RSV strains Long and A2, human type II pulmonary epithelial cell line A549, S. pneumoniae strain R6, and H. influenzae strain Rd (KW20) were obtained from the American Type Culture
Collection (Manassas, VA). Clinical isolates of S. pneumoniae and H. influenzae were described previously (Yokota et al., 2004; Ohkoshi et al., 2008). RSV was grown in HEp-2 cells. The virus titer of RSV was determined by a plaque-forming assay using HEp-2 cells as an indicator (Okabayashi et al., 2009). Fosfomycin was obtained from Meiji Seika Kaisha (Tokyo, Japan). A PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N,-trimethyl)- hexanolamine, was purchased from Calbiochem-Merck KGaA (Darmstadt, Germany). An NF-κB inhibitor, PDTC, was purchased from Sigma-Aldrich (St. Louis, MO). A phosphocholine-deficient mutant was isolated by serial passage of S. pneumoniae strain R6 in a chemically defined Vorinostat mw medium (CDM) containing decreasing concentrations of ethanolamine with each passage according to Yother et al. (1998). Briefly, approximately
106 cells were cultured in 2 mL of CDM containing 200 μg mL−1 ethanolamine for 12 h at 37 °C and then diluted 100-fold into the same medium. Following five 12-h passages in CDM containing 200 μg mL−1 ethanolamine, similar passages were performed in successively lower concentrations of ethanolamine (20, 2, 0.2, and then 0 μg mL−1). The resulting mutant was capable of growth in CDM without choline
or ethanolamine. The cell wall fraction was prepared as follows: cells grown to selleck chemicals the mid-log phase were harvested and immediately boiled with saline containing 4% SDS for 20 min. The boiled cells were disrupted by CYTH4 sonication and then centrifuged at 20 000 g for 15 min. The pellet was washed extensively with saline, and then used as a cell wall fraction. The content of choline in the cell wall preparation was determined using an enzymatic method (Assmann & Schriewer, 1985). The choline contents of cell wall fractions from R6 and the mutant were 435 nmol mg−1 and undetectable, respectively. Cell surface expression of the PAF receptor was examined by flow cytometry. A549 cells were harvested from culture flasks using a cell scraper, and then incubated with 2.5 μg mL−1 of mouse anti-PAF receptor monoclonal antibody [11A4 (clone 21); Cayman Chemical, Ann Arbor, MI] or mouse IgG2a,κ isotype control antibody (eBioscience, San Diego, CA). After incubation at 4 °C for 30 min, cells were collected by centrifugation and washed once with Dulbecco’s phosphate-buffered saline [PBS(−)]. Cell suspensions were incubated with a phycoerythrin-conjugated goat anti-mouse IgG F(ab)2 fragment antibody (1 : 100 dilution) (Abcam, Cambridge, UK) at 4 °C for 30 min, and the stained cells were assessed with a FACSCalibur (BD Bioscience, San Jose, CA). A bacterial suspension in 0.1 M NaCl–50 mM sodium carbonate buffer (pH 9.5) at 1 × 108 CFU mL−1 was prepared.