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SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli.

Hansen AM, Jin DJ - BMC Microbiol. (2012)

Bottom Line: Here we assess the effect of SspA on virulence gene expression in EHEC.We show that transcription of virulence genes including those of the LEE is decreased in an sspA mutant, rendering the mutant strain defective in forming A/E lesions.We demonstrate that the H-NS level is two-fold higher in an sspA mutant compared to wild type, and that the defects of the sspA mutant are suppressed by an hns mutation, indicating that hns is epistatic to sspA in regulating H-NS repressed virulence genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Transcription Control Section, Gene Regulation and Chromosome Biology Laboratory, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.

ABSTRACT

Background: Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestinal epithelium and causes attaching and effacing (A/E) lesions. Expression of virulence genes, particularly those from the locus of the enterocyte effacement (LEE) pathogenicity island is required for the formation of a type three secretion system, which induces A/E lesion formation. Like other horizontally acquired genetic elements, expression of the LEE is negatively regulated by H-NS. In the non-pathogenic Escherichia coli K-12 strain the stringent starvation protein A (SspA) inhibits accumulation of H-NS, and thereby allows de-repression of the H-NS regulon during the stationary phase of growth. However, the effect of SspA on the expression of H-NS-controlled virulence genes in EHEC is unknown.

Results: Here we assess the effect of SspA on virulence gene expression in EHEC. We show that transcription of virulence genes including those of the LEE is decreased in an sspA mutant, rendering the mutant strain defective in forming A/E lesions. A surface exposed pocket of SspA is functionally important for the regulation of the LEE and for the A/E phenotype. Increased expression of ler alleviates LEE expression in an sspA mutant, suggesting that the level of Ler in the mutant is insufficient to counteract H-NS-mediated repression. We demonstrate that the H-NS level is two-fold higher in an sspA mutant compared to wild type, and that the defects of the sspA mutant are suppressed by an hns mutation, indicating that hns is epistatic to sspA in regulating H-NS repressed virulence genes.

Conclusions: SspA positively regulates the expression of EHEC virulence factors by restricting the intracellular level of H-NS. Since SspA is conserved in many bacterial pathogens containing horizontally acquired pathogenicity islands controlled by H-NS, our study suggests a common mechanism whereby SspA potentially regulates the expression of virulence genes in these pathogens.

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SspA negatively affects H-NS levels in EHEC. The levels of H-NS were determined in wild type (lane 1) and sspA mutant (lane 2) derivatives of EHEC EDL933 grown to stationary phase cells by western blot. Equal amounts of total protein were resolved on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane. Levels of H-NS and Fis were detected using polyclonal antibodies against the respective proteins. Fis served as an internal control for total protein levels. The relative amounts of H-NS normalized to that of Fis are indicated by the numbers in parenthesis.
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Figure 3: SspA negatively affects H-NS levels in EHEC. The levels of H-NS were determined in wild type (lane 1) and sspA mutant (lane 2) derivatives of EHEC EDL933 grown to stationary phase cells by western blot. Equal amounts of total protein were resolved on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane. Levels of H-NS and Fis were detected using polyclonal antibodies against the respective proteins. Fis served as an internal control for total protein levels. The relative amounts of H-NS normalized to that of Fis are indicated by the numbers in parenthesis.

Mentions: Reduced virulence gene expression during the stationary phase could also be due to an increased level of H-NS in the EHEC sspA mutant as observed for H-NS-regulated genes in the E. coli K-12 sspA mutant[44]. We measured the levels of H-NS in stationary phase cells of wild type and sspA mutant EHEC strains by western analysis (Figure‚ÄČ3). Indeed, the H-NS level was two-fold higher in the sspA mutant than in the wild type, whereas the level of Fis as a control was not increased in the mutant compared to wild type. These results indicate that SspA activates the expression of EHEC virulence genes by decreasing accumulation of H-NS. Notably, such relative small change in H-NS levels was previously demonstrated to drastically affect the expression of the H-NS regulon involved in stationary phase-induced acid tolerance of E. coli K-12[44].


SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli.

