Abstract
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.
Highlights
Epsilon toxin (Etx) is the most potent clostridial toxin after botulinum and tetanus neurotoxins
To check whether a reducing agent (DTT) restores the cytotoxic activity of the mutants with paired cysteine substitutions, MTT assays were performed on MadinDarby canine kidney (MDCK) cells exposed to 6.5 nM of each of the green fluorescent protein (GFP)-Etx mutants and the results were compared to those obtained with GFP-proEtx and GFP-Etx (Figure 1E)
The present study shows that two Etx mutants designed to elude the pore formation step in the toxin’s mechanism of action have a differential in vivo effect but maintaining the binding and oligomerization abilities on target cells
Summary
Epsilon toxin (Etx) is the most potent clostridial toxin after botulinum and tetanus neurotoxins. It is produced by types B and D strains of the anaerobic bacterium Clostridium perfringens and primarily affects livestock in the form of rapidly fatal enterotoxemias, resulting in heavy economic losses [1]. The individual implication of these effects on the lethal activity of Etx is still a matter of controversy [6,7,8,9]. There is no direct evidence of human infections by Clostridium perfringens type B and D, the very low LD50 of Etx in all experimental species tested to date suggest that this toxin might be toxic to humans [10,11,12]. It has been postulated that Etx could contribute to nascent Multiple Sclerosis lesion formation [13]
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