</b>) SigA. In all panels: lanes 1–2, WT; lanes 3–4, Comp9*; lanes 5–6, Comp10*; lanes 7–8, Comp11*; lanes 9–10, Comp12*; lanes 11–12, Comp1*; lanes 13–14, Comp13; lanes 15–16, Comp14; lanes 17–18, Comp15; lanes19–20, Comp16 and lanes 21–22, Comp2. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 represent aerobic cultures; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 represent 5 days standing hypoxic cultures. Representative blots from 2 to 4 independent experiments are shown.</p