Botulinum toxin (BoNT) causes flaccid paralysis by blocking the release of neurotransmitters. BoNTs associate with neurotoxin-associated proteins to form medium and large progenitor toxin complexes. The large progenitor toxin complex serotype A-62A (L-PTC/A-62A) specifically targets intestinal M cells for invasion, whereas large progenitor toxin complex serotype B-Okra (L-PTC/B-Okra) is mainly taken up by enterocytes and exhibits higher toxicity via the oral route. Hemagglutinin (HA) is a neurotoxin-associated protein that promotes BoNT absorption from the intestine and has carbohydrate-binding and barrier-disrupting activities. In this study, we established an in vitro reconstitution and purification system for recombinant L-PTC/B-Okra and created a recombinant L-PTC/B-Okra mutant rL-PTC/B-KA with carbohydrate-binding activity but not barrier-disrupting activity. rL-PTC/B-KA showed significantly reduced oral toxicity. Our results demonstrate that the B-Okra toxin disrupts the epithelial barrier of enterocytes and exerts oral toxicity.

Botulinum toxin (BoNT) causes flaccid paralysis by blocking the release of neurotransmitters. BoNTs associate with neurotoxin-associated proteins (NAPs) to form a medium progenitor toxin complex (PTC) (M-PTC) and large PTC (L-PTC). The L-PTC serotype A-62A (L-PTC/A_62A) specifically targets intestinal M cells for invasion, whereas L-PTC serotype B-Okra (L-PTC/B_Okra), which exhibits higher oral toxicity, is mainly taken up by enterocytes. Hemagglutinin (HA) is a NAPs that promotes BoNT absorption from the intestine and has carbohydrate-binding and barrier-disrupting activities. In this study, we established an in vitro reconstitution and purification system for recombinant L-PTC/B_Okra and created a recombinant L-PTC/B_Okra mutant rL-PTC/BB-KA with carbohydrate-binding activity but not barrier-disrupting activity. rL-PTC/BB-KA significantly reduced the oral toxicity. Our results demonstrate that the toxin of B-Okra disrupts the epithelial barrier of enterocytes and exerts oral toxicity.