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Enterohemorrhagic Escherichia coli O157:H7 gene expression profiling in response to growth in the presence of host epithelia.

Jandu N, Ho NK, Donato KA, Karmali MA, Mascarenhas M, Duffy SP, Tailor C, Sherman PM - PLoS ONE (2009)

Bottom Line: Total RNA was then extracted and used for microarray analyses (Affymetrix E. coli Genome 2.0 gene chips).Microarray 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)-gamma-Jak1,2-STAT-1 signal transduction cascade.Up-regulated genes provide novel targets for use in developing strategies to interrupt the infectious process.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

ABSTRACT

Background: The 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% CO(2), and Penassay broth alone) and, second, to identify EHEC virulence factors responsible for pathogen modulation of host cell signal transduction.

Methodology/principal findings: Overnight cultures of EHEC O157:H7 were incubated for 6 hr at 37 degrees 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/significance: Microarray 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)-gamma-Jak1,2-STAT-1 signal transduction cascade. Up-regulated genes provide novel targets for use in developing strategies to interrupt the infectious process.

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Related in: MedlinePlus

EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation.Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures) at 37°C in 5% CO2. Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO2. Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 & 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p<0.01).
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pone-0004889-g006: EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation.Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures) at 37°C in 5% CO2. Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO2. Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 & 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p<0.01).

Mentions: Phenotypic analysis of the isogenic mutants was performed by employing an in vitro tissue culture infection model followed by immunoblotting. As shown in Figure 6, Panel A, unlike the parent strain, EHEC strain EDL933, ΔZ1787 did not block STAT-1 tyrosine phosphorylation in response to IFNγ stimulation. Quantification of immunoblots (Figure 6, Panel B) demonstrate significantly greater STAT-1 tyrosine phosphorylation levels in epithelial cells infected with ΔZ1787 (119.13±28.76% of uninfected control, n = 4), relative to the wild-type parent strain, EHEC strain EDL 933 (4.70±5.19% of uninfected control, n = 4). Consistent with previous observations [20], EHEC strain CL56 (1.50±14.98%, n = 4), but not EPEC O127∶H6 strain 2348/69 (98.00±3.91%, n = 4), inhibited tyrosine phosphorylation of STAT-1 following IFNγ stimulation. However, complementation of the EHEC gene Z1787 knock out only partially restored pathogen inhibition of STAT-1 tyrosine phosphorylation (Figure 6, Panel B). Similarly, insertion of gene Z1787 on a pGEM-T vector into non-pathogenic E. coli strain HB101, did not result in the commensal being able to completely subvert STAT-1 tyrosine phosphorylation by IFNγ. In addition, transcript expression of Z1787 was not altered following EHEC growth in the presence of a polarized intestinal epithelial cell line (T84 cells), relative to pathogen growth in the absence of epithelial cells (Supplemental Figure S3). Collectively, these data indicate that gene Z1787 is not the only factor mediating EHEC subversion of STAT-1 activation. Instead, as for many other microbial pathogens, multiple factors likely contribute to pathogen subversion of immune signaling.


Enterohemorrhagic Escherichia coli O157:H7 gene expression profiling in response to growth in the presence of host epithelia.

Jandu N, Ho NK, Donato KA, Karmali MA, Mascarenhas M, Duffy SP, Tailor C, Sherman PM - PLoS ONE (2009)

EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation.Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures) at 37°C in 5% CO2. Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO2. Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 & 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p<0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654852&req=5

pone-0004889-g006: EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation.Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures) at 37°C in 5% CO2. Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO2. Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 & 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p<0.01).
Mentions: Phenotypic analysis of the isogenic mutants was performed by employing an in vitro tissue culture infection model followed by immunoblotting. As shown in Figure 6, Panel A, unlike the parent strain, EHEC strain EDL933, ΔZ1787 did not block STAT-1 tyrosine phosphorylation in response to IFNγ stimulation. Quantification of immunoblots (Figure 6, Panel B) demonstrate significantly greater STAT-1 tyrosine phosphorylation levels in epithelial cells infected with ΔZ1787 (119.13±28.76% of uninfected control, n = 4), relative to the wild-type parent strain, EHEC strain EDL 933 (4.70±5.19% of uninfected control, n = 4). Consistent with previous observations [20], EHEC strain CL56 (1.50±14.98%, n = 4), but not EPEC O127∶H6 strain 2348/69 (98.00±3.91%, n = 4), inhibited tyrosine phosphorylation of STAT-1 following IFNγ stimulation. However, complementation of the EHEC gene Z1787 knock out only partially restored pathogen inhibition of STAT-1 tyrosine phosphorylation (Figure 6, Panel B). Similarly, insertion of gene Z1787 on a pGEM-T vector into non-pathogenic E. coli strain HB101, did not result in the commensal being able to completely subvert STAT-1 tyrosine phosphorylation by IFNγ. In addition, transcript expression of Z1787 was not altered following EHEC growth in the presence of a polarized intestinal epithelial cell line (T84 cells), relative to pathogen growth in the absence of epithelial cells (Supplemental Figure S3). Collectively, these data indicate that gene Z1787 is not the only factor mediating EHEC subversion of STAT-1 activation. Instead, as for many other microbial pathogens, multiple factors likely contribute to pathogen subversion of immune signaling.

Bottom Line: Total RNA was then extracted and used for microarray analyses (Affymetrix E. coli Genome 2.0 gene chips).Microarray 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)-gamma-Jak1,2-STAT-1 signal transduction cascade.Up-regulated genes provide novel targets for use in developing strategies to interrupt the infectious process.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

ABSTRACT

Background: The 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% CO(2), and Penassay broth alone) and, second, to identify EHEC virulence factors responsible for pathogen modulation of host cell signal transduction.

Methodology/principal findings: Overnight cultures of EHEC O157:H7 were incubated for 6 hr at 37 degrees 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/significance: Microarray 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)-gamma-Jak1,2-STAT-1 signal transduction cascade. Up-regulated genes provide novel targets for use in developing strategies to interrupt the infectious process.

Show MeSH
Related in: MedlinePlus