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. 2009 May;5(5):e1000415.
doi: 10.1371/journal.ppat.1000415. Epub 2009 May 1.

Bacteria-induced Uroplakin Signaling Mediates Bladder Response to Infection

Free PMC article

Bacteria-induced Uroplakin Signaling Mediates Bladder Response to Infection

Praveen Thumbikat et al. PLoS Pathog. .
Free PMC article


Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Localization of surface-expressed uroplakins on PD07i cells.
(A) 0.1 ug of bovine (lane 1, 3, 5, 7) and human (lane 2, 4, 6, 8) AUM proteins were resolved on 17% SDS-PAGE and transferred onto a nitrocellulose membrane, and detected with monospecific rabbit antisera against Ia (lane 1, 2), Ib (lane 3, 4), II (lane 5, 6), and IIIa (lane 7, 8). All the uroplakins antibodies are monospecific to a single uroplakin band in both bovine and human AUM. The surface expression of uroplakins on PD07i cells were detected using immunofluorescent staining using anti-uroplakins Ia (B), Ib (C), II (D) and IIIa (E) followed with Alexa Fluor 488-conjugated donkey anti-rabbit IgG. Propidium iodide (red) was used to stain nuclei (B–E). Double staining of uroplakins Ia (F) / II (G) / merge (H), and Ib (I) / IIIa (J) / merge (K) showed co-localization of uroplakins on the apical surface of PD07i cells. Arrows mark the co-localization of surface-expressed uroplakins (F–K).
Figure 2
Figure 2. Co-localization of uroplakins and FimH binding sites on PD07i cell surface.
The surface-expressed uroplakins on PD07i cells were detected using antisera against individual uroplakins Ia (B), Ib (E), II (H), and IIIa (K), followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG; while FimH was localized using biotinylated FimH/C complex, followed with FITC-conjugated streptavidin (A, D, G, J). Arrows mark the co-localization of surface-expressed uroplakins and FimH binding sties (A–L).
Figure 3
Figure 3. FimH induces UPIIIa phosphorylation and intracellular calcium elevation.
(A) PD07i cells were stimulated with either 10 µg/ml FimCH or 10 µg/ml BSA for 30 minutes, and UPIIIa was immunoprecipitated from 100 µg cell extract with anti-UPIII antibody. Following electrophoresis and blotting, immunoprecipitated proteins were probed with an anti-phosphothreonine antibody (P-Thr). Blots were subsequently re-probed with an anti-UPIII antibody to determine total protein loading (UP3) (B) PD07i cultures were loaded with 5 µM Fura-2 and imaged for 5 minutes at 340 nm and 380 nm using real-time video fluorescence microscopy. FimCH (10 µg/ml) was added after establishing baseline Ca2+ concentrations collection for 1 min (arrow). The trace is the mean of a representative experiment of three replicates. (C) Extracellular Ca2+ is required for FimCH-induced urothelial Ca2+ elevation. PD07i cells were loaded with 5 µM Fura-2 and imaged for 10 minutes in nominal Ca2+-free medium. Cells were then exposed to 10 µg/ml FimCH, imaged for 5 minutes followed by addition of Ca2+ to a final concentration of 2.5 mM to the buffer solution and data were acquired for another 5 min. Baseline Ca2+ concentration is represented by the dotted line. The trace is the mean of a representative experiment of three replicates. (D) FimCH-induced [Ca2+]i elevation is inhibited by pre-incubation with α-D-mannoside. FimCH was pre-incubated for 30 minutes with 25 mM α-D-mannoside or D-glucose. PDO7i cells loaded with 5 µM Fluo-4 AM were treated with 10 µg/ml of the pre-incubated FimCH. PDO7i cells were imaged at 488 nm using real-time video fluorescence microscopy and maximal fluorescence after FimCH treatment was subtracted from baseline. (E) Chelating intracellular or extracellular Ca2+ inhibits elevation of FimCH-induced [Ca2+]i. PD07i cells loaded with 5 µM Fura-2 were pre-incubated with 10 µM