Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H_C) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H_C, recombinant genes for the H_C and two hybrid H_C in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H_C of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H_C revealed that mutation of 23 residues in carboxy terminal half of H_C (1029-1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.