BoNT/E-associated protein disrupts intestinal epithelial barrier integrity to facilitate transcytosis

Epithelial and endothelial barriers play essential roles in maintaining compartmentalization within the body through specialized intercellular junctional complexes. Botulinum neurotoxin type E (BoNT/E), a ~150 kDa protein toxin produced by Clostridium botulinum, is capable of traversing the intestinal epithelial barrier despite its large molecular size. BoNT/E is produced in association with several neurotoxin-associated proteins (NAPs), which are believed to facilitate toxin stability and absorption. Among these, an approximately 80 kDa protein (P80), corresponding to OrfX2, has been identified as a BoNT/E-associated protein; however, its functional role in epithelial transport remains poorly understood.

P80 was purified from the BoNT/E progenitor toxin complex and characterized using circular dichroism spectroscopy and thermal denaturation analysis. Its biological activity was evaluated using hemagglutination assays, cytotoxicity studies, confocal microscopy, atomic force microscopy (AFM), immunofluorescence imaging, tight junction protein analysis, and transcytosis assays in Caco-2 and HT-29 intestinal epithelial cell models.

Structural analyses revealed that P80 is a stable, predominantly α-helical protein with a well-defined tertiary structure. P80 exhibited no detectable hemagglutination activity and showed minimal cytotoxicity toward intestinal epithelial cells. Cellular studies demonstrated that P80 interacts with epithelial monolayers, induces actin cytoskeletal remodeling, increases claudin-1 phosphorylation, and alters tight junction organization. AFM and confocal microscopy revealed changes consistent with transient modulation of epithelial barrier integrity. Functional transcytosis studies showed that P80 significantly enhanced the transport of co-incubated macromolecular cargo across polarized Caco-2 monolayers, resulting in approximately a fivefold increase in translocation compared with untreated controls.

These findings demonstrate that P80 functions as a non-toxic modulator of epithelial barrier permeability capable of enhancing paracellular transport of large biomolecules. Unlike classical hemagglutinin-associated neurotoxin proteins, P80 lacks hemagglutination activity while retaining the ability to reversibly alter tight junction dynamics. The results suggest that P80 contributes to BoNT/E intestinal absorption and may serve as a promising bioenhancer for improving mucosal delivery of therapeutic proteins and other hydrophilic macromolecules. Further mechanistic and in vivo studies are warranted to evaluate its translational potential.