Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
Fig 6
The role of Gb3 in the uptake and retrograde transport of OMV-delivered Stx2a.
(A, D) FACS analysis of Gb3 content in (A) PPMP-untreated (no PPMP) and PPMP-treated (+PPMP) HBMEC and (D) DLD-1 cells stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD77/Gb3 antibody or unstained (control). One representative experiment is shown for each cell line (geometric mean fluorescence ± standard deviations from three independent experiments are shown in S18A and S18B Fig). (B, E) CLSM analysis of trafficking of OMV-delivered and free Stx2a (control) into the endoplasmic reticulum (ER) and late endosomes/lysosomes (Le-Lyso) in (B) PPMP-untreated and PPMP-treated HBMEC (20 h) and (E) DLD-1 cells (4 h). The indicated single fluorescence channels are shown in the right panels (enlargements are displayed in S19 Fig) and the merged images in the left panels (green, Stx2a or OMVs, as indicated; red, PDI or CD63, as indicated; blue, nuclei; yellow, colocalized green and red signals). The percentages of colocalizations of the respective signals (white numbers) were calculated with the BioImageXD6 tool (means of colocalizations from three different samples are shown). (C) Uptake of 5791/99 OMVs by DLD-1 cells after 4 h of incubation. Untreated cells (no OMV) were a negative control. Green, OMVs; red, actin; blue, nuclei. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. Scale bars in all panels are 10 μm. (F, G) Immunoblot detection of Stx2a in isolated endoplasmic reticulum (ER) and lysosomal (Lyso) fractions of (F) PPMP-treated HBMEC and (G) DLD-1 cells after 20 h and 4 h of incubation, respectively, with the indicated samples or left untreated (no OMV); 5791/99 OMVs without cells (OMV ctrl) were a positive control.