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.

Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular 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 ß-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.

Stability of toxigenicity in proteolytic Clostridium botulinum type B upon serial passage.

Proteolytic Clostridium botulinum type B strains were investigated for stability of toxigenicity and bont/b gene upon serial passage. Strains with bont/b gene located on their plasmids showed loss or decrease of toxigenicity during serial passage. Some strains lost the bont/b gene-encoding plasmid. The stability of the plasmids varied between strains.

Molecular properties of each subcomponent in Clostridium botulinum type B haemagglutinin complex.

The role of each subcomponent of Clostridium botulinum serotype B haemagglutinin (HA), which is one component of 16S toxin, and consists of four subcomponents (HA1, 2, 3a, and 3b), was investigated. In order to identify the subcomponent contributing to the stability of a neurotoxin in the gastro-intestinal tract, each recombinant HA (rHA) subcomponent was incubated with gastro-intestinal proteases. Although rHA1 and rHA3 were stable to these proteases except for specific cleavage, rHA2 was not. Anti-free whole HA serum reacted with neither rHA2 nor HA2 in 16S toxin on both Western blot and ELISA, while anti-rHA2 serum reacted with both rHA2 and HA2 in 16S toxin on Western blots, although it did not react with 16S toxin in ELISA. Binding or haemagglutination activity against erythrocytes was found in rHA1 and rHA3, but not in rHA2. In addition, only HA1 bound to the intestinal section. These results indicate that the HA (and 16S toxin) complex is assembled in the way that HA1 and HA3 (HA3a plus HA3b) encase HA2, followed by modification with trypsin-like bacterial protease, leading to the conclusion that HA1 and HA3 act as protective factors for the neurotoxin and as attachment factors to host cells.

Comparison of electrochemiluminescence assay and ELISA for the detection of Clostridium botulinum type B neurotoxin.

We compared the ELISA and electrochemiluminescence (ECL) immunoassay technologies for the detection of botulinum type B neurotoxin (BotNT B), which requires highly sensitive techniques due to its potent biological activity. BotNT B complexes are the naturally secreted form of the toxin, approximately a third of which consists of the neurotoxin itself; they were aliquoted and frozen for this study. Results of both techniques were interpreted with the same standard statistical tests (ANOVA and Tukey). We first compared two commercial assays for BotNT B: the detection limit of the colorimetric ELISA was 1.56 ng/ml BotNT B complexes versus 0.39-0.78 ng/ml in the ECL test. We then used the same monoclonal antibody and the same polyclonal antibody, respectively purified by protein A and protein G chromatography, to optimize an in-house ELISA test and an in-house ECL test, making it possible to directly compare the two technologies without interference due to the properties of the antibodies used in the two tests. The colorimetric in-house ELISA had a detection threshold of 3.12 ng/ml versus the in-house ECL test whose detection threshold was 0.78-1.56 ng/ml. Thus, in both cases, the ECL assay was two to four times more sensitive than the colorimetric ELISA. The ECL assay was also more rapid (2.5 h for the in-house ECL versus 5 h for in-house ELISA with precoated wells). Overall, these elements can be used to compare the qualities of the two technologies, at least for the detection of protein antigens such as toxins.