Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

P. aeruginosa forms biofilms in the lungs of individuals with cystic fibrosis (CF); however, there have been no effective model systems for studying biofilm formation in the CF lung. We have developed a tissue culture system for growth of P. aeruginosa biofilms on CF-derived human airway cells that promotes the formation of highly antibiotic-resistant microcolonies, which produce an extracellular polysaccharide matrix and require the known abiotic biofilm formation genes flgK and pilB. Treatment of P. aeruginosa biofilms with tobramycin reduced the virulence of the biofilms both by reducing bacterial numbers and by altering virulence gene expression. We performed microarray analysis of these biofilms on epithelial cells after treatment with tobramycin, and we compared these results with gene expression of (i) tobramycin-treated planktonic P. aeruginosa and (ii) tobramycintreated P. aeruginosa biofilms on an abiotic surface. Despite the conservation in functions required to form a biofilm, our results show that the responses to tobramycin treatment of biofilms grown on biotic versus abiotic surfaces are different, as exemplified by downregulation of genes involved in Pseudomonas quinolone signal biosynthesis specifically in epithelial cell-grown biofilms versus plastic-grown biofilms. We also identified the gene PA0913, which is upregulated by tobramycin specifically in biofilms grown on CF airway cells and codes for a probable magnesium transporter, MgtE. Mutation of the PA0913 gene increased the bacterial virulence of biofilms on the epithelial cells, consistent with a role for the gene in the suppression of bacterial virulence. Taken together, our data show that analysis of biofilms on airway cells provides new insights into the interaction of these microbial communities with the host.

The cystic fibrosis (CF) lung is chronically inflamed and infected by Pseudomonas aeruginosa, which is a major cause of morbidity and mortality in this genetic disease. Although aerosolization of Tobramycin into the airway of CF patients improves outcomes, the lungs of CF patients, even those receiving antibiotic therapy, are persistently colonized by P. aeruginosa. Recent studies suggest that the antibiotic resistance of P. aeruginosa in the CF lung is due to the formation of drug resistant biofilms, which are defined as communities of microbes associated with surfaces or interfaces, and whose growth is facilitated by thick and dehydrated mucus in the CF lung. In this review, we discuss some of the current models used to study biofilm formation in the context of biotic surfaces, such as airway cells, as well as the contribution of host-derived factors, including DNA, actin and mucus, to the formation of these microbial communities. We suggest that better in vitro models are required, both to understand the interaction of P. aeruginosa with the host airway, and as models to validate new therapeutics, whether targeted at bacteria or host.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl ؊ channels in the apical plasma membrane. Rescue of ⌬F508-CFTR by reduced temperature or chemical means reveals that the ⌬F508 mutation reduces the half-life of ⌬F508-CFTR in the apical plasma membrane. Because ⌬F508-CFTR retains some Cl ؊ channel activity, increased expression of ⌬F508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ⌬F508-CFTR that lead to the decreased apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o؊) the ⌬F508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is an ATP binding cassette (ABC) transporter and a cAMP-regulated Cl Ϫ channel that mediates transepithelial Cl Ϫ transport in the airways, intestine, pancreas, testis, and other tissues (1-3). Cystic fibrosis (CF), a lethal genetic disease, is caused by mutations in the CFTR gene (1, 2). The most common mutation in CFTR is ⌬F508 (4, 5). ⌬F508-CFTR does not fold properly, and most of the protein is retained within the endoplasmic reticulum (ER) where it is subsequently degraded (5, 6). Several studies suggest that the ER retention of ⌬F508-CFTR is not complete, and some ⌬F508-CFTR is constitutively expressed in the plasma membrane of primary epithelial cells from individuals homozygous for the ⌬F508 mutation (7-10). Because ⌬F508-CFTR retains some Cl Ϫ channel activity when expressed in the plasma membrane (5,6,(11)(12)(13)(14), it would be desirable to increase the expression of ⌬F508-CFTR in the plasma membrane to alleviate the symptoms in CF patients. The trafficking of ⌬F508-CFTR to the plasma membrane can be increased by chemical means or reduced temperature (15-21). Yet, functional and biochemical studies in heterologous cell lines demonstrate that rescued ⌬F508-CFTR has a greatly reduced stability or halflife in the post-ER compartments, including the plasma membrane (13,(22)(23)(24). Very little is known about the apical membrane half-life of rescued ⌬F508-CFTR in polarized human airway epithelial cells. A recent study demonstrates that the functional stability of ⌬F508-CFTR in the apical membrane of differentiated respiratory epithelial cells derived from nasal polyps from individuals homozygous for the ⌬F508 mutation is decreased compared with WT-CFTR (25). Furthermore, the bioc...

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