Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin

Abstract Five monoclonal antibodies (MCA; E–8–2, 9–1, 11–2, 12–4, and 13–1) against Clostridium botulinum type E derivative toxin were prepared. Their ELISA titers were higher than or equivalent to that of conventional polyclonal antibody. Three of them (E-8–2, 12–4, and 13–1) possessed the neutralizing activity comparable to that of polyclonal antibody. The results of binding-competition experiments indicated that the monoclonal antibodies bound to different sites on the type E toxin molecule. Immunoblotting analyses demonstrated that E-8–2, 9–1, and 11–2 react to fragment I (heavy chain) of the toxin. By use of these monoclonal antibodies, it may be possible to scrutinize the structure-function relationship of botulinum toxins and cross reactions between type E and F toxins.     

Immunological Characterization of the Neurotoxin Produced by Clostridium botulinum Type A Associated with Infant Botulism in Japan

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain. This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.     

C terminal half fragment (50 kDa) of heavy chain components of Clostridium botulinum type C and D neurotoxins can be used as an effective vaccine

Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 microg), Hn (5 microg), Hc (5 microg), or a mixture of Hn (5 microg) and Hc (5 microg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10(6) the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10(5) to 10(6) MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 microg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused-or GST eliminatedpreparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 microg, is useful for vaccine trials in mice.     

Production and purification of Clostridium botulinum type C and D neurotoxin

Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay.     

In vitro evaluation methods for Clostridium botulinum type C and D vaccines

Reagents were prepared for use in ELISAs to determine the concentration of the antigenic components of Clostridium botulinum type C and D. The results obtained were compared with the L+dose assay and a good correlation was found between the two assays for measurement of the C and D neurotoxin concentration. These ELISAs were also used to determine the concentration of the neurotoxins in toxoid form. The relationship between the C neurotoxin dose, in toxoid form, and the immune response in guinea pigs could be deduced from the data obtained. The relationship for the D neurotoxin was not that clear, as the same concentration of the antigen resulted in variable potency values. However, these ELISAs can be used to formulate the concentration of the C and D components in the final bivalent vaccine. Replacement of the preliminary potency assay on the monovalent components after production with the in vitro assays will shorten the total production time of the vaccine by about 60 days. The economical and ethical implications are the reduction in the use of animals to evaluate the vaccine.     

Role of nontoxic components of serotype D botulinum toxin complex in permeation through a Caco-2 cell monolayer, a model for intestinal epithelium

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). To assess the role of nontoxic components in the oral intoxication of botulinum TCs, we investigated the permeability of serotype D strain 4947 BoNT and its various TC species through cultured Caco-2 cell monolayers. The L-TC species (complexes composed of BoNT, NTNHA, HA-70, HA-33 and HA-17) showed potent permeability through the cell layer, whereas free BoNT, M-TC (BoNT and NTNHA complexes) and M-TC/HA-70 showed little or no permeability. Cell binding tests demonstrated that HA-33/HA-17 complexes bound to cells, whereas other components did not. These findings suggest that BoNT in the 650-kDa L-TC permeates into the cell mainly in an HA-33/HA-17-mediated manner, although free BoNT can permeate into the cell. As free BoNT and M-TC were susceptible to digestion with gastrointestinal juice, it is likely that L-TC species containing HA-33 caused higher oral toxicity in mice than others. We conclude that the HA-33 subcomponent plays a critical role in the permeation of TCs into intestinal epithelium, and that other HA subcomponents protect BoNT against gastrointestinal digestion.     

Structure and action of the binary C2 toxin from Clostridium botulinum

C2 toxin from Clostridium botulinum is composed of the enzyme component C2-I, which ADP-ribosylates actin, and the binding and translocation component C2-II, responsible for the interaction with eukaryotic cell receptors and the following endocytosis. Three C2-I crystal structures at resolutions of up to 1.75 A are presented together with a crystal structure of C2-II at an appreciably lower resolution and a model of the prepore formed by fragment C2-IIa. The C2-I structure was determined at pH 3.0 and at pH 6.1. The structural differences are small, indicating that C2-I does not unfold, even at a pH value as low as 3.0. The ADP-ribosyl transferase activity of C2-I was determined for alpha and beta/gamma-actin and related to that of Iota toxin and of mutant S361R of C2-I that introduced the arginine observed in Iota toxin. The substantial activity differences between alpha and beta/gamma-actin cannot be explained by the protein structures currently available. The structure of the transport component C2-II at pH 4.3 was established by molecular replacement using a model of the protective antigen of anthrax toxin at pH 6.0. The C-terminal receptor-binding domain of C2-II could not be located but was present in the crystals. It may be mobile. The relative orientation and positions of the four other domains of C2-II do not differ much from those of the protective antigen, indicating that no large conformational changes occur between pH 4.3 and pH 6.0. A model of the C2-IIa prepore structure was constructed based on the corresponding assembly of the protective antigen. It revealed a surprisingly large number of asparagine residues lining the pore. The interaction between C2-I and C2-IIa and the translocation of C2-I into the target cell are discussed.     

