THE PROPEPTIDE OF CLOSTRIDIUM-SEPTICUM ALPHA-TOXIN FUNCTIONS AS AN INTRAMOLECULAR CHAPERONE AND IS A POTENT INHIBITOR OF ALPHA-TOXIN-DEPENDENT CYTOLYSIS

Clostridium septicum alpha toxin is activated by a proteolytic cleavag e at Arg-398 in its carboxy terminus, which yields a 41.3-kDa cytolyti cally active toxin and a 5.1-kDa propeptide. Studies were performed to determine when the propeptide dissociated from the toxin after proteo lytic activation of the protoxin (AT(pro)) and to demonstrate the chap erone activity of the propeptide, The propeptide was found to remain a ssociated with the toxin after activation with trypsin (AT(act)) when analysed by gel filtration or affinity chromatography of a polyhistidi ne-tagged derivative that contained the polyhistidine tag on the prope ptide, The affinity of the propeptide for the toxin was decreased sign ificantly when a mutation was introduced in which Var-400 was converte d to a cysteine residue. This mutation destabilized the interaction of the propeptide with the toxin and the propeptide was found to dissoci ate from the toxin under the same gel-filtration conditions used for t he wild-type toxin, The separation of the propeptide in the V400C muta nt did not affect the cytolytic activity of the toxin and therefore th e propeptide was not necessary for cytolytic activity. These data sugg ested that the propeptide did not dissociate from the main body of the toxin after proteolysis. Further analysis demonstrated that purified propeptide was a potent inhibitor of alpha toxin activity, which inhib ited the oligomerization of alpha toxin into a functional pore, These data suggest that the propeptide does not participate in the final oli gomerized complex and that oligomerization appears to displace the pro peptide from AT(act), The importance of the propeptide to the solution stability of alpha toxin was also demonstrated. When AT(pro) was acti vated in solution with trypsin a significant level (approximately 50%) of inactive aggregate formed, This aggregate, which could be removed by centrifugation at 14 000 x g, was made up of both SDS-sensitive and -resistant aggregates, suggesting that a variety of inactive aggregat es formed when the monomers interacted in solution. Significantly high er levels of haemolytic activity (approximately 16-fold) were observed when alpha toxin was proteolytically activated after membrane binding instead of in solution. These results support the role of the propept ide as an intramolecular chaperone that stabilizes the monomeric AT(pr o) and shuttles it to the membrane where it is activated by protease, oligomerizes into a pre-pore complex and forms a pore. The data sugges t that oligomerization of the toxin displaces the propeptide from the monomer form of alpha toxin and that the propeptide does not participa te in, and is not necessary to, the final cytolytic complex.