Characterization of the high affinity binding of epsilon toxin from Clostridium perfringens to the renal system

Epsilon toxin (epsilon-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 epsilon-toxin, including the specific binding to the target sensitive cells. In the present report, the binding step of epsilon-toxin to the MDCK cell line is characterized by means of an ELISA-based binding assay with recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-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 epsilon-toxin binding. The ELISA assays revealed a single binding site with a similar dissociation constant (K-d) for epsilon-toxin-GFP and epsilon-prototoxin-GFP, but a three-fold increase in B-max levels in the case of epsilon-toxin-GFP. Double staining on kidney cryoslices with lectins and epsilon-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 epsilon-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 epsilon-toxin to its receptor. (C) 2011 Elsevier B.V. All rights reserved.