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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 and B. EspPα cleaves EHEC-Hly in bacterial culture. EHEC-Hly-producing strain TA48 was grown to early log phase and supplemented either with EspPα or with the EspPα-buffer control and incubated for further 2 h. (A) Sterile supernatants were TCA-precipitated, separated in SDS-PAGE and analysed in immunoblot using anti-EHEC-Hly antibody. The arrows indicate the 107 kDa band of EHEC-Hly (white arrow) or the specific Mr ∼84 kDa and ∼34 kDa breakdown fragments of EHEC-Hly (black arrows). The very weak immunoreactive band with a slightly higher Mr than that of the ∼84 kDa specific cleavage product (#) was present in all EHEC-Hly control preparations (see also C and D) and was therefore considered a background signal. (B) The sterile culture supernatants were assayed for their haemolytic activity, which was calculated as percentage of haemolysis (see Experimental procedures). Data in (B) are presented as means ± standard deviations of three independent assays. Statistically significant differences (P < 0.01, Student's t-test) are indicated by asterisk. C–E. Cleavage and inactivation of recombinant EHEC-Hly from EHEC O157 via EspPα from EHEC O157. Immunoblot analysis using anti-EHEC-Hly antibody of (C) recombinant isolated EHEC-Hly after incubation with buffer (control) or with recombinant purified EspPα, and (D) TCA-precipitated supernatants of clones TA144 and TA145 coexpressing recombinant EHEC-Hly and either EspPα (TA145) or the non-proteolytic EspPα mutant S263A (TA144). The arrows indicate the 107 kDa band of intact EHEC-Hly (white arrow) and the Mr ∼84 kDa and the ∼82 kDa EHEC-Hly cleavage products (black arrows). (E) Haemolytic activity of sterile culture supernatants of clones TA144 and TA145 after 11 h and 13 h of growth calculated as percentage of haemolysis. Data are presented as means ± standard deviations of at least three independent assays. Statistically significant differences between haemolytic activity of TA144 and TA145 (P < 0.01, Student's t-test) are indicated by asterisks.
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fig01: A and B. EspPα cleaves EHEC-Hly in bacterial culture. EHEC-Hly-producing strain TA48 was grown to early log phase and supplemented either with EspPα or with the EspPα-buffer control and incubated for further 2 h. (A) Sterile supernatants were TCA-precipitated, separated in SDS-PAGE and analysed in immunoblot using anti-EHEC-Hly antibody. The arrows indicate the 107 kDa band of EHEC-Hly (white arrow) or the specific Mr ∼84 kDa and ∼34 kDa breakdown fragments of EHEC-Hly (black arrows). The very weak immunoreactive band with a slightly higher Mr than that of the ∼84 kDa specific cleavage product (#) was present in all EHEC-Hly control preparations (see also C and D) and was therefore considered a background signal. (B) The sterile culture supernatants were assayed for their haemolytic activity, which was calculated as percentage of haemolysis (see Experimental procedures). Data in (B) are presented as means ± standard deviations of three independent assays. Statistically significant differences (P < 0.01, Student's t-test) are indicated by asterisk. C–E. Cleavage and inactivation of recombinant EHEC-Hly from EHEC O157 via EspPα from EHEC O157. Immunoblot analysis using anti-EHEC-Hly antibody of (C) recombinant isolated EHEC-Hly after incubation with buffer (control) or with recombinant purified EspPα, and (D) TCA-precipitated supernatants of clones TA144 and TA145 coexpressing recombinant EHEC-Hly and either EspPα (TA145) or the non-proteolytic EspPα mutant S263A (TA144). The arrows indicate the 107 kDa band of intact EHEC-Hly (white arrow) and the Mr ∼84 kDa and the ∼82 kDa EHEC-Hly cleavage products (black arrows). (E) Haemolytic activity of sterile culture supernatants of clones TA144 and TA145 after 11 h and 13 h of growth calculated as percentage of haemolysis. Data are presented as means ± standard deviations of at least three independent assays. Statistically significant differences between haemolytic activity of TA144 and TA145 (P < 0.01, Student's t-test) are indicated by asterisks.

Mentions: In a first approach, we supplemented an early log-phase culture of clone TA48 producing recombinant EHEC-Hly from EHEC O157 : H7 with 5 µg ml−1 purified, recombinant EspPα from EHEC O157 : H7 or with an EspPα-buffer control (Table 2, panel A-O157 : H7-I) and continued incubation at 37°C for 2 h. Immunoblot analysis of trichloroacetic acid (TCA)-precipitated sterile supernatants using anti-EHEC-Hly polyclonal antibody demonstrated the occurrence of two immunoreactive breakdown products with relative molecular masses (Mr) of 84 ± 4 kDa and 34 ± 3 kDa in TA48 treated with EspPα (Fig. 1A, lane 2). These fragments were not present in TA48 supernatant treated with EspPα-buffer control (Fig. 1A, lane 1), indicating proteolytic cleavage of the 107 kDa large EHEC-Hly by EspPα. EspPα remained unaffected by EHEC-Hly as determined by immunoblot using an anti-EspP antibody. In addition, EspPα did not cross-react with the anti-EHEC-Hly antibody (data not shown).


