Effect of amino acid substitutions in the epitope regions of pyolysin from Arcanobacterium pyogenes.

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY, cholesterol-dependent cytolysin) family of bacterial toxins. Recently, we demonstrated that the epitopes of monoclonal antibodies (mAbs) S, H, C, and G lie in the regions of amino acids regions 55-73, 123-166, 482-506, and 482-506 of PLO, respectively, by the reaction of mAbs with truncated PLOs. In this study, we substituted the amino acids in these epitope regions of PLO by site-directed mutagenesis and examined the effect of these amino acid substitutions. Mutants I70S/R71A/L73S, Y131S/P132S, and L163S/P164S for mAbs H or S completely lost the hemolytic activity of the proteins, but these mutants still bound to erythrocyte membranes. Mutants L495S/W497S and W500S/W501S for mAbs C and G also completely lost their hemolytic activity, but still bound to erythrocyte membranes. In the undecapeptide region of PLO, the cysteine residue required for thiol activation is replaced with alanine. Therefore, we substituted Ala-492 of the undecapeptide region for Cys. The hemolytic activity of this mutant A492C decreased by adding hydrogen peroxide or storing at 4 degrees C, and the decreased hemolytic activity was restored by adding L-cysteine.