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

为了研究产气荚膜梭菌ε-毒素（ε-毒素）在中毒动物组织中的分布，将^<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。虽然这种毒素主要在大脑和肾脏中检测到，但在甲状腺、胃和唾液腺中也检测到。由于中毒后可能发生碘溶解，因此采用^<14> c标记ε-毒素进行全身放射自显影。标记的毒素仅在大脑、脊髓和肾脏中检测到。免疫组化分析显示，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。用^<125> i标记ε-毒素，研究了ε-毒素对大鼠突触体多膜的作用。虽然ε-毒素单体最初在膜中被检测到，但随着孵育时间的延长，它形成了一个大的复合物。该大型复合物由ε-毒素的七聚体组成。当毒素和失活的原毒素与膜一起孵育时，原毒素抑制了单体的七聚化，但不抑制其与膜的结合。这些结果表明，在胰蛋白酶消化激活后被切割的c端前肽不影响ε-毒素与其受体的结合，但它掩盖了七聚化所必需的区域。对培养的神经细胞进行ε-毒素细胞毒性试验。未分化细胞和已分化细胞对毒素均不敏感。建立了一种从小牛脑膜分离和鉴定ε-毒素结合受体的方法：制备大量的突触体膜，差异增溶，免疫检测如缝隙印迹法和western印迹法，亲和层析法。从小牛的脑细胞细胞膜和小牛的肾细胞膜中分离出了一些蛋白，其中包括优势的45k蛋白，似乎对毒素具有很高的亲和力。这些蛋白质的鉴定和纯化目前正在进行中。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