Our hypothesis is that P. aeruginosa decreases plasma membrane expression of CFTR in human airway epithelial cells by affecting the endocytic trafficking pathway. We hypothesize that a soluble, heat-sensitive factor secreted by P. aeruginosa decreases plasma membrane expression of wt- and deltaF508-CFTR by increasing endocytosis and/or decreasing endocytic recycling. Because identification of drugs that stimulate deltaF508-CFTR expression in the plasma membrane of airway epithelial cells is one of the major goals of CF drug discovery, it is important to know if P. aeruginosa has an effect on the plasma membrane expression of CFTR. Our preliminary studies demonstrate that P. aeruginosa causes internalization of plasma membrane wt-CFTR into endocytic vesicles in Calu-3 and MDCK cells. Furthermore, our data also indicate that P. aeruginosa reduces transepithelial CI secretion in polarized epithelial cells expressing wt-CFTR and deltaF508-CFTR. Thus, as long as CF patients are colonized with P. aeruginosa, drugs designed to increase the expression of deltaF508-CFTR in the plasma membrane may not be efficacious. Accordingly, the hypothesis to be tested is that P. aeruginosa decreases the apical plasma membrane expression of CFTR by affecting the endocytic trafficking of CFTR. To test this hypothesis we propose three specific aims:
Specific Aim # 1. Test the hypothesis that P. aeruginosa decreases plasma membrane expression of wt-CFTR by affecting the endocytic trafficking pathway. Plasma membrane expression of CFTR depends in part on the relative rates of endocytosis and endocytic recycling. Accordingly, the goal of this specific aim is to determine if P. aeruginosa decreases plasma membrane expression of wt-CFTR by regulating the endocytic trafficking pathway;
Specific Aim #2. Test the hypothesis that P. aeruginosa decreases plasma membrane expression of deltaF508-CFTR by affecting the endocytic trafficking pathway. The goal of this aim is to determine if P. aeruginosa reduces the efficacy of drugs that restore the trafficking of deltaF508-CFTR to the plasma membrane by regulating the endocytic trafficking pathway of deltaF508-CFTR;
Specific Aim # 3. Test the hypothesis that a heat-sensitive factor secreted by P. aeruginosa affects the endocytic trafficking of CFTR. The goal of this specific aim is to identify the secreted factor. We will use a combination of complementary approaches, including molecular, biochemical, functional, and proteomic to elucidate how P. aeruginosa reduces the plasma membrane expression of CFTR. We anticipate that our studies may lead to the development of drugs that block the ability of P. aeruginosa to reduce plasma membrane expression of deltaF508-CFTR, and therefore will facilitate our long-term goal, which is to develop new therapies to increase the expression of deltaF508-CFTR in the plasma membrane.
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