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.     

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.     

Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis

The dormancy (DosR) regulon of Mycobacterium tuberculosis is expressed in vitro during hypoxia and low-dose nitric oxide stimulation. Tubercle bacilli are thought to encounter these conditions in humans during latent infection. In this study, immune responses were evaluated to 25 most strongly induced DosR-regulon-encoded proteins, referred to as latency antigens. Proliferation assays were performed using M. tuberculosis-specific T-cell lines and peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients, tuberculin skin test positive (TST+) individuals and uninfected controls. All 25 latency antigens were able to induce production of interferon-gamma (IFN-gamma) by T-cell lines. Eighteen latency antigens were also recognized by PBMC of M. tuberculosis-infected individuals, which indicates expression of the DosR-regulon during natural infection. Differential analysis showed that TST+ individuals recognized more latency antigens and with a stronger cumulative IFN-gamma response than TB patients, while the opposite profile was found for culture filtrate protein-10. In particular Rv1733c, Rv2029c, Rv2627c and Rv2628 induced strong IFN-gamma responses in TST+ individuals, with 61%, 61%, 52% and 35% responders, respectively. In conclusion, several new M. tuberculosis antigens were identified within the DosR-regulon. Particularly strong IFN-gamma responses to latency antigens were observed in latently infected individuals, suggesting that immune responses against these antigens may contribute to controlling latent M. tuberculosis infection.     

The dormancy regulator DosR controls ribosome stability in hypoxic mycobacteria

It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a low-oxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria.     

Immune responses to the enduring hypoxic response antigen Rv0188 are preferentially detected in Mycobacterium bovis infected cattle with low pathology

The DosR regulon and the Enduring Hypoxic Response (EHR) define a group of M. tuberculosis genes that are specifically induced in bacilli exposed in vitro to conditions thought to mimic the environment encountered by Mycobacteria during latent infection. Although well described in humans, latent mycobacterial infection in cattle remains poorly understood. Thus, the aim of this study was to identify antigens that may potentially disclose cattle with latent M. bovis infection. To this end, we initially screened 57 pools of overlapping peptides representing 4 DosR regulon and 29 EHR antigens for their ability to stimulate an immune response in whole blood from TB-reactor cattle using IFN-γ and IL-2 as readouts. All 4 DosR regulon proteins were poorly recognized (maximum responder frequency of 10%). For the EHR antigens, both IFN-γ and IL-2 revealed similar response hierarchies, with responder frequencies ranging from 54% down to 3% depending on the given EHR antigen. Furthermore, these results demonstrated that responses in the infected cattle were largely IFN-γ biased. To support the concept for their role in latency, we evaluated if EHR antigen responses were associated with lower pathology. The EHR antigen Rv0188 was recognised predominantly in animals presenting with low pathology scores, whereas responses to ESAT-6/CFP-10 or the other EHR antigens tested were prevalent across the pathology spectrum. However, when we determined the production of additional cytokines induced by the M. bovis antigens PPD-B or ESAT-6/CFP-10, we detected significantly greater PPD-B-induced production of the pro-inflammatory cytokine IL-1β in animals recognizing Rv0188 (i.e. those with limited or no pathology). Thus, these results are consistent with the idea that responses to Rv0188 may identify a subset of animals at early stages of infection or in which disease progression may be limited.     

Proteins unique to intraphagosomally grown Mycobacterium tuberculosis

Pathogenic mycobacteria persist and replicate within phagosomes of host phagocytes by inhibiting phagosome maturation at an early endosome stage. The molecular basis for this behavior is not understood. To identify proteins of Mycobacterium tuberculosis unique to the intraphagosomal phase, mycobacteria were purified from phagosomes of infected murine bone marrow-derived macrophages and analyzed by high-resolution 2-DE and MS. Protein patterns of intraphagosomally grown M. tuberculosis were compared with those of broth-cultured mycobacteria. The analysis revealed 11 mycobacterial proteins exclusively detected in intraphagosomal mycobacteria. Some of these proteins are involved in metabolism and cell envelope synthesis, such as the lipid carrier protein Rv1627c, and the conserved hypothetical protein Rv1130 that shows homology to a virulence-associated protein of Legionella pneumophila. The relevance of these proteins as factors enabling intracellular survival of M. tuberculosis is being discussed.     

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.     

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.     

Mycobacterium tuberculosis senses host-derived carbon monoxide during macrophage infection

Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistence in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment.     

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.