Influence of Autoinducer-2 (AI-2) in Cell-Free Supernatant of Salmonella Typhimurium in Gene Expression of S. Typhimurium
Ontology highlight
ABSTRACT:
ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium
SUBMITTER: Palmy Jesudhasan
PROVIDER: E-GEOD-23761 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Salmonella enterica serovar Typhimurium is known to synthesize and respond to the cell signaling molecule, autoinducer-2 (AI-2). The luxS gene is involved in the synthesis of AI-2. We have previously shown that luxS controls a variety of bacterial processes in S. Typhimurium. In this study we identified the genes regulated by AI-2 using mRNA samples from isogenic luxS gene mutant of S. Typhimurium strain 87-26254 grown in LB media in the presence/absence of in vitro synthesized AI-2. In the presence of in vitro synthesized AI-2, 536 genes were significantly (p<0.05) regulated. Interestingly, in vitro synthesized AI-2 caused the down-regulation of 39 genes in Salmonella Pathogenicity Island-1 (SPI-1) including the transcriptional regulators hilA, hilD, and invF. Purified cDNAs from the mutant supplemented with AI-2 and mutant without AI-2 (PBS) were each labeled with Cy-3 mono-Reactive Dye and Cy-5 mono-Reactive Dye (GE Health Care, Piscataway, NJ) and were processed using a dye-swapping design. A total of eight microarray arrays, each with duplicate spots for each gene and used for mutant supplemented with AI-2.
2015-07-31 | E-GEOD-23588 | biostudies-arrayexpress
Project description:A mouse hepatoma cell line (Hepa1-6 cells, ATCC) was grown in T-25 tissue culture flasks (Corning) at 37ºC in 5% CO2 under two conditions. The first condition was the control, which contained DMEM medium (Gibco BRL) with 25 mM glucose, 4 mM glutamine, 1% penicillin-streptomycin (Sigma), and 10% CPSR-3 (Sigma). The second condition was identical to the first, however the glutamine concentration was altered periodically. Once the cells had grown to confluency, the experiment began at 9:30 PM by exchanging the medium in all flasks. The control flasks received the same medium, containing 4 mM glutamine, while the experimental flasks received identical medium with no glutamine. Twenty-four hours later, the medium was exchanged again, however this time all flasks received medium containing 4 mM glutamine. The first medium exchange was repeated at 48 hours into the experiment, while the second was repeated at 72 hours into the experiment. Hence the glutamine concentration of the experimental flasks oscillated between 0 mM and 4 mM glutamine over 96 hours, while the glutamine concentration of the control flasks remained constant at 4 mM glutamine. At regular intervals, the flasks were sampled. Total RNA was isolated, medium samples were retained, and isotopic measurements were performed from flasks fed different labelled compounds. All samples taken during the experiment were analyzed. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples from identical time points that were to be compared on DNA microarrays were then mixed. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65ºC. Following concentration, the sample was resuspended in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), 1 uL was removed for quantitation, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride, 239 mL 1-methyl-2-pyrrolidinone, and 10.7 mL of 1 M boric acid, pH 8.0. They were cleaned by rinsing in 250 mL of milli-Q water (X2), followed by 250 mL of 95% ethanol. They were finally dried using filtered air and stored in the dark at room temperature. Keywords = mouse, liver, hepatoma, cell culture, glutamine, carbon metabolism
2010-06-05 | E-GEOD-404 | biostudies-arrayexpress
Project description:Salmonella enterica serovar Typhimurium is known to synthesize and respond to the cell signaling molecule, autoinducer-2 (AI-2). The luxS gene is involved in the synthesis of AI-2. We have previously shown that luxS controls a variety of bacterial processes in S. Typhimurium. In this study we identified the genes regulated by AI-2 in the Cell-Free supernatant of S. Typhimurium using mRNA samples from isogenic luxS gene mutant of S. Typhimurium strain 87-26254 grown in LB media in the presence of S. Typhimurium wild type CFS / absence AI-2 (mutant CFS of S. Typhimurium). In the presence of AI-2 in CFS, 758 genes were significantly regulated. Interstingly, AI-2 in CFS caused the down-regulation of 39 genes in Salmonella Pathogenicity Island-1 (SPI-1).
