Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells
Fig 2
Reporter OMVs capture rapid kinetics of vesicle uptake by host cells in real time.
(A) CCF2-AM loaded Hela cells were exposed to OMVs from EHEC carrying ClyA-Bla (red), or vector control (grey) at an MOI of 1000 for 3 hours. Ratio of blue:green fluorescence) over time was plotted as mean ± stdev (n = 3). (B) Rmax was determined from data in S2A to visualize speed of uptake and is shown are means ± stdev (n = 3). Significance was determined by analysis of variance, with a Brown Forsythe test to determine equal variance. (**) p≤0.01. (C) Absolute FRET changes after 3 h were determined from data in (A) and plotted as efficiency of OMV uptake. Data shown are means ± stdev (n = 3). Significance was determined by ANOVA, with a Brown Forsythe test to determine equal variance. (**) p≤0.01. (D) CCF2-AM loaded Hela cells were imaged by confocal microscopy and merged blue/green images representative of 15 images (n = 3) are shown. Scale bars, 20 μm. (E) Hela cells incubated with cellmask orange-labelled OMVs (red) for 10 and 60 min and slice views of z-stacks were acquired by confocal microscopy. Scale bars, 10 μm.