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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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Transcript expression of EHEC O157∶H7 gene Z1787 in wild-type and mutant strains.As described in the Experimental procedures, gene deletion was performed using the Lambda Red technique [27]–[28]. Isogenic mutants were then screened and verified using reverse transcriptase polymerase chain reaction (RT-PCR). PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and bands then visualized under a UV lamp. The standard employed was a 100 bp ladder. Lane 1: negative water control; Lane 2: EHEC strain CL56; Lane 3: EPEC strain E2348/69; Lane 4: EHEC strain EDL 933 (parent to the mutant); Lane 5: ΔZ1787; Lane 6: no transcript control. [Panel A] transcript expression of EHEC gene Z1787; [Panel B] transcript expression of EHEC gapA. Primers are described in Table 2 of the Materials and Methods.
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pone-0004889-g005: Transcript expression of EHEC O157∶H7 gene Z1787 in wild-type and mutant strains.As described in the Experimental procedures, gene deletion was performed using the Lambda Red technique [27]–[28]. Isogenic mutants were then screened and verified using reverse transcriptase polymerase chain reaction (RT-PCR). PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and bands then visualized under a UV lamp. The standard employed was a 100 bp ladder. Lane 1: negative water control; Lane 2: EHEC strain CL56; Lane 3: EPEC strain E2348/69; Lane 4: EHEC strain EDL 933 (parent to the mutant); Lane 5: ΔZ1787; Lane 6: no transcript control. [Panel A] transcript expression of EHEC gene Z1787; [Panel B] transcript expression of EHEC gapA. Primers are described in Table 2 of the Materials and Methods.

Mentions: Subsequently, isogenic knock-out mutants were generated by employing the Lambda Red technique for gene deletion, as previously described [28], [29]. Gene Z1787 was selected as a candidate virulence factor to be investigated via mutational analysis due to its location on genomic island OI-50, a >100 kbp genomic island. Gene knock-outs were verified using reverse transcriptase polymerase chain reaction (RT-PCR). As shown in Figure 5, transcript expression for EHEC gene Z1787 was positive in strain CL56 and parent strain EDL933, but absent in both the knock-out, ΔZ1787 and in EPEC strain E2348/69.


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)

Transcript expression of EHEC O157∶H7 gene Z1787 in wild-type and mutant strains.As described in the Experimental procedures, gene deletion was performed using the Lambda Red technique [27]–[28]. Isogenic mutants were then screened and verified using reverse transcriptase polymerase chain reaction (RT-PCR). PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and bands then visualized under a UV lamp. The standard employed was a 100 bp ladder. Lane 1: negative water control; Lane 2: EHEC strain CL56; Lane 3: EPEC strain E2348/69; Lane 4: EHEC strain EDL 933 (parent to the mutant); Lane 5: ΔZ1787; Lane 6: no transcript control. [Panel A] transcript expression of EHEC gene Z1787; [Panel B] transcript expression of EHEC gapA. Primers are described in Table 2 of the Materials and Methods.
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Related In: Results  -  Collection

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

pone-0004889-g005: Transcript expression of EHEC O157∶H7 gene Z1787 in wild-type and mutant strains.As described in the Experimental procedures, gene deletion was performed using the Lambda Red technique [27]–[28]. Isogenic mutants were then screened and verified using reverse transcriptase polymerase chain reaction (RT-PCR). PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and bands then visualized under a UV lamp. The standard employed was a 100 bp ladder. Lane 1: negative water control; Lane 2: EHEC strain CL56; Lane 3: EPEC strain E2348/69; Lane 4: EHEC strain EDL 933 (parent to the mutant); Lane 5: ΔZ1787; Lane 6: no transcript control. [Panel A] transcript expression of EHEC gene Z1787; [Panel B] transcript expression of EHEC gapA. Primers are described in Table 2 of the Materials and Methods.
Mentions: Subsequently, isogenic knock-out mutants were generated by employing the Lambda Red technique for gene deletion, as previously described [28], [29]. Gene Z1787 was selected as a candidate virulence factor to be investigated via mutational analysis due to its location on genomic island OI-50, a >100 kbp genomic island. Gene knock-outs were verified using reverse transcriptase polymerase chain reaction (RT-PCR). As shown in Figure 5, transcript expression for EHEC gene Z1787 was positive in strain CL56 and parent strain EDL933, but absent in both the knock-out, ΔZ1787 and in EPEC strain E2348/69.

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