Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin.

Clostridium perfringens alpha-toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C-domain (251–370 residues; CP251–370) in biological activities of the toxin. The N-domain (1–250 residues; CP1–250) of the alpha-toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC-CP251–370) consisting of BcPLC and CP251–370 bound to the red cells and lysed them. Incubation of CP1–250 with CP251–370 completely complemented hemolytic and PLC activities. CP251–370 also conferred hemolytic activity on BcPLC. CP251–340 (251–340 residues) significantly stimulated PLC activity of CP1–250, but did not confer hemolytic activity on CP1–250. Kinetic analysis suggested that CP251–370 increased affinity toward the substrate of CP1–250. The results suggested that CP251–370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1–250. Acrylodan-labeled CP251–370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD)-labeled CP251–370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251–370 to a hydrophobic environment. These observations suggest that interaction of CP251–370 of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1–250.