Disruption of response regulator gene,<i>devR</i>, leads to attenuation in virulence of<i>Mycobacterium tuberculosis</i>

Malhotra, Vandana; Sharma, Deepak; Ramanathan, V. D.; Shakila, Harshavardhan; Saini, Deepak Kumar; Chakravorty, Soumitesh; Das, Taposh K.; Li, Qing; Silver, Richard F.; Narayanan, P R; Tyagi, Jaya Sivaswami

doi:10.1016/s0378-1097(04)00002-3

Cited by 114 publications

(103 citation statements)

References 24 publications

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“…In a recent study, a ⌬dosR mutant of MTB was reported to be hypervirulent in both severe combined immunodeficient and DBA mice (29). In contrast, another recent report indicated that a ⌬dosR mutant of MTB was attenuated for virulence in guinea pigs (30). Thus, although both studies implicate the An electrophoretic mobility shift assay was performed using purified DosR or DosR (D54E) and a DNA probe containing a DosR binding motif in the presence or absence of the phosphate donor acetyl phosphate (Acetyl-P).…”

Section: Resultsmentioning

confidence: 99%

Two Sensor Kinases Contribute to the Hypoxic Response of Mycobacterium tuberculosis

Roberts

Liao

Wisedchaisri

et al. 2004

Journal of Biological Chemistry

236

246

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Current estimates indicate that nearly a third of the world's population is latently infected with Mycobacterium tuberculosis. Reduced oxygen tension and nitric oxide exposure are two conditions encountered by bacilli in vivo that may promote latency. In vitro exposure to hypoxia or nitric oxide results in bacterial stasis with concomitant induction of a 47-gene regulon controlled by the transcription factor DosR. In this report we demonstrate that both the dosS gene adjacent to dosR and another gene, dosT (Rv2027c), encode sensor kinases, each of which can autophosphorylate at a conserved histidine and then transfer phosphate to an aspartate residue of DosR. Mutant bacteria lacking both sensors are unable to activate expression of DosR-regulated genes. These data indicate that DosR/DosS/DosT comprise a two-component signaling system that is required for the M. tuberculosis genetic response to hypoxia and nitric oxide, two conditions that produce reversible growth arrest in vitro and may contribute to latency in vivo.Tuberculosis (TB) 1 has placed a heavy burden on the global community for centuries, earning such morbid nicknames as The White Plague and The Captain of the Men of Death (1). The causative agent, Mycobacterium tuberculosis (MTB), kills about 2 million people annually making it a leading cause of infectious death worldwide (2, 3). The success of MTB as a pathogen is closely linked with its capacity to persist for years or decades in humans in the absence of any clinical disease symptoms. Current estimates place the number of people latently infected with MTB at nearly 2 billion, or one-third of the Earth's population (3, 4). Eradicating this enormous reservoir of latently infected carriers is complicated by several factors, including the availability, cost, and length of drug therapy required for successful treatment of latent TB.Although TB has been studied for centuries, the triggers that promote and maintain latent infections are still obscure. Two conditions frequently associated with latent TB in vivo are reduced oxygen tension and nitric oxide (NO) exposure (5, 6). Both of these stimuli can induce reversible bacterial stasis in vitro (7,8), and both are encountered by bacilli in vivo (5, 9, 10). Further, although MTB requires oxygen for growth, it can survive without oxygen for surprisingly long periods of time (11,12). Still the evidence linking hypoxia and NO to latent TB in vivo remains circumstantial. Analysis of the MTB response to these factors is needed to define the role they may play in promoting and maintaining TB latency in humans.Previous reports identified a set of 47 MTB genes that are rapidly up-regulated in response to reduced oxygen tension or NO (8, 13). Among the MTB genes induced by hypoxia or exposure to NO is the putative two-component regulatory system dosR-dosS (also called devR-devS, Rv3133c/Rv3132c 2 ) (8, 13). In bacteria, two-component response regulator systems are an important means by which a variety of environmental signals are transduced into a phenotypic respo...

