The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene

Abstract

A 26-mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the Nterminus of the Closlridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2-kb PvuII -TuqI fragment and the remaining regions of the light chain and heavy chain as a 2.4-kb Sea1 -TuqI fragment and a 3.4-kb HpaI-f'~vuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N-termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.Toxigenic strains of Clostridium botulinum, an anaerobic spore-forming bacteria, produce one or more of seven immunologically distinct neurotoxins. They act primarily at the neuromuscular junction, causing paralysis by inhibiting the release of the neurotransmitter acetylcholine [I]. The botulinum neurotoxins are synthesised as a single polypeptide chain but in their most active form comprise of two asymmetric polypeptide units; a light chain (50-59 kDa) and a heavy chain (85 -105 kDa), linked by at least one disulphide bridge [2].Although botulinum neurotoxins exhibit high degrees of similarity (when compared to each other and related tetanus and diphtheria toxins) in their molecular masses and modes of action, they still remain poorly characterised at the molecular level. Proposals for the mechanism of toxic action implicates three separate functional domains. The light chain has the pharmacological activity, while the N-terminal and C-terminal of the heavy chain mediate channel formation and toxin binding, respectively. These heavy-chain, toxin-binding functional domains have been demonstrated with diphtheria toxin [3 -51 and with tetanus toxin [6 -81. Channel forming activities have been demonstrated for the N-terminus of the heavy chain with C. botulinum type A [9, 101 and there is circumstantial Correspondence to N.

Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

“…On the other hand, a p47 gene, whose product plays an as-yet-undetermined role, although it is believed to be a regulatory one (23), is present only in type A2 gene clusters. Furthermore, the comparison of DNA sequences between boNT/A1 genes from various sources (GenBank accession numbers AF488749, AF461540, M30196, and X52066) (2,6,37,39) and the boNT/A2 gene from the Kyoto-F strain isolated in Japan (GenBank accession number X73423) (38) has revealed a 94% sequence homology between boNT/A1 and boNT/A2 genes, which is lower than the 99% sequence homology observed among boNT/A1 gene sequences. Other differences between A1 and A2 genetic clusters include the orientation and position of the positive regulatory gene botR/A (26), and the sequence of the NTNH gene (24).…”

Development of vaccines for prevention of botulism

“…We were then able to vaccinate mice with the non-toxic recombinants and test their ability to elicit protective immunity in vivo. Clones pCBA2, pCBA3, and pCBA4 (gift from Nigel Minton) containing overlapping gene fragments of the BoNTA gene [48], were the source for templates used to produce the various antigens to be examined. Smaller versions of the gene fragments were constructed by cutting with restriction enzymes.…”

Molecular characterization of two forms of nontoxic‐nonhemagglutinin components of Clostridium botulinum type A progenitor toxins

“…Nucleotides in the positions of codon degeneracy were chosen on the basis of those most commonly found in clostridial genes [11]. pAR3 was designed with the nucleotide sequence of the 5'-end of the type A neurotoxin gene [12,13]. The PCR products (about 4.6 kb) were cloned and sequenced as described in section 2.…”