Studies on the toxic action of cytolethal toxin from Clostridium septicum.

本文测定了各种动物红细胞中败血梭菌α-毒素的活性。结果表明，α-毒素的溶血活性与红细胞膜蛋白有关，而红细胞膜蛋白是不同动物体内GPI锚定蛋白的亚类。我用小鼠结缔组织细胞系L-929研究了α-毒素在哺乳动物细胞上形成寡聚体的分子机制。原毒素与非RAFT中的GPI锚定蛋白结合后被细胞表面蛋白酶激活后，被激活的毒素移动到脂筏上并寡聚，导致孔道形成。研究还表明，胆固醇在α-毒素齐聚和细胞毒性中起重要作用。α-肌动蛋白被鉴定为一种新的毒素结合蛋白，它为阐明与败血杆菌感染有关的恶性水肿等特殊症状提供了一些信息。恶性水肿是肌肉组织的一种疾病状态，最终死亡原因是心力衰竭。我证明了α-毒素在与细胞膜结合并形成孔道后与α-肌动蛋白特异性地相互作用。接种α-毒素后，原代小鼠心肌细胞和大鼠灌流心脏即刻停止跳动，心脏无坏死。这些结果表明，α-毒素可通过细胞坏死以外的其他机制诱导心脏骤停。在未来，我将在分子和细胞水平上评估心力衰竭与α-毒素与肌动蛋白结合之间的相关性。

The activity of Clostridium septicum alpha-toxin was determined in erythrocytes of various animals. It was shown that hemolytic activities of alpha-toxin associated with specific erythrocyte membrane proteins that were subsets of GPI-anchored proteins in various animal species. I examined molecular mechanisms of oligomer formation of alpha-toxin on mammalian cells using mouse connective tissue cell line, L-929. After the protoxin binds to GPI-anchored proteins in non-raft for activation by cell surface protease, activated toxin moves to lipid raft and oligomerizes leading to pore-formation. It was also shown that cholesterol played a significant role in alpha-toxin oligomerization and cytotoxicity. Alpha-actin was identified as a new toxin-binding protein, which provided some information to elucidate peculiar symptoms such as malignant edema involving C.septicum infection. Malignant edema is a disease condition of muscle tissues and the ultimate cause of the death is heart failure. I proved that alpha-toxin specifically interact with alpha-actin after binding to cell membrane and pore formation. It was observed that the heart beat has immediately stopped in primary mouse cardiac cells and rat perfused heart without necrosis after inoculation of alpha-toxin. These results indicate that alpha-toxin can induce cardiac arrest with the other mechanism than cell necrosis. In future, I will estimate relevances between heart failure and binding of alpha-toxin to actin in molecular and cellular level.