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Section: Resultsmentioning

confidence: 99%

Two Sensor Kinases Contribute to the Hypoxic Response of Mycobacterium tuberculosis

Roberts

Liao

Wisedchaisri

et al. 2004

Journal of Biological Chemistry

236

246

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“…For example, Mtb responses to NO and hypoxia were shown to be mediated by DosR/S/T and each induced the same transcription profiles (3)(4)(5). Also, Mtb dosR was shown to be essential for full virulence in guinea pigs (23), and iNOS knockout mice were demonstrated to be susceptible to Mtb infection (6). Finally, human Mtbinfected lungs were shown to produce active iNOS (7).…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Kumar¹,

Toledo

Patel

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

300

375

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A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe 3؉ ) form, whereas DosT exists in the O 2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O 2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O 2, NO, and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO, that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O 2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence.carbon monoxide ͉ dormancy ͉ nitric oxide ͉ oxygen ͉ persistence T uberculosis (TB) is a major global health burden, and current estimates suggest that one-third of the world's population (Ϸ2 billion) is latently infected with TB (1). Latency is important largely because persistent Mycobacterium tuberculosis (Mtb) are in a state of ''drug unresponsiveness'' wherein the bacilli are resistant to existing antimycobacterial drugs. A major question in the TB field is: ''what are the mechanisms that allow Mtb to persist in human tissues for decades without replicating, to then abruptly resume growth and cause disease?'' Addressing that question is essential to the development of effective therapeutic intervention strategies. Recent evidence implicates NO as an environmental trigger of mycobacterial persistence (2-5). The latter findings are particularly interesting in light of the fact that inducible NO synthase (iNOS) and therefore NO production is crucial for protection of mice against Mtb (6), and that human macrophages in Mtb-infected tissues express iNOS (2, 7). Another factor associated with latent TB is hypoxia (8). The role of oxygen tension in TB is receiving wide attention, especially because it was demonstrated that rapid withdrawal of oxygen is lethal to Mtb, whereas a gradual depletion allows time for adaptation and bacterial survival (8). Interestingly, a significant overlap exists between the gene expression profiles of Mtb cells treated with NO and that of bacilli cultured under hypoxic conditions (3-5). ...

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“…10,11,17 There is an inherent difficulty in using a coupled assay in a filter-based format owing to an inability to discriminate between the phosphorylated species of the histidine kinase and response regulator. Inhibitors of 2-component systems that inhibited the activity of AlgR1 and AlgR2 proteins regulating alginate biosynthesis in Pseudomonas aeruginosa were discovered using an algD-xylE reporter assay.…”

Section: Discussionmentioning

confidence: 99%

“…17 However, a devR knockout strain was reported by another laboratory to be hypervirulent in mice. 18 The contradictory findings in the mouse infection model 18 could be due to inherent differences in the 2 mutant strains, in the experimental models and the route of infection used (discussed in Malhotra et al 17 ). For these reasons, DevR is considered to be a crucial regulator of the dormancy response of M. tuberculosis.…”

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confidence: 99%

High-Throughput Microplate Phosphorylation Assays Based on DevR-DevS/Rv2027c 2-Component Signal Transduction Pathway to Screen for Novel Antitubercular Compounds

Saini

Tyagi

2005

SLAS Discovery

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DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phosphotransfer 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 [γ-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 [γ-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. (Journal of Biomolecular Screening 2005:215-224)

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Disruption of response regulator gene,devR, leads to attenuation in virulence ofMycobacterium tuberculosis

Cited by 114 publications

References 24 publications

Two Sensor Kinases Contribute to the Hypoxic Response of Mycobacterium tuberculosis

Two Sensor Kinases Contribute to the Hypoxic Response of Mycobacterium tuberculosis

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

High-Throughput Microplate Phosphorylation Assays Based on DevR-DevS/Rv2027c 2-Component Signal Transduction Pathway to Screen for Novel Antitubercular Compounds

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