Functional objectives are to investigate genetic molecular structure of pathogens, to define the role of gene products in pathogenic mechanisms and to perform studies directed toward development of vaccines using molecular or synthetic production of immunogenic peptides from microbial agents. Our major emphasis is focused on cloning and expression of genes relevant to the toxic components of B. pertussis. Pertussis toxin is the protective component of the whooping cough vaccine; however it toxin activity may cause the harmful side effects associated with current vaccines. The toxin is compared of five dissimilar subunits; subunit S1 contains an ADP-ribosyltransferase activity responsible for most if not all of the toxin's biological activities. We recently cloned the complete pertussis toxin operon and determined the nucleotide sequence as well as the deduced amino acid sequences of the individual subunits. A patent application for this cloned gene has been filed and several corporations have applied to license the patent for vaccine development. We are using the cloned gene to study the structure/function relationship between pertussis toxin, pathogeneses, and immunoprotection. The genes encoding each of the B. pertussis toxin subunits were directly expressed in Escherichia coli and the recombinant subunits recovered as inclusion bodies from lysed cells. The subunits react in Western blots with monoclonal and polyclonal antitoxin sera. The recombinant S1 subunit has the same enzymatic activities as native toxin; S1 subunits from both sources modify a 41 Kd membrane-associated protein, believed to be the Ni regulatory protein of the adenylate cyclase complex. Serial amino-terminal truncations of the recombinant S1 protein was accomplished by exonucleolytic digestion of the S1 gene. Analyses of these molecules allowed us to identify a specific region of the S1 subunit possessing a major epitope associated with immunoprotection and a site necessary for its enzymatic activities. These results will permit us to design specifically- mutagenized subunit genes for production of analog toxin proteins that may prove useful as recombinant vaccines against pertussis.
