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Expression of the Mycobacterium tuberculosis acr-coregulated genes from the DevR (DosR) regulon is controlled by multiple levels of regulation.

ABSTRACT: Little is known about how Mycobacterium tuberculosis regulates gene expression in response to its host environment, despite its importance as a pathogen. We previously characterized 10 acr-coregulated genes (ACGs), all of which belong to the DevR (DosR) "dormancy" regulon, and identified one to three copies of a conserved 18-bp palindromic DNA motif in the promoter of each ACG family member. In the present study, we used base substitution analyses to assess the importance of individual motif copies and to identify additional regulatory sequences in five ACG promoters. Regulation of acr, acg, Rv2623, narK2, and Rv1738 was examined by using single-copy M. tuberculosis promoter-lacZ reporter constructs in Mycobacterium bovis BCG under conditions of ambient air versus hypoxia, each in shaking versus standing shallow culture conditions. We found that regulation of these ACG promoters is more heterogeneous than expected and is controlled at multiple levels. In addition to the positive regulation previously associated with DevR (DosR) and the 18-bp ACG motif, we identified negative regulation associated with sequences in the 5' untranslated regions of acg and Rv2623 and positive regulation associated with far upstream regulatory regions of narK2 and Rv1738. The importance of individual ACG motifs varied among the promoters examined, and Rv1738 was exceptional in that its ACG motif copies were associated with negative, rather than positive, regulation under some conditions. Further understanding of this important regulon requires the identification of additional regulators that compete and/or collaborate with DevR (DosR) to regulate its individual gene members.

SUBMITTER: Vasudeva-Rao HM

PROVIDER: S-EPMC2423077 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Expression of the Mycobacterium tuberculosis acr-coregulated genes from the DevR (DosR) regulon is controlled by multiple levels of regulation.

Vasudeva-Rao Hema M HM McDonough Kathleen A KA

Infection and immunity 20080407 6

Little is known about how Mycobacterium tuberculosis regulates gene expression in response to its host environment, despite its importance as a pathogen. We previously characterized 10 acr-coregulated genes (ACGs), all of which belong to the DevR (DosR) "dormancy" regulon, and identified one to three copies of a conserved 18-bp palindromic DNA motif in the promoter of each ACG family member. In the present study, we used base substitution analyses to assess the importance of individual motif cop ...[more]

PMID: 18391009

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Project description:The DosR regulon, a set of 48 genes normally expressed in Mycobacterium tuberculosis under conditions that inhibit aerobic respiration, is controlled via the DosR-DosS/DosT two-component system. While the regulon requires induction in most M. tuberculosis isolates, for members of the Beijing lineage, its expression is uncoupled from the need for signaling. In our attempts to understand the mechanistic basis for this uncoupling in the Beijing background, we previously reported the identification of two synonymous single-nucleotide polymorphisms (SNPs) within the adjacent Rv3134c gene. In the present study, we have interrogated the impact of these SNPs on dosR expression in wild-type strains, as well as a range of dosR-dosS-dosT mutants, for both Beijing and non-Beijing M. tuberculosis backgrounds. In this manner, we have unequivocally determined that the C601T dosR promoter SNP is the sole requirement for the dramatic shift in the pattern of DosR regulon expression seen in this globally important lineage. Interestingly, we also show that DosT is completely nonfunctional within these strains. Thus, a complex series of evolutionary steps has led to the present-day Beijing DosR phenotype that, in turn, potentially confers a fitness advantage in the face of some form of host-associated selective pressure. IMPORTANCE:Mycobacterium tuberculosis strains of the Beijing lineage have been described as being of enhanced virulence compared to other lineages, and in certain regions, they are associated with the dramatic spread of multidrug-resistant tuberculosis (TB). In terms of trying to understand the functional basis for these broad epidemiological phenomena, it is interesting that, in contrast to the other major lineages, the Beijing strains all constitutively overexpress members of the DosR regulon. Here, we identify the mutational events that led to the evolution of this unique phenotype. In addition, our work highlights the fact that important phenotypic differences exist between distinct M. tuberculosis lineages, with the potential to impact the efficacy of diagnosis, vaccination, and treatment programs.

Project description: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 Mut2?devR 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/Mut2?devR/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.

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:Mycobacterium tuberculosis is a strict aerobe capable of prolonged survival in the absence of oxygen. We investigated the ability of anaerobic M. tuberculosis to counter challenges to internal pH homeostasis in the absence of aerobic respiration, the primary mechanism of proton efflux for aerobic bacilli. Anaerobic M. tuberculosis populations were markedly impaired for survival under a mildly acidic pH relative to standard culture conditions. An acidic environmental pH greatly increased the susceptibilities of anaerobic bacilli to the collapse of the proton motive force by protonophores, to antimicrobial compounds that target entry into the electron transport system, and to small organic acids with uncoupling activity. However, anaerobic bacilli exhibited high tolerance against these challenges at a near-neutral pH. At a slightly alkaline pH, which was near the optimum intracellular pH, the addition of protonophores even improved the long-term survival of bacilli. Although anaerobic M. tuberculosis bacilli under acidic conditions maintained 40% lower ATP levels than those of bacilli under standard culture conditions, ATP loss alone could not explain the drop in viability. Protonophores decreased ATP levels by more than 90% regardless of the extracellular pH but were bactericidal only under acidic conditions, indicating that anaerobic bacilli could survive an extreme ATP loss provided that the external pH was within viable intracellular parameters. Acidic conditions drastically decreased the anaerobic survival of a DosR mutant, while an alkaline environment improved the survival of the DosR mutant. Together, these findings indicate that intracellular acidification is a primary challenge for the survival of anaerobic M. tuberculosis and that the DosR regulon plays a critical role in sustaining internal pH homeostasis.IMPORTANCE During infection, M. tuberculosis bacilli are prevalent in environments largely devoid of oxygen, yet the factors that influence the survival of these severely growth-limited and metabolically limited bacilli remain poorly understood. We determined how anaerobic bacilli respond to fluctuations in environmental pH and observed that these bacilli were highly susceptible to stresses that promoted internal acidic stress, whereas conditions that promoted an alkaline internal pH promoted long-term survival even during severe ATP depletion. The DosR regulon, a major regulator of general hypoxic stress, played an important role in maintaining internal pH homeostasis under anaerobic conditions. Together, these findings indicate that in the absence of aerobic respiration, protection from internal acidification is crucial for long-term M. tuberculosis survival.

Dataset Information

Expression of the Mycobacterium tuberculosis acr-coregulated genes from the DevR (DosR) regulon is controlled by multiple levels of regulation.

Publications

Expression of the Mycobacterium tuberculosis acr-coregulated genes from the DevR (DosR) regulon is controlled by multiple levels of regulation.

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