Receptor Structure for F1C Fimbriae of Uropathogenic Escherichia coli

Bacterial strains and growth conditions.The E. coli K-12 strain HB101 used in this study was transformed with the plasmid pPIL110-54 (pACYC184 plasmid vector containing the completefoc gene cluster cloned from the uropathogenic E. coli strain AD110) (40, 51). Recombinant bacteria were grown overnight (with shaking) in liquid broth supplemented with the appropriate antibiotic (50 μg of chloramphenicol per ml).

Reagents and chemicals.Purified glycolipids, saccharides, and enzymes used were obtained from Sigma, Deisenhofen, Germany. Recombinant β-N-acetylhexosaminidase was from New England Biolabs, Beverly, Mass. Carbohydrates conjugated to bovine serum albumin (BSA) and disaccharides used as inhibitors were purchased from Dextra Laboratories (Reading, United Kingdom); Toronto Research Chemicals, North York, Canada; and Glycorex, Lund, Sweden. All chemicals used were of analytical grade.

Antisera.Polyclonal antibodies against purified F1C fimbriae were raised in chicken (Nano-Tools, Freiburg, Germany). Antiserum was adsorbed with HB101 host strain before use.

Seroagglutination.The capacity of bacteria to express F1C fimbriae was assayed by agglutinating bacteria by using chicken polyclonal anti-F1C antibody as described earlier (20). Briefly, diluted (1,000 times) antiserum (10 μl) was mixed with the same volume of liquid bacterial culture grown for 16 h (with shaking), and agglutination was observed. Nonfimbriated strain HB101 carrying plasmid vector pACYC184 was used as a negative control. Fimbriated bacteria did not show agglutination with phosphate-buffered saline (PBS).

Purification of F1C fimbriae.F1C fimbriae were harvested from recombinant strain HB101 (pPIL110-54) by using commercial blendor (Omnimixer; Waring) and were purified essentially as described by Khan and Schifferli (20). Moreover, like other fimbriae (20), F1C fimbriae were heat extractable. Fimbriated bacteria from broth cultures were pelleted by centrifugation, were suspended in 0.5 mM Tris-HCl (pH 7.4) containing 75 mM NaCl, and were treated at 60°C for 30 min in a shaking water bath. Bacteria were removed by centrifugation, and F1C fimbrial preparations were concentrated from the supernatant by using an ultrafiltration pressure cell (YM100 membranes; Amicon, Beverly, Mass.).

Biotinylation of bacteria.Overnight cultures of bacteria were pelleted at 1,940 × g for 15 min. After being washed three times with PBS bacteria were resuspended in PBS containing 0.1 mM CaCl₂ and 1 mM MgCl₂ (pH 7.6) to a final concentration of 5 × 10⁸ bacteria/ml. To this, an equal volume of PBS-CaCl₂-MgCl₂ containing 0.2 to 0.3 mg of sulfo-NHS-LC–biotin (sulfosuccinimidyl-6-[biotinamido]-hexanoate) (Pierce Chemical Company) per ml was added. Bacterial surface proteins were allowed to biotinylate for 2 h by incubating the mixture at room temperature with gentle shaking. The bacteria were collected, washed four times, resuspended to a final concentration of 2 × 10⁹bacteria/ml in PBS (pH 7.4) containing 1% BSA, and stored at 4°C. This preparation was used for 3 days.

TLC fimbriae overlay assay.TLC was performed as described earlier (12, 17, 18) with a solvent system involving chloroform-methanol-water (60:35:8). Briefly, pure glycolipids (Sigma) were separated for analytical purpose on high-performance TLC (HPTLC) aluminium-backed silica gel 60 plates (Merck, Darmstadt, Germany). Glycolipids were stained with orcinol spray (Sigma). For the overlay assay, the plates were sequentially treated for 2 min with polyisobutylmethacrylate (0.1% in hexane), were blocked with 2% BSA in Tris-buffered saline (TBS; 10 mM Tris-HCl [pH 7.4], 154 mM NaCl) for 90 min at room temperature, were washed once with TBS, were overlaid with pure F1C fimbriae (20 μg/ml in 1% BSA-TBS) for 2 h, and were washed three times with TBS–0.05% Tween 20 and once with TBS. Fimbrial antigen was detected sequentially with anti-F1C fimbrial chicken polyclonal antibody and horseradish peroxidase-conjugated anti-chicken antibody (Sigma) by conventional Western blot technique by using the DAB peroxidase substrate tablet set (Sigma) as described earlier (20).

Neuraminidase treatment of disialogangliosides separated by HPTLC.The disialoganglioside fraction containingO-acetylated derivatives of the gangliosides (GD3, disialosylparagloboside [DSPG], disialosyllacto-N-nor-hexaosylceramide [DSnHC], and disialosyllacto-N-nor-octaosylceramide [DSnOC]) purified from human leucocytes (23) were separated on five glass-backed HPTLC plates (Merck) in parallel and were fixed with polyisobutylmethacrylate as described above. One plate was used for fimbrial overlay assay and the other one was immunostained as described earlier (23) by using CDw60 monoclonal antibody M-T21, which is specific for GD3, 9-O-acetylated GD3, and terminally disialyated gangliosides of the neolacto series such as DSPG and DSnHC. In order to remove sialosyl residues linked to gangliosides, three plates were incubated overnight at 37°C with neuraminidase (10 mU) per ml of Arthrobacter ureafaciens (Boehringer GmbH, Mannheim, Germany) in 0.1% CaCl₂, were washed three times with PBS, and were blocked with 2% BSA-PBS. After the washing of blocked plates, one plate was overlaid with the monoclonal antibody 1B2 specific for the terminal Galβ1-4GlcNAc-carbohydrate structure found in glycolipids of the neolacto series and was further processed as described earlier (23). The second plate was overlaid with purified fimbriae. The third plate was used as a control where neither fimbriae nor monoclonal antibody 1B2 was overlaid. They were further processed as described above.

