Autoproteolytic cleavage mediates cytotoxicity of Clostridium difficile toxin A
Autoproteolytic cleavage mediates cytotoxicity of Clostridium difficile toxin A
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DOI：
10.1007/s00210-010-0574-x
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发表时间：
2011-03-01
影响因子：
3.6
通讯作者：
Gerhard, Ralf
中科院分区：
文献类型：
--
作者：
Kreimeyer, Isa;Euler, Friederike;Gerhard, Ralf
来自艰难梭菌的毒素A和毒素B是结肠炎相关性假膜性结肠炎的病原体。它们具有A/B结构类型，并具有肌醇六磷酸诱导的自身蛋白水解活性，将其葡糖基转移酶结构域释放到靶细胞的细胞质中。在这项研究中，我们调查的影响，细胞外和细胞内的自动蛋白水解切割TcdA的功能。细胞外裂解导致功能失活，虽然TcdA是不太容易肌醇六磷酸诱导的自蛋白水解比TcdB。产生不可切割的TcdA突变体（TcdA A541 G542 A543）以研究自身蛋白水解是否是TcdA的细胞内功能的先决条件。尽管在短期测定中关于细胞变圆的EC 50降低了约75倍，但当连续存在时，不可切割的TcdA能够在36小时后诱导完全的细胞变圆和凋亡，与野生型TcdA相当。对毒素摄取有限的研究揭示了TcdA的进行性Rac 1葡糖基化和完全细胞变圆，而不可裂解的TcdA诱导的效应是可逆的。这些研究结果表明，释放的野生型TcdA的葡糖基转移酶结构域的胞质积累和不可切割的TcdA的快速降解。总之，细胞外切割功能性地使TcdA（和TcdB）失活，而细胞内自身蛋白水解切割对于TcdA的功能不是必需的，但限定了其效力。
Toxin A and toxin B from Clostridium difficile are the causative agents of the antibiotic-associated pseudomembranous colitis. They are of an A/B structure type and possess inositol hexakisphosphate-inducible autoproteolytic activity to release their glucosyltransferase domain to the cytoplasm of target cells. In this study, we investigated the effect of extracellular and intracellular autoproteolytic cleavage on the function of TcdA. Extracellular cleavage led to functional inactivation albeit TcdA was less susceptible to inositol hexakisphosphate-induced autoproteolysis than TcdB. A non-cleavable TcdA mutant (TcdA A541 G542 A543) was generated to investigate whether autoproteolysis is a prerequisite for intracellular function of TcdA. Although the EC50 regarding cell rounding was about 75-fold reduced in short-term assay, non-cleavable TcdA was able to induce complete cell rounding and apoptosis after 36 h comparable to wildtype TcdA when continuously present. Studies with limited uptake of toxins revealed progressive Rac 1 glucosylation and complete cell rounding for TcdA, whereas the effect induced by non-cleavable TcdA was reversible. These findings argue for cytosolic accumulation of the released glucosyltransferase domain of wild-type TcdA and rapid degradation of the non-cleavable TcdA. In summary, extracellular cleavage functionally inactivates TcdA (and TcdB), whereas intracellular autoproteolytic cleavage is not essential for function of TcdA but defines its potency.