Abstract
BackgroundPseudomonas 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.ResultsPurified 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, TRAPα, 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.ConclusionThese 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.
Highlights
Pseudomonas aeruginosa is the major pathogen associated with chronic and fatal lung infections in patients with cystic fibrosis (CF)
P. aeruginosa vesicle association with lung epithelial cells is strain-dependent We examined whether vesicles from various P. aeruginosa isolates would associate with cultured human respiratory epithelial cells
Labeled vesicles (FITC-vesicles) from late log-phase cultures were incubated with A549 human lung epithelial cells and the amount of vesicles associated with host cells after incubation at 37°C was quantitated using a previously established microtiter plate assay [14]
Summary
Pseudomonas aeruginosa is the major pathogen associated with chronic and fatal lung infections in patients with cystic fibrosis (CF). Like other gram negative bacteria, P. aeruginosa release spheres of outer membrane known as outer membrane vesicles [6] They consist of entrapped periplasmic components and outer membrane constituents, including lipopolysaccharide (LPS), glycerophospholipids, and outer membrane proteins (OMPs) [7]. Considering that vesicles are armed with bacterial proteases, toxins, surface adhesins and/or invasins, vesicles present a potentially significant contributor to lung damage caused by P. aeruginosa. Since they contain many immunostimulatory compounds, it is not surprising that P. aeruginosa vesicles induce a significant IL-8 response from cultured human lung cells [8]
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