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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 is required for cell adherence and A/E lesion formation. HEp-2 cells were infected by wild type EHEC EDL933 (A) and its mutant derivatives of sspA (B), sspA pQEsspA (C), sspA pQEsspA84-86 (D), hns (E) and hns sspA (F). Bacterial adherence was examined by phase-contrast images (left panels) and the actin cytoskeleton of infected HEp-2 cells by fluorescent microscopic images (right panels). Representative images are shown. Black and white arrowheads indicate bacteria and A/E lesions, respectively.
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Figure 5: SspA is required for cell adherence and A/E lesion formation. HEp-2 cells were infected by wild type EHEC EDL933 (A) and its mutant derivatives of sspA (B), sspA pQEsspA (C), sspA pQEsspA84-86 (D), hns (E) and hns sspA (F). Bacterial adherence was examined by phase-contrast images (left panels) and the actin cytoskeleton of infected HEp-2 cells by fluorescent microscopic images (right panels). Representative images are shown. Black and white arrowheads indicate bacteria and A/E lesions, respectively.

Mentions: Since the expression of LEE-encoded genes involved in A/E lesion formation was decreased in a sspA mutant and increased in a hns sspA double mutant (Figures 1 and4), we predicted that SspA affects lesion formation in a H-NS-dependent manner. To address this, we infected HEp-2 cells with wild type, sspA, hns and hns sspA mutant derivatives of EDL933, and determined the ability of these strains to form A/E lesions in vitro. To this end we used the qualitative fluorescent actin staining (FAS) assay[53], where actin filaments are stained with FITC-phalloidin to detect A/E lesions that are visualized as condensed actin directly beneath adherent bacteria. Whereas infection with wild type EHEC was associated with the appearance of microcolonies of adherent bacteria and A/E lesion formation on 70% of the HEp-2 cells (Figure 5A), the sspA mutant was unable to adhere and form A/E lesions (Figure 5B) as determined from examination of more than 50 HEp-2 cells. The A/E lesion phenotype of the sspA mutant was restored when complementing with sspA in trans from pQEsspA (Figure 5C), whereas mutant sspA supplied from pQEsspA84-86 (Figure 5D) did not complement pedestal formation of the sspA mutant, verifying that the surface-exposed pocket is functionally important for SspA to affect virulence of EHEC. Consistent with the finding that SspA regulates LEE expression through H-NS, the sspA mutant restored the ability to form A/E lesions in the absence of hns in the hns sspA background as in the hns single mutant (Figure 5E-F). However, the hns sspA double mutant seemed to form A/E lesions to a higher degree than the hns single mutant, which indicates that SspA also affects the expression of virulence genes involved in A/E lesion formation independently of the H-NS-mediated regulation. Moreover, the finding that the cell adherence ability of the sspA mutant was restored when deleting hns indicates that a factor negatively regulated by H-NS is required for the adherence of EHEC to epithelial cells. The long polar fimbria, LpfA, which is part of the H-NS/Ler regulon and is required for cell adherence of EHEC[32,54,55], might represent such a factor. Altogether, the cell adherence and A/E lesion phenotypes of the sspA mutant are consistent with the finding that SspA positively regulates the expression of genes encoding the T3SS including those of the LEE by negatively affecting H-NS levels.


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 is required for cell adherence and A/E lesion formation. HEp-2 cells were infected by wild type EHEC EDL933 (A) and its mutant derivatives of sspA (B), sspA pQEsspA (C), sspA pQEsspA84-86 (D), hns (E) and hns sspA (F). Bacterial adherence was examined by phase-contrast images (left panels) and the actin cytoskeleton of infected HEp-2 cells by fluorescent microscopic images (right panels). Representative images are shown. Black and white arrowheads indicate bacteria and A/E lesions, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 5: SspA is required for cell adherence and A/E lesion formation. HEp-2 cells were infected by wild type EHEC EDL933 (A) and its mutant derivatives of sspA (B), sspA pQEsspA (C), sspA pQEsspA84-86 (D), hns (E) and hns sspA (F). Bacterial adherence was examined by phase-contrast images (left panels) and the actin cytoskeleton of infected HEp-2 cells by fluorescent microscopic images (right panels). Representative images are shown. Black and white arrowheads indicate bacteria and A/E lesions, respectively.
Mentions: Since the expression of LEE-encoded genes involved in A/E lesion formation was decreased in a sspA mutant and increased in a hns sspA double mutant (Figures 1 and4), we predicted that SspA affects lesion formation in a H-NS-dependent manner. To address this, we infected HEp-2 cells with wild type, sspA, hns and hns sspA mutant derivatives of EDL933, and determined the ability of these strains to form A/E lesions in vitro. To this end we used the qualitative fluorescent actin staining (FAS) assay[53], where actin filaments are stained with FITC-phalloidin to detect A/E lesions that are visualized as condensed actin directly beneath adherent bacteria. Whereas infection with wild type EHEC was associated with the appearance of microcolonies of adherent bacteria and A/E lesion formation on 70% of the HEp-2 cells (Figure 5A), the sspA mutant was unable to adhere and form A/E lesions (Figure 5B) as determined from examination of more than 50 HEp-2 cells. The A/E lesion phenotype of the sspA mutant was restored when complementing with sspA in trans from pQEsspA (Figure 5C), whereas mutant sspA supplied from pQEsspA84-86 (Figure 5D) did not complement pedestal formation of the sspA mutant, verifying that the surface-exposed pocket is functionally important for SspA to affect virulence of EHEC. Consistent with the finding that SspA regulates LEE expression through H-NS, the sspA mutant restored the ability to form A/E lesions in the absence of hns in the hns sspA background as in the hns single mutant (Figure 5E-F). However, the hns sspA double mutant seemed to form A/E lesions to a higher degree than the hns single mutant, which indicates that SspA also affects the expression of virulence genes involved in A/E lesion formation independently of the H-NS-mediated regulation. Moreover, the finding that the cell adherence ability of the sspA mutant was restored when deleting hns indicates that a factor negatively regulated by H-NS is required for the adherence of EHEC to epithelial cells. The long polar fimbria, LpfA, which is part of the H-NS/Ler regulon and is required for cell adherence of EHEC[32,54,55], might represent such a factor. Altogether, the cell adherence and A/E lesion phenotypes of the sspA mutant are consistent with the finding that SspA positively regulates the expression of genes encoding the T3SS including those of the LEE by negatively affecting H-NS levels.

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