Antibiotic-resistant Pseudomonas cepacia is an important pulmonary pathogen in children and young adults with cystic fibrosis. A critical problem in the management of infected patients is the persistence of the organism despite antibiotic therapy. A clinical isolate with an antibiotic-impermeable porin has been identified and the DNA encoding resistance has been cloned; the porin protein shows no apparent difference in protein fingerprint or mobility on SDS-PAGE compared with an isogenic fully-permeable porin. This proposal seeks to investigate four possibilities for the genetic basis of the cloned resistance determinant to encode porin-mediated permeability changes in P. cepacia: either 1. a porin-associated protein, or 2. an altered porin subunit which incorporates into heterogeneous oligomers with normal subunits, results in an - impermeable porin channel, or 3. homologous recombination occurs with replacement of the normal gene by the cloned DNA which encodes an impermeable porin, or 4. the cloned DNA regulates gene expression by insertional activation. To examine these hypotheses, this proposal will characterize porins from isogenic permeable and impermeable strains functionally, biochemically and genetically. The cloned DNA will be subcloned and analyzed. The subcloned fragment will be used to probe chloramphenicol susceptible and resistant isolates to identify homologous sequences. The DNA sequence resulting in porin-mediated outer membrane impermeability will be examined by DNA sequencing and identification of the gene product; homologous sequences that are identified will be characterized as well. Second, proteins which form transmembrane pores will be isolated from each strain and porin function and specificity determined using reconstituted membrane vesicles, liposomal swelling assays and black lipid bilayers. Next, the biochemical properties of each protein will be determined by gel electrophoresis, isoelectric focusing, peptide mapping and amino acid analysis. Finally, the structural gene for both the permeable and impermeable porins will be cloned and sequenced to confirm or identify specific differences between the proteins. This proposal has important long-term objectives because the selective outer membrane permeability of P. cepacia appears to play a critical role in the ability of this organism to survive in hostile environments, such as the lungs of a patient with cystic fibrosis. An improved understanding of the physiology of this serious, and sometimes life-threatening, pathogen may lead to the development of better therapeutic options.
