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

产气荚膜梭菌ε-毒素的活化及作用方式分析

基本信息

批准号：
09670286
负责人：
MATSUSHITA Osamu
金额：
$ 2.11万
依托单位：
Kagawa Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670286/
关键词：
Clostridium perfringens epsilon-toxin lambda-toxin mouse lethality hippocampus trypsin トリプシン ε毒素組換え毒素

项目摘要

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.

1.本文研究了λ-毒素、胰蛋白酶和糜蛋白酶对产气荚膜梭菌ε-原毒素的激活作用。小鼠致死率试验表明，用λ-毒素处理和不用λ-毒素处理的原毒素的50%致死剂量（LD_（50）D_（250）D_（2））分别为110和70，000 ng/kg体重。胰蛋白酶和胰蛋白酶+糜蛋白酶处理的原毒素LD_（50）D_（250）D_（250）分别为320和65 ng/kg体重。对每个活化的8-原毒素的N-末端氨基酸序列的测定表明，λ-毒素在原毒素N-末端的第10和11个氨基酸残基之间切割，而胰蛋白酶和胰蛋白酶加胰凝乳蛋白酶在第13和14个氨基酸残基之间切割。从分子量推断的C-末端位于原毒素C-末端的第23或30个氨基酸残基处，表明不仅N-末端肽的去除而且C-末端肽的去除负责原毒素的活化.用脑组织病理学方法观察ε-毒素的神经毒性。注射50 ng/kg的ε-毒素可引起海马神经元的损伤，海马锥体细胞出现明显的皱缩和核固缩，并丧失对微管相关蛋白2（MAP-2）的免疫反应性。Timm的锌染色显示，锌离子耗尽的苔藓层的CA 3子域含有谷氨酸作为突触递质。预先注射谷氨酸释放抑制剂或谷氨酸受体拮抗剂可保护海马免受8-毒素引起的神经元损伤。这些结果表明，8-毒素作用于谷氨酸能系统并引起谷氨酸的过度释放，导致神经元损伤。