Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis
Figure 11
Translocation of EHEC-Hly to mitochondria results in cytosolic cytochrome c release and ΔΨm decrease.
(A, B) HBMEC (A) and Caco-2 cells (B) were incubated for the times indicated with EHEC-Hly-containing OMVs TA50 or 8033 (20 µg of OMV protein containing 100 ng of EHEC-Hly) or with corresponding protein amounts of EHEC-Hly-negative OMVs TA51 or 8033c or left untreated (control). Mitochondrial and cytosolic fractions were isolated and immunoblotted with anti-cytochrome c antibody. After stripping, membranes were reprobed with antibody against porin-2 (mitochondrial marker) or actin (cytosolic marker). The sizes of immunoreactive bands are indicated. (C, D) Intensities of the cytochrome c signals in mitochondrial (mito) and cytosolic (cyto) fractions shown in panels A and B were quantified in HBMEC (C) and Caco-2 cells (D) using densitometry, expressed in arbitrary densitometric units (DU) and are presented as means ± standard deviations from three independent experiments. * Significantly decreased (p<0.05) compared to mitochondrial cytochrome c in control cells; ** significantly increased (p<0.05) compared to cytosolic cytochrome c in control cells (unpaired Student's t test). Mitochondrial cytochrome c signals in control cells (control mito) were almost identical after 8 h, 16 h and 24 h and are therefore shown as means ± standard deviations from all times. No cytochrome c was detected at any time in cytosol of control cells (control cyto). (E, F) HBMEC (E) and Caco-2 cells (F) were incubated with EHEC-Hly-containing or EHEC-Hly-free OMVs as indicated in A and B and ΔΨm was determined by uptake of TMRE using flow cytometry. Median fluorescence of OMV-treated cells was expressed as the percentage of the median fluorescence of control untreated cells (defined as 100%). The values represent means ± standard deviations from three independent experiments. * Significantly decreased (p<0.05) compared to control cells (unpaired Student's t test). Valinomycin (100 nM, 15 min) was a positive control.