Hansen AM, Jin DJ - BMC Microbiol. (2012)

SspA negatively affects H-NS levels in EHEC. The levels of H-NS were determined in wild type (lane 1) and sspA mutant (lane 2) derivatives of EHEC EDL933 grown to stationary phase cells by western blot. Equal amounts of total protein were resolved on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane. Levels of H-NS and Fis were detected using polyclonal antibodies against the respective proteins. Fis served as an internal control for total protein levels. The relative amounts of H-NS normalized to that of Fis are indicated by the numbers in parenthesis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539938&req=5

Figure 3: SspA negatively affects H-NS levels in EHEC. The levels of H-NS were determined in wild type (lane 1) and sspA mutant (lane 2) derivatives of EHEC EDL933 grown to stationary phase cells by western blot. Equal amounts of total protein were resolved on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane. Levels of H-NS and Fis were detected using polyclonal antibodies against the respective proteins. Fis served as an internal control for total protein levels. The relative amounts of H-NS normalized to that of Fis are indicated by the numbers in parenthesis.
Mentions: Reduced virulence gene expression during the stationary phase could also be due to an increased level of H-NS in the EHEC sspA mutant as observed for H-NS-regulated genes in the E. coli K-12 sspA mutant[44]. We measured the levels of H-NS in stationary phase cells of wild type and sspA mutant EHEC strains by western analysis (Figure‚ÄČ3). Indeed, the H-NS level was two-fold higher in the sspA mutant than in the wild type, whereas the level of Fis as a control was not increased in the mutant compared to wild type. These results indicate that SspA activates the expression of EHEC virulence genes by decreasing accumulation of H-NS. Notably, such relative small change in H-NS levels was previously demonstrated to drastically affect the expression of the H-NS regulon involved in stationary phase-induced acid tolerance of E. coli K-12[44].

Bottom Line: Here we assess the effect of SspA on virulence gene expression in EHEC.We show that transcription of virulence genes including those of the LEE is decreased in an sspA mutant, rendering the mutant strain defective in forming A/E lesions.We demonstrate that the H-NS level is two-fold higher in an sspA mutant compared to wild type, and that the defects of the sspA mutant are suppressed by an hns mutation, indicating that hns is epistatic to sspA in regulating H-NS repressed virulence genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Transcription Control Section, Gene Regulation and Chromosome Biology Laboratory, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.

ABSTRACT

Background: Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestinal epithelium and causes attaching and effacing (A/E) lesions. Expression of virulence genes, particularly those from the locus of the enterocyte effacement (LEE) pathogenicity island is required for the formation of a type three secretion system, which induces A/E lesion formation. Like other horizontally acquired genetic elements, expression of the LEE is negatively regulated by H-NS. In the non-pathogenic Escherichia coli K-12 strain the stringent starvation protein A (SspA) inhibits accumulation of H-NS, and thereby allows de-repression of the H-NS regulon during the stationary phase of growth. However, the effect of SspA on the expression of H-NS-controlled virulence genes in EHEC is unknown.

Results: Here we assess the effect of SspA on virulence gene expression in EHEC. We show that transcription of virulence genes including those of the LEE is decreased in an sspA mutant, rendering the mutant strain defective in forming A/E lesions. A surface exposed pocket of SspA is functionally important for the regulation of the LEE and for the A/E phenotype. Increased expression of ler alleviates LEE expression in an sspA mutant, suggesting that the level of Ler in the mutant is insufficient to counteract H-NS-mediated repression. We demonstrate that the H-NS level is two-fold higher in an sspA mutant compared to wild type, and that the defects of the sspA mutant are suppressed by an hns mutation, indicating that hns is epistatic to sspA in regulating H-NS repressed virulence genes.

Conclusions: SspA positively regulates the expression of EHEC virulence factors by restricting the intracellular level of H-NS. Since SspA is conserved in many bacterial pathogens containing horizontally acquired pathogenicity islands controlled by H-NS, our study suggests a common mechanism whereby SspA potentially regulates the expression of virulence genes in these pathogens.

Show MeSH
Related in: MedlinePlus