2015-07-31 | GSE23761 | GEO
Project description:To study the pathogenicity of Salmonella Typhimurium (ST) in heterophils, a whole Salmonella genome array was used to analyze RNA isolated from Salmonella in vitro encountered with heterophils (30 min, 1 h, 2 h, and 3 h) or medium control (NS). Dual-color, direct comparisons were carried out between heterophils-encountered ST and non-encountered controls (30 min vs. NS (30M/NS), 1 h vs. NS (1HR/NS), 2 h vs. NS (2HR/NS) and 3 h vs. NS (3HR/NS)). Each comparison includes four biological replicates. Out of 4588 genes on an array, there were 605, 758, 944 and 945 genes differentially expressed in the comparisons of 30M/NS, 1HR/NS, 2HR/NS and 3HR/NS, respectively (P < 0.001, fold-change > 2). Most of the Salmonella genes associated with invasion function were found differentially expressed at 2HR/NS and 3HR/NS comparisons. These candidate genes include TypeIII-secreted protein effector sopE2, Invasion proteins (e.g. invA, invE, invG, invH), and Invasion genes tranction activator hilA. The results of continuous transcriptional changes provide important information to elucidate the pathogenicity of ST in heterophils. Dual-color, common reference comparisons were carried out between heterophils-encountered ST and non-encountered controls (30 min vs. NS (30M/NS), 1 h vs. NS (1HR/NS), 2 h vs. NS (2HR/NS) and 3 h vs. NS (3HR/NS)). Four biological replicates, with dye swap labeling, were included in each of comparisons. Background subtracted signal intensity were collected from 16 arrays and normalized before data analysis.
2011-01-01 | E-GEOD-16399 | biostudies-arrayexpress
Project description:The Artemisia iwayomogi (Ai) has been known to inhibit the inflammatory cytokine production and the allergic reactions, and has been used to treat liver diseases. This study investigated the gene expression changes by lipopolysaccharide (LPS) stimulation in the cultured human gingival fibroblast and the gene expression changes by the Ai when challenged with LPS using a microarray chip. Human gingival fibroblast were divided into three experimental groups; â C: Control, â¡ LPS: LPS-treatment only, ⢠LPS-Ai: LPS- and Ai-treatments. Total RNA was isolated from each experimental fibroblast (3 experimental group à 1 sample of each experimental group = total 3 samples).
2010-10-23 | E-GEOD-24834 | biostudies-arrayexpress
Project description:Organisms respond to heat stress by reprogramming gene expression. Here we show that genome-wide reprogramming involves enhanced assembly of the TFIID and SAGA regulatory pathways at heat induced genes, and disassembly of the TFIID pathway at heat-repressed genes. While TFIID and SAGA are recruited to heat-induced genes, only SAGA appears to be associated with achieving maximal induction. Mot1, an ATP-dependent inhibitor of the TATA binding protein TBP, assembles at heat-induced SAGA-regulated genes, but functions to attenuate rather than promote activation. Changes in promoter occupancy of bromodomain factor Bdf1 are tightly linked to changes in TFIID occupancy, which further supports the notion that the two work together. Bdf1 is inhibitory to a number of SAGA-regulated genes and dissociates when these genes are activated, suggesting that Bdf1 normally blocks transcription complex assembly at these genes. These linkages in reprogramming of factor occupancy at promoters provide direct evidence for two functionally distinct transcription assembly pathways, and reveal unexpected cross-talk between the pathways. Keywords = Chromatin Immunoprecipitation Keywords = Microarray Keywords = TBP
2010-06-05 | E-GEOD-1373 | biostudies-arrayexpress
Project description:Transcriptional profiling of Campylobacter jejuni NCTC 11168 wild type and LuxS01 mutant strains comparing the effects of exogenously added in vitro-produced autoinducer 2 (AI-2) versus a control buffer to both strains. Two-condition experiment, addition of exogenous AI-2 to both the parent and mutant 11168 strains. Control microarrays included comparing 11168 with AI-2 free buffer vs 11168 with AI-2-free buffer and mutant with AI-2 free buffer versus mutant with AI-2 free buffer used for analyses. 3 biological replicates for each, independently grown and harvested.
