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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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A. Haemolytic activity of EHEC-Hly is cut off by EspPα cleavage. EHEC-Hly-containing sterile culture supernatant from strain TA48 was incubated for indicated time intervals (0–120 min) with EspPα (▴) or EspPα-buffer control (▵) at 37°C and residual haemolytic activity was determined and calculated as percentage of haemolysis as described in Experimental procedures. Data are presented as means ± standard deviations of three independent assays. B. EHEC-Hly associated with OMV is cleaved by EspPα. Immunoblot analysis of OMV-associated EHEC-Hly incubated with EspPα for 15 min to 26 h using anti-EHEC-Hly antibody. The white arrow indicates the 107 kDa band of intact EHEC-Hly, and the black arrows mark the Mr ∼84 kDa, the ∼82 kDa and the ∼34 kDa EHEC-Hly cleavage products. Incubation times of OMV-associated EHEC-Hly with EspPα are given at the top of the figure.
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fig02: A. Haemolytic activity of EHEC-Hly is cut off by EspPα cleavage. EHEC-Hly-containing sterile culture supernatant from strain TA48 was incubated for indicated time intervals (0–120 min) with EspPα (▴) or EspPα-buffer control (▵) at 37°C and residual haemolytic activity was determined and calculated as percentage of haemolysis as described in Experimental procedures. Data are presented as means ± standard deviations of three independent assays. B. EHEC-Hly associated with OMV is cleaved by EspPα. Immunoblot analysis of OMV-associated EHEC-Hly incubated with EspPα for 15 min to 26 h using anti-EHEC-Hly antibody. The white arrow indicates the 107 kDa band of intact EHEC-Hly, and the black arrows mark the Mr ∼84 kDa, the ∼82 kDa and the ∼34 kDa EHEC-Hly cleavage products. Incubation times of OMV-associated EHEC-Hly with EspPα are given at the top of the figure.

Mentions: To assess the functional consequences of EspPα-mediated cleavage for the biological activity of EHEC-Hly, we analysed the haemolytic activity of the sterile culture supernatant of EHEC-Hly-producing clone TA48 after the growing culture was supplemented with EspPα for 2 h (Table 2, panel A-O157 : H7-II). The EspPα treatment reduced the haemolytic activity of supernatant TA48 to 67%, compared to the EspPα-buffer control-treated sample (Fig. 1B), indicating that cleavage of EHEC-Hly eliminates its haemolytic activity. To further confirm this result, we tested culture supernatants of clones coexpressing EHEC-Hly and either EspPα (TA145) or the non-proteolytic mutant EspPα (TA144) for their haemolytic activities (Table 2, panel C-O157 : H7-II). Clone TA144 produced at different time points (11 h and 13 h) approximately fourfold higher haemolysis (57% versus 16% and 100% versus 27% respectively) than clone TA145 (Fig. 1E), demonstrating that the cleavage of EHEC-Hly by EspPα results in a significantly (P < 0.01) reduced haemolytic activity. In addition, sterile supernatant of clone TA48 containing EHEC-Hly was supplemented with 4 µg ml−1 recombinant EspPα or the respective buffer control, incubated at 37°C for different time intervals (0–120 min) and then tested for residual haemolytic activity (Table 2, panel B-O157 : H7-II). The activity of the EspPα-treated sample was reduced to 50% after about 35 min and totally abolished (1%) after 2 h as compared to its initial activity (Fig. 2A). The EHEC-Hly-containing supernatant exposed to the EspPα-buffer control also showed a reduction of haemolytic activity (from 100% to 62%) over time (Fig. 2A). This loss of activity in the control sample is due to the relatively short half-life time of the free EHEC-Hly itself, as reported previously (Aldick et al., 2009), the phenomenon that is thought to be caused by the irreversible self-aggregation of the toxin. Taken together, the rapid and complete loss of haemolytic activity of the EspPα-treated EHEC-Hly further underlines the capacity of EspPα to abolish the biological activity of EHEC-Hly by endoproteolytic cleavage.


