Clostridium perfringens epsilon toxin (ET) is a potent pore-forming
cytotoxin causing fatal
enterotoxemia in livestock. ET accumulates in brain and kidney, particularly in the renal distal-collecting ducts. ET binds and oligomerizes in
detergent-resistant membranes (DRMs) microdomains and causes cell death. However, the causal linkage between membrane permeabilization and cell death is not clear. Here, we show that ET binds and forms 220-kDa insoluble complexes in plasma membrane DRMs of renal mpkCCD(cl4) collecting duct cells.
Phosphatidylinositol-specific phospholipase C did not impair binding or the formation of ET complexes, suggesting that the receptor for ET is not GPI anchored. ET induced a dose-dependent fall in the transepithelial resistance and potential in confluent cells grown on filters, transiently stimulated Na+ absorption, and induced an inward ionic current and a sustained rise in [Ca2+]i. ET also induced rapid depletion of cellular
ATP, and stimulated the
AMP-activated protein kinase, a metabolic-sensing Ser/Thr
kinase. ET also induced mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of
apoptosis-inducing factor, a potent
caspase-independent cell death effector. Finally, ET induced cell
necrosis characterized by a marked reduction in nucleus size without DNA fragmentation. DRM disruption by
methyl-beta-cyclodextrin impaired ET oligomerization, and significantly reduced the influx of Na+ and [Ca2+]i, but did not impair
ATP depletion and cell death caused by the toxin. These findings indicate that ET causes rapid
necrosis of renal collecting duct cells and establish that
ATP depletion-mediated cell death is not strictly correlated with the plasma membrane permeabilization and ion diffusion caused by the toxin.