title: Characterization of the high affinity binding of epsilon toxin from Clostridium perfringens to the renal system.
abstract: 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.
authors.affiliation: 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.
authors.forename: Jonatan
authors.initials: J
authors.lastname: Dorca-Arévalo
authors.forename: Mireia
authors.initials: M
authors.lastname: Martín-Satué
authors.forename: Juan
authors.initials: J
authors.lastname: Blasi
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D016923:Cell Death; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003016:Clostridium perfringens / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007668:Kidney / Q000166:cytology*; D007685:Kidney Tubules, Collecting / Q000166:cytology*; D037102:Lectins / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011402:Pronase / Q000737:chemistry; D011485:Protein Binding
chemical_list: D001427:Bacterial Toxins; C039811:Clostridium perfringens epsilon-toxin; D003902:Detergents; D037102:Lectins; D049452:Green Fluorescent Proteins; D011402:Pronase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase