Pseudomonas aeruginosa outer membrane vesicles: Strain-dependent differences in composition and interactions with host cells

Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that is a major cause for morbidity and mortality in individuals with compromised lung function such as in patients with cystic fibrosis (CF). One major cause of lung injury results from the acute inflammatory response to the infection. Vesicles, consisting of periplasmic and outer membrane proteins and lipids, are secreted by P. aeruginosa as well as many other well-characterized pathogens. We characterized vesicles produced by P. aeruginosa and investigated their interactions with human respiratory cells and the consequent immune response. Vesicles were produced during log phase of growth and persisted into stationary phase. Vesicle purification protocols revealed that elastase could degrade vesicle proteins in concentrated supernatants. Vesicles were similar to outer membranes in protein profile. 2D-DIGE analysis comparing CF strain vesicles to lab strain vesicles revealed differences in protein composition and abundance. Sequencing of vesicle proteins identified the aminopeptidase PaAP (PA2939) as highly enriched protein in vesicles from CF strains. PaAP appeared to exist predominantly in a high molecular weight complex (>100 kDa). Vesicle-associated PaAP in vesicles was found to be active and exteriorly located. Since vesicles are likely to come into contact with host cells during an infection, we investigated the cellular response to vesicles from P. aeruginosa strains of different origins. Vesicles from CF strains associated with lung cells more than vesicles from lab and soil strains. The internalization of vesicles appeared to be cholesterol-dependent. Vesicles partially co-localized with both clathrin and the endoplasmic reticulum marker TRAPalpha. PaAP appear to promote vesicle association with lung cells, as association decreased when it was knocked out of a CF strain and association increased when PaAP was present in lab strain vesicles. All vesicles induced an IL-8 response from lung cells. The induction of IL-8 by vesicles was dose dependent and significantly higher than that elicited by purified LPS from the same strain, suggesting that LPS alone was not responsible for the IL-8 response. These results suggest that CF isolates of P. aeruginosa produce vesicles that are enriched in a protease that promotes the association of vesicles with lung cells, and the association of vesicles with lung cell can contribute to the inflammatory response.