Our aim is to carry out a detailed genetic and biochemical analysis of the IgA proteases of Hemophilus influenzae. We will clone and characterize IgA protease genes for H.influenzae serotypes b, c and d in E. coli K-12. The structure of the cloned genes will be studied using restriction enzymes and DNA sequence analysis. Mutant forms of the gene will be characterized in E. coli following in vivo and in vitro mutagenesis. Selected mutations, especially deletions, will be introduced into H.influenzae recipients by transformation. These IgA protease negative mutants and their protease positive parent strains will be used to study the effects of the mutations on pathogenesis using animal model systems and isolated organ cultures. We will use both biochemical and genetic approaches to study the IgA protease enzymes in order to understand the differences between type 1 (serotypes b and d) and type 2 (serotype c) proteases. We will attempt to elucidate the mechanisms by which IgA proteases are secreted in both H.influenzae and E.coli.
