Calcium enhances binding of Clostridium perfringens epsilon toxin to sulfatide

Epsilon toxin (Etx) from Clostridium perfringens is synthesized as a very low-active prototoxin form (proEtx) that becomes active upon proteolytic activation and has the capacity to cross the blood-brain barrier (BBB), thereby producing severe neurological effects. The identity and requirements of host receptors of Etx remain a matter of controversy. In the present study, we analysed the binding of proEtx or Etx to liposomes containing distearoylphosphatidylcholine (DSPC), cholesterol and sulfatide, or alternatively to detergent-solubilized lipids, using surface plasmon resonance (SPR). We also tested the influence of calcium on Etx or proEtx binding. Our findings show that the presence of sulfatide in liposomes increases both Etx and proEtx binding, and Etx binding is enhanced by calcium. These results were corroborated when SPR was conducted with immobilized toxin, since detergent-solubilized sulfatide increases its binding to Etx in the presence of calcium, but not to proEtx. Moreover, binding affinity is also affected, since the treatment of liposomes with sulfatase causes the dissociation rate constants (K_D) in both proEtx and Etx to increase, especially in the case of proEtx in the presence of calcium. In addition, protein-lipid overlay assays corroborated the calcium-induced enhancement of Etx binding to sulfatide, and to lipids extracted from sulfatide-enriched rat brain lipid rafts. In conclusion, the present work highlights the role of sulfatide as an important element in the pathophysiology of Etx and reveals the influence of calcium in the interaction of Etx, but not of proEtx, with the target membrane.