Heterogeneities of two components of C2 toxin produced by Clostridium botulinum types C and D

Botulinum C2 toxin (C2T) is composed of two dissimilar protein components, designated components I and II, which are linked with neither covalent nor noncovalent bonds. The heterogeneity of these two components of C2T produced by Clostridium botulinum type C and D strains was examined. Of 21 strains examined, 19 strains produced the two components, while the others produced neither component I nor component II. The 19 producers of C2T could be divided into three groups based on the differences in antigenicity, molecular weight and biological activity of components I and II. The results provide evidence of heterogeneity in the molecular structure of the two components of C2T, which is possibly a cause of the differences in the biological activity of the toxin observed in different strains.     

Procedures for large-scale production and purification of Clostridium botulinum C1 toxin for preparation of toxoid

Abstract Fortified cooked meat medium containing calcium carbonate (CaCO₃-FCM) supported toxin production of a strain of Clostridium botulinum type C to a level of 2 × 10⁶ mouse i.p. LD₅₀/ml. C1 toxin was purified by sequential steps of acid precipitation from 5-fold diluted culture supernatant in the presence of RNA, 2nd acid precipitation by dialysis, removal of RNA by protamine treatment, removal of excess protamine and bufferisation by ultrafiltration through Amicon PM-30 membrane, sulphopropyl-Sephadex chromatography, and Sephadex G-200 gel filtration. By these procedures, 25 mg or more of highly purified C1 toxin was constantly obtained from a lot of 600-ml culture.     

Characterization of nicking of the nontoxic-nonhemagglutinin components of Clostridium botulinum types C and D progenitor toxin

Clostridium botulinum C and D strains produce two types of progenitor toxins, M and L. Previously we reported that a 130-kDa nontoxic-nonhemagglutinin (NTNHA) component of the M toxin produced by type D strain CB16 was nicked at a unique site, leading to a 15-kDa N-terminal fragment and a 115-kDa C-terminal fragment. In this study, we identified the amino acid sequences around the nicking sites in the NTNHAs of the M toxins produced by C. botulinum type C and D strains by analysis of their C-terminal and N-terminal sequences and mass spectrometry. The C-terminus of the 15-kDa fragments was identified as Lys127 from these strains, indicating that a bacterial trypsin-like protease is responsible for the nicking. The 115-kDa fragment had mixtures of three different N-terminal amino acid sequences beginning with Leu135, Val139, and Ser141, indicating that 7–13 amino acid residues were deleted from the nicking site. The sequence beginning with Leu135 would also suggest cleavage by a trypsin-like protease, while the other two N-terminal amino acid sequences beginning with Val139 and Ser141 would imply proteolysis by an unknown protease. The nicked NTNHA forms a binary complex of two fragments that could not be separated without sodium dodecyl sulfate.     

Effects of pH on the binding of Clostridium botulinum type E derivative toxin to gangliosides and phospholipids

Abstract Clostridium botulinum type E derivative toxin directly bound to gangliosides G_T1b, G_D1a, and G_Q1b but not to G_M1 or G_D1b at pH 5.0 or above, At the same pH values, it bound to negatively charged phospholipids but not to noncharged ones. At pH 4.0, it bound to any of gangliosides and phospholipids including G_M1, G_D1a, and non-charged phospholipids. It bound to ceramide, a hydrophobic component of ganglioside and also to sphingomyelin, a phospholipid containing a ceramide moiety, only at pH 4.0. It bound to ceramide and sphingomyelin less firmly than to other phospholipids at pH 4.0. We assume that botulinum toxin adheres to the neural cell surface mainly by sialic acid-specific and charge-dependent binding possibly aided by nonspecific hydrophobic(toxin)-hydrophobic(lipids, mainly phospholipids) interaction.     

