Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells
Fig 3
Loss of Rab35 targets UPEC to late lysosomal compartment.
A. BEC-5637 cells were transfected with Rab35 expressing plasmid. 24 h later the cells were infected with UPEC. The cells were fixed at different stages of infection, permeabilised and stained for LAMP1 and were then analyzed by confocal microscopy. The results (panel a) are represented as % of UCV positive for both Rab35 and LAMP1. At least 100 UCV were counted for each experiment. A representative image is shown in panel b. DAPI {blue, host nuclei (N) or bacteria (UPEC)}, Rab35 (green), and LAMP1 (red). Arrows in the DAPI panel indicates UPEC. Zoomed panel (panel c) shows the magnified image of UCV (marked by arrows in the merged image) {(optical magnification (63x) and electronic zoom (2.5x)}. The graph (panel d) shows the quantification of the fluorescence intensity along the white line shown in the zoomed panel. Both LAMP1 and Rab35 co-localized at the UCV. B. si NT and si Rab35 cells were infected with GFP-UPEC. At 24 h post infection, the cells were stained with LysoTracker Red for 2 h; fixed and analyzed by confocal microscopy. Graph (panel a) is depicted as % bacteria co-localizing with LysoTracker Red, At least 100 bacteria were counted. Representative images are shown in panel b (si NT) and (si Rab35). *** represents p<0.001, Values shown represent mean ± standard deviation of results of three independent experiments. The graphs (panel c) show the quantification of the fluorescence intensity along the white line shown in the merged image. UPEC from si NT sample did not co-localize with LysoTracker Red; while UPEC from si Rab35 sample co-localized with LysoTracker Red. C. BEC cells were transfected with si NT or si Rab35. 24 h later the cells were transfected with RFP-Cathepsin D expressing plasmid. After another 24 h the cells were infected with GFP-UPEC. The colocalization between UPEC containing vacuole (UCV) and Cathepsin D was observed 24 h post infection by confocal microscopy. Results are represented as % of cathepsin D positive UCV (% of UCV that is positive for cathepsin D). At least 100 bacteria were counted. ** represents p < .01, Values shown represent mean ± standard deviation of results of three independent experiments. D. Cathepsin D protein expression was analyzed in si NT/ si Rab35 cells left uninfected or infected with UPEC for 4, 24 and 48 h by Western blotting. The experiment was repeated three times with similar results and a representative blot is shown (top panel). Quantitation of the Western blots using the software ImageJ is shown (bottom panel). Relative densitometric data (mature cathepsin D/GAPDH) is shown. Values shown represent means ± standard deviations of results of three experiments. (* p<0.05 vs siNT values at the corresponding time points).