25 research outputs found
The expression of Asp is controlled by temperature via an RpoE-mediated mechanism.
<p>(<b>A</b>) The activity of P<sub><i>asp</i></sub>-<i>lacZ</i> in wt, Δ<i>rpoE</i>, and Δ<i>rpoS</i> strains. The strains were grown at 30°C to the exponential phase before the bacteria were shifted to various temperatures for 0, 1, and 2 h. Then, <i>β</i>-galactosidase activity was assayed. The results show the differences relative to the level of the same strain at 0 h. The results are shown as the mean ± S.D. (<i>n</i> = 3). (<b>B</b>) qRT-PCR analysis of <i>asp</i> transcription levels in wt, Δr<i>poE</i> and <i>rpoE</i><sup>+</sup> strains that were cultivated at various temperatures. The results show the differences relative to the levels observed in the wt strain that was cultured at 22°C. The results are presented as the mean ± S.D. (<i>n</i> = 3). *, <i>P</i><0.05, and **, <i>P</i><0.01, based on ANOVA comparisons. (<b>C</b>) Western blot assays showing Asp expression in the Δr<i>poE</i> and <i>rpoE</i><sup>+</sup> strains at different temperatures. RNAP was used as the loading control for the supernatants that were obtained from the same amount of cells.</p
A σ<sup>E</sup>-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in <i>Vibrio alginolyticus</i>
<div><p>In vibrios, the expression of virulence factors is often controlled by LuxR, the master quorum-sensing regulator. Here, we investigate the interplay between LuxR and σ<sup>E</sup>, an alternative sigma factor, during the control of virulence-related gene expression and adaptations to temperature elevations in the zoonotic pathogen <i>Vibrio alginolyticus</i>. An <i>rpoE</i> null <i>V</i>. <i>alginolyticus</i> mutant was unable to adapt to various stresses and was survival-deficient in fish. In wild type <i>V</i>. <i>alginolyticus</i>, the expression of LuxR-regulated virulence factors increased as the temperature was increased from 22°C to 37°C, but mutants lacking σ<sup>E</sup> did not respond to temperature, indicating that σ<sup>E</sup> is critical for the temperature-dependent upregulation of virulence genes. Further analyses revealed that σ<sup>E</sup> binds directly to -10 and -35 elements in the <i>luxR</i> promoter that drive its transcription. ChIP assays showed that σ<sup>E</sup> binds to the promoter regions of <i>luxR</i>, <i>rpoH</i> and <i>rpoE</i> at high temperatures (e.g., 30°C and 37°C). However, at higher temperatures (42°C) that induce thermal stress, σ<sup>E</sup> binding to the <i>luxR</i> promoter decreased, while its binding to the <i>rpoH</i> and <i>rpoE</i> promoters was unchanged. Thus, the temperature-dependent binding of σ<sup>E</sup> to distinct promoters appears to underlie a σ<sup>E</sup>-controlled switch between the expression of virulence genes and adaptation to thermal stress. This study illustrates how a conserved temperature response mechanism integrates into quorum-sensing circuits to regulate both virulence and stress adaptation.</p></div
RpoE controls Asp expression via the QS regulator LuxR.
<p>(<b>A</b>) <i>luxR</i> transcriptional assays were performed in wt and Δ<i>luxO</i> strains that were grown at different temperatures. The wt and Δ<i>luxO</i> strains that carried the P<sub><i>luxR</i></sub>-<i>lacZ</i> reporter plasmid were cultured in LBS medium and assayed for <i>β</i>-galactosidase activity. The results are presented as the mean ± S.D. (<i>n</i> = 3). (<b>B</b>) Western blot assays show LuxR expression in wt, Δ<i>rpoE</i> and <i>rpoE</i><sup>+</sup> strains grown at different temperatures. Bacteria were cultured in LBS medium for 9 h and then harvested. Proteins from equal cell volumes were resolved using 12% SDS-PAGE. (<b>C</b>) Extracellular Asp activity in the <i>V</i>. <i>alginolyticus</i> strains. The bacteria were centrifuged after they were cultured in LBS medium for 9 h, and the supernatants were harvested to measure protease activity. The results are presented as the mean ± S.D. (<i>n</i> = 3). **, <i>P</i><0.01, and ***, <i>P</i><0.001, based on ANOVA comparisons. (<b>D</b>) Western blot assays were performed to analyze Asp and LuxR expression in wt and <i>rpoE-</i> and <i>luxO</i>-related mutants. pBAD33::<i>rpoE</i> was introduced into the Δ<i>rpoE</i>Δ<i>luxO</i> strain to analyze Asp and LuxR expression in the presence (+) and absence (-) of L-arabinose (Ara).</p
RpoE binds to target DNA in response to temperature changes <i>in vivo</i> and <i>in vitro</i>.
