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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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Relative expression of EHEC O157∶H7, strain CL56 housekeeping genes in response to different growth conditions.[Panel A] Signal intensities of housekeeping genes (arcA, gapA, mdh, rfbA and rpoS) from microarray chips in response to EHEC O157∶H7 grown under four different conditions. All housekeeping genes showed similar levels of expression, irrespective of the bacterial growth condition. Data are presented as means±SEM. [Panel B] qRT-PCR of the same housekeeping genes confirms comparable levels of transcript expression, irrespective of EHEC O157∶H7 growth condition. Data points from triplicates of EHEC O157∶H7 grown in contact with epithelial cells, microbial growth in minimal essential medium (+/−5% CO2) were extrapolated from a standard curve generated for each primer pair using samples of the same bacterial strain grown in Penassay broth alone. As described in the Experimental procedures, data analysis was performed using the 7500 Sequence Detection System Software package (Applied Biosystems). Black bars represent EHEC O157∶ H7 grown in the presence of epithelial cells; grey bars: pathogen grown in minimal essential medium in atmospheric conditions or in 5% CO2 (stripped bars). EHEC grown in Penassay broth are shown in white bars [Panel A].
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pone-0004889-g001: Relative expression of EHEC O157∶H7, strain CL56 housekeeping genes in response to different growth conditions.[Panel A] Signal intensities of housekeeping genes (arcA, gapA, mdh, rfbA and rpoS) from microarray chips in response to EHEC O157∶H7 grown under four different conditions. All housekeeping genes showed similar levels of expression, irrespective of the bacterial growth condition. Data are presented as means±SEM. [Panel B] qRT-PCR of the same housekeeping genes confirms comparable levels of transcript expression, irrespective of EHEC O157∶H7 growth condition. Data points from triplicates of EHEC O157∶H7 grown in contact with epithelial cells, microbial growth in minimal essential medium (+/−5% CO2) were extrapolated from a standard curve generated for each primer pair using samples of the same bacterial strain grown in Penassay broth alone. As described in the Experimental procedures, data analysis was performed using the 7500 Sequence Detection System Software package (Applied Biosystems). Black bars represent EHEC O157∶ H7 grown in the presence of epithelial cells; grey bars: pathogen grown in minimal essential medium in atmospheric conditions or in 5% CO2 (stripped bars). EHEC grown in Penassay broth are shown in white bars [Panel A].

Mentions: Housekeeping genes provided internal controls, which were used as a measure of mRNA expression levels irrespective of growth condition [24]. The expression patterns of five E. coli housekeeping genes were determined including, arcA, gapA, mdh, rfbA, and rpoS [25] during EHEC O157∶H7 growth in the presence or absence of epithelial cells. The signal intensities of these select housekeeping genes, as determined by microarray analysis, are shown in Figure 1, Panel A. Signal intensities of these housekeeping genes were comparable for samples of EHEC O157∶H7 under each of the four different growth conditions.


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)

Relative expression of EHEC O157∶H7, strain CL56 housekeeping genes in response to different growth conditions.[Panel A] Signal intensities of housekeeping genes (arcA, gapA, mdh, rfbA and rpoS) from microarray chips in response to EHEC O157∶H7 grown under four different conditions. All housekeeping genes showed similar levels of expression, irrespective of the bacterial growth condition. Data are presented as means±SEM. [Panel B] qRT-PCR of the same housekeeping genes confirms comparable levels of transcript expression, irrespective of EHEC O157∶H7 growth condition. Data points from triplicates of EHEC O157∶H7 grown in contact with epithelial cells, microbial growth in minimal essential medium (+/−5% CO2) were extrapolated from a standard curve generated for each primer pair using samples of the same bacterial strain grown in Penassay broth alone. As described in the Experimental procedures, data analysis was performed using the 7500 Sequence Detection System Software package (Applied Biosystems). Black bars represent EHEC O157∶ H7 grown in the presence of epithelial cells; grey bars: pathogen grown in minimal essential medium in atmospheric conditions or in 5% CO2 (stripped bars). EHEC grown in Penassay broth are shown in white bars [Panel A].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004889-g001: Relative expression of EHEC O157∶H7, strain CL56 housekeeping genes in response to different growth conditions.[Panel A] Signal intensities of housekeeping genes (arcA, gapA, mdh, rfbA and rpoS) from microarray chips in response to EHEC O157∶H7 grown under four different conditions. All housekeeping genes showed similar levels of expression, irrespective of the bacterial growth condition. Data are presented as means±SEM. [Panel B] qRT-PCR of the same housekeeping genes confirms comparable levels of transcript expression, irrespective of EHEC O157∶H7 growth condition. Data points from triplicates of EHEC O157∶H7 grown in contact with epithelial cells, microbial growth in minimal essential medium (+/−5% CO2) were extrapolated from a standard curve generated for each primer pair using samples of the same bacterial strain grown in Penassay broth alone. As described in the Experimental procedures, data analysis was performed using the 7500 Sequence Detection System Software package (Applied Biosystems). Black bars represent EHEC O157∶ H7 grown in the presence of epithelial cells; grey bars: pathogen grown in minimal essential medium in atmospheric conditions or in 5% CO2 (stripped bars). EHEC grown in Penassay broth are shown in white bars [Panel A].
Mentions: Housekeeping genes provided internal controls, which were used as a measure of mRNA expression levels irrespective of growth condition [24]. The expression patterns of five E. coli housekeeping genes were determined including, arcA, gapA, mdh, rfbA, and rpoS [25] during EHEC O157∶H7 growth in the presence or absence of epithelial cells. The signal intensities of these select housekeeping genes, as determined by microarray analysis, are shown in Figure 1, Panel A. Signal intensities of these housekeeping genes were comparable for samples of EHEC O157∶H7 under each of the four different growth conditions.

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