Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

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.

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

“…The recombinant toxin was used to determine the toxicity mechanism, the main susceptible cell types, preferential organs, and potential hosts [78,91,92,93]. …”

“…rETX H106P is completely non-toxic and has been validated as a safe vaccine antigen against enterotoxemia [80,92,95,97]. Despite having low or null toxicity, rETX with V56C/F118C, S156E, and Y71A mutations have not been validated as potential vaccine antigens [82,85,100].…”

Animal models to study the pathogenesis of human and animal Clostridium perfringens infections

“…In the brain of rats ETX binds to synaptosomes (Nagahama and Sakurai, 1992) via an as yet undetermined receptor, which is thought to be a sialoglycoprotein (Nagahama and Sakurai, 1991, 1992). The action of ETX on the hippocampus leads to an excessive release of glutamate, which seems to be at least in part be responsible for the neurological disorders observed in type D disease and/or ETX intoxication of several animal species (Miyamoto et al, 1998; Dorca-Arevalo et al, 2008, 2014). …”

“…Multiple animal models have been used to study the intravenous effects of ETX, including sheep (Buxton and Morgan, 1976; Uzal and Kelly, 1997), goats (Uzal and Kelly, 1997), cattle (Niilo et al, 1963; Uzal et al, 2002), mice (Finnie, 1984a and b; Fisher et al, 2006; Dorca-Arevalo et al, 2014) and rats (Finnie et al, 1999). The most significant effects of ETX are edema of the lungs and brain.…”

“…A mutant with cysteine substitutions in the membrane insertion domain of ETX has been tested in a mouse model. The mutant was not lethal and the substituted toxin did not cross the blood-brain barrier or affect renal epithelial cells (Dorca-Arevalo et al, 2014). Based on these results it was suggested that there is a direct correlation between the lethal effect of ETX with its ability to cross the blood brain barrier and its effect on renal tubular cells (Dorca-Arevalo et al, 2014).…”