2010-06-21 | E-GEOD-18427 | biostudies-arrayexpress
Project description:To determine the host response to Salmonella Typhimurium (ST) in heterophils, a whole chicken genome array was used to analyze RNA isolated from heterophils stimulated in vitro with ST (30 min, 1 h, 2 h, and 3 h) or medium control (NS). Dual-color, direct comparisons were carried between ST-stimulated and non-stimulated controls (30 min vs. NS (30M/NS), 1 h vs. NS (1HR/NS), 2 h vs. NS (2HR/NS) and 3 h vs. NS (3HR/NS)). Each comparison includes four biological replicates. There were 2791, 3744, 12947 and 12482 genes differentially expressed in the comparisons of 30M/NS, 1HR/NS, 2HR/NS and 3HR/NS, respectively (P < 0.001) , while 50, 82, 153, and 146 genes were related to immune function, respectively. More than 80% of immune-related genes overlapped between the comparisons (30M/NS vs. 1HR/NS, 1HR/NS vs. 2HR/NS, and 2HR/NS vs. 3HR/NS). These candidate genes include cytokines (e.g. Interleukin (IL) 1β, -6, -10B and -12B), chemokines (e.g. IL-8, CCL4, K60 and K203) and cluster of differentiation (CD) markers (e.g. CD44, -47 and -83). A relatively large increase in the number of differentially expressed immune-related genes was observed between 1 h and 2 h post-stimulation. The results of continuous transcriptional change provide important information to elucidate both the cellular and molecular mechanisms of ST stimulation in chickens. Dual-color, common reference comparisons were carried between ST-stimulated (30M, 1HR, 2HR and 3HR) and non-stimulated controls (NS). Four biological replicates, with dye swap labeling, were included in the comparisons of each post-stimulation time point (30M/NS, 1HR/NS, 2HR/NS and 3HR/NS). Background subtracted signal intensity were collected from 16 arrays and normalized before data analysis.
2010-04-13 | E-GEOD-14684 | biostudies-arrayexpress
Project description:Eukaryotic genes are controlled by sequence-specific DNA-binding proteins, chromatin regulators, general transcription factors, and elongation factors. Here we examine the genome-wide location of representative members of these groups and their redistribution when the Saccharomyces cerevisiae genome is reprogrammed by heat shock. As expected, assembly of active transcription complexes are coupled to eviction of H2A.Z nucleosomes, and disassembly is coupled to the return of nucleosomes. Remarkably, a large number of promoters assemble and disassemble into partial pre-initiation complexes (partial PICs), containing TFIIA, TFIID (and/or SAGA), TFIIB, TFIIE, and TFIIF. At these promoters, neither TFIIH nor RNA polymerase II are recruited, and nucleosomes are not displaced. These promoters may be preparing for additional stress that naturally accompany heat stress. For example, we find that oxidative stress, which often occurs with prolonged exposure of cells to high temperature, coverts partial PICs into full PICs. Partial PICs therefore represent novel regulated intermediates that assemble in the midst of chromatin. Keywords: other
2010-06-10 | E-GEOD-2488 | biostudies-arrayexpress
Project description:Corpus luteum (CL) is a transient endocrine tissue formed from the remnants of the ovarian follicle after ovulation. In response to gonadotropin surge, the ovulating follicle undergoes dramatic changes in expression of genes and differentiation of follicular cells into luteal cells. In several species, of the several genes that are down- regulated post ovulation, Cyp19A1 that codes for aromatase, essential for biosynthesis of estradiol- 17M-NM-2 (E2), also get down- regulated but appears to get up- regulated at later time points in the estrous cycle to have critical role in E2 secretion. In primates and rodents, higher expression and higher E2 levels has been observed in CL. Surprisingly, in the recently carried out gene expression profiling of PGF2M-NM-1- induced luteolysis studies in the bovine species [GSE27961], it was observed that expression of one of the earliest genes that was down- regulated was Cyp19A1 in the CL. However, the specific role of E2 in the regulation of CL function remains poorly defined. Thus, in the present study, efforts were made to examine the temporal changes in the global gene expression profile in the CL of pregnant rats after treatment with aromatase inhibitor (AI), Anastrozole, and E2 supplementation. The results obtained will further expand our knowledge on E2 target/responsive genes and the basic mechanism(s) that regulates the CL function. Key words: CL, E2, AI, Gene expression The objective of this study was to identify the effect of E2 deprivation and supplementation on CL function and temporal changes in the transcriptome of pregnant rat CL during day 12 to 16 of pregnancy following E2 deprivation by Anastrozole (AI, Aromatase inhibitor) treatment in pregnant rats, AI (1mg/ kg bw orally) treatment beginning on day 12 daily for 4 days. In the E2 supplementation experiments, together with AI, the rats were treated with E2 (10M-BM-5g/ day s.c.). CL tissues were collected on day 12 (no treatment; n=3 animals/ time point), day 16 (vehicle treatment; n=3 animals/ time point), day 16 (AI treatment; n=3 animals/ time point) and day 16 (AI+ E2 treatment; n=3 animals/ time point) to examine gene expression changes associated with E2 deprivation and recovery in the system. Following retrieval of CL from the ovary, corpora lutea were flash frozen in liquid nitrogen and stored at -70oC for the purpose of RNA isolation. The isolated RNA was labelled and hybridized to Affymetrix GeneChipM-BM-. Rat Gene 1.0 ST arrays as per the manufacturerM-bM-^@M-^Ys instructions.
2012-10-23 | E-GEOD-41735 | biostudies-arrayexpress