Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of E.coli grown in SAPI in the presence of AI-2, time series experiment

ABSTRACT: VS94 gene expression at different time-points in SAPI medium in absence and presence of AI-2 was studied. Autoinducer-2 (AI-2) is produced by many species of bacteria, including various commensal bacteria and is involved in inter-species communication. Since, pathogens encounter AI-2 once they enter the human gastro-intestinal tract; we studied the effects of presence of AI-2 on various phenotypes associated with infection and colonization of enterohemorrhagic Escherichia coli (EHEC) namely, chemotaxis, motility and attachment to HeLa cells. AI-2 attracted EHEC when observed in agarose plug assays and also increased EHEC motility by 1.44-fold. AI-2 also increased EHEC attachment to HeLa cells by 1.6-fold; hence, suggesting that exposure to AI-2 inside the gastro-intestinal tract can play an important role in EHEC colonization. We then investigated the global effects of AI-2 on EHEC gene expression using DNA microarrays at various time-points. We found that AI-2 controls virulence gene expression and several other groups of genes (flagellar genes, iron related genes, biofilm genes etc.) associated with virulence in a time-dependent manner. Hence, through these studies we have shown that AI-2 may be a key component in EHEC infection of human gastro-intestinal tract. Experiment Overall Design: Strain: VS94 (EHEC luxS mutant) Experiment Overall Design: Medium: SAPI Experiment Overall Design: Temperature: 37C Experiment Overall Design: Cell type: Suspension Experiment Overall Design: Chemical: 100 uM AI-2 Experiment Overall Design: Overnight cultures of VS94 were diluted in SAPI medium to a turbidity of 0.1. The cells were allowed to grow to a turbidity of 0.5 at 37 oC and 100 ÂµM AI-2 was added to the culture. In the control flasks, no AI-2 was added. The cultures were then allowed to grow for further 3.5 hours, 4 hours, 4.5 hours and 5.5 hours before cell pellets were collected by centrifugation at the above mentioned time-points and stored at -80oC. Experiment Overall Design: Total RNA was isolated from isolated cell pellets and RNA quality was assessed using gel electrophoresis. E. coli Genome 2.0 arrays (Affymetrix, California) containing 10,208 probe sets for all 20,366 genes present in four strains of E. coli, including E. coli O157:H7, were used to profile changes in gene expression using RNA samples for each treatment. Hybridization was performed for 16 hours, and the total cell intensity was scaled automatically in the software to an average value of 500. The data were inspected for quality and analyzed according to the procedures described by the manufacturer (Affymetrix: Data Analysis Fundamentals) which include using premixed polyadenylated transcripts of the B. subtilis genes (lys, phe, thr, and dap) at different concentrations. Genes were identified as differentially expressed if the expression ratio (between AI-2 and control cells at different time-points) was greater than 1.5 (based on the standard deviation between values measuring relative changes in expression) (Ren et al. 2004) and the change in the P value was less than 0.05. The differentially expressed genes were annotated using gene ontology definitions available in the Affymetrix NetAffx Analysis Center (http://www.affymetrix.com/analysis/index.affx).

ORGANISM(S): Escherichia coli

SUBMITTER: Tarun Bansal

PROVIDER: E-GEOD-9388 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:EHEC is a food-borne pathogen that colonizes human GI tract and leads to infection. To understand the process of colonization and to decipher if any factors secreted by intestinal epithelial cells help EHEC during the infection process, we studied expression of EHEC virulence gene expression when exposed to intestinal epithelial cell conditioned medium. In our study, we observed that exposure to epithelial cell conditioned medium for 1 h and 3 h increases expression of 32 out of 41 EHEC LEE virulence genes. In addition, expression of the shiga toxin 1 (Stx1) gene is up-regulated at 1 h of exposure. Also, 17 genes encoded by prophage 933W, including those for Stx2, are also upregulated at both time-points. The increase in 933W prophage expression is mirrored by a 2.7-fold increase in intracellular Stx2 phage titers. Consistent with the increase in virulence gene expression, we observed a 5-fold increase in EHEC attachment to epithelial cells when exposed to conditioned medium, suggesting that EHEC utilizes host cell molecules to increase virulence and infectivity. The molecule(s) responsible for increased EHEC virulence is heat-sensitive as heating the conditioned medium to 95oC abolishes the increase in attachment to epithelial cells. A similar decrease was observed when the conditioned medium was treated with proteinase-K to degrade the proteins. The secreted molecule(s) was found to be larger than 3 kDa and strongly suggests that the HCT-8 secreted molecule that increases EHEC virulence and colonization is a protein-based molecule. Affymetrix E. coli Genome 2.0 Arrays were used to determine the changes in EHEC virulence gene expression on exposure to intestinal epithelial cell-secreted factors (through growth in conditioned medium). Overnight cultures of EHEC were diluted in LB medium to a turbidity of 0.1 at 600 nm. The cells were allowed to grow to a turbidity of 1.0 at 600 nm at 37Â°C and the EHEC cells were then resuspended in either fresh or conditioned medium. The cultures were then allowed to grow for 1 h or 3 h before cell pellets were collected by centrifugation and stored at -80Â°C. Total RNA was isolated from the cell pellets (173) and RNA quality was assessed using gel electrophoresis. Escherichia coli Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) containing 10,208 probe sets for all 20,366 genes present in four strains of E. coli, including EHEC, were used to profile changes in gene expression using RNA samples for each treatment.

