FIG. 6.
The C-terminal region of Cpn0585 containing the Rab binding domain faces the cytosol of C. pneumoniae-infected cells. Rabbit polyclonal antibodies anti-Cpn0585 (A to D) or anti-EBs (F to I) were introduced into live HEp-2 cells infected for 24 h with C. pneumoniae by using PULSin reagent under the conditions described in Materials and Methods. After 24 h, cells were fixed and stained with an Alexa Fluor 594-conjugated F(ab′)2-goat anti-rabbit IgG. Prior to mounting coverslips onto glass slides, cells were incubated for 10 min with 1 μM SYTOX Green, which stains the nucleic acid of C. pneumoniae bacteria and HEp-2 cells. The signals from SYTOX Green (green; B and G) and Alexa Fluor 594 (red; A and F) were merged (C and H). C. pneumoniae-infected cells that incorporated (arrow) or not (arrowheads) anti-Cpn0585antibodies after PULSin treatment were evident in all experiments. To show that the anti-Cpn0585 and anti-EB antibodies were immunoreactive, a conventional indirect immunofluorescence assay was carried out in parallel using HEp-2 cells that had been infected for 48 h with C. pneumoniae. After fixation and permeabilization, the cells were stained with the anti-Cpn0585 (E) or anti-EB (J) antibodies, followed by the Alexa Fluor 594 F(ab′)2-goat anti-rabbit IgG secondary antibody. Merged signals from antibody (red) and SYTOX Green (green) stainings are shown (E and J). Note the peripheral staining of the inclusion membrane with anti-Cpn0585 (red) and the merged staining of EBs with both anti-EB and SYTOX Green (yellow). The data are from a representative of three experiments.