Differential entry of botulinum neurotoxin A into neuronal and intestinal cells

Botulinum neurotoxins (BoNTs) are often acquired from the digestive tract and specifically target neuromuscular junctions where they cause an inhibition of acetylcholine release. A transcytotic mechanism has been evidenced in epithelial intestinal cells, which delivers whole BoNTs across the intestinal barrier, whereas BoNTs enter motoneurons through a pathway that permits the translocation of light chain into the cytosol. We used fluorescent BoNT/A C-terminal part of H chain (Hc) that mediates toxin binding to cell receptors to monitor toxin entry into NG108-15 neuronal cells as well as into Caco-2 and m-IC(cl2) intestinal cells. BoNT/A Hc receptors were found to be distributed in membrane structures closely associated to cholesterol-enriched microdomains, but distinct from detergent-resistant microdomains in both cell types. BoNT/A Hc was trapped into endocytic vesicles, which progressively migrated to a perinuclear area in NG108-15 cells, and in a more scattered manner in intestinal cells. In both cell types, BoNT/A Hc entered through a dynamin- and intersectin-dependent pathway, reached an early endosomal compartment labelled with early endosome antigen 1. In neuronal cells, BoNT/A Hc entered mainly via a clathrin-dependent pathway, in contrast to intestinal cells where it followed a Cdc42-dependent pathway, supporting a differential toxin routing in both cell types.