Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin

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Abstract
The purified progenitor toxin of Clostridium botulinum type C strain 6814 (C‐6814) forms a large complex composed of 150‐kDa neurotoxin (NT), 130‐kDa nontoxic‐nonhemagglutinin (NTNHA), and hemagglutinin (HA) components. The HA component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26–21 and 17 kDa. We isolated the HA subcomponents from the progenitor toxin by chromatography in the presence of denaturants. The isolated HA subcomponents, designated as i‐HA‐33, i‐HA‐55, i‐HA‐70 and i‐HA‐33/17, were nearly homogenous on SDS/PAGE, but the HA‐17 and HA‐26–21 components were not purified. Some HA subcomponents, designated as f‐HA‐33 and f‐HA‐33/17 complex, existed free of the progenitor toxin in the culture medium and they were separately purified. Every HA subcomponent so far isolated shows binding activity to erythrocytes. The hemagglutination activities of each HA subcomponent had a titer of 25 for the f‐HA‐33/17 complex, and below 23 for the other f‐ and i‐HA subcomponents, while the parent progenitor L toxin was 28. The reconstitution of various combinations of f‐ and i‐HA subcomponents was attempted via mixing and tested for hemagglutination activity. When the i‐HA‐33/17 complex and i‐HA‐55 were mixed, the hemagglutination activity was recovered to a titer of 29, which was slightly higher than that of the parent toxin. These data imply that a combination of at least HA‐33, ‐17 and ‐55 subcomponents is required for full hemagglutination activity of the botulinum progenitor toxin, but each single HA subcomponent shows weak or no aggregation of erythrocytes.