C型肉毒毒素的制备及类毒素保护力初探

将C型肉毒梭菌经适宜条件的产毒培养后纯化,并进行相关鉴定。制备的C型肉毒毒素用分段脱毒法脱毒,并进行类毒素保护力的初步研究。以不同蛋白含量C型肉毒类毒素免疫小鼠后攻毒,结果显示,蛋白含量为0.625μg的类毒素免疫2针或蛋白含量为1.25μg的类毒素免疫1针均可保护50LD50的C型肉毒毒素攻击。蛋白含量为5μg的C型肉毒类毒素与福氏不完全佐剂配制的抗原免疫小鼠3次所得抗血清的保护力(Anti LD50/ml)为4.3×104。说明用该纯化工艺制备的C型肉毒类毒素具有很好的免疫原性,作为抗原成分用于C型肉毒疫苗和C型肉毒抗毒素的研究和生产具有较好的应用潜力。     

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I–III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X₁ (435 bp) and ORF-X₂ (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.     

Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D

Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases. The nucleic acids isolated were not digested by RNase A, but were digested by DNase I and exonuclease III, indicating that they were double-stranded DNA. On the basis of the restriction endonuclease digestion patterns on 0.8% agarose gel electrophoresis, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 kilobase pairs and that of c-203 and d-1873 was about 150 kilobase pairs. The digestion patterns of c-st, c-468, and c-n71 phage DNAs by PstI and HindIII were very similar. High homology was observed in the dot hybridization test. For other phages and nucleases, a good similarity was not observed. Only a little similarity was observed between c-203 and c-d6f phages. The existence of the structural genes for the toxin in both c-st and c-n71 phages was confirmed by the hybridization test with these phage DNAs and the oligonucleotide probe which represented the DNA sequence predicted for the N-terminal amino acids (2 to 17) of C. botulinum type C toxin. The loss of the converting ability of c-n71 phage may be caused not by the deletion of the tox+ gene but rather by the base mutation in c-st phage DNA.     

Separation and characterization of heavy and light chains from Clostridium botulinum type C toxin and their reconstitution

Clostridium botulinum type C toxin consists of a heavy and a light chain with molecular weights of 98,000 and 53,000, respectively, which are linked by one disulfide bond. The two components were separated from each other by quaternary aminoethyl Sephadex A-50 column chromatography by stepwise elution with NaCl in 27.5 mM borax-45 mM sodium dihydrogen phosphate buffer, pH 8.0, containing 5% 2-mercaptoethanol at 0 degrees C. The purified components had different amino acid compositions and antigenicities, and the toxicity of the toxin was neutralized completely by either anti-heavy chain Fab or anti-light chain Fab. the two components could be reconstituted to form an active molecule with recovered toxicity which varied according to the method used. Maximum recovery was obtained in a system in which the intersubunit S--S bond was first formed in the presence of high concentration of neutral salts, after which the concentration of salt was gradually decreased. The reconstituted preparation was highly toxic and had the same properties as the parental toxin on chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunodiffusion. By the use of three perturbants, the fractions of exposed tryptophans and tyrosines of the preparation were found to be almost the same as that of the parental toxin.     

Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D.

Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases. The nucleic acids isolated were not digested by RNase A, but were digested by DNase I and exonuclease III, indicating that they were double-stranded DNA. On the basis of the restriction endonuclease digestion patterns on 0.8% agarose gel electrophoresis, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 kilobase pairs and that of c-203 and d-1873 was about 150 kilobase pairs. The digestion patterns of c-st, c-468, and c-n71 phage DNAs by PstI and HindIII were very similar. High homology was observed in the dot hybridization test. For other phages and nucleases, a good similarity was not observed. Only a little similarity was observed between c-203 and c-d6f phages. The existence of the structural genes for the toxin in both c-st and c-n71 phages was confirmed by the hybridization test with these phage DNAs and the oligonucleotide probe which represented the DNA sequence predicted for the N-terminal amino acids (2 to 17) of C. botulinum type C toxin. The loss of the converting ability of c-n71 phage may be caused not by the deletion of the tox+ gene but rather by the base mutation in c-st phage DNA.     

Molecular characterization of GroES and GroEL homologues from Clostridium botulinum

We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947). The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies. Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue. The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced. The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins. Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage.     

B型肉毒毒素重链C端基因的表达与保护作用的初步研究

肉毒毒素是肉毒梭状杆菌产生的外毒素,有7种血清型(A～G),是目前已知的毒性最强的细菌蛋白质。人类肉毒中毒主要是由A、B和E型引起。本研究克隆了B型肉毒毒素重链C端片段(Hc),在大肠杆菌中进行了诱导表达,并用Ni离子亲和柱进行了纯化。Hc蛋白免疫后的BALB/c小鼠可以抵抗6×103LD50B型肉毒毒素攻击。