We confirmed that purified PAO1/pS41 vesicles were enriched in PaAP compared with PA01 vesicles and that S470APKO5 did not contain detectable amounts of PaAP [see Additional file 2]. Purified PAO1/pS41 vesicles associated with A549 cells more

than twice as much as PA01 vesicles, whereas S470APKO5 vesicles associated 40% less with the lung cells than S470 vesicles (Fig. 6B, C). Unfortunately, complementation of S470APKO5 was not successful since vesicles from S470APKO5 expressing PaAP from pS41 contained approximately 10-fold less PaAP and had 10-fold less aminopeptidase activity than S470 vesicles [see Additional file 3, parts A and B]. Induction of PaAP expression in S470APKO5 did not help correct the complementation GSK-3 inhibitor defect and increase the level of vesicle-bound PaAP, although the total amount of PaAP

in the supernatant was equivalent to that of S470 [see Additional file 3, part C]. As a result, it was not surprising that S470APKO5/pS41 vesicles associated with host cells to approximately the same extent as those from APKO5 (data not shown). selleckchem Collectively, these data support a dose-dependent contribution of PaAP to the Torin 2 in vivo association of vesicles with host cells. 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 Digestive enzyme 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. Discussion With these results, we have revealed several facets of interactions between P. aeruginosa vesicles and human lung epithelial cells. We have demonstrated that P. aeruginosa vesicles are internalized by epithelial cells and trafficked intracellularly so that vesicle components accumulate in the ER. We have also shown that PaAP, an enzyme more abundant in vesicles produced by many CF isolates compared with non-clinical isolates, significantly contributes to the interaction of P. aeruginosa vesicles with host cells. Internalization by host cells has been reported to occur for outer membrane vesicles from numerous species. For instance, our lab has shown previously that ETEC vesicles are internalized in an LT-dependent fashion via ganglioside GM1 in caveolin-enriched lipid rafts of epithelial cells [20].