Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase

Abstract

Background: Clostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK) cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization.

Methods: We tested the role of nSMase in the oligomerization of epsilon-toxin using specific inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining.

Results: Epsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in ACHN cells. GW4869, an inhibitor of nSMase, inhibited ceramide production induced by the toxin. GW4869 and knockdown of nSMase blocked toxin-induced cell death and oligomer formation of epsilon-toxin. Confocal microscopy images showed that the toxin induced ceramide clustering and colocalized with ceramide.

Conclusions: These results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of nSMase caused by the toxin.

General significance: Inhibitors of nSMase may confer protection against infection.

Keywords: C. perfringens epsilon-toxin; Ceramide; Neutral sphingomyelinase; Oligomer.