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.

DevR–DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 865-875 ◽  
Author(s):  
Deepak Kumar Saini ◽  
Vandana Malhotra ◽  
Deepanwita Dey ◽  
Neha Pant ◽  
Taposh K. Das ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Regulatory System ◽  
Site Directed Mutagenesis ◽  
Phosphoryl Transfer ◽  
Dependent Manner ◽  
Two Component System ◽  
Component System ◽  
Two Component

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS201) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His6-tagged proteins from Escherichia coli. DevS201 underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg2+-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His395 and Asp54 as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp8 and Asp9 residues, postulated to form the acidic Mg2+-binding pocket, and the invariant Lys104 of DevR, abrogated phosphoryl transfer from DevS201 to DevR. DevR–DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the Rv3134c–devR–devS operon was induced at the RNA level in hypoxic cultures of M. tuberculosis H37Rv and was associated with an increase in the level of DevR protein. However, in a devR mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of devS transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.

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 Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene

1998 ◽  
Vol 180 (14) ◽  
pp. 3522-3528 ◽  
Author(s):  
Shu-ichi Nakayama ◽  
Haruo Watanabe
Keyword(s):  
Response Regulator ◽  
Binding Capacity ◽  
Regulatory System ◽  
Upstream Region ◽  
Dependent Manner ◽  
Acetyl Phosphate ◽  
Two Component ◽  
Transcription In Vitro ◽  
Cognate Response Regulator

ABSTRACT virF is the master regulator which activates the virulence determinant genes of Shigella spp. such asipaBCD and virG. We previously reported that expression of virF itself is regulated in a pH-dependent manner and that cpxA, a sensor of a two-component regulatory system, is involved in this regulation (S. Nakayama and H. Watanabe, J. Bacteriol. 177:5062–5069, 1995). Disruption of cpxR, which has been thought to be the cognate response regulator of cpxA (J. Dong, S. Iuchi, H.-S. Kwan, Z. Lue, and E. C. C. Lin, Gene 136:227–230, 1993), abolishedvirF expression almost completely. Purified CpxR bound directly to the upstream region of virF. Binding capacity was enhanced when CpxR was phosphorylated by coincubation with acetyl phosphate in vitro. Furthermore, we observed that phosphorylated CpxR could activate virF transcription in vitro. These results clearly indicated that CpxR was an essential activator for virF expression and strongly suggested that the binding of phosphorylated CpxR to the target site upstream of the virF gene induced a direct activation of virF transcription.

scholarly journals The Two-Component Regulatory System senX3-regX3 Regulates Phosphate-Dependent Gene Expression in Mycobacterium smegmatis

2007 ◽  
Vol 189 (15) ◽  
pp. 5495-5503 ◽  
Author(s):  
Robert T. Glover ◽  
Jordan Kriakov ◽  
Scott J. Garforth ◽  
Anthony D. Baughn ◽  
William R. Jacobs
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Mycobacterium Smegmatis ◽  
Transcriptional Control ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Regulatory System ◽  
Expression Of Genes ◽  
Two Component ◽  
Regulated Gene Expression

ABSTRACT Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (Pi) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in Pi acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a Pi-dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis. Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon Pi availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3∼P) as a transcriptional activator of phoA. Furthermore, purified RegX3∼P bound to promoter sequences from phoA, senX3, and the high-affinity phosphate transporter component pstS, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3∼P. Based upon our findings, we propose a model for Pi-regulated gene expression mediated by SenX3-RegX3 in mycobacteria.

scholarly journals Inhibiting DosRST as a new approach to tuberculosis therapy

2020 ◽  
Vol 12 (5) ◽  
pp. 457-467 ◽  
Author(s):  
Huiqing Zheng ◽  
Robert B Abramovitch
Keyword(s):  
Nitric Oxide ◽  
Infectious Disease ◽  
Carbon Monoxide ◽  
Mycobacterium Tuberculosis ◽  
Regulatory System ◽  
New Approach ◽  
Tb Treatment ◽  
Two Component ◽  
Long Course ◽  
Tuberculosis Therapy

Progress against tuberculosis (TB) requires faster-acting drugs. Mycobacterium tuberculosis (Mtb) is the leading cause of death by an infectious disease and its treatment is challenging and lengthy. Mtb is remarkably successful, in part, due to its ability to become dormant in response to host immune pressures. The DosRST two-component regulatory system is induced by hypoxia, nitric oxide and carbon monoxide and remodels Mtb physiology to promote nonreplicating persistence (NRP). NRP bacteria are thought to play a role in the long course of TB treatment. Therefore, inhibitors of DosRST-dependent adaptation may function to kill this reservoir of persisters and potentially shorten therapy. This review examines the function of DosRST, newly discovered compounds that inhibit DosRST signaling and considers future development of DosRST inhibitors as adjunct therapies.

