pneumoniae and analysed the proteome of K. pneumoniae-derived Z-VAD-FMK mouse OMVs. Furthermore, host cell death and the inflammatory response against K. pneumoniae OMVs were investigated. Our results showed for the first time that K. pneumoniae OMVs do not induce host cell cytotoxicity, but induce the innate immune response. Klebsiella pneumoniae ATCC 13883 was purchased from the American Type Culture Collection and cultured in Luria–Bertani (LB) medium (Difco, Sparks, MD) at 37 °C. HEp-2 cells from human laryngeal epithelial cells and U937 cells from human monocytes, obtained from the Korean Cell Line Bank (Seoul, Korea), were employed. HEp-2 cells were grown in Dulbecco’s modified Eagle medium (Gibco BRL, Grand

Island, NY) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM l-glutamine, 1000 U mL−1 penicillin G and 50 μg mL−1 streptomycin at 37 °C in 5% CO2. U937 monocytes were differentiated into macrophages for 3–4 days and matured by adding 500 ng mL−1 phorbol 12-myristate

13-acetate (Sigma-Aldrich, St. Louis, MO). Macrophages were cultured in RPMI-1640 (Gibco BRL) supplemented with 10% FBS and 2 mM l-glutamine at 37 °C in 5% CO2. Confluent growth was obtained in 100-mm-diameter dishes, and the cells were routinely passaged every 3 days. OMVs were purified from bacterial culture supernatants as described previously (Wai et al., 2003; Kwon et al., 2009). Briefly, K. pneumoniae was grown in LB broth until the optical density at 600 nm (OD600) PF-562271 nmr reached 1.0 at 37 °C with shaking. After the bacterial cells were removed by centrifugation at 6000 g for 15 min, the supernatants were filtered using a QuixStand Benchtop System (GE Healthcare, Piscataway, NJ) through a 0.2-μm hollow fibre membrane (GE Healthcare) to remove residual bacteria and cellular debris. The samples were then concentrated by ultrafiltration with a QuixStand Benchtop System using a 100-kDa hollow fiber membrane (GE Healthcare). The collected OMVs were further purified by ultracentrifugation at 150 000 g for 3 h at 4 °C. Purified OMVs were resuspended in Thymidylate synthase phosphate-buffered saline (PBS), and the protein

concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). The purified OMVs were checked for sterility on blood agar plates and stored at −80 °C until use. The purified OMV samples were diluted with PBS, applied to 400-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA) and stained with 2% uranyl acetate. The samples were then visualized with a TEM (Hitachi H-7500; Hitachi, Japan) operated at 120 kV. One-dimensional electrophoresis–LC–tandem mass spectrometry (1-DE-LC-MS/MS) was performed to identify proteins in the K. pneumoniae OMVs. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel digested. The protein digests were resolved in 15 μL 0.02% formic acid in 0.