Analysis of DevR regulated genes in Mycobacterium tuberculosis

The DevRS two component system of Mycobacterium tuberculosis is responsible for its dormancy in host and becomes operative under hypoxic condition. It is experimentally known that phosphorylated DevR controls the expression of several downstream genes in a complex manner. In the present work we propose a theoretical model to show role of binding sites in DevR mediated gene expression. Individual and collective role of binding sites in regulating DevR mediated gene expression has been shown via modeling. Objective of the present work is twofold. First, to describe qualitatively the temporal dynamics of wild type genes and their known mutants. Based on these results we propose that DevR controlled gene expression follows a specific pattern which is efficient in describing other DevR mediated gene expression. Second, to analyze behavior of the system from information theoretical point of view. Using the tools of information theory we have calculated molecular efficiency of the system and have shown that it is close to the maximum limit of isothermal efficiency.     

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.     

Cognate sensor kinase‐independent activation of Mycobacterium tuberculosis response regulator DevR (DosR) by acetyl phosphate: implications in anti‐mycobacterial drug design

The DevRS/DosT two‐component system is essential for mycobacterial survival under hypoxia, a prevailing stress within granulomas. DevR (also known as DosR) is activated by an inducing stimulus, such as hypoxia, through conventional phosphorylation by its cognate sensor kinases, DevS (also known as DosS) and DosT. Here, we show that the DevR regulon is activated by acetyl phosphate under ‘non‐inducing’ aerobic conditions when Mycobacterium tuberculosis devS and dosT double deletion strain is cultured on acetate. Overexpression of phosphotransacetylase caused a perturbation of the acetate kinase‐phosphotransacetylase pathway, a decrease in the concentration of acetyl phosphate and dampened the aerobic induction response in acetate‐grown bacteria. The operation of two pathways of DevR activation, one through sensor kinases and the other by acetyl phosphate, was established by an analysis of wild‐type DevS and phosphorylation‐defective DevSH395Q mutant strains under conditions partially mimicking a granulomatous‐like environment of acetate and hypoxia. Our findings reveal that DevR can be phosphorylated in vivo by acetyl phosphate. Importantly, we demonstrate that acetyl phosphate‐dependent phosphorylation can occur in the absence of DevR’s cognate kinases. Based on our findings, we conclude that anti‐mycobacterial therapy should be targeted to DevR itself and not to DevS/DosT kinases.     

High-throughput microplate phosphorylation assays based on DevR-DevS/Rv2027c 2-component signal transduction pathway to screen for novel antitubercular compounds

DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [gamma-(32)P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [gamma-(32)P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.     

Expression of mycobacterial cell division protein, FtsZ, and dormancy proteins, DevR and Acr, within lung granulomas throughout guinea pig infection

The ability of Mycobacterium tuberculosis to persist in a dormant state is a hallmark of tuberculosis. An insight into the expression of mycobacterial proteins will contribute to our understanding of bacterial physiology in vivo. To this end, the expression of FtsZ, Acr and DevR was assessed in the lung granulomas of guinea pigs infected with M. tuberculosis. Antigen immunostaining was then compared with the detection of acid-fast bacilli (AFB) and mycobacterial DNA. Surprisingly, immunostaining for all three antigens was observed throughout the course of infection; maximum expression of all antigens was noted at 20 weeks of infection. The intensity of immunostaining correlated well with the presence of intact bacteria, suggesting that mycobacterial antigens in the extracellular fraction have a short half-life; in contrast to protein, extracellular bacterial DNA was found to be more stable. Immunostaining for bacterial division and dormancy markers could not clearly distinguish between replicating and non-replicating organisms during the course of infection. The detection of Acr and DevR from 4 weeks onwards indicates that the dormancy proteins are expressed from early on in infection. Both antigen staining and DNA detection from intact bacilli were useful for detecting intact mycobacteria in the absence of AFB.     

In silico analysis of DosR regulon proteins of Mycobacterium tuberculosis

One of the challenges faced by Mycobacterium tuberculosis (M. tuberculosis) in dormancy is hypoxia. DosR/DevR of M. tuberculosis is a two component dormancy survival response regulator which induces the expression of 48 genes. In this study, we have used DosR regulon proteins of M. tuberculosis H37Rv as the query set and performed a comprehensive homology search against the non-redundant database. Homologs were found in environmental mycobacteria, environmental bacteria and archaebacteria. Analysis of genomic context of DosR regulon revealed that they are distributed as nine blocks in the genome of M. tuberculosis with many transposases and integrases in their vicinity. Further, we classified DosR regulon proteins into eight functional categories. One of the hypothetical proteins Rv1998c could probably be a methylisocitrate lyase or a phosphonomutase. Another hypothetical protein, Rv0572 was found only in mycobacteria. Insights gained in this study can potentially aid in the development of novel therapeutic interventions.     

