Tissues reconstituted at the air-liquid interface show features of native airway epithelia. (A) After a 2- to 3-week culture, human nasal epithelial cells adhered to each other via junctional complexes (inset) and differentiated into either basal, ciliated, or goblet cells. (B) Fully polarized reconstituted epithelia expressed ezrin (green, left panel), a marker of ciliated cells, and CFTR (green, right panel) on the apical surface. (C) Freeze fracture revealed that TJ formed regular and uninterrupted belts comprising at least five fibrillar strands. (D) Immunofluorescence showed that these junctions contained both JAM-1 (green) and ZO-1 (red) proteins, which were often colocalized (yellow) and continuously surrounded each cell. Bars, 3 μm (A), 20 μm (B), 100 nm (inset), 180 nm (C), 20 μm (D). f, filter; g, goblet cell; c, ciliated cell; b, basal cell; J, junctional complex.

Production of rhamnolipids, but not elastase, is needed to promote bacterial infiltration. (A) (Left) PAO1 and the mucoid strain CHA secreted detectable levels of rhamnolipids, as visualized by the blue halo in the plate assay. In contrast, strains PT712, PAK, and PT531 did not release detectable levels of these virulence factors. Complementation of the rhlA mutation by plasmids pAKRHL and pZC6 is sufficient to release detectable levels of rhamnolipids. (Right) Elastase was produced at high levels by the PT712 mutants and to a lesser extent by PAO1. In contrast, strains PAK, PT531, and CHA did not release detectable levels of elastase in the media used for the invasion test. **, P < 0.01; ***, P < 0.001 (versus PAO1 value). Numbers of independent experiments are given on the right. (B) Pseudomonas strains MZ2 and MZ6 were derived by transforming strains PAO1 and PT712, respectively, with pZC1 (rhlA::gfp). Strain MZ2, which produced rhamnolipids, infiltrated the epithelium (upper panel). In contrast, strain MZ6, which was deficient in rhamnolipid production, was not detected within the epithelium (lower panels). Bar, 20 μm.

P. aeruginosa infiltrates the paracellular spaces in between ezrin-positive cells. (A) Profile views of control (upper left panel) and rhamnolipid-treated (lower left panel) epithelial cells immunolabeled for F-actin (blue staining) and ezrin (red staining). After exposure to rhamnolipids, the ezrin labeling decreased in the apical membrane, where it became patchy, and appeared in the basolateral domain of the cell membrane (arrows, lower left panel), indicating loss of cell polarity. Epithelia with regular, apical ezrin staining were not invaded by PAO1 (upper right panel). The bacteria (green) infiltrated intercellular spaces at sites where ezrin was observed in the basolateral membranes (arrows, lower right panel). (B) Immunostaining for goblet (mucin) (blue) and ciliated (ezrin) (red) cells revealed that, after an overnight infection by PAO1, Pseudomonas (green) was found mainly close to ezrin-positive cells. (C) After an overnight infection, electron microscopy showed that all PAO1 bacteria (arrowheads) were within the paracellular spaces. However, a few necrotic cell profiles (N), over which numerous P. aeruginosa bacteria were concentrated, were observed. (D) Shortly after exposure to rhamnolipids, PAO1 (arrowheads) was found in the paracellular spaces between ultrastructurally normal cells (upper panel). However, after 5 h, a few necrotic cell profiles were observed (lower panel) where P. aeruginosa bacteria were concentrated. Loss of polarity was evident upon paracellular infiltration. Bars, 20 μm (A and B) and 5 μm (C and D). F, filter; AD, apical domain.

l-Rhamnose cannot prevent the rhamnolipid-induced alterations of the epithelial barrier. (A) Apical pretreatment of epithelia with 2 to 0.3 mM l-rhamnose did not protect the epithelia against the significant reduction in TER that is caused by rhamnolipids. (B) Pretreatment of epithelia with 4 mM l-rhamnose also did not protect the epithelia against an overnight invasion by Pseudomonas GFP-PAO1. Actin was immunolabeled by Texas Red phalloidin to delineate the cells. Bar, 20 μm.

