Tamm-Horsfall Protein Binds to Type 1 Fimbriated Escherichia coli and Prevents E. coli from Binding to Uroplakin Ia and Ib Receptors
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m008610200
ISSN1083-351X
AutoresJoanne Pak, Yongbing Pu, Zhong-ting Zhang, David L. Hasty, Xue‐Ru Wu,
Tópico(s)Escherichia coli research studies
ResumoThe adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors. The adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors. urinary tract infection asymmetric unit membrane Tamm-Horsfall protein phosphate-buffered saline polyacrylamide gel electrophoresis The adhesion of Escherichia coli, the most common cause of urinary tract infection (UTI),1 to urothelial cells is a crucially important first step in UTI pathogenesis (1Schoolnik G.K. O'Hanley P. Lark D. Normark S. Vosti K. Falkow S. Adv. Exp. Med. Biol. 1987; 224: 53-62Crossref PubMed Scopus (6) Google Scholar, 2Stamm W.E. Hooton T.M. Johnson J.R. Johnson C. Stapleton A. Roberts P.L. Moseley S.L. Fihn S.D. J. Infect. Dis. 1989; 159: 400-406Crossref PubMed Scopus (139) Google Scholar, 3Mobley H.L. Chippendale G.R. Warren J.W. Methods Enzymol. 1995; 253: 360-367Crossref PubMed Scopus (3) Google Scholar, 4Klemm P. Schembri M. Hasty D.L. Kahane I. Ofek I. Toward Anti-Adhesin Therapy of Microbial Diseases. Plenum Press, New York1996: 81-94Google Scholar, 5Roberts J.A. Adv. Exp. Med. Biol. 1996; 462: 325-338Crossref Google Scholar). This adhesion process frequently requires filamentous surface appendages of uropathogenic E. coli that are called fimbriae, or pili. Epidemiological studies have shown that >90% of all E. coli isolates from UTI patients elaborate type 1 fimbriae (also named mannose-sensitive fimbriae) (4Klemm P. Schembri M. Hasty D.L. Kahane I. Ofek I. Toward Anti-Adhesin Therapy of Microbial Diseases. Plenum Press, New York1996: 81-94Google Scholar, 6Eisenstein B.I. Rev. Infect. Dis. 1988; 2 (suppl.): S341-S344Crossref Google Scholar, 7Schaeffer A.J. Infection. 1991; 19 Suppl. 3: S144-S149Crossref PubMed Scopus (12) Google Scholar). Although controversies existed for several years, recent investigations have unequivocally documented the importance of type 1 fimbriae as a major urovirulence factor. For instance, of the nine most common E. coli virulence factors, the genes for type 1 fimbriae emerged as the only trait common in all 203 UTI isolates examined (8Foxman B. Zhang L. Palin K. Tallman P. Marrs C.F. J. Infect. Dis. 1995; 171: 1514-1521Crossref PubMed Scopus (110) Google Scholar). In addition, 26% of the 203 strains were positive only for type 1 fimbrial genes and were negative for the eight other urovirulence factors tested, including P, S, and Dr fimbriae. In experimental mouse models, type 1 fimbriae were shown to be indispensable for bladder colonization and infection (9Connell I. Agace W. Klemm P. Schembri M. Marild S. Svanborg C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9827-9832Crossref PubMed Scopus (553) Google Scholar). Conversely, systemic immunization of mice with the FimH tip adhesin of type 1 fimbriae reduced bladder colonization of E. coli by 99% even in neutropenic mice, suggesting that blocking type 1 fimbriae could completely abolish E. coli adhesion (10Langermann S. Palaszynski S. Barnhart M. Auguste G. Pinkner J.S. Burlein J. Barren P. Koenig S. Leath S. Jones C.H. Hultgren S.J. Science. 1997; 276: 607-611Crossref PubMed Scopus (478) Google Scholar). Further evidence supporting an important role of type 1 fimbriae in UTIs has come from the identification of phenotypic variants of the type 1 fimbrial FimH adhesin. On the basis of the receptor binding specificity for monomannose residues, type 1 fimbriae can be divided into the low monomannose-binding variant (ML) and the high monomannose-binding variant (MH) (4Klemm P. Schembri M. Hasty D.L. Kahane I. Ofek I. Toward Anti-Adhesin Therapy of Microbial Diseases. Plenum Press, New York1996: 81-94Google Scholar, 11Sokurenko E.V. Courtney H.S. Abraham S.N. Klemm P. Hasty D.L. Infect. Immun. 