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The Mycobacterium tuberculosis Clp Gene Regulator is Required for in vitro Reactivation from Hypoxia-induced Dormancy

ABSTRACT: The isogenic Mtb:ΔRv2745c mutant is significantly more sensitive normoxia conditions after a period of hypoxia, relative to wild-type Mtb, implicating a role for ClgR in response to reactivation, in vivo. Both hypoxia and reaeration treatment led to dysregulation of the σH regulon in the isogenic mutant, Mtb:ΔRv2745c. Induction of clgR in Mtb did lead to Clp protease induction, indicating that clgR plays a role in differntially activating downstream genes in a condition dependent manner. Disruption of genes involved in the dosR regulon, the Enduring Hypoxia Response, lipid synthesis, and mycolic acid synthesis also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. There was also a differnetial response of genes that are known Clp protease targets, in addition to potential Clp protease targets that had similar induction patterns as known Clp protease targets.

ORGANISM(S): Mycobacterium tuberculosis CDC1551 Mycobacterium tuberculosis H37Rv

PROVIDER: GSE64065 | GEO | 2014/12/12

SECONDARY ACCESSION(S): PRJNA270020

REPOSITORIES: GEO

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The Mycobacterium tuberculosis Clp Gene Regulator is Required for in vitro Reactivation from Hypoxia-induced Dormancy

Project description:The isogenic Mtb:ΔRv2745c mutant is significantly more sensitive normoxia conditions after a period of hypoxia, relative to wild-type Mtb, implicating a role for ClgR in response to reactivation, in vivo. Both hypoxia and reaeration treatment led to dysregulation of the σH regulon in the isogenic mutant, Mtb:ΔRv2745c. Induction of clgR in Mtb did lead to Clp protease induction, indicating that clgR plays a role in differntially activating downstream genes in a condition dependent manner. Disruption of genes involved in the dosR regulon, the Enduring Hypoxia Response, lipid synthesis, and mycolic acid synthesis also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. There was also a differnetial response of genes that are known Clp protease targets, in addition to potential Clp protease targets that had similar induction patterns as known Clp protease targets. At different time points, following hypoxia (day 1, day 5, and day 7) and reaeration (1 hour, 4 hour, 6 hour, 12 hour, 24 hour, and 48 hour) treatments M. tuberculosis CDC1551 cultures, RNA was extracted and labeled with Cy5. RNA was also extracted from pre-hypoxia (t=0) time points and labeled with Cy3. These samples were cohybridized on M. tuberculosis CDC1551 whole genome microarrays using biological triplicate samples (i.e. samples derived from three distinct cultures and treatments) and thus three unique datasets were generated for each of the three time points for Mtb. Similar experiments were performed for an isogenic Rv2745c (clgR) mutant in Mtb CDC1551 which was generated by allelic exchange.

2014-12-12 | E-GEOD-64065 | biostudies-arrayexpress

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

2014-03-25 | GSE55817 | GEO

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Project description:The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. The isogenic Mtb:ΔRv2745c mutant is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb, implicating a role for ClgR in the management of intraphagosomal redox stress. Redox stress led to dysregulation of the σH regulon in the isogenic mutant, Mtb:ΔRv2745c. Induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Disruption of genes involved in sulfate assimilation also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. At different time points, (30, 60 and 90 mins, t=30, t=60 and t=90) following the addition of diamide to M. tuberculosis CDC1551 cultures, RNA was extracted and labeled with Cy5. RNA was also extracted from pre-diamide addition (t=0) time points and labeled with Cy3. These samples were cohybridized on M. tuberculosis CDC1551 whole genome microarrays using biological triplicate samples (i.e. samples derived from three distinct cultures and treatments) and thus three unique datasets were generated for each of the three time points for Mtb. Similar experiments were performed for an isogenic Rv2745c (clgR) mutant in Mtb CDC1551 which was generated by allelic exchange and a complemented strain which was generated by trans-complementation of Rv2745c in the attB locus of the mutant strain, thus restoring the wild type regulation of Rv2745c.

2014-03-25 | E-GEOD-55817 | biostudies-arrayexpress

Reaeration timecourse from a defined hypoxia model

Project description:Reaeration timecourse from a defined hypoxia model in Mycobacterium tuberculosis. The mechanism by which mycobacteria return to a replicating state upon re-exposure to favorable conditions is not well understood. In this study, we utilized reaeration from a defined hypoxia model to characterize the adaptive response of Mycobacterium tuberculosis (MTB) following a return to favorable growth conditions. Global transcriptional analysis identified the ~100 gene Reaeration Response, induced relative to both log-phase and hypoxic MTB. This response includes chaperones and proteases, as well as the transcription factor Rv2745c, a Clp protease gene regulator (ClgR) orthologue. During reaeration, genes repressed during hypoxia are also upregulated in a wave of transcription that includes genes crucial to transcription, translation and oxidative phosphorylation. Each reaerating culture compared at multiple timepoints to a sample from the same culture at seven days of hypoxia. Three or more replicates at each timepoint.

