scholarly journals Inhibiting Mycobacterium tuberculosis DosRST Signaling by Targeting Response Regulator DNA Binding and Sensor Kinase Heme

2019 ◽  
Vol 15 (1) ◽  
pp. 52-62 ◽  
Author(s):  
Huiqing Zheng ◽  
John T. Williams ◽  
Bilal Aleiwi ◽  
Edmund Ellsworth ◽  
Robert B. Abramovitch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Response Regulator ◽  
Sensor Kinase

scholarly journals The α10 helix of DevR, the Mycobacterium tuberculosis dormancy response regulator, regulates its DNA binding and activity

FEBS Journal ◽  
2016 ◽  
Vol 283 (7) ◽  
pp. 1286-1299 ◽  
Author(s):  
Atul Vashist ◽  
D. Prithvi Raj ◽  
Umesh Datta Gupta ◽  
Rajiv Bhat ◽  
Jaya Sivaswami Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Response Regulator

scholarly journals In the Staphylococcus aureus Two-Component System sae, the Response Regulator SaeR Binds to a Direct Repeat Sequence and DNA Binding Requires Phosphorylation by the Sensor Kinase SaeS

2010 ◽  
Vol 192 (8) ◽  
pp. 2111-2127 ◽  
Author(s):  
Fei Sun ◽  
Chunling Li ◽  
Dowon Jeong ◽  
Changmo Sohn ◽  
Chuan He ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Response Regulator ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Sensor Kinase ◽  
Two Component System ◽  
Component System ◽  
Direct Repeat Sequence ◽  
Two Component

ABSTRACT Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN6GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.

scholarly journals Expression, Autoregulation, and DNA Binding Properties of the Mycobacterium tuberculosis TrcR Response Regulator

2002 ◽  
Vol 184 (8) ◽  
pp. 2192-2203 ◽  
Author(s):  
Shelley E. Haydel ◽  
William H. Benjamin ◽  
Nancy E. Dunlap ◽  
Josephine E. Clark-Curtiss
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Two Component System ◽  
Mobility Shift ◽  
Rich Region ◽  
Component System ◽  
Two Component ◽  
Gel Mobility Shift

ABSTRACT The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in β-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.

scholarly journals Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the Dos dormancy regulon

2021 ◽  
Author(s):  
Ritesh R Sevalkar ◽  
Joel N Glasgow ◽  
Martin Pettinati ◽  
Marcelo A Martin ◽  
Vineel P Reddy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Response Regulator ◽  
Heme Iron ◽  
Sensor Kinase ◽  
New Paradigm ◽  
Heme Pocket ◽  
Expression Of Genes ◽  
Sensor Kinases ◽  
Dynamics Simulations ◽  
Autokinase Activity

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KD = 5.64 uM) but not the ferrous (Fe2+) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H2S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS-Fe3+. These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

scholarly journals Mycobacterium tuberculosis sensor kinase DosS modulates the autophagosome in a DosR-independent manner

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Uma S. Gautam ◽  
Smriti Mehra ◽  
Priyanka Kumari ◽  
Xavier Alvarez ◽  
Tianhua Niu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Life Cycle ◽  
Nitrogen Metabolism ◽  
Response Regulator ◽  
Infection Process ◽  
Molybdenum Cofactor ◽  
Sensor Kinase ◽  
Independent Manner ◽  
Tnf Α ◽  
Hypoxia Adaptation

Abstract Dormancy is a key characteristic of the intracellular life-cycle of Mtb. The importance of sensor kinase DosS in mycobacteria are attributed in part to our current findings that DosS is required for both persistence and full virulence of Mtb. Here we show that DosS is also required for optimal replication in macrophages and involved in the suppression of TNF-α and autophagy pathways. Silencing of these pathways during the infection process restored full virulence in MtbΔdosS mutant. Notably, a mutant of the response regulator DosR did not exhibit the attenuation in macrophages, suggesting that DosS can function independently of DosR. We identified four DosS targets in Mtb genome; Rv0440, Rv2859c, Rv0994, and Rv0260c. These genes encode functions related to hypoxia adaptation, which are not directly controlled by DosR, e.g., protein recycling and chaperoning, biosynthesis of molybdenum cofactor and nitrogen metabolism. Our results strongly suggest a DosR-independent role for DosS in Mtb.