1992; 60: 4709-4719Crossref PubMed Google Scholar, 12Sokurenko E.V. Courtney H.S. Ohman D.E. Klemm P. Hasty D.L. J. Bacteriol. 1994; 176: 748-755Crossref PubMed Google Scholar, 13Sokurenko E.V. Courtney H.S. Maslow J. Siitonen A. Hasty D.L. J. Bacteriol. 1995; 177: 3680-3686Crossref PubMed Google Scholar). Interestingly, the ML phenotype predominates in the large intestine, whereas the MH phenotype predominates in the UTI isolates, suggesting a selective advantage for certain subtypes of type 1 fimbriated E. coli in the urinary tract (14Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.-R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (301) Google Scholar). These recent results, along with numerous previous studies demonstrating the direct binding between type 1 fimbriae and the urothelium (15Iwahi T. Abe Y. Nakao M. Imada A. Tsuchiya K. Infect. Immun. 1983; 39: 1307-1315Crossref PubMed Google Scholar, 16Keith B.R. Maurer L. Spears P.A. Orndorff P.E. Infect. Immun. 1986; 53: 693-696Crossref PubMed Google Scholar, 17Fujita K. Yamamoto T. Yokota T. Kitagawa R. Infect. Immun. 1989; 57: 2574-2579Crossref PubMed Google Scholar), clearly established the functional importance of type 1 fimbriae in UTI. The established importance of type 1 fimbriae in urovirulence prompted us to search for their urothelial receptors. The apical surface of mammalian urothelia is covered by numerous rigid-looking plaques whose luminal leaflets are twice as thick as the cytoplasmic ones, hence the name asymmetric unit membrane (AUM) (18Hicks R.M. J. Cell Biol. 1965; 26: 25-48Crossref PubMed Scopus (170) Google Scholar, 19Koss L.G. Lab. Invest. 1969; 21: 154-168PubMed Google Scholar, 20Porter K.R. Bonneville M.A. An Introduction to the Fine Structure of Cells and Tissues. Lea & Febiger, New York1963Google Scholar). Since AUM constitutes >90% of the luminal surface of the urinary tract, including the proximal urethra, bladder, ureters, and renal pelvis, we explored the role of AUM in E. coli adherence. Using sucrose gradient followed by detergent washing, we isolated milligram quantities of highly purified AUMs (21Wu X.-R. Manabe M., Yu, J. Sun T.-T. J. Biol. Chem. 1990; 265: 19170-19179Abstract Full Text PDF PubMed Google Scholar, 22Wu X.-R. Lin J.-H. Walz T. Haner M., Yu, J. Aebi U. Sun T.-T. J. Biol. Chem. 1994; 269: 13716-13724Abstract Full Text PDF PubMed Google Scholar). Such isolated AUMs contain four major integral membrane proteins that were designated as uroplakins Ia (27 kDa), Ib (28 kDa), II (15 kDa), and III (47 kDa) (21Wu X.-R. Manabe M., Yu, J. Sun T.-T. J. Biol. Chem. 1990; 265: 19170-19179Abstract Full Text PDF PubMed Google Scholar, 23Yu J. Manabe M. Wu X.-R. Xu C. Surya B. Sun T.-T. J. Cell Biol. 1990; 111: 1207-1216Crossref PubMed Scopus (115) Google Scholar, 24Yu J. Lin J.-H. Wu X.-R. Sun T.-T. J. Cell Biol. 1994; 125: 171-182Crossref PubMed Scopus (166) Google Scholar). Together, these four proteins form natural two-dimensional crystals arranged in hexagonal arrays (25Vergara J.A. Longley W. Robertson J.D. J. Mol. Biol. 1969; 46: 593-596Crossref PubMed Scopus (73) Google Scholar, 26Hicks R.M. Ketterer B. Nature. 1970; 224: 1304-1305Crossref Scopus (80) Google Scholar, 27Staehelin L.A. Chlapowski F.J. Bonneville M.A. J. Cell Biol. 1972; 53: 73-91Crossref PubMed Scopus (196) Google Scholar, 28Kachar B. Liang F. Lins U. Ding M. Wu X.-R. Stoffler D. Aebi U. Sun T.