Title: Internalization of Pseudomonas aeruginosa by non-polarized and polarized epithelial cells
Description: Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population, affecting 1:2500 live births each year. The disease is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, with over 1500 mutations described so far. CFTR mutations affect multiple organs, but it is the chronic lung infections, caused by the gram-negative bacterium Pseudomonas aeruginosa , that account for most of the morbidity and more than 90% of the mortality in cystic fibrosis. The relationship between mutated CFTR molecules and P. aeruginosa lung infections remains at the center of intense research efforts. In this dissertation work, I have derived and characterized a novel, isogenic system for studying P. aeruginosa invasion of lung epithelial cells in vitro by stable transfection of a non-polarized human cystic fibrosis bronchial epithelial cell line with green fluorescent protein (GFP)-CFTR expressing plasmid. I show that in this non-polarized cell system, the expression of both CFTR and caveolin-1, a caveolar protein component without which caveolae cannot form, is necessary for efficient internalization of P. aeruginosa .

In these cells, CFTR and caveolin-1 are colocalized with P. aeruginosa as the bacteria attach, invade, and are encompassed by endocytic/vacuolar membrane. Furthermore, a parallel examination of the three-dimensional distribution of CFTR and caveolae and of Pseudomonas uptake efficiency in polarized epithelium has revealed that: (1) in polarized epithelial cell cultures, apical membrane-associated CFTR proteins are spatially separated from the basolateral membrane-localized caveolae, and P. aeruginosa invasion is minimal; (2) disruption of epithelial cell polarity via disruption of tight junctions leads to a rapid redistribution of apical membrane CFTR throughout the entire plasma membrane and correlates with significantly increased CFTR- and caveolin-1-dependent bacterial uptake; and (3) both CFTR and caveolin-2 colocalize with the attached, entering, and intracellular P. aeruginosa in cell cultures whose polarity is disrupted. Based on these results, I propose a novel, general mechanism for P. aeruginosa invasion of lung epithelial cells in which physical interaction of P. aeruginosa with caveolar CFTR is necessary for efficient bacterial uptake.