Iron acquisition via Rab35/transferrin receptor 1 pathway is critical for the intracellular survival of UPEC within BEC.

(A) UPEC-Infected BEC cells were either left untreated (UT) or treated with holotransferrin (iron), deferoxamine (DFO) or iron+DFO in serum-free conditions. The intracellular bacterial load was determined 24 h post-infection by lysing the cells in 0.1% Triton X-100 and plating on LB-agar. B BEC cells transfected with si NT, si Rab35 or si TfR were left either uninfected or infected with UPEC. Intracellular iron levels at 24 h post infection were determined by calcein-AM fluorescence as described in materials and methods. Immunoblotting was done to confirm the knock down efficiency of Rab35 and TfR siRNA (inset panel). C. BEC cells were transfected with si TfR, si Rab35 or siNT for 48 h followed by infection with UPEC. Intracellular bacterial load at different time points was determined as described before. D and E. BEC-5637 cells were transfected with 100nM each of 3’ UTR si Rab35 (D) 3’ UTR si TfR (E). 24 h following knockdown, the cells were transfected with either empty vector control or plasmids encoding Rab35 (D) or TfR (E) for 24 h. Cells were subsequently serum starved for 4 h and infected with UPEC at MOI 500. The infected cells were finally left in Gentamycin (10 μg/ml)—RPMI with 3% serum. Intracellular bacterial load was determined at 24 h. Immunoblotting was done to confirm the knockdown and over expression efficiency (inset panels). F. BEC cells were transfected with GFP-Rab35 for 24 h followed by UPEC infection for 24 h; fixed, permeabilised, stained for intracellular Transferrin Receptor and analyzed by confocal microscopy. Rab35 (green), DAPI {blue, host nuclei or bacteria (UPEC)}, and Transferrin receptor (red). Arrows in the DAPI panel indicate UPEC colonies. Panel b shows the magnified image of the UCV marked by arrows in the merged image {(optical magnification (63x) and electronic zoom (3.7x)}. The graph (panel c) shows the quantification of the fluorescence intensity along the white line shown in panel b. N, host nucleus. Both Rab35 and TfR colocalized at the UCV. The experiment was repeated three times with similar results and a representative image is shown. G. si NT and si Rab35 cells were either left uninfected or infected with UPEC for 24 h. Subsequently, the cells were loaded with Alexa 568 labelled transferrin for 20’ at 37°C. Unbound Transferrin was removed and the cells were incubated with RPMI containing unlabelled holotransferrin for the given time points. The cells were fixed and analyzed for intracellular Transferrin by confocal microscopy. Transferrin receptor recycling (Transferrin release) was measured as % of intracellular Transferrin at 0h - intracellular Transferrin at the given time point. H si NT and si Rab35 cells were either left uninfected or infected with UPEC. 24 h later the cells were fixed and stained for cell surface Transferrin Receptor and represented as Mean fluorescence intensity (MFI) and analyzed by confocal microscopy. I and J. si NT and si Rab35 BEC cells were either left uninfected or infected with UPEC. 24 h later the cells were loaded with Alexa 568 labelled transferrin for 20’ at 37°C. After removing the unbound Transferrin the cells were fixed and analyzed for intracellular Transferrin by confocal microscopy. Mean fluorescence intensity (MFI) is shown in the graph (I). The confocal microscopy experiments were repeated three times with similar results and a representative Fig for the infected samples are shown in (J). Transferrin (red). K. Infected si NT and si Rab35 cells were loaded with Bodipy 493/503 at 24 h post infection. The cells were subsequently fixed and analyzed for lipid staining by confocal microscopy and represented as MFI. Data represent mean ± standard deviation of results of three independent experiments (A, B, C, D, E, G, H, I and K). * represents p<0.05, **represents p<0.01. *** represent p<0.001. Results of (A), (C), (D) and (E) are expressed as bacterial load/ 2x10⁵ cells. si = siRNA, si NT = negative control siRNA.