-84 cells, nor were they efficiently transcytosed (8 -10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2 ) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 M brefeldin A, decreased 42% when microtubules were disrupted by 10 M nocodazole, and decreased 74% at 18°C. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A 1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17-22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not.

Experiments were done to help localize the minimum essential domain within the botulinum toxin molecule that is necessary for binding and transport across human gut epithelial cells. The data demonstrated that the neurotoxin alone, in the absence of auxiliary proteins, undergoes transcytosis. The neurotoxin by itself was examined in the single chain (unnicked serotype B) and dichain (nicked serotype B, nicked serotype A) forms, and all displayed the ability to bind and penetrate epithelial barriers. In addition, the single chain and dichain molecules were examined in the oxidized and reduced states, and again all forms were transported. To further define the minimum essential domain, experiments were done with two toxin fragments: 1) the heavy chain, which was derived from native toxin, and 2) the carboxy-terminal portion of the heavy chain, which was generated by recombinant techniques. Interestingly, both fragments were fully competent in crossing epithelial barriers. These data suggest that a polypeptide derived from the toxin could be used as a carrier domain to transport other molecules across epithelial cells. In related experiments, physiological (i.e., potassium depletion) and pharmacological (i.e., chlorpromazine) manipulations were used to implicate clathrin-coated pits/vesicles as the structures responsible for endocytosis of toxin.

Botulinum toxin is an unusually potent oral poison, which means that the toxin must have an efficient mechanism for escaping the lumen of the gut to reach the general circulation.

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn 2؉ from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn 2؉ . In contrast to soluble chelators, immobilized chelators have no effect on bound Zn 2؉ , nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn 2؉ from its coordination site in botulinum toxin is relatively slow. When exogenous Zn 2؉ is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn 2؉ can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn 2؉ does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn 2؉ or exogenous Zn 2؉ means that productive internalization does not produce conformational changes that block rebinding of cation.

Clostridium botulinum serotype A produces a neurotoxin composed of a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond. This neurotoxin is part of a ca. 900-kDa complex, formed by noncovalent association with a single nontoxin, nonhemagglutinin subunit and a family of hemagglutinating proteins. Previous work has suggested, although never conclusively demonstrated, that neurotoxin alone cannot survive passage through the stomach and/or cannot be absorbed from the gut without the involvement of auxiliary proteins in the complex. Therefore, this study compared the relative absorption and toxicity of three preparations of neurotoxin in an in vivo mouse model. Equimolar amounts of serotype A complex with hemagglutinins, complex without hemagglutinins, and purified neurotoxin were surgically introduced into the stomach or into the small intestine. In some experiments, movement of neurotoxin from the site of administration was restricted by ligation of the pylorus. Comparison of relative toxicities demonstrated that at adequate doses, complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin can be absorbed from the stomach. The potency of neurotoxin in complex was greater than that of pure neurotoxin, but the magnitude of this difference diminished as the dosage of neurotoxin increased. Qualitatively similar results were obtained when complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin were placed directly into the intestine. This work establishes that pure botulinum neurotoxin serotype A is toxic when administered orally. This means that pure neurotoxin does not require hemagglutinins or other auxiliary proteins for absorption from the gastrointestinal system into the general circulation.