Cytotoxicity of Clostridium septicum alpha-toxin:: its oligomerization in detergent resistant membranes of mammalian cells
Cytotoxicity of Clostridium septicum alpha-toxin:: its oligomerization in detergent resistant membranes of mammalian cells
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DOI：
10.1016/j.micpath.2004.09.001
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发表时间：
2004-12-01
影响因子：
3.8
通讯作者：
Kozaki, S
中科院分区：
文献类型：
--
作者：
Hang'ombe, MB;Mukamoto, M;Kozaki, S
α毒素是败血性梭菌的重要毒力因子。我们研究了α-毒素对各种哺乳动物细胞的细胞毒性，其中重组毒素在氨基末端与组氨酸标签融合。重组毒素保留了与天然形式无区别的活性。除RAW 264.7和髓系P3 U1细胞外，本研究中检查的哺乳动物有核细胞对原毒素比对胰蛋白酶化毒素更敏感。各种分子大小的细胞蛋白质与毒素相互作用。蛋白质的大小和SDS-PAGE图谱在不同细胞系中不同，但用磷脂酰肌醇特异性磷脂酶C处理后，蛋白质从细胞中释放出来。该毒素似乎靶向并利用去污剂抗性膜（DRM）进行结合和随后的寡聚化。在不连续蔗糖密度梯度中，我们通过免疫印迹证明毒素与L929细胞中含有的DRM结合并引起寡聚体形成。此外，用胆固醇相互作用剂消耗胆固醇减少毒素寡聚化并降低毒素对细胞的细胞毒性。这些结果表明α-毒素优先利用DRM进行寡聚化。(C)2004 Elsevier Ltd.保留所有权利。
Alpha-toxin is an important agent of the virulence of Clostridium septicum. We examined cytotoxicity for alpha-toxin to various mammalian cells with recombinant toxin fused with a histidine-tag at the amino-terminal. The recombinant toxin retained the activity indistinguishable from the native form. Mammalian nucleated cells examined in this study are more sensitive to the protoxin than to the trypsinized toxin, except RAW 264.7 and P3U1 cells of myeloid lineage. Cellular proteins of various molecular sizes interacted with the toxin. The size and SDS-PAGE pattern of the proteins were different among cell lines but the were liberated from the cells by the treatment with phosphatidylinositol-specific phospholipase C. The toxin appeared to target and utilize detergent resistant mernbranes (DRMs) for binding and subsequent oligomerization. In discontinuous Sucrose density gradient, we demonstrated by immunoblotting that the toxin bound to DRMs contained in L929 cells and caused the oligomer formation. Furthermore, cholesterol depletion with cholesterol-interacting agents reduced toxin oligomerization and lowered cytotoxicity of the toxin towards cells. These results suggest that alpha-toxin preferentially exploits DRMs for oligomerization. (C) 2004 Elsevier Ltd. All rights reserved.