Project description:E. coli K-12 BW25113 mutant strain yncC expression in biofilm cells relative to E. coli wild-type strain expression in biofilm cells. All samples were cultured in LB with glasswool at 37C for 15 hours and E. coli K-12 MG1655 mutant yncC colony cells vs wild type colony cells in LB plates 15h 37C. Quorum-sensing signal autoinducer 2 (AI-2) stimulates Escherichia coli biofilm formation through the motility regulator MqsR that induces expression of the putative transcription factor encoded by yncC. Here we show YncC increases biofilm formation by decreasing mucoidy (corroborated by decreased exopolysaccharide production and increased sensitivity to bacteriophage P1 infection). Differential gene expression and gel shift assays demonstrated that YncC is a repressor of the predicted periplasmic protein-encoding gene ybiM which was corroborated by the isogenic yncC ybiM double mutation which repressed the yncC phenotypes (biofilm formation, mucoidy, and bacteriophage resistance). Through nickel-enrichment microarrays and additional gel shift assays, we found that the putative transcription factor B3023 (directly upstream of mqsR) binds the yncC promoter. Overexpressing MqsR, AI-2 import regulators LsrR/LsrK, and AI-2 exporter TqsA induced yncC transcription whereas the AI-2 synthase LuxS and B3023 repressed yncC. MqsR has a toxic effect on E. coli bacterial growth which is partially reduced by the b3023 mutation. Therefore, AI-2 quorum-sensing control of biofilm formation is mediated through regulator MqsR that induces expression of the transcription factor YncC which serves to inhibit the expression of periplasmic YbiM; this inhibition of YbiM prevents it from overexpressing exopolysaccharide (causing mucoidy) and prevents YbiM from inhibiting biofilm formation. Experiment Overall Design: Strains: E. coli K-12 BW25113 wild-type, mutant yncC Experiment Overall Design: and E. coli K-12 MG1655 wild-type, mutant yncC Experiment Overall Design: Medium: LB Experiment Overall Design: Biofilm grown on glasswool or colony cells growth in LB plates Experiment Overall Design: Time: 15 hours Experiment Overall Design: Temperature: 37C Experiment Overall Design: Cell type: biofilm or colony Experiment Overall Design: For the biofilm arrays in BW25113 background: Experiment Overall Design: Overnight cultures (16 h, 2.5 mL) of wild type E. coli BW25113 and BW25113 yncC in LB and LB with kanamycin (50 Î¼g/mL), respectively, were used to inoculate 250 mL LB with 10 g of glass wool (Corning Glass Works, Corning, NY) for forming biofilm. After incubating at 37Â°C for 15 h with shaking (250 rpm), biofilm cells were prepared by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0Â°C as described before. The total RNA was isolated from biofilm cells as described previously. Experiment Overall Design: The E. coli Genechip antisense genome array (P/N 900381, Affymetrix, Santa Clara, CA) containing probes for more than 4200 open reading frames (ORFs) was used to analyze the complete E. coli transcriptome as described previously. Hybridizations were performed for 16 h and the total cell intensity was scaled automatically in the software to an average value of 630. The Gene Expression Technical Manual (Affymetrix) was followed for the procedures of DNA microarrays, and the GeneChip operating software (Affymetrix) was applied to analyze data of DNA microarrays. The data quality was assessed following the manufacturer's guidelines (GeneChip Expression Analysis: Data Analysis Fundamentals; Affymetrix) and also was based on the expected signals of E. coli BW25113 and the yncC mutant genotypes (e.g., signals of the deleted genes, araA and rhaA, were low for both BW25113 and BW25113 yncC, while the signal of yncC was low for the yncC mutant). A gene was identified as differentially-expressed when the P value based on the False Discovery Rate Method was less than 0.05 and the expression ratio was greater than threefold since the standard deviation for the expression ratio for all of genes was 2. Experiment Overall Design: for the colony arrays in MG1655 background: Experiment Overall Design: For the agar biofilm, fresh single colonies of wild-type E. coli MG1655 and MG1655 yncC were re-streaked on LB agar plates, incubated, and about 0.05 g of the colony cultures on LB plate were quickly transferred to 2-mL collection tubes. The total RNA was isolated from these colony cells as described previously. The E. coli GeneChip Genome 2.0 Array (Affymetrix, P/N 900551, Santa Clara, CA) containing 10,208 probe sets for open reading frames, rRNA, tRNA, and intergenic regions for four E. coli strains: MG1655, CFT073, O157:H7-Sakai, and O157:H7-EDL933, was used to analyze the complete E. coli transcriptome. A gene was identified as differentially-expressed when the P value based on the False Discovery Rate Method (Benjamini & Hochberg, 1995) was less than 0.05 and the expression ratio was greater than threefold since the standard deviation for the expression ratio for all of MG1655 genes (except the deleted yncC gene) was 2.

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Transcription profiling of E.coli grown in SAPI in the presence of AI-2, time series experiment

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