Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin

Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats -in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repea t domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regi ons important for the functional activities of this toxin, A previously dev eloped panel of in-frame deletion mutants of AC toxin was used to localize MAb specific epitopes on the toxin, The epitopes of these It MAbs were loca ted throughout the toxin molecule, recognizing: all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydropho bic domain, and one bound to either the hydrophobic domain or the functiona lly unidentified region adjacent to it. The remaining six MAbs recognized t he glycine/aspartate-rich repeat region. To localize these six MAbs, differ ent peptides derived from the repeat region were constructed. Two of the si x MAbs appeared to react with the repetitive motif and exhibited cross-reac tivity with Escherichia coli hemolysin, The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the ro les of these epitopes on toxin function, each MAb was screened for its effe ct on intoxication (cyclic AMP accumulation) and hemolytic activity. The tw o MAbs recognizing the distal portion of the catalytic domain blocked intox ication of Jurkat cells by AC toxin but had no effect on hemolysis, On the other hand, a MAb directed against a portion of the repeat region caused pa rtial inhibition of AC toxin-induced hemolysis without affecting intoxicati on. In addition, the MAb recognizing either the hydrophobic domain or the u nidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin, These findings extend our understanding of the regio ns necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel v irulence factor.