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

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

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

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

MS/MS-identification of proteins detected by SERPA from colorectal cancer cells exposed to hypoxia.

<p>Mapping of spots of interest resulting from the comparison described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076508#pone-0076508-g001" target="_blank">Fig.1</a> and list of identified proteins (p<0.001) obtained using lysates of HCT116 colorectal cancer cells.</p

Hypoxia integration in the SERPA strategy.

<p><b>A.</b> Workflow of the SERPA process including 2DE-gel separation of lysates from either hypoxic or normoxic tumor cells, membrane transfer, immunoblotting with the serum from either control or tumor-bearing mice, and detection of spots of interest. <b>B.</b> Typical immunoblotting patterns resulting from the incubation of 2D-resolved lysates of HCT116 cells exposed to normoxia or hypoxia, with the indicated mouse serum. In the bottom panels, proteins of the lysates are labelled with Cy dye (red) and fixed antibodies are detected with an anti-mouse secondary antibody (green spot); arrow indicates the presence of a protein exclusively detected in the lysates of hypoxic tumor cells by antibodies from the serum of tumor-bearing mice.</p

Validation of phospho-eEF2 protein as the target of autoantibodies in mice bearing colorectal HCT116 tumors.

<p><b>A.</b> Representative immunoblotting of 2D-separated lysates of hypoxic HCT116 cells with a commercial antibody against eEF2. Proteins of the lysates are labelled with Cy dye (red) and secondary antibody is conjugated to horseradish peroxidase (green spots). Positive signal is obtained for several spots of the same molecular weight but differing by their pI value. <b>B.</b> Comparison of the eEF2 spots detected with a commercial antibody against total eEF2 (top), the serum from tumor-bearing mice (middle) and a commercial antibody against phospho-Thr56 eEF2 (bottom). Spot 4 (rightmost spot) corresponds to the unphosphorylated form of eEF2 while the other spots correspond to multi-phosphorylated forms of the protein; spot 3 (second spot from the right) corresponds to the preferential monophosphorylated form of eEF2 (on Thr56).</p

Validation of hypoxia-induced phosphorylations of eEF2 in colorectal cancer cells.

<p><b>A.</b> Representative eEF2 and phospho-Thr56 eEF2 immunoblotting of HCT116 and HT29 cultured for 48 hours under hypoxia (Hx) or maintained in normoxia (Nx). <b>B.</b> Normalized expression of phospho-Thr56 eEF2 in normoxic vs hypoxic HCT116 and HT29 cells; n = 3, **p<0.01 <b>C.</b> Representative phospho-Thr56 eEF2 immunostaining of sections of HCT116 tumors in the absence (top) or the presence (bottom) of phosphatase lambda; note the complete disappearance of the phosphorylated form of eEF2 upon treatment with the phosphatase.</p

Changes in the titer of anti-phospho-eEF2 aAb as a marker of early tumor progression in mice and humans.

<p><b>A.</b> Human and mouse amino acid sequences of eEF2 in the region of Thr56. The 12 residues corresponding to the synthetic peptide (phosphorylated on Thr56) used in our immunoassay are indicated (red frame); note the perfect identity between mouse and human sequences. <b>B.</b> Detection of commercial anti-phospho-Thr56 eEF2-antibodies using our immunoassay; dashed lines show the 95% confidence band of the linear regression. <b>C.</b> Detection of anti-phospho-Thr56 eEF2 aAb in the serum of control or HCT116 tumor-bearing mice (n = 3). ***P<0.001. <b>D.</b> Time course of HCT116 tumor growth as determined by measurements of tumor diameters (n = 7 per group). <b>E.</b> Detection of anti-phospho-Thr56 eEF2 aAb at the indicated time of HCT116 tumor progression. *P<0.05, **P<0.01, ***P<0.001 (n = 6–7 per group). Note that at day 7 post-implantation, tumors are not detectable (see panel D) but a positive signal is detected in the immunoassay. <b>F.</b> Graph represents the detection of anti-phospho-Thr56 eEF2 aAb in the serum of control subjects (n = 6) and patients with adenomatous polyps (n = 14) or carcinoma (n = 9). *P<0.05, **P<0.01. Of note, K-means clustering identified two subpopulations of patients (see black and red symbols) among individuals diagnosed with adenomatous polyps (P<0.001) and carcinoma (P<0.01); the same partition was observed in 100 independent runs by varying the random initialization of K-means algorithm.</p

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

<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

Identification of the site of cleavage within Hsp90.

<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 FeCl₃ (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