Broad, long term objective of the research are: 1. The development of in vitro and in vivo model systems with which to resolve the virulence factors of CF- associated BC. 2. The design of novel therapeutic strategies based on an understanding of the unique virulence factors of CF-associated pathogens. 3. The development of precise, DNA-based systems to rapidly reveal the epidemiological relationships of clinical isolates.
Specific aims of the proposal are to: A. Determine whether the cblA pilin marker gene carried by a highly transmissible, epidemically spread lineage of CF-associated BC is also an adhesin virulence factor. B. Analogously characterize the potential virulence function of other categories of BC-encoded appendage pili. C.Exploit entirely physical strategies to identify virulence factors of CF- associated BC. D. Test the validity of RFLP- based phylogenetic and epidemiological conclusions as to the genetic- relatedness of CF- associated BC isolates.The research design and methods for achieving these goals are as follows:
For specific aims A and B: (i) cloning and sequencing of pilus adhesin genes from CF-associated BC isolates of defined epidemiological and phylogenetic relationships; (ii) use of cloned genes for construction of isogeneic mutants, and (iii) testing these clinical strains and mutant derivatives for virulence properties using matched cftr-/- and cftr+/+ primary cell lines and the CF mouse model.
For specific aim C: Identify possible virulence factors using (i) differential hybridization selection, to clone, sequence and characterize genes uniquely carried by CF- associated BC, and (ii) differential expression selection for genes that are conditionally expressed in vivo during infection of the CF patient.
For specific aim D: Use of neutral, housekeeping gene sequence analysis to determine the genetic-relatedness of CF-associated BC isolates.
