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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 upstream of H-NS in the regulatory network of virulence gene expression in EHEC. The expression of virulence genes in wild type EHEC EDL933 (lane 1), sspA (lane 2), hns (lane 3) and hns sspA (lane 4) mutant derivatives was determined by primer extension analyses using labeled DNA oligos specific to the transcripts of LEE1/ler (A), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H). In each reaction, the ompA transcript served as an internal control. Samples were prepared and analyzed as described in the legend of Figure 1. The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.
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Figure 4: SspA is upstream of H-NS in the regulatory network of virulence gene expression in EHEC. The expression of virulence genes in wild type EHEC EDL933 (lane 1), sspA (lane 2), hns (lane 3) and hns sspA (lane 4) mutant derivatives was determined by primer extension analyses using labeled DNA oligos specific to the transcripts of LEE1/ler (A), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H). In each reaction, the ompA transcript served as an internal control. Samples were prepared and analyzed as described in the legend of Figure 1. The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.

Mentions: Genetic analysis further indicated that hns mainly is epistatic to sspA in regulating H-NS-repressed virulence genes in EHEC (Figure 4). We deleted hns in EHEC wild type and sspA mutant strains as described in Methods. The EHEC hns mutant derivatives had a mucoid phenotype and a longer generation time (g) than wild type (gWT ~ 27, ghns ~ 36 min and ghns,sspA ~ 45 min). Therefore, at least two independent clones of each hns mutant derivative were used in each experiment to ensure reproducible results. The expression of LEE1-5, grlRA, map and stcE was between 4 and 26-fold higher in an isogenic hns mutant than in wild type (Figure 4A-H, compare lane 3 with 1), which is consistent with the fact that there is enough H-NS in stationary phase wild type cells (Figure 3) to partially repress those virulence genes. Although the effect of hns on cell growth will be complex, an uncontrolled expression of the LEE genes and the T3SS is likely to be detrimental to the fitness of the cell[15]. Moreover, the expression level of EHEC virulence genes in the hns sspA double mutant was within the range of the level observed for the hns single mutant (Figure 4A-H, compare lane 4 with 3). Thus, our data strongly indicate that SspA is located upstream of H-NS in the regulatory cascade controlling the virulence gene expression in EHEC. However, SspA might also directly activate virulence gene expression in addition to controlling 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 upstream of H-NS in the regulatory network of virulence gene expression in EHEC. The expression of virulence genes in wild type EHEC EDL933 (lane 1), sspA (lane 2), hns (lane 3) and hns sspA (lane 4) mutant derivatives was determined by primer extension analyses using labeled DNA oligos specific to the transcripts of LEE1/ler (A), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H). In each reaction, the ompA transcript served as an internal control. Samples were prepared and analyzed as described in the legend of Figure 1. The relative transcript levels of target genes normalized to that of ompA 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 4: SspA is upstream of H-NS in the regulatory network of virulence gene expression in EHEC. The expression of virulence genes in wild type EHEC EDL933 (lane 1), sspA (lane 2), hns (lane 3) and hns sspA (lane 4) mutant derivatives was determined by primer extension analyses using labeled DNA oligos specific to the transcripts of LEE1/ler (A), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H). In each reaction, the ompA transcript served as an internal control. Samples were prepared and analyzed as described in the legend of Figure 1. The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.
Mentions: Genetic analysis further indicated that hns mainly is epistatic to sspA in regulating H-NS-repressed virulence genes in EHEC (Figure 4). We deleted hns in EHEC wild type and sspA mutant strains as described in Methods. The EHEC hns mutant derivatives had a mucoid phenotype and a longer generation time (g) than wild type (gWT ~ 27, ghns ~ 36 min and ghns,sspA ~ 45 min). Therefore, at least two independent clones of each hns mutant derivative were used in each experiment to ensure reproducible results. The expression of LEE1-5, grlRA, map and stcE was between 4 and 26-fold higher in an isogenic hns mutant than in wild type (Figure 4A-H, compare lane 3 with 1), which is consistent with the fact that there is enough H-NS in stationary phase wild type cells (Figure 3) to partially repress those virulence genes. Although the effect of hns on cell growth will be complex, an uncontrolled expression of the LEE genes and the T3SS is likely to be detrimental to the fitness of the cell[15]. Moreover, the expression level of EHEC virulence genes in the hns sspA double mutant was within the range of the level observed for the hns single mutant (Figure 4A-H, compare lane 4 with 3). Thus, our data strongly indicate that SspA is located upstream of H-NS in the regulatory cascade controlling the virulence gene expression in EHEC. However, SspA might also directly activate virulence gene expression in addition to controlling 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