Interaction of Clostridium perfringens Epsilon Toxin with the Plasma Membrane: The Role of Amino Acids Y42, Y43 and H162

Abstract

Clostridium perfringens epsilon toxin (Etx) is a pore forming toxin that causes enterotoxaemia in ruminants and may be a cause of multiple sclerosis in humans. To date, most in vitro studies of Etx have used the Madin-Darby canine kidney (MDCK) cell line. However, studies using Chinese hamster ovary (CHO) cells engineered to express the putative Etx receptor, myelin and lymphocyte protein (MAL), suggest that amino acids important for Etx activity differ between species. In this study, we investigated the role of amino acids Y42, Y43 and H162, previously identified as important in Etx activity towards MDCK cells, in Etx activity towards CHO-human MAL (CHO-hMAL) cells, human red blood cells (hRBCs) and synthetic bilayers using site-directed mutants of Etx. We show that in CHO-hMAL cells Y42 is critical for Etx binding and not Y43 as in MDCK cells, indicating that surface exposed tyrosine residues in the receptor binding domain of Etx impact efficiency of cell binding to MAL-expressing cells in a species-specific manner. We also show that Etx mutant H162A was unable to lyse CHO-hMAL cells, lysed hRBCs, whilst it was able to form pores in synthetic bilayers, providing evidence of the complexity of Etx pore formation in different lipid environments.

Epsilon Toxin Induces Cytotoxicity by Mediating Autophagy and Apoptosis Processes via the PI3K/AKT/mTOR Signaling Pathway in A549 Cells

“…Epsilon toxin from C. perfringens is a potent cytotoxin that be found frequently in the environment and the gastrointestinal tracts of humans and animals and acts via the creating pores in the cellular membrane, leading to ion imbalances and ultimately prompting cell death [24]. Epsilon toxin has been demonstrated to target different cell types, such as lung epithelial cells [1,25,26]. Because of the vulnerability of lung epithelial cells to epsilon toxin, the A549 cell line was used as a cellular model for assessing the underlying mechanisms of cytotoxicity in vitro.…”

The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin

“…Also, unlike oligodendrocytes from wildtype mice, oligodendrocytes cultured from Mal -/mice fail to bind to ε-toxin and are toxin resistant [61]. Subsequently, other researchers have confirmed that the expression of MAL by cultured cells is associated with susceptibility to the toxin [27,28,56,[62][63][64]. MAL is associated with lipid rafts in cells, and MAL is reported to oligomerise under some conditions [65].…”

“…It is possible that ε-toxin can insert into lipid monolayers or bilayers, if the concentration of toxin is sufficiently high to drive multimer formation in the absence of a high-affinity receptor. This would explain why some researchers report that liposomes or monolayers show changes in permeability when exposed to the toxin [ 46 , 56 , 57 ].…”

“…More distantly related MAL, for example from zebra fish, appears not to bind to ε-toxin at all, and zebra fish engineered to express human MAL become toxin-sensitive [ 64 ]. It also appears that individual amino acids in ε-toxin play different roles in binding to MAL from different species [ 27 , 56 , 60 ]. The finding that MAL is found on T-cells and red cells in humans but not in other animals indicates that these cells would likely bind to ε-toxin in the blood stream, and this would then limit the amount of toxin that was available to cross the blood–brain barrier and bind to cells in the brain.…”