-T. J. Mol. Biol. 1999; 285: 595-608Crossref PubMed Scopus (112) Google Scholar). By generating antibodies to each of these proteins, we have demonstrated that all these proteins are urothelium-specific, are confined to the apical surface of the urothelium (21Wu X.-R. Manabe M., Yu, J. Sun T.-T. J. Biol. Chem. 1990; 265: 19170-19179Abstract Full Text PDF PubMed Google Scholar, 29Wu X.-R. Sun T.-T. J. Cell Sci. 1993; 106: 31-43Crossref PubMed Google Scholar), and are highly conserved morphologically and biochemically in all these mammalian species (22Wu X.-R. Lin J.-H. Walz T. Haner M., Yu, J. Aebi U. Sun T.-T. J. Biol. Chem. 1994; 269: 13716-13724Abstract Full Text PDF PubMed Google Scholar). Using an in vitro adherence system, we recently showed that type 1 fimbriatedE. coli can bind to uroplakins Ia and Ib, two major high mannose-type glycoproteins of apical urothelial plaques (30Wu X.-R. Sun T.-T. Medina J.-J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9630-9635Crossref PubMed Scopus (262) Google Scholar). The binding is highly specific, saturable, and species-conserved and can be inhibited by d-mannose. We further showed that the allelic variants of type 1 fimbriated E. coli that are prevalent in UTIs, but not those prevalent in feces, bind to the uroplakins (14Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.-R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (301) Google Scholar). 2J. Pak, D. L. Hasty, and X.-R. Wu, unpublished data.2J. Pak, D. L. Hasty, and X.-R. Wu, unpublished data. These results strongly suggest that the uroplakins can serve as the urothelial receptors for type 1 fimbriae and that these receptors can provide a selective advantage for certain E. coli strains to survive in the urinary niche. Our in vitro data showing the functional interaction between type 1 fimbriated E. coli and uroplakins have been recently confirmed morphologically by an in vivoinfection model. Using quick-freeze and deep-etch electron microscopy of infected mouse bladders, Mulvey et al. (31Mulvey M.A. Lopez-Boado Y.S. Wilson C.L. Roth R. Parks W.C. Heuser J. Hultgren S.J. Science. 1998; 282: 1494-1497Crossref PubMed Scopus (774) Google Scholar) demonstrated that the tip of type 1 fimbriae interacted directly with the central depression (3.7 nm in diameter) of hexagonal uroplakin particles. This finding parallels our previously proposed structural model of uroplakin particles in which either uroplakin Ia or Ib, the two in vitro urothelial receptors for type 1 fimbriae, occupies the inner ring surrounding the central depression of hexagonal uroplakin particles (32Wu X.-R. Medina J.-J. Sun T.-T. J. Biol. Chem. 1995; 270: 29752-29759Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Together, these in vitro and in vivo data strongly indicate that uroplakins Ia and Ib can serve as the major urothelial receptors for type 1 fimbriae. Despite these developments, little attention has been paid to the host defense factors that may interfere with E. coli adhesion to the urothelial receptors. Previous studies suggested that Tamm-Horsfall protein (THP; also named uromodulin), the most abundant protein in mammalian urine, can bind to type 1 fimbriated E. coli, thus implying a role for THP in urinary defense (33Orskov I. Ferencz A. Orskov F. Lancet. 1980; 1: 887Abstract PubMed Scopus (155) Google Scholar, 34O'Hanley P. Lark D. Falkow S. Schoolnik G. J. Clin. Invest. 1985; 75: 347-360Crossref PubMed Scopus (138) Google Scholar, 35Parkkinen J. Virkola R. Korhonen T.K. Infect. Immun. 