Department of Pathology and Experimental Therapeutics, School of Medicine, Campus of Bellvitge, Health Universitat de Barcelona Campus (HUBc), University of Barcelona, c/Feixa Llarga s/n 08907, L'Hospitalet de Llobregat, Spain.
Vet Microbiol. 2012 May 25;157(1-2):179-89. doi: 10.1016/j.vetmic.2011.12.020. Epub 2012 Jan 5.
Epsilon toxin (ε-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxaemia in livestock. In the renal system, the toxin binds to target cells before oligomerization, pore formation and cell death. Still, there is little information about the cellular and molecular mechanism involved in the initial steps of the cytotoxic action of ε-toxin, including the specific binding to the target sensitive cells. In the present report, the binding step of ε-toxin to the MDCK cell line is characterized by means of an ELISA-based binding assay with recombinant ε-toxin-green fluorescence protein (ε-toxin-GFP) and ε-prototoxin-GFP. In addition, different treatments with Pronase E, detergents, N-glycosidase F and beta-elimination on MDCK cells and renal cryosections have been performed to further characterize the ε-toxin binding. The ELISA assays revealed a single binding site with a similar dissociation constant (K(d)) for ε-toxin-GFP and ε-prototoxin-GFP, but a three-fold increase in B(max) levels in the case of ε-toxin-GFP. Double staining on kidney cryoslices with lectins and ε-prototoxin-GFP revealed specific binding to distal and collecting tubule cells. In addition, experiments on kidney and bladder cryoslices demonstrated the specific binding to distal tubule of a range of mammalian renal systems. Pronase E and beta-elimination treatments on kidney cryoslices and MDCK cells revealed that the binding of ε-toxin in renal system is mediated by a O-glycoprotein. Detergent treatments revealed that the integrity of the plasma membrane is required for the binding of ε-toxin to its receptor.
epsilon 毒素（ε-toxin）由 B 型和 D 型产气荚膜梭菌产生，会导致牲畜致命的肠毒血症。在肾脏系统中，毒素在寡聚化、形成孔和细胞死亡之前与靶细胞结合。然而，关于 epsilon 毒素细胞毒性作用的初始步骤涉及的细胞和分子机制的信息很少，包括与靶敏感细胞的特异性结合。在本报告中，通过使用重组 epsilon 毒素-绿色荧光蛋白（ε-toxin-GFP）和 ε 原毒素-GFP 的 ELISA 结合测定，对 ε-toxin 与 MDCK 细胞系的结合步骤进行了表征。此外，对 MDCK 细胞和肾冷冻切片进行了 Pronase E、去污剂、N-糖苷酶 F 和 β-消除等不同处理，以进一步表征 epsilon 毒素的结合。ELISA 测定显示，ε-toxin-GFP 和 ε-prototoxin-GFP 具有相似的解离常数（K(d)），但 ε-toxin-GFP 的 B(max)水平增加了三倍。对肾脏冷冻切片进行凝集素和 ε-prototoxin-GFP 的双重染色显示，ε-toxin 与远曲小管和收集管细胞具有特异性结合。此外，对肾脏和膀胱冷冻切片的实验表明，在一系列哺乳动物肾脏系统中，ε-toxin 特异性结合于远曲小管。对肾脏冷冻切片和 MDCK 细胞进行 Pronase E 和 β-消除处理显示，肾脏系统中 epsilon 毒素的结合是由 O-糖蛋白介导的。去污剂处理显示，质膜的完整性是 epsilon 毒素与其受体结合所必需的。