<p>(<b>A</b>) ChIP assays were used to analyze RpoE binding to the <i>luxR</i>, <i>rpoE</i> and <i>rpoH</i> promoter <i>in vivo</i>. Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using PCR with the primers P<sub><i>luxR</i></sub>chip-F/R, P<sub><i>rpoE</i></sub>-chipF/R, P<sub><i>rpoH</i></sub>-chipF/R and control-F/R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005645#ppat.1005645.s010" target="_blank">S2 Table</a>). (<b>B</b>) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene <i>gyrB</i>. Results were calculated using the ΔΔ<i>C</i><sub>T</sub> method. * <i>P</i> <0.05, ** <i>P</i> <0.01, <i>t-</i>test. (<b>C</b>) Plot showing the affinity of RpoE binding to the promoters of <i>luxR</i>, <i>rpoE</i>, and <i>rpoH</i> at different temperatures as determined using EMSA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005645#ppat.1005645.s004" target="_blank">S4 Fig</a>). The intensities of the bound DNA fragments were determined using a densitometer and plotted against the RpoE concentrations. Triplicate assays were performed, and a representative plot is shown. (<b>D</b>) The promoter strength of <i>luxR</i>, <i>rpoE</i>, and <i>rpoH</i> in the presence of similar levels of RpoE at different temperatures in <i>E</i>. <i>coli</i> DH5α cells. The three indicated promoters were fused to different fluorescence reporters and cloned into the same cloning plasmid, pMD19T. The fluorescence reads for each of the promoters after incubation in the presence of arabinose inducing pBAD-driven <i>rpoE-flag</i> expression were subtracted from the reads obtained with no arabinose and normalized to both the corresponding reads at 22°C and the densitometry values of RpoE expression. The results are presented as the mean ± S.D. (<i>n</i> = 3).</p
RpoE, AphA, and LuxR bind to the <i>luxR</i> promoter.
<p>(<b>A</b>) EMSA and DNase I footprinting analysis were used to analyze whether LuxR and AphA specifically bind to the <i>luxR</i> promoter. In EMSA assays, various concentrations of LuxR and AphA proteins were added to mixtures of poly(dI:dC) and Cy5-labeled <i>luxR</i> promoter DNA. In the DNase I footprinting assays, electropherograms of a DNase I digest of the P<i>luxR</i> promoter probe after it was incubated with or without the LuxR and AphA proteins. The level at which the respective nucleotide sequences were protected by the proteins is indicated below. (<b>B</b>) Diagram the showing promoter region of the <i>luxR</i> gene. The LuxR and AphA binding sites and the ribosome binding site (RBS) are underlined. The region protected by RpoE (RpoEB) is shadow-boxed. The well-conserved nucleotides that were bound to specific proteins are indicated with asterisks. The transcription start site (TSS) is labeled as +1. (<b>C-D</b>) EMSA assays of LuxR and RpoE mixtures binding to <i>luxR</i> promoter or mutant <i>luxR</i> promoter regions in the absence of the LBSI (P<sub><i>luxR</i></sub>ΔLBSI) or LBSII binding site (P<sub><i>luxR</i></sub>ΔLBSII). The amounts of LuxR or RpoE protein (nM) that were used are as indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. (<b>E</b>) The promoter activity is shown for P<sub><i>luxR</i></sub> and its variants that were fused to <i>lacZ</i>. The strains were grown at 22°C, 30°C, 37°C and 42°C for 9 h, and <i>β</i>-galactosidase activity was then assayed. The results are shown as the mean ± S.D. (<i>n</i> = 3).</p
RIP and anti-sigma E signaling pathways are involved in RpoE-regulated QS.
<p>(<b>A</b>) HPA digestion assays were performed to analyze extracellular Asp activity in wt and mutant strains. Bacteria were centrifuged after they were cultured in LBS medium for 9 h at various temperatures. The supernatants were harvested, and protease activity was measured. ECP activities were normalized by dividing the total level of activity by the cell density for each strain. The results are shown as the mean ± S.D. (<i>n</i> = 3). * <i>P</i> <0.05, ** <i>P</i> <0.01, <i>t-</i>test compared to the corresponding results in the wt strain at each temperature. (<b>B</b>) Western blot analysis was performed to determine the distribution of RpoE in membrane and cytoplasmic pellets in wt, Δ<i>degS</i> and Δ<i>rseA</i><sup>20-39</sup> strains at different temperatures. Bacterial cells were centrifuged after they were cultured in LBS medium for 9 h, and the cytoplasmic and membrane protein pellets were then extracted. RNAP was used as a loading control.</p
RpoE binds directly to the <i>luxR</i> promoter region.
