Recruitment of BAD by the Chlamydia trachomatis Vacuole Correlates with Host-Cell Survival
Figure 5
Time Course of BAD Phosphorylation during C. trachomatis or C. muridarum Infection
(A) Epithelial cells were transfected with a BAD expression plasmid prior to infection, as described in Materials and Methods. Cells over-expressing BAD were infected with C. trachomatis at an MOI of 1.0 for the indicated times. Cells were then collected for Western immunoblotting and analyzed for AKT phosphorylation (top row), total AKT protein (second row), total BAD protein (third row), phosphorylation of BAD on residue Ser136 (fourth row), or actin (bottom row), as described in Materials and Methods. Chlamydia infection at this MOI led to a decrease in the level of total BAD protein after 32 h of infection. For AKT residue Ser473, there was a low level of phosphorylation in uninfected cells (time 0), but there was a noticeable increase in phosphorylation for both AKT and BAD after 16 h of infection. Phosphorylation levels decreased after 32 h of infection for both BAD and AKT. Chlamydia infection had no effect on actin protein levels (bottom row). One experiment of three representative experiments performed on separate days is shown.
(B) Cells expressing endogenous levels of BAD were infected with C. trachomatis at an MOI of 1.0 for the indicated times. Cells were then collected for Western immunoblotting and analyzed for phosphorylation of BAD on residue Ser136 (top row) or actin (bottom row). The time course of BAD phosphorylation in cells expressing only endogenous BAD was similar to the time course in cells that had been transfected with the BAD expression plasmid (A). One experiment of two representative experiments performed on separate days is shown.
(C) Cells over-expressing BAD were infected with C. muridarum at an MOI of 1.0 for the indicated times. Cells were then collected for Western immunoblotting and analyzed for AKT phosphorylation (top row), total AKT protein (second row), total BAD protein (third row), phosphorylation of BAD on residue Ser136 (fourth row), or actin (bottom row). Chlamydia infection at this low MOI led to a decrease in the level of total BAD protein after 32 h of infection. BAD was phosphorylated significantly within 5 h of infection, but phosphorylation levels decreased by 8 h post-infection and were not measurable after 1 d of infection. One experiment of three representative experiments performed on separate days is shown.