Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin types C1 and D, and PCR detection of this gene from Clostridium botulinum type C organisms.

The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA. Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D. The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817. The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity). When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C. botulinum type C strains 6812 and 6814. These results suggest that some strains of C. botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.     

DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.     

A型肉毒神经毒素基因的PCR检测

目的:建立快速筛查A型肉毒毒素的PCR方法。方法:根据GenBank中报道的肉毒毒素基因序列,综合应用多种生物软件分析设计特异的检测引物,从提取的基因组DNA、热裂解产物和菌液等不同形式的模板中扩增大小为457bp的A型肉毒毒素特异基因片段,以肉毒梭菌其他血清型及破伤风梭菌为对照。结果:检测方法无交叉反应,灵敏度可达10pgDNA,3×103个菌。结论:建立的检测方法特异性强、灵敏度高,可以用于A型肉毒毒素基因的快速筛查。     

Characterization of botulinum type A neurotoxin gene: delineation of the N-terminal encoding region

A 456 basepair HindIII fragment that encoded a portion of the type A botulinum neurotoxin gene was cloned into Escherichia coli using a plasmid vector. DNA sequence analysis revealed that this botulinum DNA insert encoded an open reading frame of 35 amino acid residues of which 34 corresponded to the N-terminal residues of botulinum neurotoxin type A.     

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Phenotype and genotype of Clostridium sp. strains producing botulism neurotoxin

Neurotoxins produced by strains of Clostridium sp. are belonging to the most toxic biological substances. In the study phenotypes and genotypes of C. botulinum strains in animal studies in vivo and on the DNA level were evaluated, respectively. Additionally, the presence of genes encoding BoNT toxins of A, B, and E types among strains of Clostridium sp. were identified. In case of C. botulinum DNA was isolated from vegetative bacterial cells and from spores. Two different genes encoding two different neurotoxins harboured by three strains of Ae biotype/ae genotype, and by two strains of B biotype/be genotype were detected. Additionally, above E type C. botulinum strains, the presence of gene encoding E type neurotoxin, was found in genome of two C. baratii, two C. butyricum, and C. bifidobacterium, and C. oedematicum strains. C. bifidobacterium and C. oedematicum strains positive for presence of gene encoding E type neurotoxin, were found negative for E neurotoxin production in vivo in TN test. The study indicates that genes encoding BoNT/E neurotoxins are very common among Clostridium species. Phenotype and genotype analysis indicated co-presence of B phenotype together with be genotype and A phenotype together with ae genotype among C. botulinum strains.     

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel structural elements within the nonproteolytic clostridium botulinum type F toxin gene cluster

We sequenced for the first time the complete neurotoxin gene cluster of a nonproteolytic Clostridium botulinum type F. The neurotoxin gene cluster contained a novel gene arrangement that, compared to other C. botulinum neurotoxin gene clusters, lacked the regulatory botR gene and contained an intergenic is element between its orfX2 and orfX3 genes.     

Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.     

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10(2) cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.
Methods and results:  The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT / A or BoNT / B , were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.
Conclusions:  The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.
Significance and Impact of the Study:  The LAMP assay would be useful for detection of C. botulinum in environmental samples.     

Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani

Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.     

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 10(2)-10(3) 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).     

Characterization of Clostridium sp. RKD producing botulinum-like neurotoxin

A Gram positive, motile, rod-shaped, strictly anaerobic bacterium isolated from intestine of decaying fish was identified as Clostridium sp. RKD and produced a botulinum type B-like neurotoxin as suggested by mouse bioassay and protection with anti botulinum antibodies. The neurotoxicity was functionally characterized by the phrenic nerve hemi-diaphragm assay. Phylogenetic analysis based on 16S rDNA sequence, placed it at a different position from the reported strains of Clostridium botulinum. The strain exhibited differences from both Clostridium botulinum and Clostridium tetani with respect to morphological, biochemical and chemotaxonomic characteristics. Botulinum group specific and serotype specific primers amplified the DNA fragments of 260 and 727 bp, respectively, indicating presence of botulinum type 'B' toxin gene. Sequence of nearly 700 bp amplified using primers specific for botulinum neurotoxin type B gene, did not show any significant match in the database when subjected to BLAST search.     

Comparative analysis of C3 and botulinal neurotoxin genes and their environment in Clostridium botulinum types C and D.

