Fig. 4

Translocation of Tir into mammalian cells by SIEC-eLEE5-eEtgA. A Graphic representation of the Tir translocation process. EPEC and SIEC-eLEE5 can inject Tir into the cytoplasm of mammalian cells through injectisomes. Tir is then exposed on the surface of the cell membrane where it interacts with intimin of the bacteria, leading to the polymerization of F-actin filaments and the formation of pedestal-like structures beneath the bacterium. B Scheme of the synthetic operon eLEE5 containing the coding genes for Tir (tir), the chaperone CesT (cesT) and intimin (eae) under the control of the tac promoter (Ptac). This operon is integrated at the flu site of the chromosome. C Representative confocal fluorescence microscopy images of HeLa cells infected with the indicated bacterial strains (SIEC-eLEE5, SIEC-eLEE5-eEtgA and SIEC-eLEE5ΔescN). F-actin is stained with TRITC-conjugated phalloidin (red), and DNA and nuclei are stained with DAPI (grey). Bacteria are labeled with rabbit polyclonal anti-intimin280 and ALEXA488-conjugated anti-rabbit antibodies (green). The F-actin accumulations can be visualized as strong red fluorescence signals associated with attached bacteria, indicating the translocation of Tir protein. Bottom images are magnifications of the regions in the upper images marked with white squares