Purification and characterization of nontoxic protein complex from serotype D 4947 botulinum toxin complex.

The large-sized botulinum toxin complex (L-TC) is composed of botulinum neurotoxin (BoNT) and nontoxic proteins, e.g. nontoxic nonhemagglutinin (NTNHA) and three types of hemagglutinins (HAs; HA-33, HA-17 and HA-70). The nontoxic proteins play a critical role in L-TC oral toxicity by protecting the BoNT in the digestive tract, and facilitating absorption of the L-TC across the intestinal wall. Under alkaline conditions, the L-TC separates into BoNT and the nontoxic protein complex (NC). In this study, we established a two-step procedure to yield highly pure NC from the L-TC produced by Clostridium botulinum serotype D strain 4947 in which the NC was isolated from the L-TC by gel filtration under alkaline conditions followed by immunoprecipitation with an anti-BoNT antibody to remove contaminating BoNT from the NC fraction. Western blotting and electrophoretic analysis showed that the highly purified NC fraction had only very slight or no BoNT contamination. In addition, the purified NC fraction showed no intraperitoneal (ip) toxicity to mice at a dose of 38 ng per animal whereas the L-TC exhibited an ip median lethal dose of 0.38 ng per mouse. The highly purified NC displayed the same hemagglutination titer as the L-TC. The NC, as well as the L-TC, demonstrated cell binding and monolayer transport in the rat intestinal epithelial cell line IEC-6. Consequently, the highly purified NC can function as a "delivery vehicle" even without the BoNT.

Crystallization and preliminary X-ray analysis of the Clostridium botulinum type D nontoxic nonhaemagglutinin.

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex assembled with nontoxic nonhaemagglutinin (NTNHA) and/or haemagglutinin components. Complex formation with NTNHA is considered to be critical in eliciting food poisoning because the complex shields the BoNT from the harsh conditions in the digestive tract. In the present study, NTNHA was expressed in Escherichia coli and crystallized. Diffraction data were collected to 3.9 Å resolution. The crystal belonged to the trigonal space group P321 or P3(1)21/P3(2)21, with unit-cell parameters a = b = 147.85, c = 229.74 Å. The structure of NTNHA will provide insight into the assembly mechanism that produces the unique BoNT-NTNHA complex.

C terminal half fragment (50 kDa) of heavy chain components of Clostridium botulinum type C and D neurotoxins can be used as an effective vaccine.

Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 microg), Hn (5 microg), Hc (5 microg), or a mixture of Hn (5 microg) and Hc (5 microg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10(6) the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10(5) to 10(6) MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 microg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused-or GST eliminatedpreparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 microg, is useful for vaccine trials in mice.

Effect of nicking the C-terminal region of the Clostridium botulinum serotype D neurotoxin heavy chain on its toxicity and molecular properties.

A unique strain of Clostridium botulinum serotype D 4947 produces toxin complexes that are composed of un-nicked components, including a neurotoxin (BoNT) and auxiliary proteins. This BoNT showed aberrant elution upon Superdex gel filtration, indicating a much lower molecular weight, due to hydrophobic interaction with the column. Limited trypsin proteolysis of BoNT produces two nicks; first nick yielded a BoNT 50 kDa light chain disulfide linked to a 100 kDa heavy chain (Hc), and a second nick arose in Hc C-terminal 10 kDa. The second nick occurred in the putative binding domain of the BoNT molecule and induced alterations in its secondary structure, leading to a significant reduction of mouse toxicity in comparison with that of the fully-activated singly nicked BoNT. These results help to clarify the role of the C-terminal half of the Hc in the oral toxicity of single-chain and more complex forms of BoNT.