Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

Abstract

Background: Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells.

Results: Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPalpha, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association.

Conclusion: These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

Figure 1

Vesicles from a CF isolate associates to a greater extent with lung cells compared to PA01 vesicles. FITC-labeled vesicles (2.5 μg per well) were incubated with confluent monolayers of approximately 5 × 10⁴ A549 cells (A) or HBE cells (B) for indicated times at 37°C. Fluorescence associated with washed, solubilized cells was quantitated and correlated to vesicle amount using standard curves generated for vesicles from each strain. Experiments were done in triplicate, SEM is indicated for 2 to 7 separate experiments. At the 24 h time point, p < 0.001 for each data set.

Figure 2

S470 vesicle association with host cells is temperature-dependent. A, FITC-labeled-vesicles (2.5 μg per well) were incubated with A549 cells (5 × 10⁴ cells per well) for 24 h at 37°C (black bars), or 4°C (gray bars). SEM is indicated, n≥2, in triplicate. B and C, A549 cells alone (left panels) or incubated with 2.5 μg FITC-labeled S470 vesicles (green, right panels) for 6 h at 37°C (B) or 4°C (C). After incubation, cells were washed, labeled with AF633-WGA (blue), fixed in 2% paraformaldehyde, and visualized by confocal microscopy.

Figure 3

Vesicle components are internalized by lung cells, and internalization is inhibited by hypertonic sucrose and cyclodextrins. A, SDS-PAGE gel profiles of S470 vesicles before and after AF488 labeling. Total protein in unlabeled vesicles was visualized after SYPRO Ruby staining of the gel (R). AF488-labeled proteins were visualized by placing the unstained gel on a UV lightbox (F). The migration of molecular weight standards (kDa) and PaAP (arrow) is indicated. B, A549 cells incubated with 2.5 μg AF488-labeled S470 vesicles (green) for 1 h at 37°C. Cell surface was labeled using biotin and AF633-streptavidin (blue), fixed in 2% paraformaldehyde, and visualized by confocal microscopy. A549 cells were pretreated with 10 mM methyl-β-cyclodextrin (C), 10 mM α-cyclodextrin (D), or 0.45 M sucrose (E), for 30 minutes, and then incubated with 2.5 μg AF488-labeled S470 vesicles (green) for 1 h at 37°C. Cell surface was labeled using biotin and AF633-streptavidin (blue), fixed in 2% paraformaldehyde, and visualized by confocal microscopy. Bars indicate 25 μm.

Figure 4

Vesicles rarely co-localize with surface-associated clathrin. AF488-S470 vesicles (2.5 μg) were incubated with A549 cells for 1 h at 37°C. Cell surface was labeled using biotin and AF633-streptavidin (blue). Cells were washed, fixed, permeabilized, and probed with mouse anti-clathrin antibodies and AF555-labeled goat anti-mouse secondary antibody. Arrows indicate very occasional colocalization of clathrin and vesicle fluorescence at the cell surface.

Figure 5

Vesicles co-localize with the endoplasmic reticulum marker TRAPα. AF488-S470 vesicles (2.5 μg) were incubated with A549 cells for 1 hour at 37°C. Cell surface was labeled using biotin and AF633-streptavidin (blue). Cells were washed, fixed, permeabilized with 0.015% digitonin to release cytoplasm, and probed with anti-TRAPα primary antibody and AF555-labeled secondary antibody.

Figure 6

PaAP is abundant and active in vesicles from CF strains and promotes the association of P. aeruginosa vesicles with lung cells. A, Purified vesicles (approximately 10 μg) were TCA-precipitated and analyzed using SDS-PAGE and Coomassie staining. Previously identified proteins in PA01 vesicles and CF2 vesicles are indicated, and (*) highlights the lower molecular weight form of OprD found in PA01 [8]. The migration of molecular weight standards is indicated (kDa). B and C, Purified vesicles from the indicated strains (2.5 μg protein/well) were incubated (24 h, 37°C) with confluent monolayers of A549 cells (5 × 10⁴/well) and vesicle-host cell association was compared with S470 vesicle association within each experimental set. SEM is indicated; n = 2 in triplicate.

Figure 7

CF patients produce antibodies to PaAP. Purified outer membranes (OM) from S470 and vesicles (V) from S470 and S470APKO5 (KO) (2 μg) were separated by SDS-PAGE and stained with SYPRO Ruby (A) or transferred to PVDF and immunoblotted using sera from a CF patient and then reblotted with anti-PaAP (B). Molecular weight standards (kDa) and the migration of PaAP (arrow) are indicated.