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 and B. EspPα cleaves EHEC-Hly in bacterial culture. EHEC-Hly-producing strain TA48 was grown to early log phase and supplemented either with EspPα or with the EspPα-buffer control and incubated for further 2 h. (A) Sterile supernatants were TCA-precipitated, separated in SDS-PAGE and analysed in immunoblot using anti-EHEC-Hly antibody. The arrows indicate the 107 kDa band of EHEC-Hly (white arrow) or the specific Mr ∼84 kDa and ∼34 kDa breakdown fragments of EHEC-Hly (black arrows). The very weak immunoreactive band with a slightly higher Mr than that of the ∼84 kDa specific cleavage product (#) was present in all EHEC-Hly control preparations (see also C and D) and was therefore considered a background signal. (B) The sterile culture supernatants were assayed for their haemolytic activity, which was calculated as percentage of haemolysis (see Experimental procedures). Data in (B) are presented as means ± standard deviations of three independent assays. Statistically significant differences (P < 0.01, Student's t-test) are indicated by asterisk. C–E. Cleavage and inactivation of recombinant EHEC-Hly from EHEC O157 via EspPα from EHEC O157. Immunoblot analysis using anti-EHEC-Hly antibody of (C) recombinant isolated EHEC-Hly after incubation with buffer (control) or with recombinant purified EspPα, and (D) TCA-precipitated supernatants of clones TA144 and TA145 coexpressing recombinant EHEC-Hly and either EspPα (TA145) or the non-proteolytic EspPα mutant S263A (TA144). The arrows indicate the 107 kDa band of intact EHEC-Hly (white arrow) and the Mr ∼84 kDa and the ∼82 kDa EHEC-Hly cleavage products (black arrows). (E) Haemolytic activity of sterile culture supernatants of clones TA144 and TA145 after 11 h and 13 h of growth calculated as percentage of haemolysis. Data are presented as means ± standard deviations of at least three independent assays. Statistically significant differences between haemolytic activity of TA144 and TA145 (P < 0.01, Student's t-test) are indicated by asterisks.
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fig01: A and B. EspPα cleaves EHEC-Hly in bacterial culture. EHEC-Hly-producing strain TA48 was grown to early log phase and supplemented either with EspPα or with the EspPα-buffer control and incubated for further 2 h. (A) Sterile supernatants were TCA-precipitated, separated in SDS-PAGE and analysed in immunoblot using anti-EHEC-Hly antibody. The arrows indicate the 107 kDa band of EHEC-Hly (white arrow) or the specific Mr ∼84 kDa and ∼34 kDa breakdown fragments of EHEC-Hly (black arrows). The very weak immunoreactive band with a slightly higher Mr than that of the ∼84 kDa specific cleavage product (#) was present in all EHEC-Hly control preparations (see also C and D) and was therefore considered a background signal. (B) The sterile culture supernatants were assayed for their haemolytic activity, which was calculated as percentage of haemolysis (see Experimental procedures). Data in (B) are presented as means ± standard deviations of three independent assays. Statistically significant differences (P < 0.01, Student's t-test) are indicated by asterisk. C–E. Cleavage and inactivation of recombinant EHEC-Hly from EHEC O157 via EspPα from EHEC O157. Immunoblot analysis using anti-EHEC-Hly antibody of (C) recombinant isolated EHEC-Hly after incubation with buffer (control) or with recombinant purified EspPα, and (D) TCA-precipitated supernatants of clones TA144 and TA145 coexpressing recombinant EHEC-Hly and either EspPα (TA145) or the non-proteolytic EspPα mutant S263A (TA144). The arrows indicate the 107 kDa band of intact EHEC-Hly (white arrow) and the Mr ∼84 kDa and the ∼82 kDa EHEC-Hly cleavage products (black arrows). (E) Haemolytic activity of sterile culture supernatants of clones TA144 and TA145 after 11 h and 13 h of growth calculated as percentage of haemolysis. Data are presented as means ± standard deviations of at least three independent assays. Statistically significant differences between haemolytic activity of TA144 and TA145 (P < 0.01, Student's t-test) are indicated by asterisks.
Mentions: In a first approach, we supplemented an early log-phase culture of clone TA48 producing recombinant EHEC-Hly from EHEC O157 : H7 with 5 µg ml−1 purified, recombinant EspPα from EHEC O157 : H7 or with an EspPα-buffer control (Table 2, panel A-O157 : H7-I) and continued incubation at 37°C for 2 h. Immunoblot analysis of trichloroacetic acid (TCA)-precipitated sterile supernatants using anti-EHEC-Hly polyclonal antibody demonstrated the occurrence of two immunoreactive breakdown products with relative molecular masses (Mr) of 84 ± 4 kDa and 34 ± 3 kDa in TA48 treated with EspPα (Fig. 1A, lane 2). These fragments were not present in TA48 supernatant treated with EspPα-buffer control (Fig. 1A, lane 1), indicating proteolytic cleavage of the 107 kDa large EHEC-Hly by EspPα. EspPα remained unaffected by EHEC-Hly as determined by immunoblot using an anti-EspP antibody. In addition, EspPα did not cross-react with the anti-EHEC-Hly antibody (data not shown).

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