Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

Abstract

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), DeltaF508, leads to the absence of CFTR Cl(-) channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl(-) channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl(-) secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl(-) secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and DeltaF508-CFTR Cl(-) channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of DeltaF508-CFTR.

Targeting CAL as a Negative Regulator of ΔF508-CFTR Cell-Surface Expression

“…Previous studies have demonstrated that mature, glycosylated ⌬F508-CFTR is expressed at the apical plasma membrane both in parental CFBE41o-cells and in CFBEϩ⌬F508 cells at 37°C, and shown that significant amounts of ⌬F508-CFTR are rescued by incubation at 27°C in the transduced cell line (30,35). Other studies have shown that the CFBEϩ⌬F508 cells express functional ⌬F508-CFTR chloride channels (34).…”

“…Electrophysiology-Seven days after seeding monolayers, Ussing chamber measurements were performed essentially as described (34), except that 50 M amiloride was used. For these studies, 50 M genistein was applied apically to activate temperature-rescued ⌬F508-CFTR channels (34,35).…”

“…For these studies, 50 M genistein was applied apically to activate temperature-rescued ⌬F508-CFTR channels (34,35). Once maximal activation was achieved, 5 M 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR inh -172, EMD Biosciences, Refs.…”

Molecular typing and biological characteristics of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Brazil

“…Once chronic infection is established, P. aeruginosa is virtually impossible to eradicate and is associated with increased mortality and morbidity in CF patients. 10 Investigations of the nosocomial epidemiology of P. aeruginosa have been hampered by the inadequate discriminatory ability of classical phenotypic methods such as serotyping, phage and pyocin typing, and biotyping. 11,12 Modern DNA-based techniques, such as enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and Ribotyping, have been widely used in the epidemiological investigation of many microorganisms, including P. aeruginosa.…”

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

“…Among these is the CFTR inhibitory factor (Cif), a recently characterized P. aeruginosa protein expressed both in the PA14 strain and in clinical isolates (20,21). Originally characterized as an extracellular factor that inhibited CFTR-mediated chloride currents across polarized epithelial monolayers (22), Cif is both directly secreted and is also packaged into outer membrane vesicles that dramatically enhance delivery to airway epithelial cells (23). Further investigation revealed that Cif acts by triggering the post-endocytic degradation of CFTR, reducing its cell-surface abundance (22).…”