Differential entry of botulinum neurotoxin A into neuronal and intestinal cells

Abstract

SummaryBotulinum 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.

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

“…Dynamin is an essential protein implicated in a number of endocytic pathways, including receptormediated endocytosis and synaptic vesicle recycling (29,49). Recently, dynamin was suggested to be involved in the uptake of various other clostridial toxins (28,45,46,50). This can now be confirmed through the use of the recently developed dynamin inhibitors (31, 51-55) such as Dynasore (47) and Dyngo-4a (30).…”

“…Overall, our data establish that despite the availability of multiple cellular routes, BoNT/A primarily uses dynamin-dependent processes to enter neurons, mainly via the synaptic vesicle endocytic pathway to block synaptic transmission. The use of Alexa Fluor 488-BoNT/A-Hc to study holotoxin trafficking is warranted by several other studies using recombinant binding domains from various Clostridium neurotoxins (18,28,56). In good agreement with the recent discovery that BoNT/A binds to the intraluminal domain of the synaptic vesicle protein SV2 (17), we detected a high level of co-localization between Alexa Fluor 488-BoNT/A-Hc and VAMP2 in hippocampal neurons.…”

“…Protein Expression-The His 6 -tagged BoNT/A-Hc was purified using Co IDA-agarose (Scientifix) as described previously (28) and conjugated to Alexa Fluor 488 C 5 maleimide (Invitrogen) according to the manufacturer's instructions.…”

“…Successful conjugation of BoNT/A-Hc to Alexa Fluor 488 was checked by UV illumination of the Western blot membrane ( Fig. 1A) (28).…”

“…BoNT/A has recently been shown to enter an early endosomal compartment in a neuroblastoma cell line (28), suggesting that this trafficking pathway could also contribute to its pathogenicity. In view of the role played by dynamin-mediated fission of endocytic vesicles from the plasma membrane (29), we hypothesized that dynamin might play an important role in initiating the neuronal uptake of BoNT/A.…”