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Published on: October 18, 2019
Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin
Susan L Robertson1, Jihong Li, Francisco A Uzal
1Department 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.
Insights
Clostridium perfringens epsilon toxin (ETX) forms a non-lytic prepore at 4°C, which rapidly becomes cytotoxic upon warming to 37°C. This reveals a temperature-sensitive pore formation step crucial for ETX cytotoxicity.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Clostridium perfringens epsilon toxin (ETX) is a potent cytotoxin.
- ETX induces rapid cell death at physiological temperatures (37°C) but not at lower temperatures (4°C).
- The precise mechanism of ETX-induced cytotoxicity and its temperature dependence remain incompletely understood.
Purpose of the Study:
- To investigate the temperature-dependent steps in ETX-induced pore formation and cytotoxicity.
- To elucidate the structural intermediates involved in ETX's mechanism of action.
- To propose a revised model for ETX cytotoxicity based on experimental findings.
Main Methods:
- Utilized Madin-Darby Canine Kidney (MDCK II) cells to study ETX-cell interactions.
- Compared ETX binding, oligomerization, and pore formation at 37°C versus 4°C.
- Assessed the impact of temperature shifts on ETX-induced cytotoxicity.
- Employed pronase degradation assays to differentiate surface-bound prepore complexes from membrane-inserted pores.
- Investigated the dissociation kinetics of ETX complexes from cell membranes at different temperatures.
Main Results:
- ETX binds and oligomerizes into a complex with MDCK II cells equally well at 4°C and 37°C.
- Pore formation and cytotoxicity are strictly dependent on temperatures at or above 37°C.
- ETX complexes formed at 4°C are trapped in a surface prepore intermediate, susceptible to pronase and rapid dissociation.
- Warming cells with the 4°C-formed ETX complex to 37°C triggers rapid pore formation and cell lysis.
- ETX complexes formed at 37°C are resistant to pronase and dissociation, indicating membrane insertion and pore formation.
Conclusions:
- ETX cytotoxicity involves a temperature-sensitive step where a surface prepore intermediate transitions into an active membrane pore.
- The formation of the ETX prepore complex on the cell surface occurs independently of temperature.
- Pore insertion and subsequent cell lysis are critically dependent on achieving physiological temperatures (≥37°C).
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