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.     

Two-Component Systems Are Involved in the Regulation of Botulinum Neurotoxin Synthesis in Clostridium botulinum Type A Strain Hall

Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.     

The complete amino acid sequence of the Clostridium botulinum type-E neurotoxin, derived by nucleotide-sequence analysis of the encoding gene.

The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR). Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene. Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively. Comparisons with partial amino acid sequences of the light chain of BoNT/E from C. botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case. Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity. The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

Clostridium neurotoxins influence serotonin uptake and release differently in rat brain synaptosomes

Clostridium neurotoxins produce inhibition of both basal and K(+)-evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37 degrees C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2(+)-metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium-dependent, high-affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2(+)-metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C-terminal portion of the TeTx heavy chain (Hc-TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose-dependent manner. The IC50 of Hc-TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high-affinity site at the serotonin transporter or at other serotonin high-affinity sites. These results could indicate that TeTx and Hc-TeTx bind to different targets than BoNT/A in the plasma membrane.     

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.     

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.     

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

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.     

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.     

Sequences of the botulinal neurotoxin E derived from Clostridium botulinum type E (strain Beluga) and Clostridium butyricum (strains ATCC 43181 and ATCC 43755).

Recently, it has been shown that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755), isolated from cases of infant botulism, produce a botulinal neurotoxin type E (BoNT/E). Here we have determined the nucleotide sequences of the BoNT/E genes of these two C. butyricum strains and from C. botulinum E strain Beluga. We show that the sequences of the BoNT/E genes from the two C. butyricum strains are identical and differ in only 64 positions resulting in 39 amino acid changes (97% identity at the amino acid level) from that derived from C. botulinum. Our data suggest a transfer of the BoNT/E gene from C. botulinum to the originally nontoxigenic C. butyricum strains.     

A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell

The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin-O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca(2+)-dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca(2+)-dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.     

Insights in metabolism and toxin production from the complete genome sequence of Clostridium tetani

The decryption of prokaryotic genome sequences progresses rapidly and provides the scientific community with an enormous amount of information. Clostridial genome sequencing projects have been finished only recently, starting with the genome of the solvent-producing Clostridium acetobutylicum in 2001. A lot of attention has been devoted to the genomes of pathogenic clostridia. In 2002, the genome sequence of C. perfringens, the causative agent of gas gangrene, has been released. Currently in the finishing stage and prior to publication are the genomes of the foodborne botulism-causing C. botulinum and of C. difficile, the causative agent of a wide spectrum of clinical manifestations such as antibiotic-associated diarrhea. Our team sequenced the genome of neuropathogenic C. tetani, a Gram-positive spore-forming bacterium predominantly found in the soil. In deep wound infections it occasionally causes spastic paralysis in humans and vertebrate animals, known as tetanus disease, by the secretion of potent neurotoxin, designated tetanus toxin. The toxin blocks the release of neurotransmitters from presynaptic membranes of interneurons of the spinal cord and the brainstem, thus preventing muscle relaxation. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid, a formaldehyde-treated tetanus toxin, but nevertheless, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The World Health Organization has stated that neonatal tetanus is the second leading cause of death from vaccine preventable diseases among children worldwide. This minireview focuses on an analysis of the genome sequence of C. tetani E88, a vaccine production strain, which is a toxigenic non-sporulating variant of strain Massachusetts. The genome consists of a 2,799,250 bp chromosome encoding 2618 open reading frames. The tetanus toxin is encoded on a 74,082 kb plasmid, containing 61 genes. Additional virulence-related factors as well as an insight into the metabolic strategy of C. tetani with regard to its pathogenic phenotype will be presented. The information from other clostridial genomes by means of comparative analysis will also be explored.     

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.