Finally, sera from CF patients contained antibodies to several vesicle

proteins, and a subset of patients (3 out of 13) produced antibodies to PaAP indicating that PaAP is expressed and secreted in CF patients (Fig. 7). These findings suggest that the conditions present in infected CF lungs promote upregulation of P. aeruginosa PaAP and production of vesicles that contain PaAP. 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 buy OSI-027 weight standards (kDa) and the migration selleckchem of PaAP (arrow) are indicated. Conclusion Purified P. aeruginosa vesicles associate with human lung cells and are internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibit greater association with lung cells than vesicles from a lab strain. Vesicle internalization is temperature-dependent and inhibited by hypertonic sucrose and cyclodextrins. Vesicles also appear to be very transiently associated with clathrin-coated

pits as part of an active uptake process. After internalization, vesicle components were found to colocalize with the ER. Tested CF isolates of P. aeruginosa abundantly secrete PaAP, an aminopeptidase which is a major contributor to lung cell association. Therefore, our results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial Pifithrin-�� in vivo cells and thereby contribute to the inflammatory response during infection. Methods Bacterial strains and reagents P. aeruginosa strains used were the laboratory strain PA01 (Pf1 phage-cured from our lab collection), the soil isolate ATCC 14886 (American Type Culture Collection, isolated prior to 1958), and minimally passaged, non-mucoid cystic fibrosis clinical isolates 3-mercaptopyruvate sulfurtransferase CF2, CF3, CF4, and S470 (Duke University Hospital). A549 human lung epithelia carcinoma cells were grown according to ATCC specifications in Kaighn’s F-12K media containing 10% fetal bovine serum plus penicillin/streptomycin/fungizone. Human bronchial

epithelial (HBE) cells were derived from anonymous healthy human volunteers. HBE cells were maintained in Bronchial Epithelial Cell Growth Media supplemented with thyroid extract. Unless indicated, all reagents were purchased from VWR. Construction of PA01 overexpressing PaAP (PA01/pS41) The PA2939 gene encoding PaAP was amplified from strain S470 using the primers given in Table 1, which added an EcoRI site to the 5′ end of the sequence a HindIII site to the 3′ end of the sequence. The gene was subcloned into pBluescript and then moved to pMMB66EH (provided by Erich Lanka) to make plasmid pS41. Plasmid pS41 was moved into PA01 by triparental mating as described [45], using HB101/pS41 as the donor strain and MK616 (containing pRK2013) as the helper strain.