Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells
Fig 2
Rab35 is required for the survival of UPEC within BEC 5637.
A. BEC5637 cells were transfected with 100nM each of si Rab35 or non-targeting siRNA (si NT). 48h following knockdown, the cells were infected with UPEC at MOI 500. Intracellular bacterial load at different time points {4 h (invasion), 24 h and 48 h, was determined by lysing the cells in 0.1% Triton X-100 and plating on LB-agar as described in Materials and Methods. Results are expressed as bacterial load/ 2x105 cells. Immunoblotting was done to confirm the knock down efficiency of Rab35 siRNA (inset). B. Rab35 silencing does not enhance the efflux rate of UPEC from BEC-5637. BEC-5637 cells were transfected with 100nM each of si Rab35 or non-targeting siRNA (si NT). 48 h following knockdown, the cells were infected with UPEC at MOI 500. After gentamycin (100μg/ml) treatment, cells were washed in left in fresh culture medium containing 100mM methyl-D-mannopyranoside. At 24 h post infection, the culture medium was collected and plated for CFU counts as described in Materials and Methods. Results are expressed % exocytosis relative to siNT cells. C. Over expression of Rab35 protein leads to increase in bacterial load at 48 h post infection. BEC cells were transfected with GFP-Rab35 or control GFP vector for 24 h followed by UPEC infection (MOI 500) for another 48 h. Intracellular bacterial load was calculated as mentioned above. Results are expressed as bacterial load/ 2x105 cells. Immunoblotting was done to confirm the expression levels of Rab35 (inset). * represents p < .05; ** represents p<0.01. Values shown represent mean ± standard deviation of results of three independent experiments.