Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination.

Epsilon toxin (ET) is produced by Clostridium perfringens types B and D and causes severe neurological disorders in animals. ET has been observed binding to white matter, suggesting that it may target oligodendrocytes. In primary cultures containing oligodendrocytes and astrocytes, we found that ET (10(-9) M and 10(-7) M) binds to oligodendrocytes, but not to astrocytes. ET induces an increase in extracellular glutamate, and produces oscillations of intracellular Ca(2+) concentration in oligodendrocytes. These effects occurred without any change in the transmembrane resistance of oligodendrocytes, underlining that ET acts through a pore-independent mechanism. Pharmacological investigations revealed that the Ca(2+) oscillations are caused by the ET-induced rise in extracellular glutamate concentration. Indeed, the blockade of metabotropic glutamate receptors type 1 (mGluR1) prevented ET-induced Ca(2+) signals. Activation of the N-methyl-D-aspartate receptor (NMDA-R) is also involved, but to a lesser extent. Oligodendrocytes are responsible for myelinating neuronal axons. Using organotypic cultures of cerebellar slices, we found that ET induced the demyelination of Purkinje cell axons within 24 h. As this effect was suppressed by antagonizing mGluR1 and NMDA-R, demyelination is therefore caused by the initial ET-induced rise in extracellular glutamate concentration. This study reveals the novel possibility that ET can act on oligodendrocytes, thereby causing demyelination. Moreover, it suggests that for certain cell types such as oligodendrocytes, ET can act without forming pores, namely through the activation of an undefined receptor-mediated pathway.journal articleresearch support, non-u.s. gov't2015 Mar2014 10 31importe

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB