Mechanism of Botulinum Toxin Action

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DESCRIPTION (provided by applicant): Botulinum toxin is the etiologic agent responsible for the disease botulism. This toxin, which is generally regarded as the most poisonous of all poisons, is known to exist in seven serotypes designated A, B, C, D, E, F and G. Of these seven serotypes, three (A, B and E) account for virtually all cases of human botulism. For botulinum toxin to produce clinical poisoning, it must ordinarily cross at least two membrane barriers. Initially, the toxin binds and undergoes transcytosis across gut or airway epithelial cells. This is the mechanism by which the toxin escapes the lumen of the gut or airway to reach the general circulation. Subsequently, the toxin binds to peripheral cholinergic nerve cells, undergoes receptor-mediated endocytosis and pH-induced translocation, then acts in the cytosol as a zinc-dependent endoprotease to cleave polypeptides that are essential for exocytosis. This is the mechanism by which the toxin produces muscle weakness or flaccid paralysis, which is the characteristic outcome in botulinum toxin poisoning. The goal of the proposed work will be to select botulinum toxin type A as a prototype, to select a human gut epithelial cell line (T-84) as a prototype, and then use these two as models to gain insight into the subcellular and molecular events that account for the ability of the toxin to penetrate membrane barriers. More precisely, two approaches will be used to help elucidate the structural features of the toxin that mediate binding and transcytosis: 1.) a series of truncation mutants will be generated in an attempt to identify the minimal domain that retains the ability to cross gut epithelial cells, and 2.) a series of site-directed mutations will be generated in an attempt to localize critical amino acids within the minimal domain that are needed for binding and transcytosis. In addition, two approaches will be used to help identify subcellular components in human gut cells that transport the toxin from the apical (viz., mucosal) to the basolateral (viz., serosal) surfaces: 1.) pharmacologic techniques will be used to discriminate clathrin-coated vesicles from lipid raft vesicles, and 2.) immunologic techniques will be used to identify membrane polypeptides that are in or near the binding site for the toxin. It is anticipated that the combined products of this proposed research will provide a clear understanding of botulinum toxin penetration of human cell membranes, with the added benefit of generating a potential vaccine against the toxin.