Characterization of the binding of botulinum type B 16S toxin to human intestinal epithelial cells

Botulinum neurotoxin produced by Clostridium botulinum type B is a complex of 12S and 16S toxins. 12S toxin consists of a neurotoxin and a nontoxic non-HA (NTNH). The 16S toxin consists of a neurotoxin, an NTNH, and a hemagglutinin (HA). Food-borne botulism is caused by these complex toxins, which are ingested orally and absorbed from the digestive tract across the epithelial barrier lining the gut. Here we show that the type B 16S toxin, but not the 12S toxin or the neurotoxin, binds to the T84 human intestinal epithelial cell line. We also demonstrate that the HA moiety in the 16S toxin mediates the toxin binding to the cells. The carbohydrates containing a galactose moiety inhibited the binding of the 16S toxin to the T84 cells, and neuraminidase treatment of the cells increased the 16S toxin binding. The binding of the 16S toxin to the neuraminidase-treated cells was also inhibited by carbohydrates containing a galactose moiety. These results suggest that the type B 16S toxin binds to human intestinal epithelial cells via the galactose moiety in the carbohydrate chain on the cell surface

Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine

Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90–120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined

The botulinum toxin complex meets E-cadherin on the way to its destination

Botulinum neurotoxin (BoNT) causes the disease botulism, which is characterized by flaccid paralysis in humans and animals. The metalloprotease activity of BoNT inhibits neurotransmitter release at neuro-muscular junctions. In most cases, poisoning occurs when BoNT is ingested. Therefore, BoNT must pass through the epithelial barrier of the gastrointestinal tract to enter the systemic circulation and reach the target site. BoNT forms large protein complexes by associating with non-toxic components referred to as non-toxic non-hemagglutinin (NTNH) and hemagglutinin (HA). These proteins protect BoNT from the low pH and proteases in the digestive tract. We recently determined that HA has an unexpected function of disrupting the intercellular epithelial barrier by directly binding to E-cadherin. HA binds to E-cadherin and disrupts its function in a species-specific manner, and this interaction is essential to disrupt tight junctions. This activity is thought to facilitate the absorption of BoNT through the paracellular route of the intestinal epithelium in susceptible species