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Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis.

ABSTRACT: The DevR/DosR regulator is believed to play a key role in dormancy adaptation mechanisms of Mycobacterium tuberculosis in response to a multitude of gaseous stresses, including hypoxia, which prevails within granulomas. DevR activates transcription by binding to target promoters containing a minimum of two binding sites. The proximal site overlaps with the SigA -35 element, suggesting that DevR-SigA interaction is required for activating transcription. We evaluated the roles of 14 charged residues of DevR in transcriptional activation under hypoxic stress. Seven of the 14 alanine substitution mutants were defective in regulon activation, of which K191A, R197A, and K179A+K168A (designated K179A*) mutants were significantly or completely compromised in DNA binding. Four mutants, namely, E154A, R155A, E178A, and K208A, were activation defective in spite of binding to DNA and were classified as positive-control (pc) mutants. The SigA interaction defect of the E154A and E178A proteins was established by in vitro and in vivo assays and implies that these substitutions lead to an activation defect because they disrupt an interaction(s) with SigA. The relevance of DevR interaction to the transcriptional machinery was further established by the hypoxia survival phenotype displayed by SigA interaction-defective mutants. Our findings demonstrate the role of DevR-SigA interaction in the activation mechanism and in bacterial survival under hypoxia and establish the housekeeping sigma factor SigA as a molecular target of DevR. The interaction of DevR and RNA polymerase suggests a new and novel interceptable molecular interface for future antidormancy strategies for Mycobacterium tuberculosis.

SUBMITTER: Gautam US

PROVIDER: S-EPMC3911168 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis.

Gautam Uma S US Sikri Kriti K Vashist Atul A Singh Varshneya V Tyagi Jaya S JS

Journal of bacteriology 20131206 4

The DevR/DosR regulator is believed to play a key role in dormancy adaptation mechanisms of Mycobacterium tuberculosis in response to a multitude of gaseous stresses, including hypoxia, which prevails within granulomas. DevR activates transcription by binding to target promoters containing a minimum of two binding sites. The proximal site overlaps with the SigA -35 element, suggesting that DevR-SigA interaction is required for activating transcription. We evaluated the roles of 14 charged residu ...[more]

PMID: 24317401

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Project description:DevR/DosR is a well-characterized regulator in Mycobacterium tuberculosis which is implicated in various processes ranging from dormancy/persistence to drug tolerance. DevR induces the expression of an ~48-gene dormancy regulon in response to gaseous stresses, including hypoxia. Strains of the Beijing lineage constitutively express this regulon, which may confer upon them a significant advantage, since they would be 'pre-adapted' to the environmental stresses that predominate during infection. Aerobic DevR regulon expression in laboratory-manipulated overexpression strains is also reported. In both instances, the need for an inducing signal is bypassed. While a phosphorylation-mediated conformational change in DevR was proposed as the activation mechanism under hypoxia, the mechanism underlying constitutive expression is not understood. Because DevR is implicated in bacterial dormancy/persistence and is a promising drug target, it is relevant to resolve the mechanistic puzzle of hypoxic activation on one hand and constitutive expression under 'non-inducing' conditions on the other. Here, an overexpression strategy was employed to elucidate the DevR activation mechanism. Using a panel of kinase and transcription factor mutants, we establish that DevR, upon overexpression, circumvents DevS/DosT sensor kinase-mediated or small molecule phosphodonor-dependent activation, and also cooperativity-mediated effects, which are key aspects of hypoxic activation mechanism. However, overexpression failed to rescue the defect of C-terminal-truncated DevR lacking the ?10 helix, establishing the ?10 helix as an indispensable component of DevR activation mechanism. We propose that aerobic overexpression of DevR likely increases the concentration of ?10 helix-mediated active dimer species to above the threshold level, as during hypoxia, and enables regulon expression. This advance in the understanding of DevR activation mechanism clarifies a long standing question as to the mechanism of DevR overexpression-mediated induction of the regulon in the absence of the normal environmental cue and establishes the ?10 helix as an universal and pivotal targeting interface for DevR inhibitor development.

Project description:BackgroundThe 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 findingsM. 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/significanceA 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.

Project description:Two-component regulatory systems allow bacteria to respond adequately to changes in their environment. In response to a given stimulus, a sensory kinase activates its cognate response regulator via reversible phosphorylation. The response regulator DevR activates a state of dormancy under hypoxia in Mycobacterium tuberculosis, allowing this pathogen to escape the host defense system. Here, we show that OsdR (SCO0204) of the soil bacterium Streptomyces coelicolor is a functional orthologue of DevR. OsdR, when activated by the sensory kinase OsdK (SCO0203), binds upstream of the DevR-controlled dormancy genes devR, hspX, and Rv3134c of M. tuberculosis. In silico analysis of the S. coelicolor genome combined with in vitro DNA binding studies identified many binding sites in the genomic region around osdR itself and upstream of stress-related genes. This binding correlated well with transcriptomic responses, with deregulation of developmental genes and genes related to stress and hypoxia in the osdR mutant. A peak in osdR transcription in the wild-type strain at the onset of aerial growth correlated with major changes in global gene expression. Taken together, our data reveal the existence of a dormancy-related regulon in streptomycetes which plays an important role in the transcriptional control of stress- and development-related genes. IMPORTANCE Dormancy is a state of growth cessation that allows bacteria to escape the host defense system and antibiotic challenge. Understanding the mechanisms that control dormancy is of key importance for the treatment of latent infections, such as those from Mycobacterium tuberculosis. In mycobacteria, dormancy is controlled by the response regulator DevR, which responds to conditions of hypoxia. Here, we show that OsdR of Streptomyces coelicolor recognizes the same regulatory element and controls a regulon that consists of genes involved in the control of stress and development. Only the core regulon in the direct vicinity of dosR and osdR is conserved between M. tuberculosis and S. coelicolor, respectively. Thus, we show how the system has diverged from allowing escape from the host defense system by mycobacteria to the control of sporulation by complex multicellular streptomycetes. This provides novel insights into how bacterial growth and development are coordinated with the environmental conditions.

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Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis.

Publications

Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis.

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