Fig. 2. Pneumococcal CPS expression on EcN ΔflhD cells.

A Western blot using anti-CPS14 (left panel) and anti-O6 antisera (middle panel), with sliver staining (right panel) as the loading control. The bacterial strains used in this experiment are shown with lane numbers at the lower right. For CPS14- and O6-probed western blot, the whole cells of all examined E. coli strains were standardized at an OD₆₀₀ of 4.0, and the whole cells of all examined S. pneumoniae strains were standardized an OD₆₀₀ of 8.0 using SDS-PAGE sample buffer. Twenty microliters of each sample was applied to the 12.5% polyacrylamide SDS-PAGE, electro-transferred onto a PVDF membrane, and probed with an anti-lipid A-core oligosaccharide antibody. For silver staining, 10-fold diluted whole-cell samples were prepared, i.e., whole cells of E. coli and S. pneumoniae were standardized an OD₆₀₀ of 0.4 and 0.8, respectively, and twenty microliters of each sample was applied to the 12.5% polyacrylamide SDS-PAGE. Asterisk and dagger on CPS14-probed western blot membrane (left panel) denote CPS14-specific signals detected in the wells and the stacking gel of SDS-PAGE gel, respectively, shown in lanes 7 (S. pneumoniae, strain ATCC 700676 [serotype 14]) and 8 (S. pneumoniae, strain KSP1094 [serotype 14]). B Amounts of exogenous pneumococcal CPS14 (left panel) and E. coli original O6 (right panel) in whole cells of EcNΔflhD/pWSK129 (vector control, n = 3) and EcNΔflhD/pNLP80 (CPS14⁺, n = 3). All whole-cell samples used for analysis were collected from independent experiments. Data are expressed as the mean ± SD from results obtained in three independent experiments. *p ≤ 0.05, when performed with a Mann–Whitney U-test. C Immuno-FE-SEM. EcN ΔflhD cells harboring CPS14⁺ vector and the vector control, and S. pneumoniae strain ATCC 700676 (serotype 14) were probed with anti-CPS14 antibody (α-CPS14) or non-immunized rabbit serum (Control Ab). Primary antibodies were used at 1:500 dilutions. Anti-rabbit IgG antibody conjugated with 12-nm colloidal gold were used as the secondary antibody at 1:50 dilutions. Shown are representative images with scale bars indicated at the lower right. Bars: 200 nm. D Immuno-TEM. Whole cells of EcN ΔflhD harboring CPS14⁺ vector and the vector control were incubated with α-CPS14 or the control Ab. The primary antibodies were used at 1:500. Anti-rabbit IgG antibody conjugated with 12-nm colloidal gold was used as the secondary antibody at 1:50 dilutions. Shown are representative images with scale bars indicated at the lower right. Bars: 200 nm. E Subcellular fractionation of CPS14-expressing EcN ΔflhD cells. Subcellular fractions (cytoplasm [CP], inner membrane [IM], periplasm [PP], and outer membrane [OM]) were probed with α-CPS14, and with antisera against the following subcellular marker proteins: Crp [CP], RodZ [IM], DsbA [PP], and OmpA [OM]. Subcellular fractions were also stained with CBB. A 20-fold concentrated cytoplasmic fraction (CP), a 100-fold concentrated inner membrane fraction (IM), a 20-fold concentrated periplasmic fraction (PP), and a 100-fold concentrated outer membrane fraction (OM) were prepared from the bacterial suspensions standardized to OD₆₀₀ = 2.0. Ten microliter of each sample was analyzed by SDS-PAGE.