The Residue Threonine 82 of DevR (DosR) Is Essential for DevR Activation and Function in Mycobacterium tuberculosis Despite Its Atypical Location

The DevR (DosR) response regulator initiates the bacterial adaptive response to a variety of signals, including hypoxia in in vitro models of dormancy. Its receiver domain works as a phosphorylation-mediated switch to activate the DNA binding property of its output domain. Receiver domains are characterized by the presence of several highly conserved residues, and these sequence features correlate with structure and hence function. In response regulators, interaction of phosphorylated aspartic acid at the active site with the conserved threonine is believed to be crucial for phosphorylation-mediated conformational change. DevR contains all the conserved residues, but the structure of its receiver domain in the unphosphorylated protein is strikingly different, and key threonine (T82), tyrosine (Y101), and lysine (K104) residues are placed uncharacteristically far from the D54 phosphorylation site. In view of the atypical location of T82 in DevR, the present study aimed to examine the importance of this residue in the activation mechanism. Mycobacterium tuberculosis expressing a DevR T82A mutant protein is defective in autoregulation and supports hypoxic induction of the DevR regulon only very weakly. These defects are ascribed to slow and partial phosphorylation and the failure of T82A mutant protein to bind cooperatively with DNA. Our results indicate that the T82 residue is crucial in implementing conformational changes in DevR that are essential for cooperative binding and for subsequent gene activation. We propose that the function of the T82 residue in the activation mechanism of DevR is conserved in spite of the unusual architecture of its receiver domain.     

Sphingosine kinase 1 (SK1) is recruited to nascent phagosomes in human macrophages: inhibition of SK1 translocation by Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with phagosomes containing live M.tb. To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1G82D, and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1S225A). The magnitude and kinetics of translocation of SK1G82D and SK1S225A to latex bead phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation.     

TLR-4/microRNA-125a/NF-κB signaling modulates the immune response to Mycobacterium tuberculosis infection

Tuberculosis (TB), caused by Mycobacterium tuberculosis, could lead to kinds of clinical disorders and remains a leading global health problem, resulting in great morbidity and mortality worldwide. Previous studies have firmly demonstrated that M. tuberculosis (M.tb) has evolved to utilize different mechanisms to evade or attenuate the host immune response, such as regulation of immune-related genes by modulation of miRNAs of host or bacteria. However, the knowledge of functions of miRNAs during M.tb infection remains limited. Here, we reported that a host microRNA, miR-125a, was significantly up-regulated by M.tb infection in both RAW264.7 and THP-1cells, in a TLR4 signaling-dependent manner. Subsequently, our results demonstrated that miR-125a was a negative regulator of NF-kB pathway by directly targeting TRAF6, resulting in the suppression of cytokines, attenuation of immune response and promotion of M.tb survival. Taken together, our findings provide a novel detailed molecular mechanism in which miR-125a was enhanced to inhibit inflammatory cytokines secretion and attenuate the immune response during M.tb infection in RAW264.7 and THP-1 cells, and suggest an intrinsic a promising anti-M.tb therapeutic target.     

Differential immune response of adipocytes to virulent and attenuated Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tb) takes advantage of various cell types, allowing it to remain in the host for long periods. Because adipocytes have been proposed as niches for dormant M. tb in the latent state, understanding the interaction of virulent M. tb with adipocytes is important. We compared changes in cytokine secretion from 3T3-L1 murine adipocytes infected with virulent M. tb H37Rv (V-M. tb) and attenuated M. tb H37Ra (A-M. tb) strains. Both strains maintained non-replicating states within adipocytes until 10 days post-infection. Adipocytes infected with V-M. tb secreted lower levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12p40, IL-6, and IL-17, and lower levels of nitric oxide than those infected with A-M. tb. In contrast, the anti-inflammatory cytokines, IL-10 and IL-4, were markedly induced in V-M. tb-infected adipocytes versus those infected with A-M. tb at an early time point. Heat-killed or formalin-fixed bacteria induced lower levels of cytokines and no difference was observed between strains. Moreover, V-M. tb induced a high level of necrosis versus A-M. tb in conjunction with increased levels of LHD. These results suggest that V-M. tb regulates cytokine expression in its favor, increasing cytokines necessary for immune evasion and decreasing those required for protective immunity.     

Novel drug target strategies against Mycobacterium tuberculosis

The resurgence of drug resistant tuberculosis (TB) is a significant global healthcare challenge. Mycobacterium tuberculosis (MTB), TB's causative agent, evades the host immune system and drug regimes by entering prolonged periods of non-proliferation or dormancy. In infected individuals, the immune system sequesters MTB into structures called granulomas where the bacterium survives by shifting into a non-replicative state. Although still not well understood, progress has been made in characterizing the genetic program of MTB, activated by DosR (DevR) signal transduction that allows adaptation to the hypoxic, nutrient limiting granuloma microenvironment. Recent work, especially the identification genes involved in regulatory networks and the Enduring Hypoxic Response (EHR), hold promise for developing new drugs targeting dormancy phase MTB.