Mycobacterium tuberculosis growth following aerobic expression of the DosR regulon

The Mycobacterium tuberculosis regulator DosR is induced by multiple stimuli including hypoxia, nitric oxide and redox stress. Overlap of these stimuli with conditions thought to promote latency in infected patients fuels a model in which DosR regulon expression is correlated with bacteriostasis in vitro and a proxy for latency in vivo. Here, we find that inducing the DosR regulon to wildtype levels in aerobic, replicating M. tuberculosis does not alter bacterial growth kinetics. We conclude that DosR regulon expression alone is insufficient for bacterial latency, but rather is expressed during a range of growth states in a dynamic environment.     

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.     

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.     

Mycobacterium tuberculosis DosR regulon gene Rv0079 encodes a putative, 'dormancy associated translation inhibitor (DATIN)'

Mycobacterium tuberculosis is a major human pathogen that has evolved survival mechanisms to persist in an immune-competent host under a dormant condition. The regulation of M. tuberculosis metabolism during latent infection is not clearly known. The dormancy survival regulon (DosR regulon) is chiefly responsible for encoding dormancy related functions of M. tuberculosis. We describe functional characterization of an important gene of DosR regulon, Rv0079, which appears to be involved in the regulation of translation through the interaction of its product with bacterial ribosomal subunits. The protein encoded by Rv0079, possibly, has an inhibitory role with respect to protein synthesis, as revealed by our experiments. We performed computational modelling and docking simulation studies involving the protein encoded by Rv0079 followed by in vitro translation and growth curve analysis experiments, involving recombinant E. coli and Bacille Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations concerning the interaction of the protein with the ribosomes are supportive of its role in regulation/inhibition of translation. We propose that the protein encoded by locus Rv0079 is a 'dormancy associated translation inhibitor' or DATIN.     

DosS responds to a reduced electron transport system to induce the Mycobacterium tuberculosis DosR regulon

The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.     

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.     

The W-Beijing lineage of Mycobacterium tuberculosis overproduces triglycerides and has the DosR dormancy regulon constitutively upregulated

The Beijing family of Mycobacterium tuberculosis strains has been associated with epidemic spread and an increased likelihood of developing drug resistance. The characteristics that predispose this family to such clinical outcomes have not been identified, although one potential candidate, the phenolic glycolipid PGL-tb, has been shown to mediate a fulminant lethal disease in mice and rabbits due to lipid-mediated immunosuppression. However, PGL-tb is not uniformly expressed throughout the Beijing lineage and may not be the only unique virulence trait associated with this family. In an attempt to define phenotypes common to all Beijing strains, we interrogated a carefully selected set of isolates representing the five extant lineages of the Beijing family. Comparison of lipid production in this set revealed that all Beijing strains accumulated large quantities of triacylglycerides in in vitro aerobic culture. This accumulation was found to be coincident with upregulation of Rv3130c, whose product was previously characterized as a triacylglyceride synthase. Rv3130c is a member of the DosR-controlled regulon of M. tuberculosis, and further examination revealed that several members of this regulon were upregulated throughout this strain family. The upregulation of the DosR regulon may confer an adaptive advantage for growth in microaerophilic or anaerobic environments encountered by the bacillus during infection and thus may be related to the epidemiological phenomena associated with this important strain lineage.     

Whole genome shotgun sequencing of one Colombian clinical isolate of Mycobacterium tuberculosis reveals DosR regulon gene deletions

Several genomes of different Mycobacterium tuberculosis isolates have been completely sequenced around the world. The genomic information obtained have shown higher diversity than originally thought and specific adaptations to different human populations. Within this work, we sequenced the genome of one Colombian M.?tuberculosis virulent isolate. Genomic comparison against the reference genome of H37Rv and other strains showed multiple deletion and insertions that ranged between a few bases to thousands. Excluding PPE and PG-PGRS genes, 430 proteins present changes in at least 1 amino acid. Also, novel positions of the IS6110 mobile element were identified. This isolate is also characterized by a large genomic deletion of 3.6?kb, leading to the loss and modification of the dosR regulon genes, Rv1996 and Rv1997. To our knowledge, this is the first report of the genome sequence of a Latin American M.?tuberculosis clinical isolate.     

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.     

The Mycobacterium DosR regulon structure and diversity revealed by comparative genomic analysis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), which claims approximately two million people annually, remains a global health concern. The non‐replicating or dormancy like state of this pathogen which is impervious to anti‐tuberculosis drugs is widely recognized as the culprit for this scenario. The dormancy survival regulator (DosR) regulon, composed of 48 co‐regulated genes, is held as essential for Mtb persistence. The DosR regulon is regulated by a two‐component regulatory system consisting of two sensor kinases—DosS (Rv3132c) and DosT (Rv2027c), and a response regulator DosR (Rv3133c). The underlying regulatory mechanism of DosR regulon expression is very complex. Many factors are involved, particularly the oxygen tension. The DosR regulon enables the pathogen to persist during lengthy hypoxia. Comparative genomic analysis demonstrated that the DosR regulon is widely distributed among the mycobacterial genomes, ranging from the pathogenic strains to the environmental strains. In‐depth studies on the DosR response should provide insights into its role in TB latency in vivo and shape new measures to combat this exceeding recalcitrant pathogen. J. Cell. Biochem. 114: 1–6, 2012. © 2012 Wiley Periodicals, Inc.     

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis

The amidinourea 8918 was recently reported to inhibit the type II phosphopantetheinyl transferase (PPTase) of Mycobacterium tuberculosis (Mtb), PptT, a potential drug‐target that activates synthases and synthetases involved in cell wall biosynthesis and secondary metabolism. Surprisingly, high‐level resistance to 8918 occurred in Mtb harboring mutations within the gene adjacent to pptT, rv2795c, highlighting the role of the encoded protein as a potentiator of the bactericidal action of the amidinourea. Those studies revealed that Rv2795c (PptH) is a phosphopantetheinyl (PpT) hydrolase, possessing activity antagonistic with respect to PptT. We have solved the crystal structure of Mtb's phosphopantetheinyl hydrolase, making it the first phosphopantetheinyl (carrier protein) hydrolase structurally characterized. The 2.5 Å structure revealed the hydrolases' four‐layer (α/β/β/α) sandwich fold featuring a Mn‐Fe binuclear center within the active site. A structural similarity search confirmed that PptH most closely resembles previously characterized metallophosphoesterases (MPEs), particularly within the vicinity of the active site, suggesting that it may utilize a similar catalytic mechanism. In addition, analysis of the structure has allowed for the rationalization of the previously reported PptH mutations associated with 8918‐resistance. Notably, differences in the sequences and predicted structural characteristics of the PpT hydrolases PptH of Mtb and E. coli's acyl carrier protein hydrolase (AcpH) indicate that the two enzymes evolved convergently and therefore are representative of two distinct PpT hydrolase families.     

Catalase-peroxidase (Mycobacterium tuberculosis KatG) catalysis and isoniazid activation

Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.