Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Simplified purification method for Clostridium botulinum type E toxin

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Sporulation and C2 toxin production by Clostridium botulinum type C strains producing no C1 toxin.

All of the 8 strains that were previously assumed to be nontoxigenic Clostridium botulinum type C were re-examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions. One per cent glucose added to trypticase peptone medium enhanced C2 toxin production. The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 10(4)/ml, no C2 toxicity was demonstrated. The C2 toxin was produced only during sporulation and not during vegetative growth.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.     

Observations on toxin and hemagglutinin produced by Clostridium botulinum type C.

In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.     

Observations on toxin and hemagglutinin produced by Clostridium botulinum type C.

In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Repression of toxin production by tryptophan in Clostridium botulinum type E

Of the seven amino acids required by Clostridium botulinum type E, tryptophan is the most essential and may provide the cell with nitrogen. The addition of excess tryptophan (10–20 mM) or other nitrogenous nutrients to minimal growth medium markedly decreased toxin formation but did not affect growth in C. botulinum type E. On the other hand, the addition of an enzymatic digest of casein (NZ Case) stimulated toxin formation and overcame repression by tryptophan. Immunoblots of proteins in culture fluids using antibodies to type E toxin indicated that tryptophan-repressed cultures produced less neurotoxin protein. Inhibitors of neurotoxin did not accumulate in cultures grown in minimal medium supplemented with high tryptophan. The results suggest that tryptophan availability in foods or in the intestine may be important for toxin formation by C. botulinum type E.