Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells
Fig 5
Iron supplementation is unable to support intracellular UPEC survival in the absence of Rab35 or TfR.
A. siNT, siRab35 or siTfR treated BEC cells were maintained in serum-free conditions 4 h prior to infection and were subsequently left in serum free medium for the rest of the duration of the experiment. Cells were infected with UPEC at MOI 500 for 2 h. Following infection, and gentamycin treatment, the cells were washed and left in RPMI with gentamycin (10μg/ml) and treated with 50μg/ml holotransferrin or left untreated for further 24 h. The cells were then lysed and plated for enumeration of intracellular bacteria by cfu assay. Results are expressed as % change in bacterial load/ 2x105 cells. Values shown represent means ± standard deviations of results of three experiments. (** p<0.01 vs untreated siNT values). B. UPEC infection upregulates TfR at the protein level. BEC cells were infected with UPEC and TfR protein expression was analyzed at 4, 24 and 48 h post infection by Western blot analysis. Quantitation of the Western blots using the software ImageJ is shown (bottom panel). Relative densitometric data (TfR/GAPDH) is shown. Values shown represent means ± standard deviations of results of three experiments. (* p<0.05 vs 0 h time point values).