The C3 exoenzyme gene is located on a bacteriophage in Clostridium botulinum types C and D (M. R. Popoff, D. Hauser, P. Boquet, M. W. Eklund, and D. M. Gill, Infect. Immun. 59:3673-3679, 1991). A derivative CN phage from phage C of C. botulinum Stockholm (C-St) (K. Oguma, H. Iida, and K. Inoue, Jpn. J. Microbiol. 19:167-172, 1975), isolated as neurotoxin negative, also does not produce exoenzyme C3. The botulinal neurotoxin C1 gene is present on the CN phage but contains a stop mutation in the DNA region encoding the N-terminal part of the heavy chain (codon 553). The putative truncated botulinal neurotoxin C1 protein was not recovered in a C. botulinum strain harboring the CN phage. We found that the C3 gene is localized on a 21.5-kbp DNA fragment flanked by the core motif 5'-AAGGAG-3' in DNAs of phage C of C. botulinum 468 (C-468), C-St phage, and phage D of C. botulinum 1873 (D-1873). The 21.5-kbp DNA fragment is deleted in CN phage DNA, and the motif 5'-AAGGAG-3' is present only in one copy at the deletion junction, but the deletion in the CN phage could be nonspecific, since this phage was obtained by nitrosoguanidine treatment. These findings could indicate that the C3 gene is localized on a 21.5-kbp mobile element. C. botulinum type C strain 003-9 produces a C3 exoenzyme (Y. Nemoto, T. Namba, S. Kozaki, and S. Narumiya, J. Biol. Chem. 266:19312-19319, 1991), and Staphylococcus aureus E1 produces a related C3 enzyme which is named epidernmal cell differentiation inhibitor (S. Inoue, M. Sugai, Y. Murooka, S. Y. Paik, Y. M. Hong, H. Oghai, and H. Suginaka, Biochem. Biophys. Res. Comm. 174:459-464, 1991) and which shares 80.6 and 56.6% similarity, respectively with the C3 enzymes from C-468 or C-St and D-1873 phages athe amino acid level. The features of the putative 21.5-kbp transposon were not found in C. botulinum 003-9 and S. aureus E1, as determined by analysis of the C3 and epidermal cell differentiation inhibitor gene-flanking DNA regions. These data suggest a common ancestral origin and divergent evolution of the C3 genes in these three groups of bacterial strains and dissemination of a 21.5-kbp element carrying the C3 gene C-468, C-St, and D-1873 phages.     

The complete nucleotide sequence of the gene coding for botulinum type C1 toxin in the C-ST phage genome

Two DNA fragments, 3 kbp and 7.8kbp, which encode the type C1 botulinum neurotoxin gene, were obtained from toxigenic bacteriophage DNA by treatment with a restriction enzyme. They were cloned into the plasmid vectors for nucleotide sequence determination. The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000. The amino acid sequence of the C1 toxin has a few regions highly homologous with tetanus toxin.     

Conserved structure of genes encoding components of botulinum neurotoxin complex M and the sequence of the gene coding for the nontoxic component in nonproteolyticClostridium botulinum type F

For investigation of the genes of proteins associated in vivo with botulinum neurotoxin (BoNT), polymerase chain reaction (PCR) experiments were carried out with oligonucleotide primers designed to regions of the nontoxic-nonhemagglutinin (NTNH) gene ofClostridium botulinum type C. The primers were used to amplify a DNA fragment from genomic DNA ofC. botulinum types A, B, E, F, G and toxigenic strains ofClostridium barati andClostridium butyricum. The amplified product from all of these strains hybridized with an internal oligonucleotide probe, whereas all nontoxigenic clostridia tested gave no PCR product and showed no reaction with the probe. TheNTNH gene was shown to be located upstream of the gene encoding BoNT, thereby revealing a conserved structure for genes encoding the proteins of the M complex of the progenitor botulinum toxin in these organisms. The sequence of theNTNH gene of nonproteolyticC. botulinum type F was determined by PCR amplification and sequencing of overlapping cloned fragments. NTNH/F showed 71% and 61% identity with NTNH ofC. botulinum type E and type C respectively.     

Genome analysis of Clostridium botulinum type A by pulsed-field gel electrophoresis.

Genomic DNA from type A Clostridium botulinum was digested with restriction endonucleases that cut at rare sites, and the large fragments were separated by pulsed-field gel electrophoresis. Of 15 restriction enzymes tested, MluI, RsrII, SmaI, NruI, KspI, NaeI, and XhoI generated satisfactory digestion patterns of genomic DNA of various C. botulinum strains, enabling the use of the method for genomic fingerprinting. The genomes of four group I (type A) C. botulinum strains examined had similar restriction patterns. However, each strain had unique digestion patterns, reflecting genotypic differences. The genome size of C. botulinum strain 62A was estimated to be 4,039 +/- 40 kbp from the summation of restriction fragments from MluI, RsrII, and SmaI digestions. Genes encoding proteins involved in the toxinogenicity of C. botulinum, including neurotoxin, hemagglutinin A, and genes for a temperate phage, as well as various transposon Tn916 insertion sites in C. botulinum 62A, were mapped by pulsed-field gel electrophoresis. The genes encoding neurotoxin and hemagglutinin A-1, were located on the same fragment in several cases, indicating their probable physical linkage. The macrorestriction analysis established here should be useful for genetic and epidemiological studies of C. botulinum.     

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.     

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.