<p>(<b>A</b>) EMSAs were performed with purified RpoE, and the <i>luxR</i> promoter region was analyzed. The amount of RpoE protein (nM) that was used is indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. The shifts were verified to be specific in experiments in which we added a 5- to 50-fold excess of unlabeled specific DNA and non-specific competitor DNA (poly(dI:dC)). (<b>B</b>) Plot showing the affinity of RpoE to the <i>luxR</i> promoter. The densitometric intensities of bound DNA fragments were plotted against RpoE concentrations. The arrow indicates the concentration of RpoE that caused half-maximal binding (<i>K</i><sub>d</sub>). (<b>C</b>)Footprinting analysis of RpoE binding to a binding site in the <i>luxR</i> promoter. Electropherograms of a DNase I digest of the P<sub><i>luxR</i></sub> promoter probe (400 ng) after incubation with 0 or 400 nM RpoE. The respective nucleotide sequences that were protected by His-RpoE are indicated below, and the specific -10 and -35 regions are underlined. (<b>D-E</b>) <i>In vitro</i> transcription was performed using a P<sub><i>rpoE</i></sub> template, (<b>D</b>) a P<sub><i>luxR</i></sub> template (<b>E</b>), NTP, and RNAP core enzyme as well as RpoE. Various concentrations of purified LuxR were added into the reaction mixture to determine its effect on <i>luxR</i> transcription (<b>E</b>). The transcripts were purified, reverse-transcribed (RT, +) and detected using PCR. As a control, the same purified transcripts were also treated using the same process but without reverse transcriptase (RT, -).</p
RpoE is involved in the temperature-dependent <i>in vivo</i> virulence of <i>V</i>. <i>alginolyticus</i>.
<p>(<b>A-D</b>) Virulence was analyzed in the <i>rpoE</i> mutant in zebrafish. A series of dilutions of the wt and <i>rpoE</i> mutant strains were intramuscularly (i.m.) inoculated into fish that were acclimated at 22°C or 30°C for 4 weeks. A total of 30 fish were used for each of the dilutions. The infected fish were cultivated at 22°C or 30°C and monitored for 7 days. <i>P</i> values were calculated using Kaplan-Meier survival analysis with a log rank test. (<b>E</b>) CI assays for the Δ<i>rpoE</i> strain against the wt (<i>lacZ</i><sup>+</sup>), which was a wt strain carrying <i>lacZ</i> following the <i>glmS</i> locus. The CI values of the 1:1 Δ<i>rpoE</i> vs. wt (<i>lacZ</i><sup>+</sup>) inoculums were first tested in LBS medium at 30°C for 24 h. Then, 1:1 [the indicated strains vs. wt (<i>lacZ</i><sup>+</sup>)] inoculums were i.m. administered into zebrafish and cultivated at 30°C for 24 h before the fish were anesthetized and their cells were numerated on IPTG plates. ***, <i>P</i><0.001, based on ANOVA followed by Bonferroni’s multiple-comparison post-test to compare the data to the values for the corresponding wt/wt (<i>lacZ</i><sup>+</sup>) samples.</p
Transcriptome Profiling Reveals Th17-Like Immune Responses Induced in Zebrafish Bath-Vaccinated with a Live Attenuated <i>Vibrio anguillarum</i>
<div><p>Background</p><p>A candidate vaccine, live attenuated <i>Vibrio anguillarum</i> developed in our laboratory could prevent vibriosis of fish resulted from <i>V. anguillarum</i> and <i>V. alginolyticus</i>. To elucidate the molecular mechanisms underlying the vaccine protection, we used microarray technology to compare the spleen transcriptomes of bath-vaccinated and unvaccinated zebrafish at 28 days post vaccination.</p><p>Principal Findings</p><p>A total of 2164 genes and transcripts were differentially expressed, accounting for 4.9% of all genes represented on the chip. In addition to iron metabolism related to the innate immunity and the signaling pathways, these differentially expressed genes also involved in the adaptive immunity, mainly including the genes associated with B and T cells activation, proliferation and expansion. Transcription profiles of Th17-related transcription factors, cytokines and cytokine receptors during 35 days post-vaccination implied that Th17 cells be activated in bath-vaccinated zebrafish.</p><p>Conclusion/Significance</p><p>The transcriptome profiling with microarray revealed the Th17-like immune response to bath-vaccination with the live attenuated <i>V. anguillarum</i> in zebrafish.</p></div
Top 20 of Statistically significant KEGG pathways in response to vaccination.
<p>Top 20 of Statistically significant KEGG pathways in response to vaccination.</p