Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins
Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins
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DOI：
10.1111/cmi.12321
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发表时间：
2014-11-01
影响因子：
3.4
通讯作者：
Popoff, Michel R.
中科院分区：
文献类型：
--
作者：
Genth, Harald;Pauillac, Serge;Popoff, Michel R.
大梭菌葡糖基化毒素（LCGT）由艰难梭菌、产气荚膜梭菌、诺氏梭菌和索氏梭菌的产酶菌株产生。而大多数C. Sordellii菌株仅产生致死毒素（TcsL），C. Sordellii菌株VPI 9048共产生出血毒素（TcsH）和TcsL。在此，提供了TcsH-9048和TcsL-9048的序列，表明这两种毒素都保留了保守的LCGT特征，并且TcsL和TcsH与来自C的毒素A（TcdA）和毒素B（TcdB）高度相关。艰难梭菌菌株VPI 10463。用重组LCGT转移酶结构域（rN）和一组广泛的小GTP酶研究毒素的底物谱。rN-TcsH-9048和rN-TcdA-10463优选葡萄糖基化Rho-GTPases，但也在一定程度上葡萄糖基化Ras-GTPases。在这方面，rN-TcsH-9048和rN-TcdA-10463与各自的全长TcsH-9048和TcdA-10463不同，全长TcsH-9048和TcdA-10463仅葡糖基化Rho-GTP酶。rN-TcsL-9048和全长TcsL-9048葡糖基化Rho-和Ras-GTP酶，而rN-TcdB-10463和全长TcdB-10463仅葡糖基化Rho-GTP酶。用全长TcsH-9048或TcdA-10463处理的Vero细胞也显示Ras的葡糖基化，尽管程度低于Rho-GTP酶。因此，使用对应于自动蛋白水解切割结构域的重组转移酶结构域对底物光谱进行体外分析，比全长毒素更精确地预测体内底物。除TcdB-1470外，所有LCGT均引起小GTdR RhoB表达增加，其在用TcsL同种型处理的细胞中表现出细胞保护活性，但在用TcdA、TcdB和TcsH处理的细胞中表现出促凋亡活性。所有的LCGT诱导的pY 118-桩蛋白和pS144/141-PAK 1/2的快速去磷酸化之前，肌动蛋白丝解聚，表明解体的焦点粘连是一个早期事件，导致肌动蛋白细胞骨架的解体。
Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co-produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH-9048 and TcsL-9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN-TcsH-9048 and rN-TcdA-10463 glucosylated preferably Rho-GTPases but also Ras-GTPases to some extent. In this respect, rN-TcsH-9048 and rN-TcdA-10463 differ from the respective full-length TcsH-9048 and TcdA-10463, which exclusively glucosylate Rho-GTPases. rN-TcsL-9048 and full length TcsL-9048 glucosylate both Rho- and Ras-GTPases, whereas rN-TcdB-10463 and full length TcdB-10463 exclusively glucosylate Rho-GTPases. Vero cells treated with full length TcsH-9048 or TcdA-10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto-proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB-1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro-apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.