Enterohemorrhagic Escherichia coli O157∶H7 Gene Expression Profiling in Response to Growth in the Presence of Host Epithelia

Abstract

BackgroundThe pathogenesis of enterohemorrhagic Escherichia coli (EHEC) O157∶H7 infection is attributed to virulence factors encoded on multiple pathogenicity islands. Previous studies have shown that EHEC O157∶H7 modulates host cell signal transduction cascades, independent of toxins and rearrangement of the cytoskeleton. However, the virulence factors and mechanisms responsible for EHEC-mediated subversion of signal transduction remain to be determined. Therefore, the purpose of this study was to first identify differentially regulated genes in response to EHEC O157∶H7 grown in the presence of epithelial cells, compared to growth in the absence of epithelial cells (that is, growth in minimal essential tissue culture medium alone, minimal essential tissue culture medium in the presence of 5% CO2, and Penassay broth alone) and, second, to identify EHEC virulence factors responsible for pathogen modulation of host cell signal transduction.Methodology/Principal FindingsOvernight cultures of EHEC O157∶H7 were incubated for 6 hr at 37°C in the presence or absence of confluent epithelial (HEp-2) cells. Total RNA was then extracted and used for microarray analyses (Affymetrix E. coli Genome 2.0 gene chips). Relative to bacteria grown in each of the other conditions, EHEC O157∶H7 cultured in the presence of cultured epithelial cells displayed a distinct gene-expression profile. A 2.0-fold increase in the expression of 71 genes and a 2.0-fold decrease in expression of 60 other genes were identified in EHEC O157∶H7 grown in the presence of epithelial cells, compared to bacteria grown in media alone.Conclusion/SignificanceMicroarray analyses and gene deletion identified a protease on O-island 50, gene Z1787, as a potential virulence factor responsible for mediating EHEC inhibition of the interferon (IFN)-γ-Jak1,2-STAT-1 signal transduction cascade. Up-regulated genes provide novel targets for use in developing strategies to interrupt the infectious process.

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7

“…Conversely, another study identified only 131 differentially expressed genes following 6 h of O157 : H7 co-incubation with HEp-2 cells compared to unexposed O157 : H7 (Jandu et al, 2009). These data, however, were generated from pooled RNA that included both adherent and non-adherent O157 : H7 (Jandu et al, 2009). Here, after a 1.5-fold cutoff was applied, above which the change in expression is considered meaningful (Hughes et al, 2000), we found 206 genes to be differentially expressed between two phylogenetically distinct O157 : H7 strains exposed to epithelial cells, but prior to attachment.…”

“…One study, which examined the transcriptome of O157 : H7 after 3 h of attachment to HT-29 epithelial cells, describes 611 upregulated and 384 downregulated O157 : H7 genes relative to the same strain grown in media alone (Kim et al, 2009). Conversely, another study identified only 131 differentially expressed genes following 6 h of O157 : H7 co-incubation with HEp-2 cells compared to unexposed O157 : H7 (Jandu et al, 2009). These data, however, were generated from pooled RNA that included both adherent and non-adherent O157 : H7 (Jandu et al, 2009).…”

“…One study demonstrated transcription of~8 % of the genome and downregulation of LEE gene expression following association of O157 : H7 with erythrocytes, compared to O157 : H7 grown in medium alone (Dahan et al, 2004). Another study compared the transcriptomes of O157 : H7 co-incubated with epithelial cells to the O157 : H7 transcriptome without epithelial cell exposure; differential expression of 131 genes was observed, one of which has important immunomodulatory roles (Jandu et al, 2009). While these studies have focused on within-strain differences, little is known about the variation in gene expression between phylogenetically distinct O157 : H7 strains, upon exposure but preceding adherence to host epithelial cells, and how that relates to cell attachment.…”

Enterohemorrhagic Escherichia coli O157:H7 Shiga Toxins Inhibit Gamma Interferon-Mediated Cellular Activation

“…HEp-2 epithelial cells (ATCC CCL-23) were used as a model epithelial cell line, as previously described (25). Briefly, cells were grown in minimal essential medium (MEM) containing 15% (vol/vol) fetal bovine serum (FBS), 2% (vol/vol) sodium bicarbonate, 2.5% (vol/ vol) penicillin-streptomycin, and 1% (vol/vol) amphotericin B (all from Invitrogen, Burlington, Ontario, Canada).…”

An efficient design strategy for a whole-cell biosensor based on engineered ribosome binding sequences

“…Luciferase activity was measured using the E1500 luciferase assay system and Glomax-Muti microplate multimode reader (Promega, USA) in accordance with the supplier's instructions. A housekeeping gene, rpoS, was used as an internal standard to calculate the relative quantity of each luc reporter gene mRNA [24]. The levels of rpoS mRNA with induction and the luc mRNA with and without induction were calculated relative to that of rpoS without induction.…”