Bordetella pertussis causes a severe respiratory illness (pertussis, or whooping cough) characterized by specific colonization and destruction of ciliated respiratory epithelial cells. Although the cytopathology has been known since 1912, the pathophysiologic mechanisms have not been satisfactorily explained. In 1982, we reported the identification of a B. pertussis toxin, tracheal cytotoxin (TCT), which reproduces in vitro the specific ciliated cell damage seen in humans. The objective of this research proposal is to study the production of TCT and its role in the classic pertussis primary cytopathology. E. coli carrying a chimeric plasmid consisting of the Co1E1 origin of replication, RP4 conjugation genes, and Tn5 (a transposon coding for kanamycin resistance) will be conjugated with B. pertussis. Tn5 insertion mutants which do not produce TCT or which produce an aberrant toxin will be identified with polyclonal and monoclonal antibody screens (solid phase radioimmunoassay). These mutants will be then tested by assaying tracheal cell cultures for inhibition of DNA synthesis and by examining tracheal organ cultures and mice for cytopathology. Mutant chromosomal DNA will be digested with a restriction enzyme which does not clave within Tn5, ligated to pBR322, and transformed into E. coli. Recombinant plasmid DNA from these clones will be nick-translated and hybridized to plaques of a B. pertussis genomic library to identify the wild type gene sequences associated with TCT production. These sequences will facilitate the future identification of mRNA species relevant to TCT production and studies of how TCT synthesis is regulated. By understanding factors or conditions that control the genes involved in TCT production, it may be easier to design and test ways of altering toxin expression and perhaps to develop more effective prophylaxis for humans. In addition, we are planning preliminary studies to determine if TCT binds to a specific receptor on host cells. Identification of a TCT receptor will be the first step in evaluating the mechanism of action of TCT and the molecular basis of its specific effect on cells of the respiratory tract.
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