Clostridium botulinum type B strain produces two forms of progenitor toxin, 16S and 12S. The 12S toxin is formed by association of a neurotoxin (NTX) and a non-toxic non-haemagglutinin (NTNH), and the 16S toxin is formed by conjugation of the 12S toxin with a haemagglutinin (HA). HA consists of four subcomponents designated HA1, HA2, HA3a and HA3b. When mice were immunized with formalin-detoxified NTX, 12S or 16S, a significantly greater amount of anti-NTX antibody (Ab) was produced in the mice injected with 16S than in NTX-or 12S-injected mice. Immunization with NTX mixed with HA1 and/or HA3b also increased the anti-NTX Ab production, whereas NTX mixed with HA2 did not, indicating that HA1 and HA3b have adjuvant activity. This was further confirmed by immunizing mice with human albumin (Alb) alone or Alb mixed with either HA1 or HA3b. When mouse-spleen cells were stimulated with NTX, 16S or different HA subcomponents, 16S, HA1, HA3b and the mixture of HA1 and HA3 significantly increased interleukin 6 (IL6) production compared with NTX alone. Transcription of IL6 mRNA was low after stimulation with NTX alone, but increased to 16S-stimulation levels when NTX was mixed with HA1 or HA3b. In flow cytometry using labelled Abs against CD3 and CD19, the percentage of CD19 cells was higher following stimulation with 16S or NTX mixed with HA1 or HA3b compared with stimulation with NTX. The percentage of CD3 cells remained unchanged. These results suggest strongly that HA1 and HA3b demonstrate adjuvant activity via increasing IL6 production.

Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA). The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes. In type C, these bindings are dependent on sialic acid. The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b. To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST). These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes. GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not. GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding. TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin [Inoue, K. et al. (1999). Microbiology 145, 2533–2542]. On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3. Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity. Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.

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Haemagglutinin (HA) activity of

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.Clostridium botulinum strains produce immunologically distinct neurotoxins (serotypes A to G). The molecular masses of neurotoxin types A to G are approximately 150 kDa. The neurotoxins are produced as a single form and become dichain-form light (50-kDa) and heavy (100-kDa) chains by cleavage with proteases such as trypsin at about one-third of the distance from the amino terminus, and the toxic activity of the dichain form becomes fully activated (1). In culture fluid and food with acidic conditions, the neurotoxins associate with nontoxic components and form large complexes designated progenitor toxins. Under alkaline conditions, the progenitor toxins dissociate into neurotoxin and nontoxic components (10, 18). The progenitor toxins are found in three forms with molecular masses of 900 kDa (19S), 500 kDa (16S), and 300 kDa (12S) (14). The 12S toxin is composed of a neurotoxin and a nontoxic component having no hemagglutinin (HA) activity (designated nontoxic non-HA [NTNH]), whereas the 16S and 19S toxins are composed of a neurotoxin, NTNH, and HA. The serotype A strain produces three forms of toxins (19S, 16S, and 12S). Type B, C, and D strains produce the 16S and the 12S toxins. We purified different-sized progenitor toxins from serotype A, C, and D cultures (2,4,6,12,13) and demonstrated that (i) the 19S toxin is a dimer of the 16S toxin; (ii) HA consists of four subcomponents with molecular masses of 52 to 53, 33 to 35, 19 to 23, and 15 to 17 kDa, designated here as HA3b, HA1, HA3a, and HA2, respectively; and (iii) NTNH of the 12S toxin has a cleavage site(s) at the N-terminal region.Recently, serotype A and B progenitor toxins have been used for treating patients with strabismus, blepharospasm, nystagmus, facial spasm, spastic aphonia, and many other forms of dystonia (9, 11). In both toxin types, progenitor toxins are used because they are easily obtained and are more stable than neurotoxin. The treatment is very effective but has a serious side effect for some patients in whom antiprogenitor toxins, including antineurotoxin antibodies, are produced after several injections. It seems that using neurotoxin alone is better than using the progenitor toxin (a complex of the neurotoxin and a nontoxic component). Furthermore, it has been reported that serotype B toxin, which is used therapeutically at present, is partially cleaved (16) and therefore the toxin is not fully activated. In this paper, we report a simple procedure for largescale purification of botu...