Chemical inhibition of the Mycobacterium tuberculosis DosRST two-component regulatory system by targeting sensor kinase heme and response regulator DNA binding
Ontology highlight
ABSTRACT:
ORGANISM(S): Mycobacterium tuberculosis
PROVIDER: GSE115892 | GEO | 2018/09/10
REPOSITORIES: GEO
Similar Datasets
Project description:Mycobacterium tuberculosis can exist in the actively growing state of the overt disease or in a latent quiescent state that can be induced, among other things, by anaerobiosis. Eradication of the latent state is particularly difficult with the available drugs and requires prolonged treatment. DevS is a member of the DevS-DevR two-component regulatory system that is thought to mediate the cellular response to anaerobiosis. Here we report the cloning, expression, and initial characterization of a truncated version of DevS (DevS642) containing only the N-terminal GAF sensor domain (GAF-A) and of the full-length protein DevS. The DevS truncated construct quantitatively binds heme in a 1:1 stoichiometry, and the complex of the protein with ferrous heme reversibly binds O2, NO, and CO. UV-vis and resonance Raman spectroscopy of the wild-type protein and the H149A mutant confirm that His149 is the proximal ligand to the heme iron atom. While the heme-CO complex is present as two conformers in the GAF-A domain, a single set of [Fe-C-O] vibrations is observed with the full-length protein, suggesting that interactions between domains within DevS influence the distal pocket environment of the heme in the GAF-A domain.
| S-EPMC2518089 | biostudies-literature
Project description:The purpose of this study was to discover and characterize small molecule inhibitors of the M. tuberculosis DosRST two-component regulatory pathway. The impact of three novel DosRST regulon inhibitors on gene expression was studied in wild type M. tuberculosis and a DosRS mutant strain.
2016-12-19 | GSE76566 | GEO
Project description:Chemical inhibition of the Mycobacterium tuberculosis DosRST two-component regulatory system by targeting sensor kinase heme and response regulator DNA binding
| PRJNA476612 | ENA
Project description:Mycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, DosRST, that enables Mtb to sense host immune cues and establish a state of nonreplicating persistence (NRP). NRP bacteria are tolerant to several antimycobacterial drugs in vitro and are thought to play a role in the long course of tuberculosis therapy. 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 sensor 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 pairwise 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.
| S-EPMC6970277 | biostudies-literature
Project description:In Mycobacterium tuberculosis, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Several mutations, present in phoR of Mycobacterium canettii relative to M. tuberculosis, impact the expression of the PhoP regulon and the pathogenicity of the strains. Here, we analyse by RNA-seq the expression profile of PhoP-regulated genes between the two M. tuberculosis strains H37Rv and HN878 and the two M. canettii isolates STB-Ks and STB-Kr.
2023-05-24 | GSE229680 | GEO
Project description:The mechanisms utilized by Mycobacterium tuberculosis to establish, maintain, or reactivate from latent infection in the host are largely unknown but likely include genes that mediate adaptation to conditions encountered during persistence. Previously, a two-component signal transduction system, mprAB, was found to be required in M. tuberculosis for establishment and maintenance of persistent infection in a tissue- and stage-specific fashion. To begin to characterize the role of this system in M. tuberculosis physiology and virulence, a functional analysis of the mprA and mprB gene products was initiated. Here, evidence is presented demonstrating that sensor kinase MprB and response regulator MprA function as an intact signal-transducing pair in vitro and in vivo. Sensor kinase MprB can be autophosphorylated, can donate phosphate to MprA, and can act as a phospho-MprA phosphatase in vitro. Correspondingly, response regulator MprA can accept phosphate from MprB or from small phosphodonors including acetyl phosphate. Mutagenesis of residues His249 in MprB and Asp48 in MprA abolished the ability of these proteins to be phosphorylated in vitro. Introduction of these alleles into Mycobacterium bovis BCG attenuated virulence in macrophages in vivo. Together, these results support a role for the mprAB two-component system in M. tuberculosis physiology and pathogenesis. Characterization of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis during acute and/or persistent infection in the host.
| S-EPMC308901 | biostudies-literature
Project description:The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii-like ancestor, remain poorly investigated. In MTB, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M. canettii relative to MTB, impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M. canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M. canettii strain, displays lower expression of PhoP-induced genes than MTB. Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M. canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M. canettii strains than in M. tuberculosis. Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host.
| S-EPMC10348564 | biostudies-literature
Project description:The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.
| S-EPMC5638771 | biostudies-literature
Project description:Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.
| S-EPMC7036368 | biostudies-literature
Project description:BackgroundHeme is typically a major iron source for bacteria, but little is known about how bacteria of the Leptospira genus, composed of both saprophytic and pathogenic species, access heme.ResultsIn this study, we analysed a two-component system of the saprophyte Leptospira biflexa. In vitro phosphorylation and site-directed mutagenesis assays showed that Hklep is a histidine kinase which, after autophosphorylation of a conserved histidine, transfers the phosphate to an essential aspartate of the response regulator Rrlep. Hklep/Rrlep two-component system mutants were generated in L. biflexa. The mutants could only grow in medium supplemented with hemin or delta-aminolevulinic acid (ALA). In the pathogen L. interrogans, the hklep and rrlep orthologous genes are located between hemE and hemL genes, which encode proteins involved in heme biosynthesis. The L. biflexa hklep mutant could be complemented with a replicative plasmid harbouring the L. interrogans orthologous gene, suggesting that these two-component systems are functionally similar. By real-time quantitative reverse transcription-PCR, we also observed that this two-component system might influence the expression of heme biosynthetic genes.ConclusionThese findings demonstrate that the Hklep/Rrlep regulatory system is critical for the in vitro growth of L. biflexa, and suggest that this two-component system is involved in a complex mechanism that regulates the heme biosynthetic pathway.
| S-EPMC2266917 | biostudies-literature