Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Botulinum Neurotoxin (BoNT) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a mean residue weight ellipticity of ?1.8 × 10⁵° cm²/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the T_m for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.     

Enhancement of the endopeptidase activity of botulinum neurotoxin by its associated proteins and dithiothreitol.

Botulinum neurotoxins type A (BoNT/A), the most toxic substance known to man, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs), possibly through a polycistronic expression of a clustered group of genes. The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract. We now report that NAPs also potentiate the Zn2+ endopeptidase activity of BoNT/A in both in vitro and in vivo assays against its known intracellular target protein, 25 kDa synaptosomal associated protein (SNAP-25). While BoNT/A exhibited no protease activity prior to reduction with dithiothreitol (DTT), the BoNT/A complex exhibited a high protease activity even in its nonreduced form. Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment. Structural features of BoNT/A change considerably upon disulfide reduction, as revealed by near-UV circular dichroism spectroscopy. BoNT/A in the reduced form adopts a more flexible structure than in the unreduced form, as also indicated by large differences in DeltaH values (155 vs 248 kJ mol-1) of temperature-induced unfolding of BoNT/A.     

Hemagglutinin-33 of type A botulinum neurotoxin complex binds with synaptotagmin II

Botulinum neurotoxin type A (BoNT/A), the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. NAPs are known to protect BoNT against adverse environmental conditions and proteolytic digestion. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs that is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. However, it is not known whether Hn-33 plays any role other than the protection of BoNT. Using immunoaffinity column chromatography and pull-down assays, we have now discovered that Hn-33 binds to synaptotagmin II, the putative receptor of botulinum neurotoxin. This finding provides important information relevant to the design of novel anti-botulism therapeutic agents targeted to block the entry of botulinum neurotoxin into nerve cells.     

Analysis of the Botulinum Neurotoxin Type F Gene Clusters in Proteolytic and Nonproteolytic Clostridium botulinum and Clostridium barati

Comparison of genes encoding type F botulinum neurotoxin progenitor complex in strains of proteolytic Clostridium botulinum strain Langeland, nonproteolytic Clostridium botulinum strain 202F, and Clostridium barati strain ATCC 43256 reveals an identical organization of genes encoding a protein of molecular mass of approx. 47 kDa (P-47), nontoxic-nonhemagglutinin (NTNH) and botulinum toxin (BoNT). Although homology between the protein components of the complexes encoded by these different species all producing botulinum neurotoxin type F is considerable (approx. 69–88% identity), exceptionally high homology is observed between the C-termini of the P-47s (approx. 96% identity) and the NTNHs (approx. 94% identity) encoded by Clostridium botulinum type F strain Langeland and Clostridium botulinum type A strain Kyoto. Such a region of extremely high sequence identity is strongly indicative of recombination in these strains synthesizing botulinum neurotoxins of different antigenic types. Received: 13 April 1998 / Accepted: 9 May 1998     

High level expression,purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E

Botulinum neurotoxin type E heavy chain consists of two domains: N-terminal half as a translocation domain and C-terminal half (Hcc) as a binding domain. In this research a synthetic gene fragment encoding the binding domain of botulinum neurotoxin type E (BoNT/E-Hcc) was highly expressed in Escherichia coli by pGEX4T-1 vector. After purification, the recombinant BoNT/E-Hcc was evaluated by SDS-PAGE and western blot (immunoblot) analysis. Average yields obtained in this research were 3.7 mg recombinant BoNT/E-Hcc per liter of bacterial culture. The recombinant protein was injected in mice for study of its protection ability against botulinum neurotoxin type E challenges. The challenge studies showed that, vaccinated mice were fully protected against 10⁴ × minimum lethal dose of botulinum neurotoxin type E.     

Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD₅₀]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD₅₀) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD₅₀), and 117 pg/ml for BoNT/F (less than 1 LD₅₀) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.     

Expression and stability of the nontoxic component of the botulinum toxin complex

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex associated with nontoxic-nonhemagglutinin (NTNHA) and/or hemagglutinin components. In the present study, high-level expression of full-length (1197 amino acids) rNTNHA from C. botulinum serotype D strain 4947 (D-4947) was achieved in an Escherichia coli system. Spontaneous nicking of the rNTNHA at a specific site was observed during long-term incubation in the presence of protease inhibitors; this was also observed in natural NTNHA. The rNTNHA assembled with isolated D-4947 BoNT with molar ratio 1:1 to form a toxin complex. The reconstituted toxin complex exhibited dramatic resistance to proteolysis by pepsin or trypsin at high concentrations, despite the fact that the isolated BoNT and rNTNHA proteins were both easily degraded. We provide definitive evidence that NTNHA plays a crucial role in protecting BoNT, which is an oral toxin, from digestion by proteases common in the stomach and intestine.     

Subunit Stoichiometry of the Clostridium botulinum Type A Neurotoxin Complex Determined Using Denaturing Capillary Electrophoresis

A denaturing capillary electrophoresis method was developed to evaluate the subunit stoichiometry of the Clostridium botulinum type A neurotoxin complex. The results indicate that the neurotoxin complex contains single copies of the 150 kDa neurotoxin and the non-toxic non-hemagglutinating subunits as well as 5–6 HA17, 4–5 HA23, 3–4 HA48, and 8–9 HA34 subunits. The calculated molecular mass for a complex with this stoichiometry is between 880 and 1,000 kDa. The molecular mass of the intact complex was determined using size-exclusion HPLC (SE-HPLC) and SE-HPLC in conjunction with multi-angle laser light scattering detection. Based on a comparison to a mixture of standard proteins, SE-HPLC analysis yielded a molecular mass of 880 kDa while light scattering analysis indicated a weight average molecular mass of 925 ± 45 kDa. The close agreement between the molecular mass values determined by the three approaches supports the subunit stoichiometry proposed for the C. botulinum type A neurotoxin complex.     