BAPTA-AM, 4 mM EGTA, 60 µM 2-APB, 10 µM nifidepine (Ndp), or equivalent amounts of DMSO for 30 minutes, followed by washing and exposure to 10 µg/ml FimCH. Maximal [Ca2+]i was subtracted from baseline [Ca2+]i. Statistical significance is indicated at *p<0.05 or **p<0.001 and data are represented as mean±SEM. (F) Knockdown of UPIIIa expression inhibits FimCH-induced Ca2+ elevation. Control cells (PDO7isiTNFR2) or PD07isiUPIII cells were loaded with Fura-2 and imaged upon addition of 10 µg/ml FimCH followed by post-acquisition analysis. All experiments were repeated at least three times, and statistical significance is indicated at *p<0.05 and data are represented as mean±SEM.
Figure 4
Figure 4. FimCH-induced Ca2+ elevation involves T244 mediated signaling.
(A) The cytoplasmic tail of UPIIIa comprises of 52 amino acids with two predicted CK2 and one protein kinase C phosphorylation motif (underlined). T244 and S282 (indicated by asterisks) were mutated to A/E and Y266 (indicated by pound) to F to identify the phosphorylation site involved in FimCH-mediated Ca2+ signaling. (B) Mouse bladder sections were stained with P3 antibodies (red, i; see text) and AU1 antibody (green, ii), and the images were merged to confirm specificity (iii). COS-7 cells were infected with UPIb (iv) or UPIIIa adenovirus (v) and stained for UPIIIa protein with P3 antibodies. COS-7 cells co-infected with UPIb and UPIIIa viruses were stained with P3 antibodies (red, vi and vii) or P3 and AU1 (viii). Arrows indicate staining consistent with surface expression (vi–viii). Arrows indicate staining consistent with surface expression (vi–viii). (C) T244 mediates FimCH-induced Ca2+ elevation. Data acquired as in (D) were subject to post–acquisition analysis by subtracting maximal Ca2+ elevation following addition of 10 µg/ml FimCH from baseline Ca2+ concentration in COS-7 cultures. COS-7 cultures were previously infected with recombinant adenoviruses encoding UPIb and either wild type UPIIIa or UPIIIa variants or LacZ. At 24 h post-infection, traces of FimCH-induced Ca2+ elevation were acquired and reveal a requirement for T244 mediated signaling. (D) Representative traces are shown for T244 (i), S282(ii) and T266(iii) along with WT. All experiments were repeated three or more times, and statistical significance is indicated at *p<0.05 or **p<0.001 and data are represented as mean±SEM.
Figure 5
Figure 5. CK2 phosphorylates UPIIIa cytoplasmic domain and is required for FimCH-induced Ca2+ elevation.
(A) UPIIIa cytoplasmic domain is phosphorylated by CK2 in vitro. Recombinant human CK2 (50 U) exhibited autophosphorylation (lane 1) that was blocked by 10 µM of the CK2 inhibitor TBB (lane 2). A fusion protein of the C-terminal domain of uroplakin IIIa with glutathione-S-transferase (UP3C-GST, arrow) served as a concentration-dependent substrate for phosphorylation by CK2 (lanes 3–6 containing 0.01 µg, 0.10 µg, 0.25 µg, or 1.00 µg UP3C-GST, respectively). CK2-mediated phosphorylation of 0.10 µg UP3C-GST was inhibited by TBB (lane 7). (B) Inhibition of CK2 abrogates FimCH-induced calcium elevation. Urothelial cells were loaded with Fura-2 AM and pretreated with 10 µM TBB (or equivalent concentration of vehicle (DMSO)) for 30 minutes, treated with FimCH and followed by imaging. Statistical significance is indicated at *p<0.05 and data are represented as mean±SEM. (C) COS-7 cells were infected with recombinant adenoviruses and FimCH-induced calcium was quantified as in (B) in the presence or absence of TBB. (D) RNA silencing of CK2 inhibits FimCH-induced calcium elevation in urothelial cells. PD07i cells transfected with siCK2 showed significantly reduced elevation in calcium as measured by the change in Fura-2 340/380 ratio compared to transfection with negative control siRNA. All experiments were repeated three or more times, and statistical significance is indicated by *p<0.05, **p<0.01.
