Analysis of the activation and mode of action of Clostridium perfringens ε-toxin

1.The activation of Clostridium perfringens epsilon-prototoxin(ε-prototoxin)byλ-toxin，trypsin and chymotrypsin was examined。The mouse lethality test showed that the 50%lethal doses(LD I D 250个D 2)of the prototoxin with and withoutλ-toxin treatment were 110 and 70，000 ng/kg of body weight，respectively.LD D 250个D 2 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight，respectively.Determination of the N-terminal amino acid sequence of each activated 8-prototoxin revealed thatλ-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin，while trypsin and trypsin plus chymotrypsin did so between the 13th and 14th amino acid residues。The C-terminus deduced from the molecular weight is located at the 23th or 30th amino acid residue from the C-terminus of the prototoxin，suggesting that removal of not only N-but also C-terminal peptides is responsible for the activation of the prototoxin.2。The neurotoxicity ofε-toxin was examined by histological examination of the rat brain。Injection ofε-toxin at a sublethal dose，50 ng/kg，caused neuronal damage predominantly in the hippocampus：pyramidal cells in the hippocampus showed marked shrinkage and karyopyknosis，and the cells lost the immunoreactivity to microtubule-associated protein2(MAP-2)。Timm‘s zinc staining revealed that zinc ions were depleted in the mossy layers of the CA3subfield containing glutamate as a synaptic transmitter.Prior injection of either a glutamate-release inhibitor or glutamate-receptor antagonist protected the hippocampus from the neuronal damage caused by 8-toxin.These results suggest that8-toxin acts on the glutamatergic system and evokes excessive release of glutamate，leading to neuronal damage.

1. The activation of Clostridium perfringens epsilon-prototoxin (ε-prototoxin) by λ-toxin, trypsin and chymotrypsin was examined. The mouse lethality test showed that the 50% lethal doses (LDィイD250ィエD2) of the prototoxin with and without λ-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. LDィイD250ィエD2 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. Determination of the N-terminal amino acid sequence of each activated 8-prototoxin revealed that λ-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin did so between the 13th and 14th amino acid residues. The C-terminus deduced from the molecular weight is located at the 23th or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N- but also C-terminal peptides is responsible for the activation of the prototoxin.2. The neurotoxicity of ε-toxin was examined by histological examination of the rat brain. Injection of ε-toxin at a sublethal dose, 50 ng/kg, caused neuronal damage predominantly in the hippocampus: pyramidal cells in the hippocampus showed marked shrinkage and karyopyknosis, and the cells lost the immunoreactivity to microtubule-associated protein 2 (MAP-2). Timm's zinc staining revealed that zinc ions were depleted in the mossy layers of the CA3 subfield containing glutamate as a synaptic transmitter. Prior injection of either a glutamate-release inhibitor or glutamate-receptor antagonist protected the hippocampus from the neuronal damage caused by 8-toxin. These results suggest that 8-toxin acts on the glutamatergic system and evokes excessive release of glutamate, leading to neuronal damage.