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

Abstract

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.

Translocation and dissemination to target neurons of botulinum neurotoxin type B in the mouse intestinal wall

“…Botulinum complexes formed by BoNT and associated nontoxic proteins (ANTPs) including haemagglutinin (HA) components have reported to pass across the epithelial barrier upon HA-mediated opening of intercellular junctions. BoNT/A seems to preferentially use specific subsets of intestinal cells such as crypt enteroendocrine cells to enter the intestinal mucosa (Couesnon et al, 2012). However, purified BoNT has also been shown to undergo transcytosis through intestinal epithelial cells without the help of HAs (Fujinaga et al, 1997;Maksymowych and Simpson, 1998;Park and Simpson, 2003;Maksymowych and Simpson, 2004;Nishikawa et al, 2004;Ahsan et al, 2005;Couesnon et al, 2008).…”

Probiotic Microorganisms Inhibit Epithelial Cell Internalization of Botulinum Neurotoxin Serotype A

“…Previous work showed that the majority of toxin absorption occurs in the mouse upper small intestine [7,32]. One model for the transit of BoNTs suggests that the holotoxin itself can transcytose through the intestinal epithelium [33,34,35]. A second model for the BoNT absorption from the epithelium implicates the hemagglutin proteins (HA) in this process by binding cell surface receptors on the apical side, transcytosis, and potential disruption of the epithelial barrier at the basolateral side to allow for paracellular transport of the toxins in certain situations [33,36].…”

Endomicroscopy and electromyography of neuromuscular junctions in situ

“…Perhaps by increasing the photon yield of fluorescent ligands bound to the BoNTx/A heavy chain, or to its receptor in nerve terminal membranes, will enable this attractive molecular target to be exploited more effectively and to achieve its full potential for applications to CEM imaging. Quantum yield of fluorescence could be increased by uniting HcBoNTx/A with other conjugates 66. Alternatively, targeting fluorescent ligands to other receptors in presynaptic terminals could improve the contrast and intensity of the motor nerve terminal fluorescence.…”