Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

Abstract

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

The Cytotoxicity of Epsilon Toxin from Clostridium perfringens on Lymphocytes Is Mediated by MAL Protein Expression

“…Interestingly, MAL protein has been related to a defined membrane lipid composition, basically, in glycosphingolipid (mainly galactosylceramide and sulfatide)-enriched domains (39). Removal of the sulfate group significantly impairs Etx cytotoxic activity in MDCK cells, suggesting a close relationship between MAL protein, sulfatide, and Etx (19). Moreover, MAL protein has been involved in myelin biogenesis, probably in the vesicular transport of sulfatide to the membrane, forming myelin (39,40).…”

“…It is assumed that the specific action of Etx on sensitive cells relies on the presence of an Etx receptor so that it may selectively bind the cell surface before the formation of the oligomer. In spite of the proposed role of membrane lipids in the recognition of cell targets by Etx or the affinity of Etx to cell targets (19)(20)(21), a set of proteins which can account for the full and highly sensitive effect of the toxin has been explored as potential receptors for Etx. Among them, the most promising candidates are the hepatitis A virus cellular receptor 1 (HAVCR1) (18) and the myelin and lymphocyte (MAL) protein (22).…”

The crystal structure of SnTox3 from the necrotrophic fungusParastagonospora nodorumreveals a unique effector fold and insights into Kex2 protease processing of fungal effectors

“…In light of the structural differences and the lack of the rim and stem domain, 344 SnTox3 alone would be unable to form a membrane-spanning pore analogous to PFTs. Despite 345 these differences, we tested whether SnTox3 would associate with lipids using a simple lipid 346 overlay assay, as reported for several bacterial PFTs (Savva et al, 2013;Gil et al, 2015). No 347 binding of SnTox3 Kex2 was observed to any of the membrane lipids tested (Fig.…”

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

“…The first study that assessed the cloning, sequencing, and expression of ETX in E. coli served as a base for further studies that sought to determine its structure, toxicity, interaction with host cells, and immunogenicity [22,82,89,90]. The recombinant toxin was used to determine the toxicity mechanism, the main susceptible cell types, preferential organs, and potential hosts [78,91,92,93].…”