Identification and characterization of functional subunits of <i>Clostridium botulinum</i> type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes

Fujinaga, Yukako; Inoue, Kaoru; Nomura, Takako; Sasaki, Junzo; Marvaud, Jean-Christophe; Popoff, Michel R.; Kozaki, Shunji; Oguma, Keiji

doi:10.1016/s0014-5793(00)01147-9

Cited by 103 publications

(89 citation statements)

References 20 publications

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“…At present, the nontoxic components of the botulinum progenitor toxin are considered to be critical to elicit food poisoning: the NTNHA protects the NT from acidic conditions and proteases in the stomach (6) and the HA component facilitates effective absorption of the progenitor toxin to the epithelial cells in the intestine (31,32). Thus the botulinum progenitor toxin is a unique example of a protein complex where nontoxic components protect the NT against the gastrointestinal tract.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin

Kouguchi¹,

Watanabe²,

Sagane³

et al. 2002

Journal of Biological Chemistry

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Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17. Botulinum neurotoxin (NT)1 is produced by the anaerobic, Gram-positive bacterium Clostridium botulinum as seven related but serologically distinct proteins, designated by the seven serotypes A through G, and is known to be a potent toxin. After ingestion of NT-contaminated food, the NT passes through the gastrointestinal tract and ultimately reaches the neuromuscular junctions. NT binds to the presynaptic membrane and is internalized by receptor-mediated endocytosis into the nerve cell where it cleaves specific sites on its target proteins (synaptobrevin/vesicle-associated membrane protein, syntaxin, and SNAP-25) through its Zn 2ϩ -endopeptidase activity and then blocks the docking and fusion of synaptic vesicles, leading to the inhibition of neurotransmitter release (1, 2). This process causes muscular paralysis in human and animals leading to the botulinum disease state.The NT molecule (ϳ150 kDa) is ordinarily part of a complex formed by noncovalent association with other proteins, including a single nontoxic-nonhemagglutinin (NTNHA) subunit and/or a member of a family of hemagglutinin (HA) proteins (3, 4). The complex, designated as the progenitor toxin, is found in three forms with molecular masses of 900 kDa (LL toxin for type A), 500 kDa (L toxin for types A to D and G), and 300 kDa (M toxin for types A to F) depending on the serotype (5, 6). Previous experiments have demonstrated that the progenitor toxin complex protects the toxin during exposure to harsh conditions. Most proteins are degraded into short peptides and amino acids in the stomach and small intestine during the process of digestion. However, the progenitor toxin is exposed to the acidic (pH 2) gastric juice containing the protease pepsin in the stomach and then enters the small intestine, where it enco...

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Section: Discussionmentioning

confidence: 99%

In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin

Kouguchi¹,

Watanabe²,

Sagane³

et al. 2002

Journal of Biological Chemistry

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“…Only the 16 S toxin bound obviously to the epithelial cells and time to death after injection with 16 S toxin was measurably shorter compared with 12 S toxin or neurotoxin [13]. Furthermore, in a way comparable to 16 S toxin, recombinant proteins of HA1 and HA3 could bind both epithelial cells and erythrocytes [14,15]. Some monoclonal antibodies against HA1 reduced the 16 S toxin binding to the epithelial cells and neutralised the oral toxicity of small amounts of 16 S toxin [16].…”

Section: Introductionmentioning

confidence: 99%

Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component

Mahmut¹,

Inoue

Fujinaga³

et al. 2002

Journal of Medical Microbiology

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Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C-and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C-or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C-and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

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“…One of the reasons for this phenomenon is the protective effect of NAPs toward BoNT in the toxin complex against the low pH and proteases in the digestive tract (Sakaguchi et al, 1984). Meanwhile, the direct interaction of HA proteins with the intestinal epithelium via cell-surface glycoconjugates (Fujinaga et al, 1997(Fujinaga et al, , 2000Kojima et al, 2005;Nakamura et al, 2007) has been reported. In addition, in a human intestinal epithelial cell line (HT-29), the cell surface sialic acid-containing Olinked glycoproteins that are recognized by the type C 16S toxin have been shown to induce the internalization of the toxin into the cells (Nishikawa et al, 2004;Uotsu et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins – differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C

et al. 2009

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Orally ingested botulinum neurotoxin (BoNT) causes food-borne botulism, but BoNT must pass through the gut lining and enter the bloodstream. We have previously found that type B haemagglutinin (HA) proteins in the toxin complex play an important role in the intestinal absorption of BoNT by disrupting the paracellular barrier of the intestinal epithelium, and therefore facilitating the transepithelial delivery of BoNT. Here, we show that type A HA proteins in the toxin complex have a similar disruptive activity and a greater potency than type B HA proteins in the human intestinal epithelial cell lines Caco-2 and T84 and in the canine kidney epithelial cell line MDCK I. In contrast, type C HA proteins in the toxin complex (up to 300 nM) have no detectable effect on the paracellular barrier in these human cell lines, but do show a barrier-disrupting activity and potent cytotoxicity in MDCK I. These findings may indicate that type A and B HA proteins contribute to the development of food-borne botulism, at least in humans, by facilitating the intestinal transepithelial delivery of BoNTs, and that the relative inability of type C HA proteins to disrupt the paracellular barrier of the human intestinal epithelium is one of the reasons for the relative absence of food-borne human botulism caused by type C BoNT.

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Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes

Cited by 103 publications

References 20 publications

In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin

In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin

Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component

Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins – differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C

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