The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin

Abstract

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination

“…Because MAL is required for ε-toxin binding to myelin in mice, we choose to evaluate the role of MAL in oligodendrocyte susceptibility to ε-toxin (37). MAL is a proteolipid expressed during late myelination in the CNS and plays a role in proper maintenance of CNS paranodes (38).…”

F199E substitution reduced toxicity ofClostridium perfringensepsilon toxin by depriving the receptor binding capability

“…Some proteins have been suggested as potential receptors for ETX such as hepatitis A virus receptor 1 (HAVCR1) 17 and the myelin and lymphocyte protein (MAL). 29 However, there is no sufficient evidence to confirm the identity of the receptor of ETX.…”

“…This finding is consistent with those of previous studies, which have shown that there is a putative protein receptor located at detergent resistant membrane (DRMs) in the apical cell membrane. 7,24,29,39 However, previous studies also showed that epsilon toxin was capable of forming channels in lipid bilayers without the need of a receptor, although with less efficiency. 21,25 We also found that rETX could occasionally form slight heptamer in PBS without cells.…”

“…Actually, it is not clear if rETX F199E is defective in binding other target cells such as the cerebellar granule cells, because it is not known whether ETX exploits a single receptor shared by all possible targets. For instance, MAL is a likely receptor candidate expressed by MDCK and brain oligodendrocytes 29 but not expressed by neurons. However, some of neurons such as the cerebellar granule cells are ETX targets.…”

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

“…Rumah et al [84] showed binding of ETX to its target cells and its cytotoxic activity on mammalian cells are dependent on myelin and lymphocyte (MAL) protein. The pores formed by ETX include seven monomers of the toxin and some other proteins from the cell membrane, such as caveolin-1 and -2.…”