7 research outputs found

    Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen <i>Brucella melitensis</i> 16 M

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    Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen <i>Brucella melitensis</i>, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of <i>B. melitensis</i> 16 M. A Δ<i>vjbR</i> mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of <i>Brucella</i>. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the <i>B. melitensis</i> 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in <i>Brucella</i> and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of <i>Brucella</i> strains to the host environment

    Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen <i>Brucella melitensis</i> 16 M

    No full text
    Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen <i>Brucella melitensis</i>, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of <i>B. melitensis</i> 16 M. A Δ<i>vjbR</i> mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of <i>Brucella</i>. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the <i>B. melitensis</i> 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in <i>Brucella</i> and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of <i>Brucella</i> strains to the host environment

    Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen <i>Brucella melitensis</i> 16 M

    No full text
    Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen <i>Brucella melitensis</i>, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of <i>B. melitensis</i> 16 M. A Δ<i>vjbR</i> mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of <i>Brucella</i>. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the <i>B. melitensis</i> 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in <i>Brucella</i> and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of <i>Brucella</i> strains to the host environment

    Identification of the site of cleavage within Hsp90.

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    <p>(A) Silver stained SDS-PAGE gel showing both C-term and N-term protein fragments of Hsp90β after 30 min incubation with A/M (2 mM/10 µM) supplemented with ADP (0.2 mM) and FeCl<sub>3</sub> (0.5 mM). Asterisks show the presence of contaminants. (B) Mass spectrometry analysis of cleaved Hsp90β. Deconvoluted spectra of the small N-term fragment gave a precise monoisotopic mass of 13777.1122 daltons. (C) The picture illustrates the location of the cleavage site occurring in Hsp90β. The IGQFGVGFYS motif corresponding to a conserved amino acid sequence in several Hsp90 proteins is highlighted in red. (D) Degradation of various Hsp90 client proteins in K562 cells treated with A/M (2 mM/10 µM).</p

    Hsp90 Is Cleaved by Reactive Oxygen Species at a Highly Conserved N-Terminal Amino Acid Motif

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    <div><p>Hsp90 is an essential chaperone that is necessary for the folding, stability and activity of numerous proteins. In this study, we demonstrate that free radicals formed during oxidative stress conditions can cleave Hsp90. This cleavage occurs through a Fenton reaction which requires the presence of redox-active iron. As a result of the cleavage, we observed a disruption of the chaperoning function of Hsp90 and the degradation of its client proteins, for example, Bcr-Abl, RIP, c-Raf, NEMO and hTert. Formation of Hsp90 protein radicals on exposure to oxidative stress was confirmed by immuno-spin trapping. Using a proteomic analysis, we determined that the cleavage occurs in a conserved motif of the N-terminal nucleotide binding site, between Ile-126 and Gly-127 in Hsp90β, and between Ile-131 and Gly-132 in Hsp90α. Given the importance of Hsp90 in diverse biological functions, these findings shed new light on how oxidative stress can affect cellular homeostasis.</p> </div

    Formation of Hsp90 protein radicals following oxidative stress.

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    <p>(A) Immuno-spin trapping blots showing the formation of nitrone adducts in Hsp90α (200 µg) incubated with DMPO (1 mM) and exposed to oxidative stress generated by Glox (60 mM glucose/1 U/ml glucose oxidase) supplemented with ADP (0.2 mM) and FeCl<sub>3</sub> (0.5 mM). Blank and Ctrl mean, respectively, that DMPO and Glox were omitted. (B) Same as (A) but with Hsp90β. (C) Proposed mechanism leading to oxidative polypeptide cleavage according to Stadman et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040795#pone.0040795-Berlett1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040795#pone.0040795-Stadtman2" target="_blank">[19]</a>.</p

    Hsp90 cleavage by A/M requires the presence of ionic iron and ADP.

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    <p>K562 cell lysates (100 µg) were incubated for 1 h in the absence (Ctrl) or in the presence of A/M (2 mM/10 µM) supplemented with ADP (0.2 mM) and FeCl<sub>3</sub> (0.5 mM). Different concentrations of ADP (A), FeCl<sub>3</sub> (B) and MgCl<sub>2</sub> (C) were tested, as indicated. Hsp90 was detected with an anti-C terminus antibody from Santa Cruz Biotechnology (Hsp90 α/β, clone F-8).</p
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