SopB is required for PI(3)P localization to SCVs and an elevation of cellular PIP. (A and B) Cells were transfected with 2FYVE-GFP and infected with WT S. typhimurium (A) or a sopb deletion mutant (B). Left panels indicate 2FYVE-GFP relative to the RFP-expressing bacteria (red) in the merged images in the right panels. Insets are enlarged from the indicated areas (dashed boxes). Cells were analyzed using confocal microscopy. These images correspond to <15 min of infection. (C) Cells were cotransfected with PH(Akt)-RFP and 2FYVE-GFP before infection by WT S. typhimurium. Images were acquired at 1-min intervals. The time after infection of each frame is indicated. Insets are enlarged from the dashed boxes. Bars, 10 μm. (D) Cells were transfected with 2FYVE-GFP, infected with WT or sopB mutant, and analyzed as in A and B. Images were acquired at 1-min intervals for at least 1 h. Colocalization of the RFP-expressing bacteria with 2FYVE-GFP during the course of infection is shown. Data are means ± SEM of four separate experiments for WT bacteria (137 SCVs analyzed) and three separate experiments for sopB mutant (76 SCVs analyzed). (E) Phosphoinositide levels were quantified before (control) and after infection with either WT or sopB mutant. Lipids were extracted and analyzed by HPLC. The amount of the phosphoinositides is given as the percentage of PI in the same sample. PIP refers to the sum of PIPs. PIP2 refers to the sum of PIP2s. Data are means ± SEM of five separate experiments. The p-values are shown.
