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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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Increased ler expression overcomes repression of LEE in an sspA mutant. The expressions of virulence genes in wild type EHEC EDL933 (lane 1), the sspA mutant harboring the empty vector pACYC184 (pVector) (lane 2) and pACYCler (pler) expressing ler from its native promoters (lane 3) were determined by primer extension analyses using labeled DNA oligos specific to LEE1/ler (A), LEE2/espZ (B), LEE4/sepL (C), grlRA (D) and stcE (E) along with the ompA-specific oligo as a 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 2: Increased ler expression overcomes repression of LEE in an sspA mutant. The expressions of virulence genes in wild type EHEC EDL933 (lane 1), the sspA mutant harboring the empty vector pACYC184 (pVector) (lane 2) and pACYCler (pler) expressing ler from its native promoters (lane 3) were determined by primer extension analyses using labeled DNA oligos specific to LEE1/ler (A), LEE2/espZ (B), LEE4/sepL (C), grlRA (D) and stcE (E) along with the ompA-specific oligo as a 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: A decreased expression of ler in the sspA mutant (Figure 1A) could account for the apparent transcriptional repression of LEE2-5, grlRA, map and stcE (Figure 1B-H) because Ler positively controls those genes. Thus, we examined whether supplying ler in trans from the plasmid pACYCler would alleviate the expression of Ler-regulated genes in an sspA mutant (Figure 2). Our results showed that transcript levels of LEE1, LEE2, LEE4, grlRA and stcE were all increased in the sspA mutant harboring pACYCler and exceeded that in wild type with up to about 9-fold (Figure 2A-E, compare lanes 1 and 3). These results are consistent with the explanation that a reduced expression of ler in the sspA mutant leads to an insufficient amount of Ler to antagonize H-NS-mediated repression of those virulence genes.


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)

Increased ler expression overcomes repression of LEE in an sspA mutant. The expressions of virulence genes in wild type EHEC EDL933 (lane 1), the sspA mutant harboring the empty vector pACYC184 (pVector) (lane 2) and pACYCler (pler) expressing ler from its native promoters (lane 3) were determined by primer extension analyses using labeled DNA oligos specific to LEE1/ler (A), LEE2/espZ (B), LEE4/sepL (C), grlRA (D) and stcE (E) along with the ompA-specific oligo as a 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 2: Increased ler expression overcomes repression of LEE in an sspA mutant. The expressions of virulence genes in wild type EHEC EDL933 (lane 1), the sspA mutant harboring the empty vector pACYC184 (pVector) (lane 2) and pACYCler (pler) expressing ler from its native promoters (lane 3) were determined by primer extension analyses using labeled DNA oligos specific to LEE1/ler (A), LEE2/espZ (B), LEE4/sepL (C), grlRA (D) and stcE (E) along with the ompA-specific oligo as a 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: A decreased expression of ler in the sspA mutant (Figure 1A) could account for the apparent transcriptional repression of LEE2-5, grlRA, map and stcE (Figure 1B-H) because Ler positively controls those genes. Thus, we examined whether supplying ler in trans from the plasmid pACYCler would alleviate the expression of Ler-regulated genes in an sspA mutant (Figure 2). Our results showed that transcript levels of LEE1, LEE2, LEE4, grlRA and stcE were all increased in the sspA mutant harboring pACYCler and exceeded that in wild type with up to about 9-fold (Figure 2A-E, compare lanes 1 and 3). These results are consistent with the explanation that a reduced expression of ler in the sspA mutant leads to an insufficient amount of Ler to antagonize H-NS-mediated repression of those virulence genes.

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