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. 2012 Nov 16;287(47):39578-88.
doi: 10.1074/jbc.M112.380287. Epub 2012 Sep 25.

Surfactant Protein D Inhibits Adherence of Uropathogenic Escherichia Coli to the Bladder Epithelial Cells and the Bacterium-Induced Cytotoxicity: A Possible Function in Urinary Tract

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Surfactant Protein D Inhibits Adherence of Uropathogenic Escherichia Coli to the Bladder Epithelial Cells and the Bacterium-Induced Cytotoxicity: A Possible Function in Urinary Tract

Yuichiro Kurimura et al. J Biol Chem. .
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Abstract

The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca(2+)-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.

Figures

FIGURE 1.
FIGURE 1.
Expression of human SP-D. A–G, immunostaining of SP-D and uroplakin Ia in human tissue. Human lung (A and B), bladder (C–E), and prostate (F and G) were fixed by 3% formaldehyde and paraffin-embedded. The sections were stained with an anti-human SP-D polyclonal antibody (A, C, and F), control antibody (B, D, and G), or anti-uroplakin Ia polyclonal antibody (E) and observed under light microscopy. Representative specimens are shown from two independent experiments. Scale bars, 100 μm. H, detection of SP-D in human urine. The concentrated urine, native SP-D, and recombinant SP-D (rSP-D) were boiled in SDS-PAGE sample buffer under reducing conditions. SP-D expression in urine was analyzed by immunoblot using an anti-SP-D polyclonal (Ab) antibody as described under “Experimental Procedures.” Representative results are shown from three independent experiments. WB, Western blot.
FIGURE 2.
FIGURE 2.
Increased expression of SP-D mRNA in the bladder of the UPEC infection model in mice. A, J96 (108 cfu) or saline was injected into the bladder of female C57BL/6 mice, and the external urethral orifices were clamped for 2 h. Twenty-four hours after infection, the bladders from infected and non-infected mice and the lungs from non-infected mice were excised and used for detection of SP-D mRNA by RT-PCR as described under “Experimental Procedures.” Representative results are shown. B, densitometric analysis of the PCR bands shown as the ratio of SP-D/GAPDH. The data shown are the means ± S.D. (error bars) from three separate experiments. *, p < 0.05 when compared with uninfected bladder.
FIGURE 3.
FIGURE 3.
Recombinant human SP-D binds to UPEC. A, the indicated concentrations of SP-D were incubated at 37 °C for 2 h with UV-killed J96 coated onto microtiter wells (107 cfu/well) in the presence of 2 mm CaCl2 (open circles), 2 mm EDTA (closed circles), or 0.2 m α-methyl-d-mannoside (open squares). The SP-D binding to the solid phase UPEC was detected using an anti-human SP-D polyclonal antibody as described under “Experimental Procedures.” The data shown are the means ± S.D. (error bars) from three to six separate experiments. **, p < 0.01 when compared with 2 mm CaCl2. B, J96 (109 cfu) was incubated with or without 2 μg of SP-D in the presence of 2 mm CaCl2, 5 mm EDTA, or 0.2 m α-methyl-d-mannoside (MM) at 37 °C for 1 h. After the incubation, the mixture of J96 and protein was washed and centrifuged. The bacterial pellet obtained was subjected to SDS-PAGE, and blotting analysis was performed to detect SP-D co-sedimented with the bacteria as described under “Experimental Procedures.” As a positive control, recombinant SP-D (20 ng) was loaded. Representative results are shown from three independent experiments. cont., control.
FIGURE 4.
FIGURE 4.
SP-D agglutinates UPEC in a Ca2+-dependent manner. A and B, EGFP-expressing J96 (109 cfu) was incubated with or without SP-D (20 μg/ml) in the presence of 5 mm CaCl2. After the incubation, the bacteria were observed by fluorescence microscopy (original magnification, ×400). Scale bars, 50 μm. C, J96 (5 × 108 cfu) was mixed with or without SP-D (20 μg/ml) in a cuvette containing 5 mm CaCl2 or 5 mm EDTA, and the cuvette was left at rest for the indicated length of time. At each time point, bacterial precipitation was determined by measuring the absorbance at 660 nm. The data shown are the means ± S.D. (error bars) from three separate experiments. **, p < 0.01 when compared with the absorbance at time 0.
FIGURE 5.
FIGURE 5.
Co-incubation of UPEC and SP-D inhibits the bacterial adherence to urothelial cells and its cytotoxicity. EGFP-expressing J96 (106 cfu/well) was incubated with or without SP-D (10 μg/ml) at 37 °C for 1 h. After the incubation, the complex of J96 and SP-D was isolated and further incubated with 5637 cells. A–C, UPEC adherence. The 5637 cells were stained with 0.5% (w/v) concanavalin A and observed by fluorescence microscopy (A and B). Scale bars, 50 μm. The percentage of J96-adherent cells is shown (C). The data shown are the means ± S.D. (error bars) from three separate experiments. **, p < 0.01 when compared with control. D–F, urothelial cell injury. The 5637 cells were stained with 25 μg/ml ethidium bromide and 5 μg/ml acridine orange to examine the permeability of cell membranes (D and E). The percentage of cells stained with ethidium bromide in total cells counted is shown (F). The data shown are the means ± S.D. (error bars) from three separate experiments. *, p < 0.05 when compared with the control.
