Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan

Abstract

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 food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.

FIG. 1

SDS-PAGE of 111/NT and Okra/NT in the presence of DTT. A sample (2 μg of each neurotoxin per lane) was applied to a 10% polyacrylamide gel. The two minor bands in Okra/NT were derivatives of the heavy chain (14). The positions of molecular mass standards are shown on the left. H, heavy chain; L, light chain.

FIG. 2

Immunoblotting analyses of 111/NT (I) and Okra/NT (F) with MAbs. The MAb used is shown on top of the lane. The data presented are representative of MAbs reacting to the same fragment.

FIG. 3

Scatchard analyses of ¹²⁵I-111/NT (A) and ¹²⁵I-Okra/NT (B) binding to rat brain synaptosomes. Synaptosomes (10 μg of protein) were incubated at 37°C for 30 min with increasing concentrations of ¹²⁵I-labeled toxin in the absence or presence of excess unlabeled toxin. Specific binding was plotted after correction for nonspecific binding. The data presented are from one experiment and are representative of three experiments with similar results. A binding saturation curve is shown in an inset in each panel.

FIG. 4

Binding of ¹²⁵I-labeled toxins to recombinant amino-terminal domain of synaptotagmins 1 (Stg1N) and 2 (Stg2N) associated with ganglioside GT1b. (A) Dose dependence of ¹²⁵I-labeled 111/NT (solid symbols) and ¹²⁵I-labeled Okra/NT (open symbols) to Stg1N (triangles) and Stg2N (circles) incorporated into lipid vesicles with ganglioside GT1b. The recombinant synaptotagmin (10 ng of protein) reconstituted into lipid vesicles with ganglioside GT1b (2 ng of NeuAc) was incubated at 37°C for 30 min with increasing concentrations of ¹²⁵I-labeled toxin. Values are the means ± standard errors from three experiments. Error bars smaller than the symbols were omitted. (B) Scatchard plot of the binding data shown in panel A, except for ¹²⁵I-labeled 111/NT binding to the lipid vesicles containing Stg1N and ganglioside GT1b, for which the values were too low to show in the panel.

FIG. 5

Dose dependence of proteolysis of recombinant VAMP-2 by 111/NT and Okra/NT. (A) Samples were treated at 37°C for 1 h with reduced 111/NT (solid circles) or reduced Okra/NT (open circles) at different concentrations and electrophoresed on a 15% polyacrylamide gel. The gel was stained with Coomassie brilliant blue and quantified by densitometry. Results are expressed as a percentage of the initial VAMP-2 content. The data presented are the means of three independent experiments. (B) SDS-PAGE profile of VAMP-2 after incubation with or without reduced 111/NT (I) and reduced Okra/NT (F). The positions of VAMP-2 and the toxin-induced fragment are indicated by solid and open arrowheads, respectively.