1988; 56: 2623-2630Crossref PubMed Google Scholar, 36Reinhart H.H. Obedeanu N. Sobel J.D. J. Infect. Dis. 1990; 162: 1335-1340Crossref PubMed Scopus (29) Google Scholar). These studies were, however, not performed in the context of well defined E. coli strains or in the presence of the cognate urothelial receptors. It was also unclear whether THP possesses a single, well conserved, high-mannose chain that is necessary for type 1 fimbrial binding (37Williams J. Marshall R. van Halbeek H. Vliegenthart J.F.G. Carbohydr. Res. 1984; 134: 141-155Crossref PubMed Scopus (50) Google Scholar, 38Hard K. van Zadelhoff G. Moonen P. Kamerling J.P. Vliegenthart F.G. Eur. J. Biochem. 1992; 209: 895-915Crossref PubMed Scopus (273) Google Scholar) and whether soluble urinary THP is capable of interacting at all with the fimbriae (35Parkkinen J. Virkola R. Korhonen T.K. Infect. Immun. 1988; 56: 2623-2630Crossref PubMed Google Scholar). There have also been questions regarding the specificity of the THP-E. coliinteraction as non-mannose-specific P fimbriae were found to bind THP (39Leeker A. Kreft B. Sandmann J. Bates J. Wasenauer G. Muller H. Sack K. Kumar S. Exp. Nephrol. 1997; 5: 38-46PubMed Google Scholar). In addition, it remains uncertain what the relative contribution of THP would be to urinary defense compared with other urinary proteins. Finally, it is unclear how THP interacts with the two recently identified major phenotypic FimH variants (14Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.-R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (301) Google Scholar). We have undertaken this study to address some of these questions and to examine the potential role of THP in host urinary tract defense. The fimbrial expression of clinical and recombinant E. colistrains was determined by yeast aggregation and hemagglutination as previously described (14Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.-R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (301) Google Scholar, 30Wu X.-R. Sun T.-T. Medina J.-J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9630-9635Crossref PubMed Scopus (262) Google Scholar). Thus, J96 (O4, K6), a human pyelonephritis isolate, expresses both type 1 (MH variant of FimH) and P (PapG1 and PapG3) fimbriae (34O'Hanley P. Lark D. Falkow S. Schoolnik G. J. Clin. Invest. 1985; 75: 347-360Crossref PubMed Scopus (138) Google Scholar, 40Stromberg N. Marklund B.-I. Lund B. Ilver D. Hamers A. Gaastra W. Karlsson K.-A. Normark S. EMBO J. 1990; 9: 2001-2010Crossref PubMed Scopus (218) Google Scholar). P678-54, a minicell-producingE. coli K12 derivative, expresses no fimbriae. SH48 and HU849, two recombinant strains derived by transfecting the non-fimbriated P678-54 strain with J96 genomic DNA fragments, express type 1 and P fimbriae (PapG1), respectively (41Hull R.A. Gill R.E. Hsu P. Minshew B.H. Falkow S. Infect. Immun. 1981; 33: 933-938Crossref PubMed Google Scholar). IA2 (O6, H−), a clinical isolate from a patient with acute UTI, expresses the PapG2-type P fimbriae (42Clegg S. Infect. Immun. 1982; 38: 739-744Crossref PubMed Google Scholar). KB54 and KB91, two recombinant strains obtained by transfecting afimH-null E. coli AAEC191A strain withfimH genes isolated from UTI and intestine, respectively, express a high monomannose-binding variant (MH) and a low monomannose-binding variant (ML), respectively (14Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.