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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 positively affects LEE expression in stationary phase cells. Primer extension analyses on total RNA extracted from wild type EHEC EDL933 (lane 1), the sspA mutant (lane 2) and the sspA mutant complemented with wild type sspA (lane 3) or mutant sspA84-86 (lane 4) as indicated, grown in LB at 37°C to stationary phase (OD600 ~ 3.0). The Labeled DNA oligos specific to the transcripts of LEE1/ler (A and I), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H) were used. The ompA transcripts, detected with a labeled ompA-specific DNA oligo, served as internal control for the primer extension reaction. Wild type and mutant SspA were expressed from pQEsspA and pQEsspA84-86 respectively in the absence of induction at similar levels. The transcripts LEE1-5, map, grlRA, stcE and the control transcript ompA are indicated. The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.
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Figure 1: SspA positively affects LEE expression in stationary phase cells. Primer extension analyses on total RNA extracted from wild type EHEC EDL933 (lane 1), the sspA mutant (lane 2) and the sspA mutant complemented with wild type sspA (lane 3) or mutant sspA84-86 (lane 4) as indicated, grown in LB at 37°C to stationary phase (OD600 ~ 3.0). The Labeled DNA oligos specific to the transcripts of LEE1/ler (A and I), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H) were used. The ompA transcripts, detected with a labeled ompA-specific DNA oligo, served as internal control for the primer extension reaction. Wild type and mutant SspA were expressed from pQEsspA and pQEsspA84-86 respectively in the absence of induction at similar levels. The transcripts LEE1-5, map, grlRA, stcE and the control transcript ompA are indicated. The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.

Mentions: To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927[52] and measured transcription of LEE- (LEE1-5, grlRA and map) and non-LEE-encoded (stcE encoded by pO157) genes (Figure 1). Wild type and sspA mutant strains were grown in LB medium to stationary phase with similar growth rates (data not shown). Total RNA was isolated and transcript abundance was measured by primer extension analyses using labeled DNA oligos specific to each transcript of interest and ompA, which served as internal control for total RNA levels. Results revealed that transcript levels of LEE1-5, grlRA, map and stcE were reduced by up to 8-fold in the sspA mutant compared to wild type (Figure 1A-H, lanes 1 and 2). The expression of these genes was restored when the sspA mutant was supplied with wild type sspA in trans from pQEsspA[43] (Figure 1A-H, lane 3). However, the expression of ler and other virulence genes tested (grlRA, espZ, sepL and stcE) remained repressed when the sspA mutant strain was supplied with mutant sspA from pQEsspA84-86[45], which expresses SspA containing the triple alanine substitution in the surface-exposed pocket (Figure 1I and data not shown). These results indicate that SspA positively affects stationary phase-induced expression of both LEE- and non-LEE-encoded virulence genes in EHEC. Moreover, the mode of action of SspA is likely similar in E. coli K-12 and EHEC as the surface-exposed pocket of SspA also is required for SspA to affect the expression of EHEC 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)

SspA positively affects LEE expression in stationary phase cells. Primer extension analyses on total RNA extracted from wild type EHEC EDL933 (lane 1), the sspA mutant (lane 2) and the sspA mutant complemented with wild type sspA (lane 3) or mutant sspA84-86 (lane 4) as indicated, grown in LB at 37°C to stationary phase (OD600 ~ 3.0). The Labeled DNA oligos specific to the transcripts of LEE1/ler (A and I), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H) were used. The ompA transcripts, detected with a labeled ompA-specific DNA oligo, served as internal control for the primer extension reaction. Wild type and mutant SspA were expressed from pQEsspA and pQEsspA84-86 respectively in the absence of induction at similar levels. The transcripts LEE1-5, map, grlRA, stcE and the control transcript ompA are indicated. 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 1: SspA positively affects LEE expression in stationary phase cells. Primer extension analyses on total RNA extracted from wild type EHEC EDL933 (lane 1), the sspA mutant (lane 2) and the sspA mutant complemented with wild type sspA (lane 3) or mutant sspA84-86 (lane 4) as indicated, grown in LB at 37°C to stationary phase (OD600 ~ 3.0). The Labeled DNA oligos specific to the transcripts of LEE1/ler (A and I), LEE2/espZ (B), LEE3/mpc (C), LEE4/sepL (D), LEE5/tir (E), map (F), grlRA (G) and stcE (H) were used. The ompA transcripts, detected with a labeled ompA-specific DNA oligo, served as internal control for the primer extension reaction. Wild type and mutant SspA were expressed from pQEsspA and pQEsspA84-86 respectively in the absence of induction at similar levels. The transcripts LEE1-5, map, grlRA, stcE and the control transcript ompA are indicated. The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.
Mentions: To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927[52] and measured transcription of LEE- (LEE1-5, grlRA and map) and non-LEE-encoded (stcE encoded by pO157) genes (Figure 1). Wild type and sspA mutant strains were grown in LB medium to stationary phase with similar growth rates (data not shown). Total RNA was isolated and transcript abundance was measured by primer extension analyses using labeled DNA oligos specific to each transcript of interest and ompA, which served as internal control for total RNA levels. Results revealed that transcript levels of LEE1-5, grlRA, map and stcE were reduced by up to 8-fold in the sspA mutant compared to wild type (Figure 1A-H, lanes 1 and 2). The expression of these genes was restored when the sspA mutant was supplied with wild type sspA in trans from pQEsspA[43] (Figure 1A-H, lane 3). However, the expression of ler and other virulence genes tested (grlRA, espZ, sepL and stcE) remained repressed when the sspA mutant strain was supplied with mutant sspA from pQEsspA84-86[45], which expresses SspA containing the triple alanine substitution in the surface-exposed pocket (Figure 1I and data not shown). These results indicate that SspA positively affects stationary phase-induced expression of both LEE- and non-LEE-encoded virulence genes in EHEC. Moreover, the mode of action of SspA is likely similar in E. coli K-12 and EHEC as the surface-exposed pocket of SspA also is required for SspA to affect the expression of EHEC 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