A novel multiplex PCR method for Clostridium botulinum neurotoxin type A gene cluster typing

A rapid, simple and sensitive multiplex PCR method for boNT/A gene cluster typing was developed by combining the results of BoNT/A subtype (boNT/A1 or /A2) gene detection with ha33 and/or p47 gene detection. Ten isolates associated with infant botulism in Japan were examined and divided into boNT/A gene cluster types 2 and 3 by origin (honey feeding or not) and period (1986–1987 or 1999–2007). It is suggested that this multiplex PCR method will be be useful for epidemiological studies of botulism.     

Sequence of the gene encoding type F neurotoxin of Clostridium botulinum

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.     

Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences

Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences. The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene. Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame. The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain.     

A型肉毒神经毒素(BoNT/A)基因序列分析及其B细胞表位预测

比较GenBank中4个不同毒株的A型肉毒神经毒素（BoNT／A）的基因组序列,发现其基因序列的一致性高迭92．2％-99．9％。基于保守性高的BoNT／A氨基酸序列,根据BioSun和LaserGene软件包中的表住分析相关参数,辅以对BoNT／A蛋白的二级结构的分析,综合预测BoNT／A的B细胞表位。结果表明,BoNT／A轻链的142-150、284-292区段,轻链的284-292,重链的440-450、465-480、538-549、699-710、751-760、1087-1095、1224-1231、1263-1270区段是B细胞表位优势区的可能性较大。多参数方案井结合不同软件综合预测BoNT／A蛋白的B细胞表位,为进一步实验鉴定BoNT／A的B细胞表位及其多表位疫苗设计和研究奠定了基础。     

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.     

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.     

Cloning of a Clostridium botulinum type B toxin gene fragment encoding the N-terminus of the heavy chain

Two lambda gt11 clones of the toxin gene of Clostridium botulinum type B were identified by the monoclonal antibody specific to the heavy chain of type B toxin. Neither of the expressed fusion proteins from the lysates of lysogenic E. coli Y1089 showed any botulinal toxic activity. One of the clones hybridized to the oligonucleotide probe which was synthesized according to the amino acid sequence of N-terminus of heavy chain. The sequence analysis revealed that highly homologous regions in N-terminus of heavy chain exist among botulinum neurotoxins (type A, B) and tetanus toxin on the amino acid sequence level.     

Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.     

A型肉毒毒素轻链基因的克隆及其结构分析

以献报道的A型肉毒毒素基因全序列为标准,设计并合成一对引物,自肉毒梭菌基因组中扩增出肉毒毒素轻链基因片段,并将扩增产物与pGEM—T载体在体外连接,构建测序重组质粒,进行测序和基因结构分析。PCR扩增获得了产物为1 364bp的DNA片段,测序结果与DNA数据库对照检索分析证明,此基因片段与GenBank中的A型肉毒毒素LC基因的一致性达99．9％以上,可以认为克隆的基因为A型肉毒毒素LC基因。     

Synergistic capture of Clostridium botulinum type A neurotoxin by scFv antibodies to novel epitopes

A non‐immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (H_c)] with the goal of identifying scFv to novel epitopes. To do this, an antibody‐mediated labeling strategy was used in which antigen‐binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the H_c. Twenty unique scFv clones were isolated that bound H_c. Of these, 3 also bound to full‐length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep‐MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A‐specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep‐MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep‐MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support. Biotechnol. Bioeng. 2011;108: 2456–2467. © 2011 Wiley Periodicals, Inc.     

Clinical application of Clostridium botulinum type A neurotoxin purified by a simple procedure for patients with urinary incontinence caused by refractory destrusor overactivity

Type A neurotoxin of Clostridium botulinum was purified by a simple procedure using a lactose gel column. This procedure was previously reported for type B neurotoxin. Hemagglutinin-positive toxins (19S and 16S) were bound to the column under acid conditions, and the neurotoxin alone was dissociated from these hemagglutinin-positive toxins by changing the pH of the column to an alkaline condition. The toxicity of this purified toxin preparation was retained for at least 1 year at -30 degrees C by supplementing it with either 0.1% albumin or 0.05% albumin plus 1% trehalose. This preparation was used to treat 18 patients with urinary incontinence caused by refractory idiopathic and neurogenic detrusor overactivity; 16 of the patients showed excellent improvement. Improvements started within 1 week after injection in most cases and lasted 3-12 months [corrected]     

Identification of structural genes for Clostridium botulinum type C neurotoxin-converting phage particles

The structural genes for strain C-Stockholm (c-st) phage particles, a representative type C toxin-converting phage of Clostridium botulinum, have been determined. First, by determining the N-terminal amino acid sequences of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands of c-st phage particles, it became clear that four proteins, 14, 25, 32 and 42 kDa, are the products of the ORFs, cst166, cst165, cst160 and cst164, respectively, of the c-st phage genome. The Western blot analyses reacting these phage bands with an antiphage serum prepared previously indicated that the products of cst165 and cst160 are the main proteins of the phage particles. Then, six candidates for the phage structural proteins, including cst165 and cst160 gene products, were prepared as recombinant proteins. Also, the protein corresponding to the cst164 gene product was excised from SDS-PAGE gels. The antibodies against these seven proteins were prepared in rabbits, and finally, the reaction of these antibodies to the c-st phage particles was analyzed by electron microscopy. It was concluded that a sheath protein and a head protein of the c-st phage are the products of genes cst160 and cst165, respectively, and that these two proteins are conserved in the other three converting phages, but not in the nonconverting phage.     

F型肉毒梭菌培养液可滤性实验的初步研究

目的通过探索F型肉毒梭菌培养液可滤性实验,为现有生产工艺提供科学的优化方案。方法分别采用不同型号的过滤器,通过不同的过滤组合对F型肉毒梭菌培养液进行澄清过滤。结果采用K700P过滤器(过滤精度6.0～15.0μm)对F型肉毒梭菌培养液进行一级澄清过滤,采用50P过滤器(过滤精度0.5～0.85μm)对料液进行二级澄清过滤,对F型肉毒梭菌培养液具有良好的澄清效果。结论可滤性实验的探索对于F型肉毒梭菌培养液批生产量的扩大具有切实可行的指导意义。     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

A型产气荚膜梭菌α-毒素基因的克隆与核苷酸序列分析

利用聚合酶链式反应(PCR)技术,从A型产气莫膜梭菌染色体基因组中扩增了1．2kb的α毒素基因。通过T4 DNA连接酶,将纯化的PCR产物与载体pGEM-T连接,转化受至体菌JM109中,经NcoI/EcoRI和BamHI/EcoRI双酶切分析,证明重组质粒pXCPA02中含有A型产气荚膜棱菌α毒素全基因。经核苷酸序列分析,明确了克隆的α毒素基因在重组质粒中的连接向位且核苷酸序列是正确的。     

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.     

Plasmid encoded neurotoxin genes in Clostridium botulinum serotype A subtypes

Clostridium botulinum, an important pathogen of humans and animals, produces botulinum neurotoxin (BoNT), the most poisonous toxin known. We have determined by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations that the genes encoding BoNTs in strains Loch Maree (subtype A3) and 657Ba (type B and subtype A4) are located on large (approximately 280 kb) plasmids. This is the first demonstration of plasmid-borne neurotoxin genes in Clostridium botulinum serotypes A and B. The finding of BoNT type A and B genes on extrachromosomal elements has important implications for the evolution of neurotoxigenicity in clostridia including the origin, expression, and lateral transfer of botulinum neurotoxin genes.     

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.