The respiratory epithelium provides a major defense mechanism against the many pathogens that are inadvertently inhaled, responding with the expression of chemokines such as IL-8 and mucin. In cystic fibrosis, there is excessive inflammatory response to these organisms, particularly P. aeruginosa, which initiates the airway inflammation characteristic of this disease. In the experiments described, the ligands and receptors involved in this host-pathogen interaction will be defined in detail. Caveolae, spatially organized clusters of membrane glycolipids, scaffolding proteins, and signaling kinases appear to be important in mediating bacterial attachment and directing the epithelial IL-8 response. The components of caveolae that present the GalNAcGal bacterial receptor will be established, and how they may differ in cells with CFTR mutations will be explored. As several different P. aeruginosa ligands interact with epithelial components, additional receptors will be sought, including those which may activate other epithelial signaling cascades. Many of the known P. aerugunosa ligands are glycosylated, such as pilin, flagellin, and lipopolysaccharide (LPS). Exactly how glycosylation affects the adhesin function of these gene products or their ability to stimulate the host immune response is not well established. By using genetically defined mutants of P. aeruginosa, and comparing glycosylated and non-glycosylated ligands, especially flagella, we will determine how glycosylation affects bacterial-epithelial interactions, and if these carbohydrate structures may serve as targets for the development of therapeutic strategies. LPS is an important glycosylated virulence factor that activates both immune and epithelial cells. The role of specific carbohydrate components of P. aeruginosa LPS in pathogenesis will be examined and how LPS carbohydrate side chains alter host pathogen interactions in normal and CF cells will be explored. Other bacterial virulence factors, such as phospholipases, are important is activating epithelial responses. P. aeruginosa express several PLC'S which can interact with membrane components and activate cytokine signaling. Using genetically defined mutants, the contribution of PLC to epithelial damage in normal and CF cells will be examined.
