Studies on neurotropism of Clostridium perfringens epsilon-toxin and molecular mechanism of its toxicity toward neuronal cells

为了检测产气荚膜梭菌ε-毒素（ε-毒素）在中毒动物组织中的分布，将13 <125>I标记的ε-毒素静脉注射到小鼠体内，然后进行全身放射自显影。虽然毒素主要在大脑和肾脏中检测到，但它也在甲状腺，胃和唾液腺中。由于中毒后可能发生碘解，因此使用13 C标记的ε-毒素进行全身放射自显影<14>。标记的毒素仅在大脑、脊髓和肾脏中检测到。免疫组织化学分析显示，毒素主要分布在海马和肾小球。然而，其他区域也被染色，这些组织中的蛋白质显示与抗体交叉反应。已经尝试通过使用预吸附的多克隆或单克隆抗体来增加免疫反应性的特异性。ε-毒素对大鼠突触体的作用 ...更多信息 用~（13）<125>I标记的ε-毒素对膜进行了研究。虽然最初在膜中检测到ε-毒素单体，但其随着孵育时间形成大的复合物。大复合物由ε-毒素的七聚体组成。当毒素和失活的原毒素与膜孵育时，单体的七聚化而不是其与膜的结合被原毒素剂量依赖性地抑制。这些结果表明，C-末端前肽，这是裂解后激活胰蛋白酶消化，不影响ε-毒素与其受体的结合，而它掩盖了一个区域所必需的七聚化。测试几种培养的神经元细胞的ε-毒素细胞毒性。无论是未分化的细胞还是分化的细胞对毒素都不敏感。建立了从牛脑细胞膜分离鉴定ε-毒素结合受体的方法：大量制备突触体膜，差异溶解，免疫学检测，如狭缝印迹和western印迹，亲和层析。从小牛脑细胞膜和小牛肾细胞膜中分离出了一些蛋白质，其中包括主要的45 K蛋白，它们似乎对毒素具有高亲和力。这些蛋白质的鉴定和纯化正在进行中。少

In order to examine distribution of Clostridium perfringens epsilon-toxin (ε-toxin) in tissues of intoxicated animals, ^<125>I-labelled ε-toxin was injected intravenously into a mouse, and then subjected to whole body autoradiography. Although the toxin was detected mainly in the brain and kidneys, it was also in the thyroids, stomach, and salivary glands. Since iodolysis might have occurred after the intoxication, the whole body autoradiography was performed using ^<14>C-labelled ε-toxin. The labelled toxin was detected exclusively in the brain, spinal cord and kidneys. Analysis by immunohistochemistry revealed that the toxin was distributed mainly in the hippocampus and the glomerulus. However, other areas were also stained, and proteins in these tissues were shown to be cross-reactive with the antibody. An attempt has been made to increase the specificity of the immunoreactivity by using pre-adsorbed polyclonal or monoclonal antibodies. The action of ε-toxin on the rat synaptosomal … More membrane was investigated by using ^<125>I-labelled ε-toxin. While the ε-toxin monomer was initially detected in the membrane, it formed a large complex with incubation time. The large complex was shown to consist of a heptamer of ε-toxin. When the toxin and inactive protoxin were incubated with the membrane, the heptamerization of the monomer but not its association with the membrane was inhibited dose-dependently by the protoxin. These results suggest that a C-terminal propeptide, which was cleaved upon activation by tryptic digestion, does not affect the binding of ε-toxin to its receptor, while it masks a region necessary for the heptamerization. Several cultured neuronal cells were tested for ε-toxin cell toxicity. Either undifferentiated or differentiated cells were not sensitive to the toxin. A method for the isolation and identification of an ε-toxin binding receptor from the membrane fraction of the calf brain have been established : preparation of large quantities of the synaptosomal membrane, differential solubilization, immunological detection such as slot blotting and western blotting, and affinity chromatography. A few proteins including predominant 45 K protein, which seemed to possess a high affinity to the toxin, were isolated from the calf brain cell membrane and also from the calf kidney cell membrane. Identification and purification of these proteins are now in progress. Less