The object of the proposed research is to understand the genetic and molecular nature of microbial determinants that contribute to the pathogenesis of Bordetella pertussis infection and disease. Cloned Bordetella DNA sequences have been isolated encompassing the operon, Vir, which specifies a trans-acting inducer required for the coordinate expression of the genes associated with B. pertussis virulence. We propose to define this gene by genetic crosses and characterize the Vir gene product(s). In addition, we have successfully cloned and expressed genes specifying the B. pertussis filamentous hemagglutinin (Fha) in E. coli K-12 as well as the gene(s) specifying pertussis toxin. These virulence determinants will be characterized genetically, and their products examined by in vitro biosynthetic models and by DNA sequencing. We have, in addition, devised a simple method to return modified, cloned genetic sequences from E. coli K-12 back to Bordetella. By this means, we propose to precisely determine the regulatory mechanism of Vir as well the precise contribution of the virulence determinants to the pathogenesis of infection as well as the severe encephalopathy which is occasionally associated with current pertussis immunization.
