The long-term goal of this project is to understand the molecular and cellular basis of the hypersusceptibility of cystic fibrosis (CF) patients to chronic infection with Pseudomonas aeruginosa. This infection is responsible for greater than 80 percent of the morbidity and mortality that occurs in CF patients. A key component of this hypersusceptibility is the role and function of the cystic fibrosis transmembrane conductance regulator (CFTR) in host resistance to P. aeruginosa. CFTR in epithelial cell membranes binds directly to P. aeruginosa and mediates bacterial internalization, an interaction critical for microbial clearance in individuals with wild-type CFTR. Although the major components of the ligand-receptor interaction have been identified the work proposed in this application will explore more of the complexities of this interaction. One focus will be on bacterial factors that provoke the cellular response leading to ingestion of whole P. aeruginosa cells and LPS. A second focus will be on the CFTR-dependent eukaryotic cellular responses to P. aeruginosa infection. The first set of aims will encompass: a) identification of P. aeruginosa proteins that provoke translocation of CFTR from cytoplasmic stores to plasma membranes, by identifying and measuring the ability of purified bacterial products to provoke membrane localization of CFTR and determining their role in the pathogenic process using cell culture and animal models of infection; b), a detailed structural analysis of the chemical components of the P. aeruginosa LPS involved in binding to CFTR using NMR and mass spectrometry techniques.
The second aim will focus on the cellular activation and signaling molecules elicited in response to the P. aeruginosa-CFTR interaction and their role in host resistance to infection. Specific areas of investigation encompass: a) studies using FACS and confocal microscopy on how CFTR extracts and internalizes the LPS from the bacterial outer membrane leading to NF-kappa B and other eukaryotic cellular responses critical for coordination of innate immunity; b) the molecular and genetic factors involved in progression of cells with wild-type CFTR to apoptosis which does not occur comparably in CF cells; and c) the role of the Fas-Fas Ligand system in CFTR-controlled apoptosis and resistance to P. aeruginosa infection. From these studies we anticipate ascertaining how wild-type CFTR coordinates immunity to P. aeruginosa lung infection and how this process is defective in CF patients. Such insights may lead to interventions to prevent the common occurrence of chronic P. aeruginosa infection in CF patients.
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