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Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin.

Robertson SL, Li J, Uzal FA, McClane BA - PLoS ONE (2011)

Bottom Line: The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C.Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface.Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Clostridium perfringens epsilon toxin (ETX) rapidly kills MDCK II cells at 37°C, but not 4°C. The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C. However, the complex formed in MDCK cells treated with ETX at 4°C has the potential to form an active pore, since shifting those cells to 37°C results in rapid cytotoxicity. Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface. Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore. In addition, the ETX complex rapidly dissociated from MDCK II cells at 4°C, but not 37°C; this result is consistent with the ETX complex being resistant to dissociation at 37°C because it has inserted into membranes, while the ETX prepore readily dissociates from cells at 4°C because it remains on the membrane surface. These results support the identification of a prepore stage in ETX action and suggest a revised model for ETX cytotoxicity, i) ETX binds to an unidentified receptor, ii) ETX oligomerizes into a prepore on the membrane surface, and iii) the prepore inserts into membranes, in a temperature-sensitive manner, to form an active pore.

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Analysis of temperature effects on ETX binding and complex formation in MDCK II cells.A) ETX complex formation. MDCK II cells were treated with 10 µg of AF448-ETX for 60 min at 4°C or 37°C and then electrophoresed and quantitatively imaged (see Materials and Methods). Arrows indicate, as specified, migration of the ∼155 kDa ETX complex or the ∼30 kDa ETX monomer. B) Quantitative analysis of ETX binding at 4°C vs. 37°C. MDCK II cells were treated with 10 µg of AF488-ETX for 60 min at 4°C or 37°C. After electrophoresis of cell lysates, total AF488-ETX present in the gel i.e. AF488-ETX bound in complex or as monomer, was quantified by fluorescence scanning. Shown are the average results from 4 gel scans. C) Quantitative analysis of ETX complex formation. MDCK II cells were treated with 10 µg of ETX for 60 min at 4°C or 37°C. The amount of fluorescence specifically present in gel regions containing AF488-ETX bound in the ETX complex was determined. Shown are the mean values from 4 experiments.
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pone-0022053-g003: Analysis of temperature effects on ETX binding and complex formation in MDCK II cells.A) ETX complex formation. MDCK II cells were treated with 10 µg of AF448-ETX for 60 min at 4°C or 37°C and then electrophoresed and quantitatively imaged (see Materials and Methods). Arrows indicate, as specified, migration of the ∼155 kDa ETX complex or the ∼30 kDa ETX monomer. B) Quantitative analysis of ETX binding at 4°C vs. 37°C. MDCK II cells were treated with 10 µg of AF488-ETX for 60 min at 4°C or 37°C. After electrophoresis of cell lysates, total AF488-ETX present in the gel i.e. AF488-ETX bound in complex or as monomer, was quantified by fluorescence scanning. Shown are the average results from 4 gel scans. C) Quantitative analysis of ETX complex formation. MDCK II cells were treated with 10 µg of ETX for 60 min at 4°C or 37°C. The amount of fluorescence specifically present in gel regions containing AF488-ETX bound in the ETX complex was determined. Shown are the mean values from 4 experiments.

Mentions: Having obtained evidence supporting ETX binding at 4°C, the current study next conducted the first head-to-head quantitative comparison of ETX binding levels and complex formation levels at 4°C vs. 37°C. When quantitative fluorescence analysis was performed on gels containing samples of MDCK II cells treated with AF488-ETX at either 4°C or 37°C (Fig. 3A), similar amounts of bound toxin were detected at either 4°C or 37°C (Fig. 3B). This analysis further determined that, i) ∼70–90% of the AF488-ETX bound to MDCK II cells was localized in the ETX complex and ii) nearly equivalent amounts of ETX complex had formed in MDCK II cells at both temperatures (Fig. 3C).


Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin.

Robertson SL, Li J, Uzal FA, McClane BA - PLoS ONE (2011)

Analysis of temperature effects on ETX binding and complex formation in MDCK II cells.A) ETX complex formation. MDCK II cells were treated with 10 µg of AF448-ETX for 60 min at 4°C or 37°C and then electrophoresed and quantitatively imaged (see Materials and Methods). Arrows indicate, as specified, migration of the ∼155 kDa ETX complex or the ∼30 kDa ETX monomer. B) Quantitative analysis of ETX binding at 4°C vs. 37°C. MDCK II cells were treated with 10 µg of AF488-ETX for 60 min at 4°C or 37°C. After electrophoresis of cell lysates, total AF488-ETX present in the gel i.e. AF488-ETX bound in complex or as monomer, was quantified by fluorescence scanning. Shown are the average results from 4 gel scans. C) Quantitative analysis of ETX complex formation. MDCK II cells were treated with 10 µg of ETX for 60 min at 4°C or 37°C. The amount of fluorescence specifically present in gel regions containing AF488-ETX bound in the ETX complex was determined. Shown are the mean values from 4 experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140917&req=5

pone-0022053-g003: Analysis of temperature effects on ETX binding and complex formation in MDCK II cells.A) ETX complex formation. MDCK II cells were treated with 10 µg of AF448-ETX for 60 min at 4°C or 37°C and then electrophoresed and quantitatively imaged (see Materials and Methods). Arrows indicate, as specified, migration of the ∼155 kDa ETX complex or the ∼30 kDa ETX monomer. B) Quantitative analysis of ETX binding at 4°C vs. 37°C. MDCK II cells were treated with 10 µg of AF488-ETX for 60 min at 4°C or 37°C. After electrophoresis of cell lysates, total AF488-ETX present in the gel i.e. AF488-ETX bound in complex or as monomer, was quantified by fluorescence scanning. Shown are the average results from 4 gel scans. C) Quantitative analysis of ETX complex formation. MDCK II cells were treated with 10 µg of ETX for 60 min at 4°C or 37°C. The amount of fluorescence specifically present in gel regions containing AF488-ETX bound in the ETX complex was determined. Shown are the mean values from 4 experiments.
Mentions: Having obtained evidence supporting ETX binding at 4°C, the current study next conducted the first head-to-head quantitative comparison of ETX binding levels and complex formation levels at 4°C vs. 37°C. When quantitative fluorescence analysis was performed on gels containing samples of MDCK II cells treated with AF488-ETX at either 4°C or 37°C (Fig. 3A), similar amounts of bound toxin were detected at either 4°C or 37°C (Fig. 3B). This analysis further determined that, i) ∼70–90% of the AF488-ETX bound to MDCK II cells was localized in the ETX complex and ii) nearly equivalent amounts of ETX complex had formed in MDCK II cells at both temperatures (Fig. 3C).

Bottom Line: The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C.Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface.Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Clostridium perfringens epsilon toxin (ETX) rapidly kills MDCK II cells at 37°C, but not 4°C. The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C. However, the complex formed in MDCK cells treated with ETX at 4°C has the potential to form an active pore, since shifting those cells to 37°C results in rapid cytotoxicity. Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface. Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore. In addition, the ETX complex rapidly dissociated from MDCK II cells at 4°C, but not 37°C; this result is consistent with the ETX complex being resistant to dissociation at 37°C because it has inserted into membranes, while the ETX prepore readily dissociates from cells at 4°C because it remains on the membrane surface. These results support the identification of a prepore stage in ETX action and suggest a revised model for ETX cytotoxicity, i) ETX binds to an unidentified receptor, ii) ETX oligomerizes into a prepore on the membrane surface, and iii) the prepore inserts into membranes, in a temperature-sensitive manner, to form an active pore.

Show MeSH
Related in: MedlinePlus