Figure 6.

Pre-infection/recovery/reinfection and its impact on KY-2 cells and chlamydial infectivity. (a) For Western blot, KY-2 cells were pre-infected (MOI 40) for 72 h, washed, recovered for 72 h and then reinfected. At different time points, cells were lysed and pellet fractions of corresponding culture supernatants were collected. β-actin served as a loading control. Primary infection of KY-2 cells is shown in the upper part of (a). (b) Flow cytometry of target cell killing by pre-infected/recovered and/or non-infected KY-2 cells. Adherent KY-2 cells were co-cultured for 4 h with YAC-1 or RMA-S cells with an effector/target ratio (E:T) of 10:1 and 20:1, respectively. The suspension target cells were carefully separated from KY-2 cells by aspiration and stained with PI. For controls, fixed KY-2 cells were mixed with target cells immediately before staining. The graph shows the fold increase of permeabilized target cells after co-cultivation with KY-2 cells compared to the control cell mix (mean values from three measurements ± SD, *p < 0.05 and **p < 0.01 vs. control (non-infected), n = 3). (c) Flow cytometry of the infectivity of culture supernatants (supern.) from infected epithelial and KY-2 cells. KY-2 cells (non-infected and recovered after primary pre-infection) and MN-R cells were infected (MOI 30) or not for 48 h. Culture supernatants were used for incubation with BGM reporter cells. The graph shows the relative amount of chlamydia-positive cells (48 hpi). (d) Flow cytometry of epithelial cell infection after treatment of EBs with granzyme B. EBs were incubated for 4 h at 37 °C with proteolytically activated granzyme B or left untreated. EBs were washed and used for the infection of MN-R cells (48 hpi, MOI 30). The result is depicted as a histogram plot (right panel) (***p < 0.001 vs. control (non-infected), n = 3). (a) depicts cropped blots obtained by each protein evaluation. Full-length blots are shown in the Supplementary Fig. S14.