Bacterial invasion of the reconstituted epithelia. (A) Addition of GFP-PAO1 failed to result in adhesion of the bacteria to the apical surface of the epithelia for up to 8 h (top left panel, en face and profile views). In contrast, when the exposure was prolonged to 16 h to allow for activation of the QS systems, bacterial infiltration was evident (top right panel, en face and profile views). Strains deficient in the two QS systems (PT531) or in rhlA (PT712), as well as strain PAK, failed to infiltrate the reconstituted epithelium (profile views). In contrast, epithelia were readily invaded by the cystic fibrosis strain CHA, the TTSS-deficient strain CHAexsA, and the lasR-deficient strain PT498. The red immunostaining of actin delineates the cell profiles. Bar, 20 μm. (B) TER significantly decreased as a function of time after exposure of the apical surface of epithelia to pathogen-free supernatants of P. aeruginosa strain PAO-1, as well as to those of strain PT712, provided the latter was supplemented with purified rhamnolipids. This decrease was also observed with supernatants of the mutated P. aeruginosa strains PT531 and PT712 but was less drastic than the effects observed with supernatants containing rhamnolipids (PAO1 and PT712 plus purified rhamnolipids). Data are means ± SEMs from four independent experiments. **, P < 0.01; ***, P < 0.001 (versus control value).

Purified rhamnolipids decrease the permeability of epithelia and promote their invasion by P. aeruginosa without altering cell viability. (A) TER was not altered by 15 μg/ml rhamnolipids but was markedly decreased by ≥50-μg/ml concentrations of these factors. The rapidity of this change increased with the concentration of rhamnolipids. (B) After treatment with 150 μg/ml rhamnolipids, the permeability of the reconstituted epithelia to [³H]inulin also increased significantly as a function of time. (C) Under these conditions, the viability of epithelial cells was not affected, as evaluated by the MTT assay. (D) Staining with the LIVE/DEAD viability and cytotoxicity kit revealed that 99.5% of rhamnolipid-treated epithelial cells incorporated calcein (green), indicating cell viability (middle panel), a proportion similar to that observed in control untreated epithelia (top panel). In contrast, treatment with 0.1% saponin for 1 h induced 90% cell death, as indicated by the staining with ethidium bromide (red) (lower panel). Bar, 20 μm. (E) Addition of GFP-PAO1 to epithelia previously exposed for 60 min to 150 μg/ml rhamnolipids resulted in adhesion of the fluorescent bacteria to the surfaces of epithelial cells (upper left panel) and in invasion by numerous pathogens (lower left panel). The red immunostaining of actin, used to delineate the cell periphery, indicates that a minority of epithelial cells were in contact with P. aeruginosa and suggests that the bacteria did not enter the cells. Quantitative analysis (right panel) confirmed that, under the conditions we used, 10% of the cells contacted about 6 bacteria (stippled bars). **, P < 0. 01; ***, P < 0.001 (versus control value [solid bars]).

Rhamnolipids initially bind to the apical membrane and progressively enter the basolateral membrane. (A) Apical treatment of the reconstituted epithelia with FITC-rhamnolipids resulted, within 60 min, in a drastic drop in the TER, which was comparable to that caused by unlabeled rhamnolipids. In contrast, addition of labeled or unlabeled l-rhamnose did not alter the transepithelial resistance. (B) The loss of transepithelial resistance induced by rhamnolipids was rapidly rescued after the molecules were washed off from the apical surface of the epithelia. (C) Initially, labeled rhamnolipids were found associated with the apical membranes of epithelial cells. With time, the apical labeling (top left panel) decreased and the labeling of the basolateral membranes of the reconstituted epithelia increased (lower left and right panels). Bar, 20 μm. Actin (red) was labeled by phalloidin Texas Red to delineate the cells.

Rhamnolipids alter tight-junction architecture. (A) Control epithelial cells (time zero) featured uninterrupted belts of TJ fibrils, running in parallel, which separated the basolateral domain (BLD) of the cell membrane from the apical domain (AD). This organization was progressively altered as a function of time after rhamnolipid treatment. Thus, by 120 min, TJ belts showed reduced numbers of strands. At later time points (≥240 min), TJ belts featured strands with loose ends (arrowheads) or strands that encircled domains of the cell membrane (asterisks). At this time point, TJ belts were interrupted and no longer separated the apical and basolateral membrane domains (bottom panel). Bar, 200 nm. (B) Quantitative analysis revealed that the number of TJ strands decreased with time after rhamnolipid treatment, whereas the number of fibrils showing loose ends, i.e., not connected to other fibrils, increased. As a result of these changes, the area occupied by TJ fibrils was rapidly reduced after rhamnolipid treatment but returned to control levels within 4 h. Data are means ± SEMs for the number of measurements (one measurement per TJ belt) given at the bottoms of the bars. **, P < 0. 01; ***, P < 0.001 (versus control value).