2010-07-28 | E-GEOD-21590 | biostudies-arrayexpress

Reaeration timecourse from a defined hypoxia model

2010-07-28 | GSE21590 | GEO

Interactome analysis by shotgun proteomics identifies a novel substrate for Mycolicibacterium smegmatis unfoldase ClpC1

Project description:Tuberculosis remains a leading cause of worldwide infectious mortality and one of the top ten leading causes of death overall. While advances in public health have contributed to a reduction in tuberculosis cases, the prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) (MDR-TB) infections has created an urgent need to exploit novel drug targets. One such target is the ClpC1P1P2 protease, which degrades folded cytosolic proteins through the cooperation of the ATP-dependent unfoldase ClpC1 and the ClpP1P2 peptidase. Both protease components are strictly essential for Mtb viability and are validated therapeutic targets. However, efforts to develop anti-Mtb compounds are constrained by a limited understanding of Clp protease function and essentiality. Thus, it is crucial to identify physiological substrates and pathways regulated by this protease. In this study, we identify cellular proteins that interact with the ClpC1 unfoldase in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate for Mtb. Using a FLAG-tagged ClpC1 variant with mutations within the Walker B motifs, candidate ClpC1 interaction partners were captured by co-immunoprecipitation and identified by mass spectrometry-based proteomics (LC-MS/MS). Notably, our work reveals a novel proteolytic substrate, 5-oxoprolinase, which is recognized by ClpC1P1P2 via an N-terminal degradation sequence.

2022-12-12 | PXD030385 | Pride

Hypoxia decreases the pathogenicity of Pseudomonas aeruginosa by reducing expression of multiple virulence factors.

Project description:Hypoxia is a feature of the microenvironment during P. aeruginosa infection in several disease states. We confirm that the pathogenicity of P. aeruginosa derived from sites of acute infection is higher than those derived from sites of chronic infection. Hypoxia attenuated the pathogenicity of acute but not chronic strains implicating a role for hypoxia in controlling virulence. Mass spectrometric analysis revealed reduced expression of multiple virulence factors in hypoxia including exotoxin A, alkaline protease and proteins important in the synthesis of pyoverdine. Inhibition of pyoverdine production by iron supplementation mimicked the effects of hypoxia. Finally, strains of P. aeruginosa which lacks a functional pseudomonas prolyl-hydroxylase domain containing protein (PPHD) do not respond to hypoxia implicating a possible role for PPHD as an oxygen-sensing determinant of pathogenicity. Understanding how hypoxia influences bacterial virulence will identify new targets for anti–infective therapy against P. aeruginosa.

2017-06-06 | PXD004421 | Pride

Expression data from E. faecalis Fsr mutants during GBAP induction

Project description:To rigorously characterize the Fsr regulon, we compared gene expression in V583 isogenic mutants in Fsr genes and each of the Fsr-regulated protease genes using microarrays and purified GBAP, the quorum-sensing molecule. We used microarrays to identified the genes that are regulated directly by Fsr Quorum sensing system.

2013-05-01 | GSE42036 | GEO

Protease-activated receptor-2 promotes osteogenesis in skeletal mesenchymal stem cells at the expense of adipogenesis

Project description:Osteoblast-like cells isolated from the calvariae of wildtyope and protease-activated receptor-2 null were treated with unconditioned medium (Control) or medium conditionedby the prostate cancer cell line MDA-PCa-2b (MDA). RNAseq analysis was performed to identify differntially gene expression in wildtype and protease-activated receptor-2 null in response to factors released by prostate cancer cells.

2019-08-16 | GSE135865 | GEO

Time-resolved inducible knock-down of Clp protease subunits in tobacco identifies putative protease substrates

Project description:The Clp protease complex in plants is the one with the highest complexity through all life kingdoms. It is composed of 14 nuclear- and plastid-encoded subunits which are allocated in different rings across the complex. The proteolytic ring (P-ring; ClpP3-ClpP6) and non-proteolytic ring (R-ring; ClpR1-ClpR4 and ClpP1) form the core complex playing an essential role in protein degradation and complex stabilization, respectively. The chaperones, ClpC1, ClpC2 and ClpD are involved in substrate folding and unfolding while accessory proteins, ClpT1 and ClpT2, are important to stabilize the core complex assembly. Adaptor proteins of the complex, ClpS and ClpF, recognize protease targets. Finding substrates of proteases is one of the remaining open questions in the protease field and specifically in the Clp protease. Several attempts were made analyzing the change of protein abundance in null clp mutants by proteomic analysis. However, null clp mutants are associated with massive accumulation of pleiotropic effects, as for example impaired growth and delayed in plant development, making impossible the detection of potential Clp substrates. To address this question, we design an ethanol (EtOH) inducible strategy to silence the expression of several subunits of the Clp protease in tobacco, and follow the change in protein abundance through the EtOH time-course. In this way, we distinguish between primary and secondary effects and determine time-resolved changes in proteins stability. Time-resolved proteomic analysechloroplast processes affected by the repression of Clp activity (e.g., photosynthesis and protein homeostasis).

2022-02-22 | PXD008305 | Pride

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