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)

A. Haemolytic activity of EHEC-Hly is cut off by EspPα cleavage. EHEC-Hly-containing sterile culture supernatant from strain TA48 was incubated for indicated time intervals (0–120 min) with EspPα (▴) or EspPα-buffer control (▵) at 37°C and residual haemolytic activity was determined and calculated as percentage of haemolysis as described in Experimental procedures. Data are presented as means ± standard deviations of three independent assays. B. EHEC-Hly associated with OMV is cleaved by EspPα. Immunoblot analysis of OMV-associated EHEC-Hly incubated with EspPα for 15 min to 26 h using anti-EHEC-Hly antibody. The white arrow indicates the 107 kDa band of intact EHEC-Hly, and the black arrows mark the Mr ∼84 kDa, the ∼82 kDa and the ∼34 kDa EHEC-Hly cleavage products. Incubation times of OMV-associated EHEC-Hly with EspPα are given at the top of the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: A. Haemolytic activity of EHEC-Hly is cut off by EspPα cleavage. EHEC-Hly-containing sterile culture supernatant from strain TA48 was incubated for indicated time intervals (0–120 min) with EspPα (▴) or EspPα-buffer control (▵) at 37°C and residual haemolytic activity was determined and calculated as percentage of haemolysis as described in Experimental procedures. Data are presented as means ± standard deviations of three independent assays. B. EHEC-Hly associated with OMV is cleaved by EspPα. Immunoblot analysis of OMV-associated EHEC-Hly incubated with EspPα for 15 min to 26 h using anti-EHEC-Hly antibody. The white arrow indicates the 107 kDa band of intact EHEC-Hly, and the black arrows mark the Mr ∼84 kDa, the ∼82 kDa and the ∼34 kDa EHEC-Hly cleavage products. Incubation times of OMV-associated EHEC-Hly with EspPα are given at the top of the figure.
Mentions: To assess the functional consequences of EspPα-mediated cleavage for the biological activity of EHEC-Hly, we analysed the haemolytic activity of the sterile culture supernatant of EHEC-Hly-producing clone TA48 after the growing culture was supplemented with EspPα for 2 h (Table 2, panel A-O157 : H7-II). The EspPα treatment reduced the haemolytic activity of supernatant TA48 to 67%, compared to the EspPα-buffer control-treated sample (Fig. 1B), indicating that cleavage of EHEC-Hly eliminates its haemolytic activity. To further confirm this result, we tested culture supernatants of clones coexpressing EHEC-Hly and either EspPα (TA145) or the non-proteolytic mutant EspPα (TA144) for their haemolytic activities (Table 2, panel C-O157 : H7-II). Clone TA144 produced at different time points (11 h and 13 h) approximately fourfold higher haemolysis (57% versus 16% and 100% versus 27% respectively) than clone TA145 (Fig. 1E), demonstrating that the cleavage of EHEC-Hly by EspPα results in a significantly (P < 0.01) reduced haemolytic activity. In addition, sterile supernatant of clone TA48 containing EHEC-Hly was supplemented with 4 µg ml−1 recombinant EspPα or the respective buffer control, incubated at 37°C for different time intervals (0–120 min) and then tested for residual haemolytic activity (Table 2, panel B-O157 : H7-II). The activity of the EspPα-treated sample was reduced to 50% after about 35 min and totally abolished (1%) after 2 h as compared to its initial activity (Fig. 2A). The EHEC-Hly-containing supernatant exposed to the EspPα-buffer control also showed a reduction of haemolytic activity (from 100% to 62%) over time (Fig. 2A). This loss of activity in the control sample is due to the relatively short half-life time of the free EHEC-Hly itself, as reported previously (Aldick et al., 2009), the phenomenon that is thought to be caused by the irreversible self-aggregation of the toxin. Taken together, the rapid and complete loss of haemolytic activity of the EspPα-treated EHEC-Hly further underlines the capacity of EspPα to abolish the biological activity of EHEC-Hly by endoproteolytic cleavage.

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