title: Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.
abstract: Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Ivie
authors.forename: Mark S
authors.initials: MS
authors.lastname: McClain
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D030041:Hepatitis A virus / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001427:Bacterial Toxins; C039811:Clostridium perfringens epsilon-toxin; D011991:Receptors, Virus