FIGURE 6.
FIGURE 6.
Preincubation of urothelial cells with SP-D inhibits UPEC adherence and cell injury. The 5637 cells were incubated with or without SP-D (10 μg/ml) at 37 °C for 1 h. After the incubation, the cells were washed to remove non-adherent SP-D and infected with EGFP-expressing J96 (106 cfu/well) at 37 °C for 1 h. A–C, UPEC adherence. The 5637 cells were stained with 0.5% (w/v) concanavalin A and observed by fluorescence microscopy (A and B). Scale bars, 50 μm. The percentage of J96-adherent cells is shown (C). The data shown are the means ± S.D. (error bars) from three separate experiments. **, p < 0.01 when compared with the control. D–F, urothelial cell injury. The 5637 cells were stained with 25 μg/ml ethidium bromide and 5 μg/ml acridine orange to examine the permeability of cell membranes (D and E). The percentage of cells stained with ethidium bromide in total cells counted is shown (F). The data shown are the means ± S.D. (error bars) from three separate experiments. *, p < 0.02 when compared with control.
FIGURE 7.
FIGURE 7.
SP-D and FimH bind to UPIa. A, the 293T cells were transfected with FLAG-tagged uroplakin Ia with or without V5-tagged uroplakin II (Tf(UPs): Ia/II or Ia) or with the p3XFLAG-CMV-14 expression vector and pcDNA3.1 vector (Tf(UPs): −). The cell surface proteins were biotinylated as described under “Experimental Procedures.” The cell lysates were subjected to immunoprecipitation (IP) using an anti-FLAG antibody or anti-V5 antibody or pulled down using streptavidin-agarose. The immunoprecipitates were analyzed by Western blot (Blot) using an anti-FLAG antibody, anti-V5 antibody, or concanavalin A (ConA). B and C, the 293T cells were co-transfected with FLAG-tagged uroplakin Ia and V5-tagged uroplakin II (UPIa: +) or with FLAG-tagged BAP and the pcDNA3.1 vector (UPIa: −). Forty hours after transfection, the cells were lysed, and the cell lysate was incubated with the anti-FLAG antibody coated onto microtiter wells. The binding of SP-D (B) and receptor-binding domain of FimH (C) to uroplakin Ia captured by the anti-FLAG antibody was analyzed as described under “Experimental Procedures.” In some experiments, 0.2 m α-methyl-d-mannoside (Man) was added instead of CaCl2. The data shown are means ± S.D. (error bars) from three to six separate experiments. **, p < 0.01 when compared with the indicated samples. D, SP-D competes with FimH for the binding to uroplakin Ia. The 293T cells were transfected with FLAG-tagged uroplakin Ia and V5-tagged uroplakin II (UPIa: +) or with a p3XFLAG-CMV-14 expression vector (UPIa: −). Forty hours after transfection, the cell lysates were incubated with an anti-FLAG monoclonal antibody coated onto microtiter wells. After the incubation, SP-D (1 μg; open squares) or BSA (1 μg; closed squares) was added to the wells and incubated for 1 h. After the wells were washed, the His-tagged FimH-RBD (100 ng) was incubated for 2 h at 37 °C. The binding of FimH-RBD to uroplakin Ia was detected using the anti-His polyclonal antibody followed by incubation with HRP-labeled anti-rabbit IgG as described under “Experimental Procedures.” The data shown are the means ± S.D. (error bars) from four separate experiments. **, p < 0.01 when compared with the experiments with UPIa (+) and SP-D (−).
FIGURE 8.
FIGURE 8.
Exogenous administration of SP-D attenuates UPEC adherence to the bladder and dampens UPEC-induced inflammation in mice. An inoculum containing 5 × 107 cfu of J96 with or without SP-D (50 μg/ml) was injected transurethrally into the bladders of female C57BL/6 mice, and the external urethral orifices were clamped for 2 h. Three independent experiments with three mice in each group were performed. A, UPEC adherence. After euthanasia, the bladders were excised, washed to remove non-adherent UPEC, and then homogenized. The bladder homogenates were serially diluted in PBS and plated onto LB agar for colony counts. The data are the means ± S.D. (error bars) of three independent experiments. **, p < 0.01 when compared with the absence of SP-D. B, UPEC-induced histological changes of the bladder. Mice were euthanized at 48 h after administration of UPEC with or without SP-D as described above. The bladders were fixed with 15% buffered formalin phosphate and embedded in paraffin. Bladder sections were stained with hematoxylin-eosin and observed under light microscopy. Scale bars, 40 μm. Representative results are shown from three independent experiments. Cont, control.

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