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

2019 ◽  
Vol 111 (5) ◽  
pp. 1182-1194 ◽  
Author(s):  
Saurabh Sharma ◽  
Priyanka Kumari ◽  
Atul Vashist ◽  
Chanchal Kumar ◽  
Malobi Nandi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Design ◽  
Response Regulator ◽  
Sensor Kinase ◽  
Acetyl Phosphate

scholarly journals Inhibition of Mycobacterium tuberculosis DosRST two-component regulatory system signaling by targeting response regulator DNA binding and sensor kinase heme

2018 ◽  
Author(s):  
Huiqing Zheng ◽  
Bilal Aleiwi ◽  
Edmund Ellsworth ◽  
Robert B. Abramovitch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Treatment Time ◽  
Response Regulator ◽  
Transcriptional Profiling ◽  
Regulatory System ◽  
Dependent Manner ◽  
Triacylglycerol Synthesis ◽  
Two Component ◽  
Long Course

AbstractMycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, DosRST, that enables Mtb to sense host immune cues and establish a state of non-replicating persistence (NRP). NRP bacteria are tolerant to several anti-mycobacterial drugs and are thought to play a role in the long course of tuberculosis (TB) therapy. Therefore, small molecules that inhibit Mtb from establishing or maintaining NRP could reduce the reservoir of drug tolerant bacteria and function as an adjunct therapy to reduce treatment time. Previously, we reported the discovery of six novel chemical inhibitors of DosRST, named HC101A-106A, from a whole cell, reporter-based phenotypic high throughput screen. Here, we report functional and mechanism of action studies of HC104A and HC106A. RNAseq transcriptional profiling shows that the compounds downregulate genes of the DosRST regulon. Both compounds reduce hypoxia-induced triacylglycerol synthesis by ~50%. HC106A inhibits Mtb survival during hypoxia-induced NRP, however, HC104A did not inhibit survival during NRP. An electrophoretic mobility assay shows that HC104A inhibits DosR DNA binding in a dose-dependent manner, indicating that HC104A may function by directly targeting DosR. In contrast, UV-visible spectroscopy studies suggest HC106A directly targets the histidine kinase heme, via a mechanism that is distinct from the oxidation and alkylation of heme previously observed with artemisinin (HC101A). Synergistic interactions were observed when DosRST inhibitors were examined in pair-wise combinations with the strongest potentiation observed between artemisinin paired with HC102A, HC103A, or HC106A. Our data collectively show that the DosRST pathway can be inhibited by multiple distinct mechanisms.

scholarly journals Phosphatase-defective DevS sensor kinase mutants permit constitutive expression of DevR-regulated dormancy genes in Mycobacterium tuberculosis

2020 ◽  
Vol 477 (9) ◽  
pp. 1669-1682
Author(s):  
Priyanka Kumari ◽  
Sudhir Kumar ◽  
Kohinoor Kaur ◽  
Umesh Datta Gupta ◽  
Sameer Suresh Bhagyawant ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Response Regulator ◽  
Constitutive Expression ◽  
Culture Conditions ◽  
Negative Control ◽  
Adaptation To Hypoxia ◽  
Sensor Kinase ◽  
Site Mutation ◽  
Aerobic Conditions ◽  
Mutant Strains

The DevR–DevS/DosR–DosS two-component system of Mycobacterium tuberculosis, that comprises of DevS sensor kinase and DevR response regulator, is essential for bacterial adaptation to hypoxia by inducing dormancy regulon expression. The dominant phosphatase activity of DevS under aerobic conditions enables tight negative control, whereas its kinase function activates DevR under hypoxia to induce the dormancy regulon. A net balance in these opposing kinase and phosphatase activities of DevS calibrates the response output of DevR. To gain mechanistic insights into the kinase-phosphatase balance of DevS, we generated alanine substitution mutants of five residues located in DHp α1 helix of DevS, namely Phe-403, Gly-406, Leu-407, Gly-411 and His-415. For the first time, we have identified kinase positive phosphatase negative (K+P−) mutants in DevS by a single-site mutation in either Gly-406 or Leu-407. M. tuberculosis Gly-406A and Leu-407A mutant strains constitutively expressed the DevR regulon under aerobic conditions despite the presence of negative signal, oxygen. These mutant proteins exhibited ∼2-fold interaction defect with DevR. We conclude that Gly-406 and Leu-407 residues are individually essential for the phosphatase function of DevS. Our study provides new insights into the negative control mechanism of DevS by demonstrating the importance of an optimal interaction between DevR and DevS, and local changes associated with individual residues, Gly-406 and Leu-407, which mimic ligand-free DevS. These K+P− mutant strains are expected to facilitate the rapid aerobic screening of DevR antagonists in M. tuberculosis, thereby eliminating the requirement for hypoxic culture conditions.

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