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Uroplakin Ia is the urothelial receptor for uropathogenic Escherichia coli: evidence from in vitro FimH binding

¹ Skirball Institute of Biomolecular Medicine, Departments of
² Biochemistry,
³ Pharmacology,
⁵ Microbiology,
⁶ Dermatology and
⁷ Urology, Kaplan Comprehensive Cancer Center, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
⁴ Institut für Molekularbiologie und Biophysik, Eidenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland

View larger version (42K): [in a new window]   Fig. 1. Biotinylation of recombinant FimH-FimC complex. (A) Detection of the biotinylated FimH-FimC complex after the proteins were electrophoretically separated and transferred onto a nitrocellulose membrane. Note the detectability of as little as 16 ng of the complex per lane. (B) Competitive binding between the biotinylated and unlabeled FimH-FimC to immobilized -D-mannose. Note that a one-to-one dilution of the biotinylated FimH-FimC with unlabeled protein resulted in 50% reduction in the binding of the labeled protein to immobilized mannose, indicating that the mannose-binding activity of the recombinant protein was not affected by biotinylation. (C) Typical ultrastructure of negatively stained mouse urothelial plaques showing two-dimensional crystals of 16 nm uroplakin particles.

View larger version (47K): [in a new window]   Fig. 2. In vitro binding of type 1-fimbriated E. coli and FimH-FimC protein complex to mouse and bovine uroplakins. Purified bovine (A) and mouse (B) urothelial plaque proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Lane 1: coomassie blue staining showing the positions of uroplakins Ia, Ib, II and III; note the excellent resolution of the mouse 24 kDa uroplakin Ia and 29 kDa uroplakin Ib, in comparison with the poorly resolved bovine 27 kDa uroplakin Ia and 28 kDa uroplakin Ib. Lane 2: binding of biotinylated FimH-FimC to uroplakins; note the selective binding to mouse uroplakin Ia with no detectable binding to uroplakin Ib. Lane 3: same as in lane 2 but the binding was carried out in the presence of 1 mM -D-mannose; note the complete inhibition of the FimH-FimC binding to uroplakin Ia. Lane 4: same as in lane 2 but done in the presence of 1 mM galactose; note the lack of inhibition. Lane 5: binding of 35S-labeled, type 1-piliated E. coli (strain SH48) to urothelial plaque proteins; note again the selective binding to mouse uroplakin Ia. Lane 6: binding of a similarly labeled, but nonfimbriated E. coli (strain P678) to urothelial proteins; note the complete lack of binding. Positions of uroplakins Ia, Ib, II and III are marked. MW: molecular weight standards.

View larger version (60K): [in a new window]   Fig. 3. Identification of the major mouse FimH binding urothelial plaque protein by mass spectrometry and cDNA cloning. The 24 kDa protein identified as the major FimH and bacterial binding protein of purified mouse urothelial plaque was gel-purified, microsequenced and cDNA-cloned. (A) Identification of the major mouse FimH-binding protein as uroplakin Ia. The cDNA-deduced amino acid sequence of the mouse (mou) FimH binding protein is aligned here with those of human (hum) and bovine (bov) uroplakin Ias. The predicted N-glycosylation sites and the putative transmembrane domains are shaded in gray and underlined, respectively. Asterisks indicate amino acids that vary among the species. (B) Sequencing of a major tryptic peptide using mass spectrometry. The sequence of this peptide was derived from the y series ions and is indicated on the top from right (N-terminal) to left (C-terminal).

View larger version (29K): [in a new window]   Fig. 4. Carbohydrate heterogeneity among mouse uroplakins and the identification of the mouse uroplakin Ia glycosylation site. (A) Carbohydrate heterogeneity among mouse uroplakins as assessed by deglycosylation and lectin binding. Mouse uroplakins dissolved in SDS were incubated in the absence (–) or presence (+) of PNGase F before they were resolved by SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies to specific uroplakins (lanes 1, 4, 5 and 6 as indicated), FimH (lane 2), ConA (lane 3), or erythrina cristagalli lectin (ECL), a lectin that has specificity for glucosamine (lane 7). Note the reduction in the apparent sizes of mouse uroplakins Ia, Ib and III after deglycosylation (lanes 1, 4 and 6); the recognition of uroplakin Ia, but not uroplakin Ib, by FimH (lane 2) and ConA (lane 3); and the ablation of the uroplakin Ia binding by deglycosylation. The molecular weights of standard proteins are indicated on the left. (B) Mapping of the glycosylation site of mouse uroplakin Ia utilizing the fact that the glycosylated asparagine can be derivatized to a partially 18O-labeled aspartic acid (D*) during deglycosylation. The upper panel shows the full MS/MS spectrum of the doubly charged precursor ion (m/z 1443.625 centroid monoisotopic) with the peptide sequence indicated by y series ions. The lower panel is an expanded segment of the upper panel. Upon partial 18O-labeling, fragment ions containing the 18O-label appeared as doublets that were readily differentiated from the unlabeled ones. UP, uroplakin.

View larger version (156K): [in a new window]   Fig. 5. Selective binding of FimH (A) and antibodies to uroplakins (B) to the umbrella cells of mouse urothelium. Sections of mouse bladder were incubated with biotinylated FimH-FimC (A) or rabbit antibodies to total uroplakins (B), and then with FITC-conjugated streptoavidin or goat anti-rabbit IgG. Nuclei were counterstained with a blue Hoechst dye. Note the similar staining of urothelial umbrella cells by FimH-FimC and antibodies to uroplakins.

View larger version (17K): [in a new window]   Fig. 6. Effects of sugars and pH on the binding of FimH to mouse urothelial plaques. The wells of 96-well plates were coated with purified mouse urothelial plaques, quenched with bovine serum albumin and incubated with biotinylated FimH-FimC, in the presence or absence of galactose or mannose and at various pHs. (A) Typical saturation curve for the binding of FimH-FimC to mouse urothelial plaques. The solid line represents a best fit from nonlinear regression. This is a typical curve representative of results from 10 experiments, yielding Kd values in the range of 110-160 nM (correlation coefficient r=0.9948). (B) The binding is completely abolished by -D-mannose (, but not by galactose (). (C) Binding of the FimH to mouse urothelial plaques at pH 4.2-7.0. pH 4.2 (), 5.0 (), 6.0 () and 7.0 (). (D) Similar binding at pH 7-9. pH 7.0 (), 7.5 (), 8.0 () and 9.0 ().