Abstract
Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis (botulism), which in most cases enter the organism via the digestive tract and then disseminate into the blood or lymph circulation to target autonomic and motor nerve endings. The passage way of BoNTs alone or in complex forms with associated nontoxic proteins through the epithelial barrier of the digestive tract still remains unclear. Here, we show using an in vivo model of mouse ligated intestinal loop that BoNT/B alone or the BoNT/B C-terminal domain of the heavy chain (HCcB), which interacts with cell surface receptors, translocates across the intestinal barrier. The BoNT/B or HCcB translocation through the intestinal barrier occurred via an endocytosis-dependent mechanism within 10-20 min, because Dynasore, a potent endocytosis inhibitor, significantly prevented BoNT/B as well as HCcB translocation. We also show that HCcB or BoNT/B specifically targets neuronal cells and neuronal extensions in the intestinal submucosa and musculosa expressing synaptotagmin, preferentially cholinergic neurons and to a lower extent other neuronal cell types, notably serotonergic neurons. Interestingly, rare intestinal epithelial cells accumulated HCcB suggesting that distinct cell types of the intestinal epithelium, still undefined, might mediate efficient translocation of BoNT/B.
Highlights
Clostridium botulinum produces potent neurotoxins which are responsible for severe neuroparalytic illness in man and animals resulting from inhibition of spontaneous and nerve-evoked acetylcholine (ACh) release at cholinergic nerve endings
HCc domain of clostridial neurotoxins (BoNTs and tetanus neurotoxin), which interacts with cell receptors, has already been extensively used to investigate internalization and intracellular trafficking of the neurotoxins in neuronal cells, as well as toxin translocation through intestinal epithelial cells (Lalli et al, 2003, Maksymowych et al, 2004, Bohnert et al, 2005, Roux et al, 2006, Harper et al, 2011, Restani et al, 2012, Lam et al, 2015)
Albeit the in vivo experiments at different incubation time periods did not allow to determine a precise kinetics of the toxin passage through the intestinal barrier, they permitted to evidence the progressive translocation of HCcB lasting 5 to 20 min from the intestinal lumen to target structures in the submucosa and musculosa
Summary
Clostridium botulinum produces potent neurotoxins (botulinum neurotoxins, BoNTs) which are responsible for severe neuroparalytic illness (botulism) in man and animals resulting from inhibition of spontaneous and nerve-evoked acetylcholine (ACh) release at cholinergic nerve endings. In infant botulism and some adult cases, ingested C. botulinum spores develop in the intestinal content and produce the toxin in situ (Tacket et al, 1989, Sobel, 2005). In both forms, foodborne botulism and botulism by intestinal colonization, BoNT escapes the gastro-intestinal tract to reach the target cholinergic nerve endings, possibly through the blood and lymph circulation (Maksymowych et al, 1999). Botulinum complexes formed by BoNT and associated non-toxic proteins (ANTPs) including hemagglutinin (HA) components have reported to pass across the epithelial barrier upon HA-mediated opening of intercellular junctions. We investigated the passage of BoNT/B in the mouse intestinal mucosa since it is a major cause of foodborne botulism and infant botulism in various countries (Fox et al, 2005, Peck, 2009, Malaska, 2014, Mazuet et al, 2014)
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