PknE, a serine/threonine protein kinase from Mycobacterium tuberculosis has a role in adaptive responses.

Serine/threonine protein kinases (STPK) play a major role in the physiology and pathogenesis of Mycobacterium tuberculosis. Here, we have examined the role of pknE, a STPK in the adaptive responses of M. tuberculosis using a deletion mutant ΔpknE. The survival of ΔpknE was assessed in the presence of stress (pH, surfactant and cell wall-damaging agents) and anti-tuberculosis drugs. ΔpknE had a defective growth in pH 7.0 and lysozyme (a cell wall-damaging agent) with better survival in pH 5.5, SDS and kanamycin (a second-line anti-tuberculosis drug). Furthermore, ΔpknE was reduced in cell size during growth in liquid media and exhibited hypervirulence in a guinea pig model of infection. In conclusion, our data suggest that pknE plays a role in adaptive response of M. tuberculosis regulating cellular integrity and survival.

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 (DevR(N)-Kan) and in Mut2ΔdevR background (deletion mutant). They were compared for their hypoxia adaptation and virulence properties. Diverse phenotypes were noted; basal level expression (∼5.3±2.3 µM) when induced to levels equivalent to WT levels (∼25.8±9.3 µM) 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 (∼3.3±1.2 µM) partially restored hypoxic adaptation functions in Comp2, but not in Comp1* bacteria that co-expressed DevR(N)-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/Mut2ΔdevR/other Comp strains, indicating a plasticity in expression pathways that is determined by the magnitude of signaling perturbation through DevR(N)-Kan. CONCLUSIONS/SIGNIFICANCE: A minimum DevR concentration of ∼3.3±1.2 µM (as in Comp2 bacteria) is required to support HspX expression in the standing culture hypoxia model. The relative intracellular concentrations of DevR and DevR(N)-Kan appear to be critical for determining dormancy regulon induction, hypoxic adaptation and virulence. Dysregulated DevR(N)-Kan-mediated signaling selectively triggers the PSR in bacteria expressing no/very low level of DevR. Our findings illustrate the important role of appropriate two-component-mediated signaling in pathogen physiology and the resilience of bacteria when such signaling is perturbed.

Co-expression of DevR and DevR(N)-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.