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.

The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue.

The effect of Clostridium perfringens alpha-toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated. The toxin-induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased. However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin. However, the toxin did not hydrolyze solubilized distearoyl-L-a-phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC. These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC. The N-terminal domain of alpha-toxin (AT1-246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl-L-a-phosphatidylcholine (DOPC). H148G bound to the liposomes, but AT,.2 did not. However, the C-terminal domain (AT251-370) conferred binding to liposomes and the membrane-damagingactivityonAT1-246. These observations suggest that the membrane-damaging action of alpha-toxin is due to the binding of the C-terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N-terminal domain.

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the Nterminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.