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Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.

Matsumura T, Sugawara Y, Yutani M, Amatsu S, Yagita H, Kohda T, Fukuoka S, Nakamura Y, Fukuda S, Hase K, Ohno H, Fujinaga Y - Nat Commun (2015)

Bottom Line: Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2).HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers.Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamada-oka, Suita, Osaka 565-0871, Japan.

ABSTRACT
To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

No MeSH data available.


Related in: MedlinePlus

L-PTC is taken up by Peyer’s patch M cells.(a) Schematic representation of botulinum neurotoxin complexes. (b) Various concentrations of toxins were intragastrically (M-PTC 6.0 pmol: 1.72 μg, 60 pmol: 17.2 μg, L-PTC 0.6 pmol: 0.45 μg, 6 pmol: 4.5 μg, BoNT 60 pmol: 9.0 μg) or intraperitoneally (M-PTC 0.013 fmol: 3.85 pg, 0.13 fmol: 38.5 pg, L-PTC 0.013 fmol: 10 pg, 0.13 fmol: 100 pg, BoNT 0.013 fmol: 2.01 pg, 0.13 fmol: 20.1 pg) administered to mice (n=6 per group). (c) Alexa Fluor 488-labelled toxins (green) were injected into ligated mouse intestinal loops containing PPs, and incubated for 2 h. Whole-mount intestinal villous epithelium (VE) regions and FAE regions were stained with Alexa Fluor 568-labelled phalloidin (red). (d) Alexa Fluor 488-labelled toxins or NAPs (NTNHA/HA, green) were injected into ligated mouse intestinal loops; FAE regions were stained with rhodamine-labelled UEA-1 (red). x–z images in lower panels correspond to the positions indicated by dotted lines in the x–y images. Scale bars, 100 μm (c), 10 μm (d). The data in c,d are representative of three independent experiments.
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f1: L-PTC is taken up by Peyer’s patch M cells.(a) Schematic representation of botulinum neurotoxin complexes. (b) Various concentrations of toxins were intragastrically (M-PTC 6.0 pmol: 1.72 μg, 60 pmol: 17.2 μg, L-PTC 0.6 pmol: 0.45 μg, 6 pmol: 4.5 μg, BoNT 60 pmol: 9.0 μg) or intraperitoneally (M-PTC 0.013 fmol: 3.85 pg, 0.13 fmol: 38.5 pg, L-PTC 0.013 fmol: 10 pg, 0.13 fmol: 100 pg, BoNT 0.013 fmol: 2.01 pg, 0.13 fmol: 20.1 pg) administered to mice (n=6 per group). (c) Alexa Fluor 488-labelled toxins (green) were injected into ligated mouse intestinal loops containing PPs, and incubated for 2 h. Whole-mount intestinal villous epithelium (VE) regions and FAE regions were stained with Alexa Fluor 568-labelled phalloidin (red). (d) Alexa Fluor 488-labelled toxins or NAPs (NTNHA/HA, green) were injected into ligated mouse intestinal loops; FAE regions were stained with rhodamine-labelled UEA-1 (red). x–z images in lower panels correspond to the positions indicated by dotted lines in the x–y images. Scale bars, 100 μm (c), 10 μm (d). The data in c,d are representative of three independent experiments.

Mentions: Botulinum neurotoxin (BoNT), which is produced by Clostridium botulinum and related species, is a potent metalloprotease toxin consisting of a large protein (~150 kDa) that binds neuronal cells1. On entering the cytoplasm of these cells, it cleaves SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) required for vesicle fusion, thereby inhibiting neurotransmitter release and causing paralysis1. BoNTs are produced along with one or more neurotoxin-associated proteins (NAPs) that non-covalently associate with BoNT to form progenitor toxin complexes (PTCs)2 (Fig. 1a). The NAPs include non-toxic non-hemagglutinin (NTNHA) and hemagglutinin (HA). HA is composed of three different subcomponents: HA1, HA2 and HA3 (alternatively, HA-33, HA-17 and HA-70, respectively)3. HA1 and HA3 have carbohydrate-binding activities4. C. botulinum type A1 strains produce M-PTC, L-PTC and LL-PTC simultaneously2. M-PTC contains BoNT and NTNHA5, whereas L-PTC consists of BoNT, NTNHA and HA67. LL-PTC is assumed to be a dimer of L-PTC8, and dilution of concentrated LL-PTC leads to dissociation into L-PTC9. Ingestion of foods contaminated with PTCs causes food-borne botulism, the most common form of botulism in adults10. The presence of NAPs in PTCs drastically increases BoNT toxicity following oral administration2. At least three mechanisms possibly involved in this phenomenon have been reported: protection of BoNT by NTNHA and HA against degradation in the gastrointestinal tract211; promotion of binding to intestinal epithelial cells through the carbohydrate-binding activity of HA12 and disruption of the epithelial barrier via an interaction between HA and E-cadherin13141516.


Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.

Matsumura T, Sugawara Y, Yutani M, Amatsu S, Yagita H, Kohda T, Fukuoka S, Nakamura Y, Fukuda S, Hase K, Ohno H, Fujinaga Y - Nat Commun (2015)

L-PTC is taken up by Peyer’s patch M cells.(a) Schematic representation of botulinum neurotoxin complexes. (b) Various concentrations of toxins were intragastrically (M-PTC 6.0 pmol: 1.72 μg, 60 pmol: 17.2 μg, L-PTC 0.6 pmol: 0.45 μg, 6 pmol: 4.5 μg, BoNT 60 pmol: 9.0 μg) or intraperitoneally (M-PTC 0.013 fmol: 3.85 pg, 0.13 fmol: 38.5 pg, L-PTC 0.013 fmol: 10 pg, 0.13 fmol: 100 pg, BoNT 0.013 fmol: 2.01 pg, 0.13 fmol: 20.1 pg) administered to mice (n=6 per group). (c) Alexa Fluor 488-labelled toxins (green) were injected into ligated mouse intestinal loops containing PPs, and incubated for 2 h. Whole-mount intestinal villous epithelium (VE) regions and FAE regions were stained with Alexa Fluor 568-labelled phalloidin (red). (d) Alexa Fluor 488-labelled toxins or NAPs (NTNHA/HA, green) were injected into ligated mouse intestinal loops; FAE regions were stained with rhodamine-labelled UEA-1 (red). x–z images in lower panels correspond to the positions indicated by dotted lines in the x–y images. Scale bars, 100 μm (c), 10 μm (d). The data in c,d are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4339894&req=5

f1: L-PTC is taken up by Peyer’s patch M cells.(a) Schematic representation of botulinum neurotoxin complexes. (b) Various concentrations of toxins were intragastrically (M-PTC 6.0 pmol: 1.72 μg, 60 pmol: 17.2 μg, L-PTC 0.6 pmol: 0.45 μg, 6 pmol: 4.5 μg, BoNT 60 pmol: 9.0 μg) or intraperitoneally (M-PTC 0.013 fmol: 3.85 pg, 0.13 fmol: 38.5 pg, L-PTC 0.013 fmol: 10 pg, 0.13 fmol: 100 pg, BoNT 0.013 fmol: 2.01 pg, 0.13 fmol: 20.1 pg) administered to mice (n=6 per group). (c) Alexa Fluor 488-labelled toxins (green) were injected into ligated mouse intestinal loops containing PPs, and incubated for 2 h. Whole-mount intestinal villous epithelium (VE) regions and FAE regions were stained with Alexa Fluor 568-labelled phalloidin (red). (d) Alexa Fluor 488-labelled toxins or NAPs (NTNHA/HA, green) were injected into ligated mouse intestinal loops; FAE regions were stained with rhodamine-labelled UEA-1 (red). x–z images in lower panels correspond to the positions indicated by dotted lines in the x–y images. Scale bars, 100 μm (c), 10 μm (d). The data in c,d are representative of three independent experiments.
Mentions: Botulinum neurotoxin (BoNT), which is produced by Clostridium botulinum and related species, is a potent metalloprotease toxin consisting of a large protein (~150 kDa) that binds neuronal cells1. On entering the cytoplasm of these cells, it cleaves SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) required for vesicle fusion, thereby inhibiting neurotransmitter release and causing paralysis1. BoNTs are produced along with one or more neurotoxin-associated proteins (NAPs) that non-covalently associate with BoNT to form progenitor toxin complexes (PTCs)2 (Fig. 1a). The NAPs include non-toxic non-hemagglutinin (NTNHA) and hemagglutinin (HA). HA is composed of three different subcomponents: HA1, HA2 and HA3 (alternatively, HA-33, HA-17 and HA-70, respectively)3. HA1 and HA3 have carbohydrate-binding activities4. C. botulinum type A1 strains produce M-PTC, L-PTC and LL-PTC simultaneously2. M-PTC contains BoNT and NTNHA5, whereas L-PTC consists of BoNT, NTNHA and HA67. LL-PTC is assumed to be a dimer of L-PTC8, and dilution of concentrated LL-PTC leads to dissociation into L-PTC9. Ingestion of foods contaminated with PTCs causes food-borne botulism, the most common form of botulism in adults10. The presence of NAPs in PTCs drastically increases BoNT toxicity following oral administration2. At least three mechanisms possibly involved in this phenomenon have been reported: protection of BoNT by NTNHA and HA against degradation in the gastrointestinal tract211; promotion of binding to intestinal epithelial cells through the carbohydrate-binding activity of HA12 and disruption of the epithelial barrier via an interaction between HA and E-cadherin13141516.

Bottom Line: Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2).HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers.Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamada-oka, Suita, Osaka 565-0871, Japan.

ABSTRACT
To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

No MeSH data available.


Related in: MedlinePlus