Periodate oxidation.Microtiter wells containing either asialo-GM₁ (GgO₄Cer) or asialo-GM₂(GgO₃Cer) were washed twice with PBS, incubated with 10 mM sodium metaperiodate in PBS (pH 7.1) at 37°C for 90 min (31), washed four times with PBS, blocked with 2% BSA-PBS at 37°C for 2 h, and then overlaid with twofold serially diluted bacteria as described below. Bacterial binding was determined as mentioned below.

Solid-phase binding and binding inhibition assay.Polyvinyl chloride plates (Falcon; Becton Dickinson) were coated with glycolipids per well, (2 μg of glucosylceramide, 2 μg of galactosylceramide 1 and 2-[hydroxylated and nonhydroxylated fatty acids], 2 μg of sulfatide, 2 μg of lactosylceramide [LacCer], 2 μg of Gb₃Cer, 2 μg of GM1, 2 μg of asialo-GM₁(GgO₄Cer), 1 μg of asialo-GM₂(GgO₃Cer), 2 μg of paragloboside) in chloroform-methanol (1:9 [vol/vol]). The solvent was allowed to evaporate overnight at room temperature, and the wells were blocked with 2% BSA-PBS (pH 7.37) for 2 h at 37°C and were washed three times with PBS. For each glycolipid, a serial dilution of F1C fimbriated E. coliHB101(pPIL110-54), control strain HB101(pACYC184), or purified F1C fimbriae was first tested to determine the numbers of bacteria and the quantity of purified fimbriae required to obtain 50% binding. The number of fimbriated HB101(pPIL110-54) and nonfimbriated control strain HB101(pACYC184) was determined by measuring absorbance at a wavelength of 550 nm, with a standardized chart correlating absorbance with viable counts. After removal of the blocking solution and washing, 100 μl of serially diluted bacterial suspensions (2 × 10⁸bacteria in the first well) or purified fimbrial solution (10 μg of fimbriae in the first well) in PBS–0.5% BSA was added to each well, and plates were incubated for 2 h at 37°C. After wells were washed as previously described, rabbit anti-E. colipolyclonal antibody (Biodesign International, Kennebunk, Maine) in PBS–0.5% BSA (1:2,000) (or for wells with fimbriae, anti-F1C chicken antibody [polyclonal; 1:400]) was added for 1 h at 37°C (100 μl/well). Following another washing step, peroxidase-conjugated goat anti-rabbit immunoglobulin G (Dako, Hamburg, Germany) or anti-chicken immunoglobulin G whole molecule (Sigma) in PBS–1% BSA (1:2,000) was added for 1 h at 37°C (100 μl/well). Following a final wash, the bound enzyme was detected by the addition of 100 μl of substrate (Pierce ImmunoPure TMB Substrate Kit) per well for 5 to 30 min. The reaction was stopped by adding 100 μl of 2 M H₂SO₄ to each well. Absorbance was measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Similar experiments were also performed by using biotinylated bacteria, and binding was detected with horseradish peroxidase-conjugated streptavidine (Pierce Chemical Company) and its substrate as above. The control wells were treated in the same manner except that blank control wells had no bacteria or fimbriae.

The binding inhibition assay was performed only with asialo-GM₁ (GgO₄Cer)- and asialo-GM₂ (GgO₃Cer)-coated microtiter plates. For the binding inhibition assays, bacteria at the number (3 × 10⁷/well) giving 50% binding were preincubated with twofold serially diluted carbohydrates for 1 h at room temperature. The mixtures were transferred to the glycolipid-containing microtiter plates, and bacterial binding was determined as described above. Percentages of inhibition were calculated by the following equation: percent inhibition = { 1 − [(A₄₅₀ of the test well −A₄₅₀ of the blank control well) / (A₄₅₀ of the 100% binding control well −A₄₅₀ of the blank control well)]} × 100. For this calculation, the 100% binding control wells had no carbohydrate inhibitors and the blank control well had neither bacteria nor inhibitors. Each strain was tested in three separate experiments, and in each experiment, 30 determinations of bacterial or fimbrial adherence were performed in parallel.

To establish the binding affinities of E. coliHB101(pPIL110-54), control strain HB101(pACYC184), and purified fimbriae to different glycolipids, twofold serially diluted glycolipids were dried in microtiter plates as mentioned above. After blocking and washing were carried out as described above, bacteria (3 × 10⁷/well) or purified fimbriae (1.5 μg/well) giving 50% binding was incubated as described above. Bacterial or fimbrial binding was determined as described above.

Enzymatic treatment of asialo-GM₁(GgO₄Cer).Purified asialo-GM₁(GgO₄Cer) (Sigma) was immobilized on ELISA plates and was sequentially treated with bovine β-galactosidase (β-Gal) (Sigma) and then with recombinant β-N-acetylhexosaminidase (New England Biolabs). Asialo-GM₁ (GgO₄Cer) was suspended in methanol (10 μg/ml), and 100 μl of the suspension was added to wells of microtiter plates. After evaporation of methanol, immobilized glycolipids were incubated with 0.4 U of β-Gal in Tris buffer [10 mM Tris, 1.7 M (NH₄)₂SO₄, 10 mM MgCl₂, pH 7.3] for 2 h at 37°C. The wells were washed with PBS, and the glycolipids were further treated with 10 U of β-N-acetylhexosaminidase in citrate buffer (50 mM sodium citrate, pH 4.5) per well for 6 h at 37°C in a shaking water bath. After performing washing and blocking steps, bacterial or fimbrial binding was determined as described above.