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

Differentially expressed genes in Mut1* bacteria.

<p>Relative aerobic transcript levels (Mean ± SD) of PSR genes in Mut1* and Comp1* strains with respect to that in WT bacteria were calculated from C_t values determined by RT reverse transcriptase-PCR analysis. Data from three independent cultures is shown.</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

Survival of H37Rv (WT), mutant (Mut1* and Mut2Δ<i>devR</i>) and various complemented <i>M. tb</i> strains.

<p>The survival of various <i>M. tb</i> strains was assessed under aerobic and hypoxic conditions over a period of 50 days. The mean CFU ± SD determined from three independent cultures is shown as % survival with respect to CFU on day zero.</p

Genes significantly upregulated or downregulated during hypoxia in WT, Mut1* and Comp1* strains.

<p>Fold change 5 day/0 day (Hypoxia/Aerobic) was calculated. Significant differential regulation: ≥1.5-fold induction, ≤0.66-fold repression (P≤0.05).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

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