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Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα.

Brockmeyer J, Aldick T, Soltwisch J, Zhang W, Tarr PI, Weiss A, Dreisewerd K, Müthing J, Bielaszewska M, Karch H - Environ. Microbiol. (2011)

Bottom Line: In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed.We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors.Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Chemistry, University of Münster, Corrensstrasse 45, Münster, Germany. jbrockm@uni-muenster.de

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Chronology of EHEC-hlyA and espP expression in EHEC strains during contact with human intestinal epithelial cells. HCT-8 monolayers were infected with overnight cultures of EHEC strains producing EHEC-Hly together with either EspPα (O157 : H7 strain EDL933 and O26 : H11 strain 5236/96) (A and B) or EspPβ (O6 : HNT strain 3503/98 and O163 : H19 strain 36/03) (C and D) for 2–24 h as indicated. Bacteria were harvested by centrifugation, RNA was isolated and transcription levels of EHEC-hlyA and espP were determined using RT-PCR and normalized to gapA. Upregulation of each gene expression relative to 2 h time point was determined using Student's t-test with *P < 0.05 and **P < 0.001. Data are means ± standard deviations from two independent assays.
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fig04: Chronology of EHEC-hlyA and espP expression in EHEC strains during contact with human intestinal epithelial cells. HCT-8 monolayers were infected with overnight cultures of EHEC strains producing EHEC-Hly together with either EspPα (O157 : H7 strain EDL933 and O26 : H11 strain 5236/96) (A and B) or EspPβ (O6 : HNT strain 3503/98 and O163 : H19 strain 36/03) (C and D) for 2–24 h as indicated. Bacteria were harvested by centrifugation, RNA was isolated and transcription levels of EHEC-hlyA and espP were determined using RT-PCR and normalized to gapA. Upregulation of each gene expression relative to 2 h time point was determined using Student's t-test with *P < 0.05 and **P < 0.001. Data are means ± standard deviations from two independent assays.

Mentions: To investigate the expression of both virulence factors under conditions mimicking the situation during human infection, we developed an intestinal cell infection assay. Human ileo-caecal epithelial cells (HCT-8) were infected with overnight cultures of the same set of EHEC strains tested above and incubated at 37°C under static conditions for 2 h, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h. Relative transcription of EHEC-hlyA and espP mRNA was again normalized to gapA (Fig. 4). In the presence of intestinal epithelial cells, expression of both EHEC-hlyA and espP was significantly upregulated compared with LB media. After 12 h, EHEC-hlyA expression increased by two- to fivefold relative to the starting conditions (2 h time point). Similarly, espP was significantly upregulated after 12 h leading to 15- to 20-fold increase; in strain 5236/96 (O26 : H11) upregulation reached even > 35-fold (Fig. 4). Again, no significant difference between strains expressing proteolytically active EspPα and inactive EspPβ was observed.


Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα.

Brockmeyer J, Aldick T, Soltwisch J, Zhang W, Tarr PI, Weiss A, Dreisewerd K, Müthing J, Bielaszewska M, Karch H - Environ. Microbiol. (2011)

Chronology of EHEC-hlyA and espP expression in EHEC strains during contact with human intestinal epithelial cells. HCT-8 monolayers were infected with overnight cultures of EHEC strains producing EHEC-Hly together with either EspPα (O157 : H7 strain EDL933 and O26 : H11 strain 5236/96) (A and B) or EspPβ (O6 : HNT strain 3503/98 and O163 : H19 strain 36/03) (C and D) for 2–24 h as indicated. Bacteria were harvested by centrifugation, RNA was isolated and transcription levels of EHEC-hlyA and espP were determined using RT-PCR and normalized to gapA. Upregulation of each gene expression relative to 2 h time point was determined using Student's t-test with *P < 0.05 and **P < 0.001. Data are means ± standard deviations from two independent assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472028&req=5

fig04: Chronology of EHEC-hlyA and espP expression in EHEC strains during contact with human intestinal epithelial cells. HCT-8 monolayers were infected with overnight cultures of EHEC strains producing EHEC-Hly together with either EspPα (O157 : H7 strain EDL933 and O26 : H11 strain 5236/96) (A and B) or EspPβ (O6 : HNT strain 3503/98 and O163 : H19 strain 36/03) (C and D) for 2–24 h as indicated. Bacteria were harvested by centrifugation, RNA was isolated and transcription levels of EHEC-hlyA and espP were determined using RT-PCR and normalized to gapA. Upregulation of each gene expression relative to 2 h time point was determined using Student's t-test with *P < 0.05 and **P < 0.001. Data are means ± standard deviations from two independent assays.
Mentions: To investigate the expression of both virulence factors under conditions mimicking the situation during human infection, we developed an intestinal cell infection assay. Human ileo-caecal epithelial cells (HCT-8) were infected with overnight cultures of the same set of EHEC strains tested above and incubated at 37°C under static conditions for 2 h, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h. Relative transcription of EHEC-hlyA and espP mRNA was again normalized to gapA (Fig. 4). In the presence of intestinal epithelial cells, expression of both EHEC-hlyA and espP was significantly upregulated compared with LB media. After 12 h, EHEC-hlyA expression increased by two- to fivefold relative to the starting conditions (2 h time point). Similarly, espP was significantly upregulated after 12 h leading to 15- to 20-fold increase; in strain 5236/96 (O26 : H11) upregulation reached even > 35-fold (Fig. 4). Again, no significant difference between strains expressing proteolytically active EspPα and inactive EspPβ was observed.

Bottom Line: In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed.We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors.Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Chemistry, University of Münster, Corrensstrasse 45, Münster, Germany. jbrockm@uni-muenster.de

Show MeSH
Related in: MedlinePlus