The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System
Fig 5
EspC controls pore formation mediated by the T3SS during cell infection.
(A-C) Cells were challenged with EPEC strains for 45 min in presence of the fluorescent membrane-impermeant dye LY and analyzed by epifluorescence microscopy. (A) Representative micrographs of fixed TC7 cells showing DAPI staining (right) or LY fluorescence (middle panels). Binary images were generated by thresholding images corresponding to the LY fluorescence (right panels). LY positive cells were scored from binary images and the average percentage of LY cells / total cells ± SEM is indicated for each samples in TC7 cells (B) or HeLa cells (C). (D-E) Cells were loaded with the fluorescent dye calcein prior to bacterial challenge for 45 min. (D) Representative micrographs of pseudocolored fluorescence images of cells challenged with the indicated bacteria. Dashed lines indicate contours delineated from phase contrast images of cells with fluorescence intensity below the applied threshold. (E) The relative percentage of calcein leakage was calculated after normalization to cells challenged with the T3SS-deficient ΔescN strain (Experimental Procedures). The total number of analysed cells (n) and number of experiments (N) is indicated. *: p ≤ 0.05; **: p ≤ 0,01. Scale bar: 20 μm.