Clostridium perfringens type F strains cause
gastrointestinal disease when they produce a pore-forming toxin named C. perfringens
enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause
necrosis. Since
necrosis or apoptosis sometimes involves receptor-interacting
serine/
threonine-
protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those
kinases are important for CPE-induced apoptosis or
necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and
necrosis in Caco-2 cells. Those findings suggested that the form of
necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage
kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those
kinases also reduced MLKL oligomerization during treatment with high CPE concentrations.
Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating
calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or
calpain inhibitors can be explored as potential
therapeutics against CPE effects in vivoIMPORTANCEC. perfringens type F strains are a common cause of
food poisoning and
antibiotic-associated
diarrhea. Type F strain virulence requires production of C. perfringens
enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause
necrosis while low
enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting
serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and
necrosis in Caco-2 cells, while mixed-lineage
kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced
necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells.
Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or
calpain are useful
therapeutics against CPE-mediated diseases.