Figure 6
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.
Figure 7
Figure 7. UPIIIa mediates FimH-induced urothelial apoptosis.
FimH-induced caspase 3/7 activation requires UPIIIa (A). Caspase 3/7 was measured in culture extracts by cleavage of fluorogenic substrate. Induction of caspase 3/7 activity by NU14 (MOI 500) was significantly inhibited in PD07siUPIII cultures (*p<0.05) compared to control, and no induction of caspase 3/7 was detected in response to NU14-1. (B) PD07siTNFR2 cells or PD07siUPIII cells were treated with 10 µg/ml FimCH for 4 h. Apoptosis was visualized using fluorescent annexin V staining (green) and propidium iodide (red) staining. Annexin staining was quantified by manual counting of fluorescently labeled cells relative to brightfield images (see Methods). Annexin-positive cells were significantly reduced in siUPIII cells relative to siTNFR2 cells (p<0.05). (C) In experiments in the presence of inhibitors, PD07i cells treated with 10 µg/ml FimCH showed reduced annexin staining when treated with the CK2 inhibitor TBB compared to DMSO (p<0.05). (D) Organotypic raft cultures of PD07i or PD07siUPIII cells were incubated with NU14 or NU14-1 for 4 h at an MOI of 100. After processing, frozen sections were stained for TUNEL (green) and counterstained with DAPI (blue). Prominent TUNEL staining was induced by NU14 only in PD07i tissue. Images are representative of staining patterns obtained from TUNEL assay of four different raft cultures. (E) UPEC-induced apoptosis was also assessed in bladder sections using TUNEL staining (green). In in vivo experiments female C57BL/6 mice were infected with 108 NU14 or NU14-1 for 6 hours in the presence of DMSO (i) or 10 µM TBB (ii). Bladder sections from DMSO treated animals exhibited foci of apoptosis (arrows) that were reduced or absent in TBB-treated animals. In H&E stained bladder sections from DMSO (iii) and TBB (iv) treated mice, infiltration of large numbers of inflammatory cells (white arrows) were observed in response to infection with 108 NU14 for 2 hours, suggesting an intact innate immune response. Images are of representative sections and scale bars represent 50 µm. All experiments were repeated two or more times, and statistical significance is indicated at *p<0.05 and data are represented as mean±SEM.
Figure 8
Figure 8. Model of UPIIIa mechanisms of UPEC pathogenesis.
(A) UTI pathogenesis in the bladder occurs in distinct kinetic phases. Immediate early host responses include UPIIIa phosphorylation and increased [Ca2+]i (this study), PI3K activation , actin rearrangement , and increased cAMP . Subsequent early host responses include UPEC internalization , NFκB-dependent chemokine production and modulation , urothelial apoptotic cascades , and bacterial clearance mediated by neutrophils . Late events include adaptive responses that confer protective immunity and establishment of stable UPEC reservoirs within the urothelium . (B) UPEC interaction with uroplakins leads to phosphorylation of the UPIIIa cytoplasmic tail by CK2 and the initiation of two signaling cascades. Elevation in intracellular calcium from intra- and extracellular stores and the associated recruitment of other signaling molecules activates host cell cytoskeletal elements and the endocytosis of UPEC. UPIIIa phosphorylation activates an unknown signaling intermediate that initiates intrinsic and extrinsic apoptotic cascades. Pro-survival signals initiated by TLR activation may shift the balance away from UPIIIa-induced pro-apoptotic signals in those cells where UPEC successfully establish stable intracellular populations.

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