-R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (301) Google Scholar). KB18, a negative control strain, expresses a nonfunctional FimH mutant. All strains were cultured in Luria-Bertani medium at 37 °C for 16 h in methionine- and cysteine-free Dulbecco's modified Eagle's medium (glucose-free; Life Technologies, Inc.) for 2 h and then in Dulbecco's modified Eagle's medium containing [35S]methionine and [35S]cysteine (PerkinElmer Life Sciences) for 2 h. The labeled E. coli cells were washed four times in phosphate-buffered saline (PBS) and stored in 30% glycerol in PBS at −70 °C until use. Human THP was purified from pooled fresh urine samples collected from three healthy male donors by three rounds of precipitation in 0.58m NaCl and solubilization and dialysis in distilled water (43Afonso A.M. Carbohydr. Res. 1981; 89: 309-319Crossref PubMed Scopus (25) Google Scholar). Mouse THP was purified from pooled fresh urine of BALB/c and 129/svj strains using the same method. The yield and purity of THP were assessed by SDS-PAGE followed by silver nitrate staining (see below), and both human and mouse THPs migrated as a single band. AUM was purified from bovine urinary bladders by first isolating total urothelial membranes with gradient centrifugation and then treating them with 2% Sarkosyl followed by 25 mm NaOH (21Wu X.-R. Manabe M., Yu, J. Sun T.-T. J. Biol. Chem. 1990; 265: 19170-19179Abstract Full Text PDF PubMed Google Scholar, 22Wu X.-R. Lin J.-H. Walz T. Haner M., Yu, J. Aebi U. Sun T.-T. J. Biol. Chem. 1994; 269: 13716-13724Abstract Full Text PDF PubMed Google Scholar). AUM was quantified using bicinchoninic acid reagent (Pierce) in the presence of 1% SDS. On SDS gel, purified AUM contained four major proteins: 27-kDa uroplakin Ia, 28-kDa uroplakin Ib, 15-kDa uroplakin II, and 47-kDa uroplakin III (21Wu X.-R. Manabe M., Yu, J. Sun T.-T. J. Biol. Chem. 1990; 265: 19170-19179Abstract Full Text PDF PubMed Google Scholar, 24Yu J. Lin J.-H. Wu X.-R. Sun T.-T. J. Cell Biol. 1994; 125: 171-182Crossref PubMed Scopus (166) Google Scholar, 29Wu X.-R. Sun T.-T. J. Cell Sci. 1993; 106: 31-43Crossref PubMed Google Scholar, 44Lin J.-H. Wu X.-R. Kreibich G. Sun T.-T. J. Biol. Chem. 1994; 269: 1775-1784Abstract Full Text PDF PubMed Google Scholar). For the bacterial overlay assay, total urinary proteins or purified THP was resolved by SDS-PAGE (15% acrylamide, 120:1 acrylamide/bisacrylamide ratio), electrophoretically transferred onto nitrocellulose membrane, and reacted with [35S]methionine-labeled E. coli strains reconstituted in 3% bovine serum albumin and 0.1% NaN3 in PBS. After washing in PBS, the binding was visualized by autoradiography. For the microtiter well binding assay, purified THP was dissolved in distilled water and incubated in 96-well polystyrene microtiter plates at room temperature for 30 min and at 4 °C overnight. After washing, the microtiter wells were blocked with 3% bovine serum albumin in PBS for 2 h, and immobilized THP was then incubated with [35S]methionine-labeled E. coli strains reconstituted in 3% bovine serum albumin and 0.1% NaN3 in PBS at room temperature for 2 h. After washing four times in PBS, the bound bacteria were dissolved in 1% SDS and quantified using scintillation counting. All binding was performed in triplicate. Purified human THP was reduced andS-carboxymethylated according to van Rooijen et al. (45van Rooijen J.J. Voskamp A.R. Kamerling J.P. Vliegenthart J.F. Glycobiology. 1999; 9: 21-30Crossref PubMed Scopus (85) Google Scholar). THP was dissolved in 1 m Tris-HCl (pH 8.25) containing a final concentration of 50 mm dithiothreitol (Sigma), 6 m guanidine chloride, and 1 mm EDTA. After incubation at 37 °C for 2 h, the mixture was supplemented with iodoacetic acid (Sigma) to a final concentration of 100 mm. The reaction proceeded in the dark for 30 min and was stopped by the addition of 200 mm β-mercaptoethanol followed by dialysis against distilled water. For deglycosylation, reduced and carboxymethylated THP was digested with endoglycosidase H (0.05 units) or with N-glycosidase F (2500 units) in 50 mm phosphate buffer containing 0.5% SDS, 1% Nonidet P-40, 1% β-mercaptoethanol, 10 mm EDTA, and 0.05% NaN3 at 37 °C for 16 h. After electrophoresis, the polyacrylamide gel was prefixed with 50% methanol