Figure 1

Localization of Cpn0146, 0147, 0284 & 0285 in C. pneumoniae -infected cells. HeLa cells were infected with C. pneumoniae AR39 organisms at an MOI of 0.5 in the presence of 2 μg/ml of cycloheximide for 72 hours. The infected cultures grown on coverslips were processed for various immunostainings. (A) Cpn0146 was probed with a mouse antiserum (pAb, panel a) and monoclonal antibodies (mAb clones 3G2, panel b, 2F9, panel c, 11F9, panel d) while Cpn0147 by pAb (panel g) and mAbs 2G4 (panel h), 4B6 (panel i) and 7H10 (panel j), all of which were raised with the correspodning GST fusion proteins and visualized with a Cy3-conjugated goat anti-mouse IgG (red). Control mouse antibodies against IncA (2B12.1, IgG1; panel e), MOMP (GZD1E8, IgG1, panel f), CPAFcp (EB3.1, IgG1, panel k), and HSP60 (BC7.1, IgG1, panel l) were used to visualize the corresponding antigens. A rabbit anti-AR39 antiserum (R12AR39) together with a Cy2-conjugated goat anti-rabbit IgG (green) was used to visualize the C. pneumoniae organisms and Hoechst to visualize DNA. (B) The AR39 organism-infected cell samples were co-stained with the anti-Cpn0147 mAb 7H10 (IgG1, lambda; panel a; red), the anti-incA mAb 2B12.1 (IgG1, kappa; panel b; green) and DNA Hoechst dye (panel c; blue). The triple Images of the immunostainings were obtained using an AX70 fluorescence microscope equipped with a CCD camera. For confocal microscopic observation (panels e to h), the inclusions were visualized with the rabbit antiserum R12AR39 in combination with a goat anti-mouse IgG conjugated with Cy5 (blue, panel g). The images were acquired sequentially one color at a time and overlayed in tri-color using a confocal microscope (Olympus, provided by the UTHSCSA imaging core). Note that the anti-Cpn0146 and 0147 antibodies detected strong inclusion membrane signals similar to and partially overlapped with that obtained with the anti-IncA but not the other reference antibodies. (C) The labelings of Cpn0284 and 0285 with the corresponding pAbs were carried out similarly as described in (A). Note that the anti-Cpn0284 and 0285 antibodies labeled strong signals inside small but not large inclusions (panels a-d for Cpn0284 and e-h for Cpn0285; white arrows pointing to large inclusions) while the anti-MOMP mAb GZD1E8 labeled all inclusions regardless of size (panels i and l).