Co-expression of DevR and DevR(N)-Aph proteins is associated with hypoxic adaptation defect and virulence attenuation of Mycobacterium tuberculosis

Abstract

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.

Figure 1. Effect of DevR_N-Kan and full-length DevR co-expression on DevR regulon gene expression.

M. tb lysates were electrophoresed and subjected to immunoblot analysis using polyclonal antibodies to HspX (top panel), DevR (middle panel) and SigA (bottom panel). Lanes 1, 3, 5, 7, 9, 11 and 13 represent aerobic culture and lanes 2, 4, 6, 8, 10, 12 and 14 represent 5 days standing hypoxic cultures. Anti-HspX immunoblots were developed for longer periods to visualize HspX in Comp1 bacterial lysates. Representative blots from 2 to 4 independent cultures are shown.

Figure 2. Comparison of promoter activity using GFP reporter assay.

GFP fluorescence in M. tb WT and Mut1 cultures carrying operon (pOperon-2) and devR (pdevR-2) promoter constructs under aerobic and hypoxic conditions.

Figure 3. DevR_N-Kan competes efficiently with full-length DevR for phosphosignal from DevS.

Reaction mixtures containing purified DevS∼P (5 µM) plus DevR_N-Kan (0.83 to 30 µM) and full-length DevR (5 µM) proteins were incubated at 25°C for 2 minutes. Samples were analyzed by 15% SDS-PAGE and subjected to phosphorimaging (top panel) and Coomassie blue staining (bottom panel).

Figure 4. Survival of M. tuberculosis strains cultured in vitro.

WT, Mut1 and Comp1 strains were grown under hypoxic (A) and aerobic (B) conditions for upto 50 days in Dubos Tween Albumin medium as described. The mean CFU ± SD determined from three independent cultures is shown as % survival with respect to number of bacteria on day zero. ▪, WT; □, Mut1 and ▿, Comp1.

Figure 5. Virulence of passaged M. tuberculosis strains in guinea pigs.

(A) Pictorial representation of lungs and spleens. (B) CFU in lungs and spleens are expressed as mean ± SD. *, ** represent P<0.05 in comparison to WT and Mut1, respectively.

Figure 6. Intracellular survival of M. tuberculosis strains.

THP-1 cells were infected with various strains (A, passaged and B, laboratory cultured strains) at a m.o.i of 1∶50 (bacterium: macrophage) and the number of intracellular viable bacteria was determined over 7 days. Results are given as the mean ± SD of 3 independent experiments. (Inset) Infectivity of the three strains in THP-1 cells as determined by 2 separate experiments. *, ** P<0.05 indicate significant differences in infectivity between Comp1 vs. WT and Mut1, respectively. •, WT; ○, Mut1 and ▴, Comp1.