and 7% acetic acid and then incubated with 10% glutaraldehyde for 30 min. After extensive washing with distilled water, the gel was exposed to a solution containing 20% silver nitrate, 0.4% NaOH, 0.1% NH4OH, and 2% ethanol for 6 min. The gel was washed with distilled water for 1 h and developed in a solution containing 0.005% citric acid, 0.02% formaldehyde, and 10% ethanol. The reaction was stopped by incubating the gel with 10% acetic acid. For Western blotting, proteins resolved by SDS-PAGE were electrophoretically transferred onto nitrocellulose membrane and incubated first with an anti-human THP polyclonal antibody (BIODESIGN International) and then with a secondary antibody conjugated with peroxidase. The membrane was developed in a diaminobenzidine/H2O2 solution. To identify urinary proteins that can potentially serve as defense factors against E. coli adherence, we examined the interaction between type 1 fimbriated E. coli and total urinary proteins using the gel overlay assay. Pooled fresh urine samples from healthy male donors were immediately denatured and reduced in SDS/β-mercaptoethanol solution to minimize protein degradation and aggregation and subsequently analyzed by SDS-PAGE. Silver nitrate staining revealed two major protein species at 90 and 65 kDa, along with many minor proteins at a lower molecular mass range (Fig.1 A). When duplicate samples resolved by SDS-PAGE were electrotransferred onto nitrocellulose membrane and reacted with [35S]methionine-labeled type 1 fimbriated E. coli (strain SH48, MH type), the bacteria reacted specifically with the 90-kDa protein band, with very little binding to any other urinary proteins (Fig. 1 B). On the basis of the predominance and the molecular mass range, we speculated that the 90-kDa protein was THP (46Kumar S. Muchmore A. Kidney Int. 1990; 37: 1395-1401Abstract Full Text PDF PubMed Scopus (247) Google Scholar). This was proven to be the case, as immunoblotting using a polyclonal antibody raised against human THP specifically reacted with the 90-kDa band (Fig. 1 C). The fact that as little as 20 &l of the unconcentrated urine contained sufficient amounts of THP to bind a detectable number of type 1 fimbriated E. coli cells suggested that this protein is the major urinary protein that can potentially block E. coliadherence to urothelial receptors (see below). It has been recently documented that two major phenotypic variants exist for type 1 fimbriae based on their binding specificity for the monomannose residues: the low monomannose-binding (ML) and high monomannose-binding (MH) variants (14Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.-R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (301) Google Scholar). They predominate in different niches, with 80% of fecal E. coli expressing the ML phenotype and >70% of UTI isolates expressing the MH phenotype. A previous in vitro adherence assay showed that the MH (UTI) variant bound to the purified urothelial plaques containing uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae, in significantly greater numbers than the ML (fecal) variant (14Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.-R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (301) Google Scholar). This provides an explanation for the selective advantage of the urothelium for the MH variant and raises the interesting possibility that the two variants might bind differentially to THP (47Sokurenko E.V. Chesnokova V. Doyle R.J. Hasty D.L. J. Biol. Chem. 1997; 272: 17880-17886Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). To further study this possibility, we performed an in vitroadherence assay to test the binding between a panel of E. coli strains and purified human THP. THP, shown