Role of Cftr in Host Defence Against Pseudomonas Aeruginosa

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial anion channel which is mutated in people with cystic fibrosis (CF). Loss of CFTR function in the airways leads to bacterial infection, chronic inflammation and eventually respiratory failure. Interactions between CFTR and the most common CF pathogen, Pseudomonas aeruginosa (PA), are not well understood. In this study we have studied the dynamical and functional effects of bacterial infection on CFTR in human primary airway epithelial cells. A new analysis based on the well-established k-space image correlation spectroscopy technique (kICS) was used to extract the mean-square-displacement (MSD) of fluorescently labeled CFTR (GFP-CFTR) molecules in time. Under resting conditions, GFP-CFTR formed sub-resolution clusters that were homogeneously distributed in sub-confluent cells. kICS analysis revealed the presence of two dynamically distinct populations, one which was freely diffusing (Ddiff = 1.5 ±0.3 x10-2 μm2/s) and other which was spatio-temporally confined (Dconfine = 10 ±2 x10-3 μm2/s). The confined population was dependent on cholesterol and sphingomyelin. When epithelial cells were exposed to PA, small CFTR clusters aggregated into large platforms (0.5-3 μm). This was accompanied by a significant reduction in CFTR dynamics (from Ddiff = 0.2 ±0.2 x10-2 μm2/s to Dconfine = 2.5 ±0.4 x10-3 μm2/s). Pre-treatment with lipid raft disrupting reagents prevented platforms from forming upon treatment with PA and the subsequent reduction of CFTR dynamics. CFTR platform formation is also triggered by several host defence mechanisms and inflammatory factors even in the absence of bacteria, therefore it may help protect the host epithelial cell against invasion by pathogens.