Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis
Figure 5
EHEC-Hly separates from OMVs during intracellular trafficking.
(A) HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs for the times indicated and analyzed using CLSM. OMVs were stained using mouse anti-E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green), and EHEC-Hly (EHly) was stained using rabbit anti-EHEC-Hly antibody and Cy3-conjugated goat anti-rabbit IgG (red). Nuclei were stained with DRAQ5 (blue). Pictures were taken and processed as described in the legend to Figure 4. Colocalized red and green signals appear in yellow (examples are indicated by white arrows). Red arrows indicate examples of red signal of EHEC-Hly dissociating from OMVs during time. The percentages of colocalization between OMVs and EHEC-Hly were calculated using BioImageXD6 colocalization tool and are indicated by white numbers (averages from at least five different samples). (B) HBMEC and Caco-2 cells were incubated for 24 h with EHEC-Hly-free OMVs from strains TA51 or 8033c and stained as described in panel A. (C) HBMEC and Caco-2 cells were incubated for 24 h with 20 mM TRIS-HCl (OMV buffer) instead of OMVs and stained for OMVs and EHEC-Hly as described in panel A or stained with secondary antibodies in the absence of primary antibodies. Scale bars in all panels are 10 µm.