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。检查了λ-毒素，胰蛋白酶和胰胆红蛋白的激活，渗透性卵巢蛋白酶 - 蛋白毒素（ε-蛋白毒素）的激活。小鼠致死性测试表明，有和不接受λ-毒素治疗的原始毒素的50％致命剂量（LDII D250 D2）分别为110 ng/kg的体重。用胰蛋白酶和胰蛋白酶加胰胆红素处理的原始毒素的LDII D250 D2分别为320和65 ng/kg体重。测定每个活化的8-蛋白毒素的N末端氨基酸序列，表明在原始毒素的N末端保留了第10和11个氨基酸之间裂解的λ-毒素，而胰蛋白酶和胰蛋白酶和胰蛋白酶的胰蛋白酶则在13th至14th氨基酸之间保留。从分子量推导的C末端位于原始毒素的C末端的第23或30氨基酸，这表明不仅去除N-，而且C-末端胡椒剂的去除是原始毒素的激活。2。通过对大鼠脑的组织学检查检查ε-茶素的神经毒性。将ε-茶素注射为50 ng/kg，主要在海马中引起神经元损伤：显示的海马中的锥体细胞标志着明显的收缩和karypopy氏症，并且细胞对微管相关蛋白2（MAP-2）失去了对微蛋白酶相关蛋白2的免疫反应性。 Timm的锌染色表明，锌离子在含有谷氨酸作为突触发射机的CA3子场的苔藓层中耗尽。事先注射谷氨酸释放抑制剂或谷氨酸 - 受体拮抗剂可以保护海马免受8毒素引起的神经元损伤。这些结果表明，8毒素作用于谷氨酸系统，并唤起谷氨酸的过度释放，从而导致神经元损伤。