to be a single species by silver nitrate staining (see Figs. 4 A and6 A), was immobilized on microtiter wells and incubated with equivalent numbers of each [35S]methionine-labeledE. coli strain (Fig. 2). The first experiment tested isogenic strains representing the two major phenotypic variants of type 1 fimbriae. Although KB91 and KB54, which express the ML and MH adhesins, respectively, aggregatedSaccharomyces cerevisiae equally well (data not shown), KB54 bound to THP four times better compared with KB91. This result suggests that it is the unmodified terminal mannose, most likely a moiety within the high-mannose residues, but not the bulky complex-type sugars, of THP that is responsible for the binding. Not surprisingly, the negative control strain, KB18, which expresses a nonfunctional mutant adhesin, showed little binding (Fig. 2 A). The second experiment examined strains generated in the E. coli P678-54 background, with SH48 expressing type 1 fimbriae (MH type), HU849 expressing PapG1-type P fimbriae, IA2 expressing PapG2-type P fimbriae, and P678 expressing no fimbriae. High-level binding to THP was observed only with the type 1 fimbriated SH48 strain, with no significant binding of the P or non-fimbriated E. colistrains (Fig. 2 B). With both sets of strains, the binding to bovine serum albumin was consistently at a background level. These results strongly suggest that the binding of E. coli to THP is specific for type 1 fimbriated E. coli. Since THP preferentially binds to the MH variant of type 1 fimbriae, which is prevalent in UTIs, this lends further support that THP may play an important role in urinary tract defense.Figure 6Type 1 fimbriated E. colibinds to both human and mouse THPs. A, SDS-PAGE analysis of purified human and mouse THPs. THP was isolated from BALB/c (lane 2) and 129/svj (lane 3) mouse urine using the NaCl precipitation method; 1 &g of each sample was analyzed by SDS-PAGE and silver staining. Lane 1 is a human THP sample (control). Note that the molecular mass of mouse THP is slightly higher than that of human THP. B, comparative binding of E. coli strains to purified human and mouse THPs.35S-Labeled isogenic E. coli strains expressing no fimbriae (None), type 1 fimbriae (Type 1: SH48, MH type), PapG1-type P fimbriae (P (G-1)), or PapG2-type P fimbriae (P (G-2)) (2 × 105cpm/strain) were incubated with 1 &g of immobilized human or mouse THP. The number of E. coli cells bound to THP is indicated as (counts/min) × 10−4. Note that type 1 fimbriated E. coli bound to THP isolated from both species equally efficiently, suggesting that the high-mannose residues in THP are highly conserved.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2In vitro adherence of E. coli strains to purified human THP. A, three isogenic E. coli strains expressing a mutant FimH adhesin (KB18), an ML FimH adhesin (KB91), and an MH FimH adhesin (KB54) were metabolically labeled with [35S]methionine/cysteine. Equal amounts of the labeled bacteria (2 × 105 cpm) were incubated with the same amount of immobilized THP (1 &g/well). Note the high-level binding with the MH strain. The open bars indicate bovine serum albumin (BSA; 1 &g/microtiter well) as a negative control. B, four isogenic strains generated in a different genetic background and expressing MH-type FimH (SH48), PapG1 (HU849), PapG2, and no fimbriae (P678) were incubated with THP as described for A. Note that only the FimH-expressing strain bound to THP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further examine the binding specificity between type 1 fimbriated E. coli and THP, we performed two saturation binding assays (Fig.3). In the first, we incubated increasing
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