Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

Abstract

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.

Author Summary

Food-borne botulinum neurotoxin (BoNT) poisoning results in fatal muscle paralysis. But how can BoNT–a large protein released by the bacteria clostridia–survive the hostile gastrointestinal (GI) tract to gain access to neurons that control muscle contraction? Here, we report the complete structure of a bimodular ∼760 kDa BoNT/A large progenitor toxin complex (L-PTC), which is composed of BoNT and four non-toxic bacterial proteins. The architecture of this bacterial machinery mimics an Apollo lunar module, whereby the “ascent stage” (a ∼290 kDa module) protects BoNT from destruction in the GI tract and the 3-arm “descent stage” (a ∼470 kDa module) mediates absorption of BoNT by binding to host carbohydrate receptors in the small intestine. This new finding has helped us identify the carbohydrate-binding sites and the monosaccharide IPTG as a prototypical oral inhibitor, which extends survival following lethal BoNT/A intoxication of mice. Hence, pre-treatment with small molecule inhibitors based on carbohydrate receptor mimicry can provide temporary protection against BoNT entry into the circulation.