Figure 3

EscU auto-cleavage is required for Tir translocation, actin pedestal formation and maximal intimate EPEC adherence. (A) Fluorescent microscopy images of HeLa cells following a three-hour infection with various EPEC strains. Phalloidin staining (red) was used to detect F-actin. All EPEC strains contain a plasmid that encodes GFP (green). Note the strong F-actin enrichment (red signal) within the boxed insets. This experiment was performed twice and representative merged images are shown. (B) Quantification of intimately adherent bacteria using a binding index. The bacterial binding index was defined as the percentage of cells with at least five bound bacteria that co-localized to actin pedestals. At least 50 cells were counted per sample. (C) A Tir-TEM1 effector translocation assay was used to quantify translocation levels during infection of HeLa cells. HeLa cells were infected with the indicated bacterial strains, washed twice to remove non-adherent bacteria and then loaded with the cell permeable fluorescent β-lactamase substrate CCF2/AM. Blue and green (460 and 530 nm) signals were detected with a plate reader and the fluorescence ratio (460/530 nm) corrected for background is shown for the indicated strains. An immunoblot of whole cell lysates with anti-TEM1 antibodies demonstrated equivalent amounts of β-lactamase in the five strains with pTir-bla (inset). The presented translocation assay data are averages of triplicate values of the results from three independent experiments.