Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B
Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B
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DOI：
10.1111/j.1365-2958.2011.07549.x
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发表时间：
2011-03-01
影响因子：
3.6
通讯作者：
Aktories, Klaus
中科院分区：
文献类型：
--
作者：
Genisyuerek, Selda;Papatheodorou, Panagiotis;Aktories, Klaus
P>艰难梭菌毒素A和B结合真核靶细胞，被内吞，加工进入细胞质后将其n端葡萄糖基转移酶结构域传递给细胞。虽然葡萄糖基转移酶、自动加工和细胞结合结构域已经明确，但毒素传递的结构特征尚不清楚。在这里，我们研究了决定毒素B的膜插入、孔形成和易位的结构决定因素。缺失分析显示，在Vero细胞中，毒素B的1501-1753氨基酸覆盖了一个很大的区域，对细胞毒性是必不可少的。因此，由氨基酸1-1550和白喉毒素的受体结合域组成的嵌合毒素引起了细胞毒性作用。毒素B的大n端部分（氨基酸1-829）对于孔的形成不是必需的（通过哺乳动物细胞中的86Rb+释放来测量）。利用毒素B的c端截短片段进行的研究表明，氨基酸残基1-990仍然能够诱导大脂质囊泡释放荧光染料，并导致黑色脂质膜的电导率增加。因此，我们在氨基酸残基830和990中定义了毒素B的最小成孔区域。此外，我们发现在这个区域内，氨基酸对谷氨酸970和谷氨酸976在毒素B的孔形成中起着至关重要的作用。
P>Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by 86Rb+ release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.