Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoc lonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two ant ibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were d erived from recombinant Escherichia coli by various deletions from the N-te rminus. Strong hemolytic activity was recognized in truncates of PLO follow ing the deletion of 30 or 55 amino acids, but not in the truncate with dele tion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies wit hin the undecapeptide region (amino acids 487-505) of the C-terminus of PLO , which seems to be the binding site to erythrocytes. In contrast, the epit opes for mAbs S and H, which showed strong neutralizing activity, were foun d to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N- terninal region of PLO, in particular, the region of amino acids 55-74 is i mportant for hemolytic activity. (C) 2001 Elsevier Science B.V. All rights reserved.