Comparative role of neurotoxin-associated proteins in the structural stability and endopeptidase activity of botulinum neurotoxin complex types A and E

Seven serotypes of botulinum neurotoxins, the most toxic substances known to mankind, are each produced by different strains of Clostridium botulinum along with a group of neurotoxin-associated proteins (NAPs). NAPs play a critical role in the toxicoinfection process of botulism in addition to their role in protecting the neurotoxin from proteolytic digestion in the GI tract as well as from adverse environmental conditions. In this study we have investigated the effect of temperature on the structural and functional stability of BoNT/A complex (BoNT/AC) and BoNT/E complex (BoNT/EC). Although the NAPs in the two complexes are quite different, both groups of NAPs activate the endopeptidase activities of their BoNTs without any need to reduce the disulfide bonds between light and heavy chains of respective BoNTs. BoNT/AC attains optimum enzyme activity at the physiological temperature of 37 degrees C whereas BoNT/EC is maximally active at 45 degrees C, and this is accompanied by conformational alterations in its polypeptide folding at this temperature, leading to favorable binding with its intracellular substrate, SNAP-25, and subsequent cleavage of the latter. BoNT/A in its complex form is found to be structurally more stable against temperature whereas BoNT/E in its complex form is functionally better protected against temperature. Based on the analysis of isolated NAPs we have observed that the structural stability of the BoNT/AC is contributed by the NAPs. In addition to the unique structural conditions in which the enzyme remains active, functional stability of botulinum neurotoxins against temperature plays a critical role in the survival of the agent in cooked food and in food-borne botulism.     

Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A–G) gene complex

Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A–G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A–G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n = 27). Using purified DNA, the assay had a sensitivity of 4–95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 10²–10³ CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days).     

Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia

Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins. Botulinum neurotoxins associate with neurotoxin‐associated proteins (NAPs) forming large complexes that are protected from the harsh environment of the gastrointestinal tract. However, it is still unclear how BoNT complexes as large as 900 kDa traverse the epithelial barrier and what role NAPs play in toxin translocation. In this study, we examined the transit of BoNT serotype A (BoNT/A) holotoxin, complex and recombinantly purified NAP complex through cultured and polarized Caco‐2 cells and, for the first time, in the small mouse intestine. Botulinum neurotoxin serotype A and NAPs in the toxin complex were detectable inside intestinal cells beginning at 2 h post intoxication. Appearance of the BoNT/A holotoxin signal was slower, with detection starting at 4–6 h. This indicated that the holotoxin alone was sufficient for entry but the presence of NAPs enhanced the rate of entry. Botulinum neurotoxin serotype A detection peaked at approximately 6 and 8 h for complex and holotoxin, respectively, and thereafter began to disperse with some toxin remaining in the epithelia after 24 h. Purified HA complexes alone were also internalized and followed a similar time course to that of BoNT/A complex internalization. However, recombinant HA complexes did not enhance BoNT/A holotoxin entry in the absence of a physical link with BoNT/A. We propose a model for BoNT/A toxin complex translocation whereby toxin complex entry is facilitated by NAPs in a receptor‐mediated mechanism. Understanding the intestinal uptake of BoNT complexes will aid the development of new measures to prevent or treat oral intoxications.     

Characterization of the Genes Encoding the Botulinum Neurotoxin Complex in a Strain of Clostridium botulinum Producing Type B and F Neurotoxins

The organization of the clusters of genes encoding proteins of the botulinum neurotoxin (BoNT) progenitor complex was elucidated in a strain of Clostridium botulinum producing type B and F neurotoxins. With PCR and sequencing strategies, the type B BoNT-gene cluster was found to be composed of genes encoding BoNT/B, nontoxic nonhemagglutinin component (NTNH), P-21, and the hemagglutinins HA-33, HA-17, and HA-70, whereas the type F BoNT-gene cluster has genes encoding BoNT/F, NTNH, P-47, and P-21. Comparative sequence analysis showed that BoNT/F in type BF strain 3281 shares highest homology with BoNT/F of non-proteolytic (group II) C. botulinum whereas NTNH and P-21 in the type F cluster of strain 3281 are more similar to the corresponding proteins in proteolytic (group I) type F C. botulinum. These findings indicate diverse evolutionary origins for genes encoding BoNT/F and its associated non-toxic proteins, although the genes are contiguous. By contrast, sequence comparisons indicate that genes encoding BoNT/B and associated non-toxic proteins in strain 3281 possess a similar evolutionary origin. It was demonstrated that the genes present in the BoNT/B gene cluster of this type BF strain show exceptionally high homology with the equivalent genes in the silent BoNT/B gene cluster of C. botulinum type A(B), possibly indicating their common ancestry. Received: 30 March 1998 / Accepted: 21 May 1998     

Cloning,Expression, Purification,and Characterization of Biologically Active Recombinant Hemagglutinin-33, Type A Botulinum Neurotoxin Associated Protein

Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA⁺ OrfX⁻) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA⁻ OrfX⁺) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA⁺ OrfX⁻ cluster (69A and 32A) and one strain with the HA⁻ OrfX⁺ cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.     

Differentiation of Clostridium botulinum Serotype A Strains by Multiple-Locus Variable-Number Tandem-Repeat Analysis

Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.     

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.