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

AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1851-1857 ◽  
Author(s):  
Nicole Gliese ◽  
Viola Khodaverdi ◽  
Max Schobert ◽  
Helmut Görisch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethanol Oxidation ◽  
Response Regulator ◽  
Regulatory System ◽  
Pyrroloquinoline Quinone ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Cassette ◽  
Two Component ◽  
Ethanol Dehydrogenase ◽  
Oxidation System

The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c 550 and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.

scholarly journals The Two-Component Regulatory System TCS08 Is Involved in Cellobiose Metabolism of Streptococcus pneumoniae R6

2006 ◽  
Vol 189 (4) ◽  
pp. 1342-1350 ◽  
Author(s):  
Stuart J. McKessar ◽  
Regine Hakenbeck
Keyword(s):  
Streptococcus Pneumoniae ◽  
Response Regulator ◽  
Regulatory System ◽  
Phosphotransferase System ◽  
Two Component System ◽  
Transcription Profiles ◽  
Regulatory Systems ◽  
Two Component ◽  
Cognate Response Regulator ◽  
Gram Positive Cocci

ABSTRACT The two-component system TCS08 is one of the regulatory systems that is important for virulence of Streptococcus pneumoniae. In order to investigate the TCS08 regulon, we have analyzed transcription profiles of mutants derived from S. pneumoniae R6 by microarray analysis. Since deletion mutants are often without a significant phenotype, we constructed a mutation in the histidine kinase HK08, T133P, in analogy to the phosphatase mutation T230P in the H box of the S. pneumoniae CiaH kinase described recently (D. Zähner, K. Kaminski, M. van der Linden, T. Mascher, M. Merai, and R. Hakenbeck, J. Mol. Microbiol. Biotechnol. 4:211-216, 2002). In addition, a deletion mutation was constructed in rr08, encoding the cognate response regulator. The most heavily suppressed genes in the hk08 mutant were spr0276 to spr0282, encoding a putative cellobiose phosphoenolpyruvate sugar phosphotransferase system (PTS). Whereas the R6 Smr parent strain and the Δrr08 mutant readily grew on cellobiose, the hk08 mutant and selected mutants with deletions in the PTS cluster did not, strongly suggesting that TCS08 is involved in the catabolism of cellobiose. Homologues of the TCS08 system were found in closely related streptococci and other gram-positive cocci. However, the genes spr0276 to spr0282, encoding the putative cellobiose PTS, represent a genomic island in S. pneumoniae and homologues were found in Streptococcus gordonii only, suggesting that this system might contribute to the pathogenicity potential of the pneumococcus.

scholarly journals Regulation of igaA and the Rcs System by the MviA Response Regulator in Salmonella enterica

2009 ◽  
Vol 191 (8) ◽  
pp. 2743-2752 ◽  
Author(s):  
Clara B. García-Calderón ◽  
Josep Casadesús ◽  
Francisco Ramos-Morales
Keyword(s):  
Membrane Protein ◽  
Salmonella Enterica ◽  
Response Regulator ◽  
Regulatory System ◽  
Enteric Bacteria ◽  
Lon Protease ◽  
Dependent Manner ◽  
Independent Manner ◽  
Serovar Typhimurium

ABSTRACT IgaA is a membrane protein that prevents overactivation of the Rcs regulatory system in enteric bacteria. Here we provide evidence that igaA is the first gene in a σ70-dependent operon of Salmonella enterica serovar Typhimurium that also includes yrfG, yrfH, and yrfI. We also show that the Lon protease and the MviA response regulator participate in regulation of the igaA operon. Our results indicate that MviA regulates igaA transcription in an RpoS-dependent manner, but the results also suggest that MviA may regulate RcsB activation in an RpoS- and IgaA-independent manner.

scholarly journals Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

2014 ◽  
Vol 197 (5) ◽  
pp. 861-871 ◽  
Author(s):  
Kumiko Kurabayashi ◽  
Yuko Hirakawa ◽  
Koichi Tanimoto ◽  
Haruyoshi Tomita ◽  
Hidetada Hirakawa
Keyword(s):  
Phosphate Uptake ◽  
Response Regulator ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Carbon Substrate ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Two Component ◽  
Biological Cost

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagicEscherichia coli(EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression oftorR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression ofglpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.

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