Takuhiro Matsumura

The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption.

The type B botulinum neurotoxin (BoNT) elicits flaccid paralysis and death in humans by intoxicating peripheral nerves after oral absorption. Here, we examine the function of the haemagglutinin (HA), a non‐toxic component of the large 16S BoNT complex. We find that the HA acts in the intestine to disrupt epithelial barrier function by opening intercellular tight and adherens junctions. This allows transport of BoNT and other large solutes into the systemic circulation and explains how the type B BoNT complexes are efficiently absorbed. In vitro, HA appears to act on the epithelial cell via the basolateral membrane only, suggesting the possibility of another step in the absorptive process. These studies show that the 16S BoNT complex is a multifunctional protein assembly equipped with the machinery to efficiently breach the intestinal barrier and act systemically on peripheral nerves.

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.

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.

Uptake of Botulinum Neurotoxin in the Intestine

Foodborne and intestinal botulism are the most common forms of human botulism; both result from the absorption of botulinum neurotoxin (BoNT) from the digestive tract into the circulation. BoNT is a large protein toxin (approximately 150 kDa), but it is able to pass through the epithelial barrier in the digestive tract. Recent cellular and molecular biology studies have begun to unravel the mechanisms by which this large protein toxin crosses the intestinal epithelial barrier. This review provides an overview of current knowledge relating to the absorption of botulinum toxins (BoNT and BoNT complex) from the gastrointestinal tract, with particular emphasis on the interaction of these toxins with the intestinal epithelial barrier.

Crystal Structure of Clostridium botulinum Whole Hemagglutinin Reveals a Huge Triskelion-shaped Molecular Complex

Background: Botulinum HA is a component of the botulinum neurotoxin complex and dramatically increases the oral toxicity of the complex. Results: The crystal structure of botulinum HA reveals that 12 subcomponents of HA constitute a huge triskelion-shaped molecule. Conclusion: The complex is functionally and structurally separable into two parts. Significance: This is the first crystal structure of the whole botulinum HA complex. Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of β-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.

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

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.

A novel function of botulinum toxin-associated proteins: HA proteins disrupt intestinal epithelial barrier to increase toxin absorption.

Food-borne botulinum neurotoxin (BoNT) in the gastrointestinal lumen must cross an epithelial barrier to reach peripheral nerves to mediate its toxicity. The detailed mechanism by which BoNT traverses this barrier remains unclear. We found that hemagglutinin (HA) proteins of type B BoNT complex play an important role in the intestinal absorption of BoNT, disrupting the paracellular barrier of intestinal epithelium, which facilitates transepithelial delivery of BoNT both in vitro and in vivo (Matsumura, T., et al., 2008. Cell. Microbiol. 10, 355-364). We also found that type A HA proteins have a similar disrupting activity with a greater potency than type B HA proteins in the human intestinal epithelial cell lines Caco-2 and T84. 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. These results may indicate that types A and B HA contribute to develop the food-borne human botulism by facilitating the intestinal transepithelial delivery of BoNTs.

Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.

Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3

Botulinum neurotoxin is conventionally divided into seven serotypes, designated A–G, and is produced as large protein complexes through associations with non‐toxic components, such as hemagglutinin (HA) and non‐toxic non‐HA. These non‐toxic proteins dramatically enhance the oral toxicity of the toxin complex. HA is considered to have a role in toxin transport through the intestinal epithelium by carbohydrate binding and epithelial barrier‐disrupting activity. Type A and B HAs disrupt E‐cadherin‐mediated cell adhesion, and, in turn, the intercellular epithelial barrier. Type C HA (HA/C) disrupts the barrier function by affecting cell morphology and viability, the mechanism of which remains unknown. In this study, we identified GM3 as the target molecule of HA/C. We found that sialic acid binding of HA is essential for the activity. It was abolished when cells were pre‐treated with an inhibitor of ganglioside synthesis. Consistent with this, HA/C bound to a‐series gangliosides in a glycan array. In parallel, we isolated clones resistant to HA/C activity from a susceptible mouse fibroblast strain. These cells lacked expression of ST‐I, the enzyme that transfers sialic acid to lactosylceramide to yield GM3. These clones became sensitive to HA/C activity when GM3 was expressed by transfection with the ST‐I gene. The sensitivity of fibroblasts to HA/C was reduced by expressing ganglioside synthesis genes whose products utilize GM3 as a substrate and consequently generate other a‐series gangliosides, suggesting a GM3‐specific mechanism. Our results demonstrate that HA/C affects cells in a GM3‐dependent manner.

Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption: Multivalency effect of HA

Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E‐cadherin binding. It is known that HA forms a three‐arm structure, in which each of three protomers has three carbohydrate‐binding sites and one E‐cadherin‐binding site. A three‐arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier‐disrupting activity. We prepared type B full‐length HA (three‐arm form) and mini‐HA, which is a deletion mutant lacking the trimer‐forming domain. Size‐exclusion chromatography analysis showed that mini‐HA exists as dimers (two‐arm form) and monomers (one‐arm form), which are then separated. We examined the multivalency effect of HA on the barrier‐disrupting activity, the E‐cadherin‐binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco‐2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier‐disrupting activity in Caco‐2 cells. We found that basal cell surface attachment and binding ability to immobilized E‐cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier‐disrupting activity.

Crystallization and preliminary crystallographic studies of the HA3 subcomponent of the type B botulinum neurotoxin complex

The haemagglutinin subcomponent HA3 of the type B botulinum neurotoxin complex, which is important in toxin absorption from the gastrointestinal tract, has been expressed, purified and subsequently crystallized in two crystal forms at different pH values. Form I belonged to space group R32, with unit-cell parameters a = b = 357.4, c = 249.5 Å, α = β = 90, γ = 120°. Form II belonged to space group I4(1)32, with unit-cell parameters a = b = c = 259.0 Å, α = β = γ = 90°. Diffraction data were collected from these crystals to a resolution of 3.0 Å for both form I and form II.