Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection (original) (raw)
Figure 6
CK2 mediated FimH-dependent UPEC invasion in vitro and in vivo.
(A) NU14 invades PD07i cells. PD07i cultures were infected with NU14-GFP (MOI 100; green) and stained for extracellular E. coli (red). (B) FimH mediates UPEC adherence to PD07i cells. Urothelial cells were infected with NU14 or NU14-1 at an MOI of 10, and infection proceeded for 2 h followed by washing. Cell lysates were plated on LB-agar to determine adherent bacteria. (C) FimH promotes UPEC invasion. PD07i cells were infected as above, washed and incubated in culture medium with 100 µg/ml gentamicin for 30 minutes followed cell lysis and plating onto agar. (D) NU14 invades 5637 cells infected with UPIIIa variants. Adenoviruses expressing various UPIIIa phosphorylation variants were used to infect 5637 cells followed by quantification of invasive bacteria. Inhibition of CK2 abrogates UPEC invasion but not adherence to urothelial cells (E&F). Adherence and invasion assays were performed as above with some modifications. Urothelial cells were infected with NU14 in the presence of 10 µM TBB, 1 µM wortmannin or vehicle control at an MOI of 100, medium was replaced after 1 hour with inhibitor-free media and infection was allowed to proceed for a further 1 hour followed by quantification of adherence and invasion as above. (G) Inhibition of CK2 using transfected siRNA does not affect adherence of NU14 to urothelial cells but reduces bacterial invasion of the bladder (H). In vivo TBB administration significantly reduced NU14 invasion of the bladder in female C57BL/6 mice (I). Mice were catheterized transurethrally with 108 CFU of NU14 bacteria in PBS with TBB (10 µM) or a vehicle control (DMSO). Infection was allowed to proceed for 2 h followed by bladder removal and incubation in PBS with 100 µg/ml gentamicin ex vivo for 30 minutes at 37°C. Bladders were washed in antibiotic-free PBS and homogenized for quantification of bacterial colonization. With the exception of (I), all experiments were repeated three or more times, results are expressed as mean±SEM with statistical significance indicated at *p<0.05.