Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant DeltaF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Figure 1

Determination that CFTR is the likely epithelial cell receptor for internalizing P. aeruginosa. (A) Enhanced bacterial uptake by murine epithelioid cells transfected with cDNA for wild-type human CFTR compared with uptake by cells expressing either no CFTR (parental) or ΔF508 human CFTR, as indicated in the legend. (B) Inhibition of uptake of P. aeruginosa ingestion by human CFT1-LCFSN-transformed airway epithelial cells in the presence of membranes from the C127 cells or purified wild-type human CFTR, as indicated in the legends. PgP indicates insect cell membranes expressing P-glycoprotein from Leishmania, and control represents insect cell membranes not expressing foreign proteins. Bars represent the means of 6–9 replicates, and error bars represent SD. Asterisks (∗) indicate points significantly different from others in the same groups by ANOVA (P < .001; P < .01 for all pairwise comparisons by Fisher’s probable least-squares difference).

Figure 2

Effect of mAbs to CFTR on inhibition of internalization of three strains of P. aeruginosa. Legend indicates specificity of the mAbs. (A) P. aeruginosa strain 324. (B) P. aeruginosa strain PAO1. (C) P. aeruginosa strain 149. Bars indicate the means of 6–9 replicate determinations, and error bars represent SD. Asterisks (∗) indicate the inhibitors shown by multiple comparisons to significantly decrease internalization (P ≤ 0.001, ANOVA; P ≤ 0.01 Fisher’s probable least-squares difference for pairwise comparisons).

Figure 3

Effect of synthetic peptides of CFTR on inhibition of internalization of three strains of P. aeruginosa. Legend indicates peptide used as inhibitor. (A) P. aeruginosa strain 324. (B) P. aeruginosa strain PAO1. (C) P. aeruginosa strain 149. Points indicate the means of 6–9 replicate determinations, and error bars represent SD. Asterisks (∗) indicate the inhibitors shown by multiple comparisons to significantly decrease internalization (P ≤ 0.001; ANOVA, P ≤ 0.01 Fisher’s probable least-squares difference for pairwise comparisons). (D) Binding of carboxy-terminal biotinylated peptide comprising amino acids 103–117 of CFTR to purified complete or incomplete-core LPS from P. aeruginosa, P. aeruginosa cells, or control E. coli cells.

Figure 4

Effect of addition of synthetic peptides to the bacterial inoculum on P. aeruginosa infection in neonatal mice. (A) Amount of P. aeruginosa internalized by lung cells 24 hr after infection. (B) Total amount of P. aeruginosa found in lungs 24 hr after infection. Box plots indicate (from bottom to top) the 10th, 25th, 50th (median), 75th, and 90th percentiles. Circles above or below the 90th or 10th percentile indicate individual points outside this range. There were 12–14 total lung samples used in each group. For both groups of comparisons (A and B), the overall differences were significant at P < 0.001 using the Kruskal–Wallis nonparametric ANOVA test, and the difference between the group receiving the first-domain peptide and the other three groups was significant at P < .001 using the Dunn procedure for pairwise comparisons.

Figure 5

Electron microscopic visualization of CFTR interacting with P. aeruginosa. (A and B) P. aeruginosa cells are seen with accumulations of CFTR at a single point on the bacterial surface (arrow). (Bar = 0.1 μM; gold particles are 30 nm.) (C) Epithelial cells not ingesting P. aeruginosa showed primarily intracytoplasmic CFTR (open arrows) and membrane CFTR usually only bound to one or two gold particles (closed arrows) and, on a rare occasion, small aggregations of gold particles (closed arrowhead). (Bar = 0.5 μM; gold particles are 30 nm.) (D) Control where the primary antibody to CFTR was omitted showing epithelial cell with internalized P. aeruginosa bacteria (indicated by “b” on figure). (Bar = 0.1 μM.) (E–G) Epithelial cells not treated with methanol (used to permeabilize the cells in A–D for intracytoplasmic antibody reactions) showed P. aeruginosa in intracytoplasmic enclosures with only a portion of the bacterial cell surface attached to the vesicle membrane (arrow), similar to the accumulation of CFTR on the bacterial surface (A and B). (Bar = 0.1 μM in E; 0.5 μM in F and G.) (H) Epithelial cells homozygous for the ΔF508 CFTR mutation could not be seen ingesting P. aeruginosa. (Bar = 1.0 μM.)

Figure 6

Immunofluorescence of CFTR in lung sections of mice infected with P. aeruginosa. (Bars = 100 μM.) (A) CFTR visualized by mAb CF3 and fluoresceinated secondary antibody in the airway epithelium of mice infected with P. aeruginosa 24 hr previously. (B) Hematoxylin and eosin stain of a lung section from a neonatal mouse infected with P. aeruginosa. (C) Visualization of CFTR by mAb CF3 and fluoresceinated secondary antibody in the airway epithelium of mice infected with P. aeruginosa 24 hr previously was inhibited if the mAb was first incubated with the first extracellular domain synthetic peptide (amino acids 103–117 of CFTR). (D) CFTR could not be visualized by mAb CF3 and fluoresceinated secondary antibody in the airway epithelium of mice infected 24 hr previously with P. aeruginosa along with a synthetic peptide corresponding to the first predicted extracellular domain of CFTR. This synthetic peptide inhibited CFTR-mediated epithelial cell internalization of P. aeruginosa and the subsequent clearance of the bacterium from the lung (see Fig. 4). (E) Staining of uninfected mouse lung section with mAb CF3 and fluoresceinated secondary antibody. Little to no CFTR is seen in the epithelium. (F) Hematoxylin and eosin stain of a lung section from an uninfected neonatal mouse.