The preliminary research objective of this laboratory is the determination of the immunogenic and functional domains of the exotoxin A molecule from Pseudomonas aeruginosa. To accomplish this, we have taken a combined immunochemical, biochemical and genetic approach. Specifically, we have produced a library of toxin-specific monoclonal antibodies which are being used as site-specific probes for the detailed analysis of this protein. Several of these antibodies inhibit specific functions of the toxin molecule. Using specific monoclonal reagents in conjunction with genetically altered toxin molecules we are conducting a comparative analysis of toxin structure and function. We hope to determine: (1) the location of the immunogenic epitopes required to develop host immunity to toxin; (2) the location of the receptor-specific epitope(s) of toxin; (3) the NAD- dependent ADP-ribosyltransferase active site of toxin; (4) location of the membrane translocation domain of toxin. The project outlined above has very specific objectives, but the potential applications of the expected results are numerous and significant. First, a detailed knowledge of functional regions of this protein will allow an in-depth analysis of the correlation between structure and function for microbial toxins, since the complete amino acid sequence of this protein is known. Knowledge of the specific immunogenic epitopes of the toxin will provide the basis for the development of synthetic peptides corresponding to regions known to elicit the formation of anti- toxin in vivo. As such, the system described (a lethal toxin well- characterized with respect to susceptible cell lines and animal model systems) will provide the means to effectively test the plausibility of the use of synthetic peptides in vaccine development.
