©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glycoconjugate Receptors for P-fimbriated Escherichia coli in the Mouse
AN ANIMAL MODEL OF URINARY TRACT INFECTION (*)

Boel Lanne (§) , Britt-Marie Olsson , Per- Jovall , Jonas öm , Henrik Linder (¶) , Britt-Inger Marklund (**) , Jörgen Bergström , Karl-Anders Karlsson

From the (1) Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, S-413 90 Göteborg, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glycosphingolipids were isolated from kidneys, urethers, and bladders (including urethrae) of C3H/HeN mice. Binding was studied of a clinical isolate and recombinant strains of uropathogenic P-fimbriated Escherichia coli to these glycolipids. A series of receptor-active glycolipids with Gal4Gal in common, previously shown to be recognized by these bacteria, was identified by use of specific monoclonal antibodies, fast-atom bombardment and electron-impact mass spectrometry, and proton nuclear magnetic resonance spectroscopy: galabiosylceramide (Gal4GalCer), globotriaosylceramide (Gal4Gal4GlcCer), globoside (GalNAc3Gal4Gal4GlcCer), the Forssman glycolipid (GalNAc3GalNAc3Gal4Gal4GlcCer), Gal4GlcNAc6(Gal3)GalNAc3Gal4Gal4GlcCer, and Gal4(Fuc3)GlcNAc6(Gal3)GalNAc3Gal4Gal4GlcCer.

The binding pattern for mouse kidney glycolipids differed from that for kidney glycolipids of man and monkey. In particular, the dominant 8-sugar glycolipid in the mouse was not detected in the primates. A second difference was found in the binding of E. coli to kidney glycoproteins on blots after electrophoresis; the mouse showed distinct receptor-active bands while human and monkey did not. These differences may be of relevance when using the mouse as a model for clinical urinary tract infection of man.


INTRODUCTION

An important factor in the pathogenesis of bacterial infections is the ability of the bacteria to adhere to host tissues (1) , often by means of specific binding of bacterial adhesins to host cell carbohydrates (2, 3) . P-fimbriated Escherichia coli are important in human urinary tract infections and are known to adhere to the epithelial cells (4, 5, 6) . The adhesion has been shown to depend on Gal4Gal-containing glycolipids on the host cells (5, 7) and a number of glycolipid isoreceptors with Gal4Gal in terminal or internal positions have been identified (8) . Recently, it was demonstrated for human red blood cells that Gal4Gal was absent from glycoproteins and exclusively present in the glycolipid form (9) . The molecular genetics and biogenesis of P-fimbriae have been extensively studied, including the sequence of the three classes of adhesin, I, II, and III (10, 11) , which have slightly different affinities for various isoreceptors of glycolipids (12, 13, 14, 15) . With respect to human and dog urinary tract infection, clinical isolates of E. coli differ in adhesin class, which is related to different glycolipid patterns of the two species. In isolates of human origin, the class II adhesin predominates. This adhesin preferentially binds to globoside,() a glycolipid that dominates in human urinary tract. E. coli isolates from dog infections, however, express mostly the class III adhesin, which binds more strongly to the Forssman glycolipid, a major glycolipid in dog kidney.

The mouse is currently used as a model of human urinary tract infection()(16, 17, 18) . It is therefore of interest to investigate the carbohydrate basis for E. coli adhesion in this animal. Thus, model C3H/HeN mice were analyzed for the presence of receptor-active glycoconjugates in various parts of the urinary tract using both clinical isolates and recombinant strains of uropathogenic E. coli. It was found that the isoreceptor pattern differed significantly from that found in human and monkey urinary tract with respect to both glycolipids and glycoproteins.


EXPERIMENTAL PROCEDURES

Isolation of Glycolipids

Female C3H/HeN mice (original breeding stock, Charles River Laboratories, Margate, Kent, UK) were kept at the animal facilities at the Department of Infection and Immunology, Astra Arcus, S-181 85 Södertälje, Sweden, where the model infections were performed.The mice were used at 8-10 weeks of age.

The animals were anesthetized with diethyl ether and tissues were removed using sterile instruments. Kidneys from 60 animals, and urethers, bladders, and urethrae (in one piece) from 10 animals were obtained. The tissues were immediately frozen using cryostat spray and stored at -70 °C until analysis was performed. Before lipid extraction the urethers were separated from bladders plus urethrae. Lipids were extracted with a Soxhlet apparatus using chloroform/methanol (2:1 for 24 h, and 1:9 for 24 h). The combined extracts were subjected to mild-alkaline hydrolysis (0.2 M KOH in methanol, 3 h), dialyzed against water, and purified on a silica straight-phase column. No further purification of lipids from urethers and bladders plus urethrae, 2.1 and 2.7 mg, respectively, was carried out, while the purification of the mouse kidney preparation continued as described by Karlsson (19) . The kidney glycosphingolipids were separated into two main fractions, a neutral one containing uncharged lipids, 8.5 mg, and an acid fraction containing gangliosides and other charged lipids.

Fractionation of neutral glycolipids was achieved by HPLC chromatography on a Spherisorb S10W silica column, 250 8-mm inner diameter, particle size 10 µm (Phase Separation Ltd., Queensferry, UK). The glycolipid mixture, 6 mg, was applied and eluted with a gradient starting with chloroform/methanol/water, 60:35:8 (by volume unless otherwise stated), and ending with 10:10:3, 2 ml/min. Mild hydrolysis of acid glycosphingolipids to remove sialic acid was achieved in 1 ml of acetic acid/water, 1:100 (300 µg of lipid), 100 °C, 1 h.

Reference glycosphingolipids were obtained as follows: globotriaosylceramide, globoside, and P1 glycolipid from human erythrocytes (20) , Leand Lefrom human small intestine (21) , the Forssman glycolipid (22) and Le(23) from dog intestine, and gangliotetraosylceramide from feces of axenic mouse (24) . Reference mixtures of glycolipids were obtained from dog intestine, calf brain, human kidneys, human erythrocytes (blood group A), and human primary liver cancer, which contains Lestructures (20) .

For analysis of glycolipid mixtures using bacteria and antibodies, the glycolipids were separated on TLC with chloroform/methanol/water, 60:35:8, on aluminum backed silica nano plates of 0.2-mm phase thickness (Merck, Germany). Chemical detection was done with anisaldehyde (25) or resorcinol staining (26) . The procedure used for incubation of TLC plates with labeled biological reagents has been described (27) . Radioactivity was detected by autoradiography (Kodak XAR-5 film, Eastman Kodak).

Isolation and Analysis of Mouse and Monkey Kidney Proteins

Dried residues (20 mg) remaining after the lipid extraction of C3H/HeN mice and cynomolgus monkey ( Macaca fascicularis) kidneys were mixed with 1 ml of 50 m M Tris-HCl buffer (pH 8.0) containing 2.5% SDS. The mixture was incubated at room temperature overnight (gentle shaking), heated to 95 °C for 10 min, and then centrifuged at 10,000 g for 10 min. To extract residual lipids from the SDS-solubilized material, this was treated with 1-butanol/water, 9:1, three times. Prior to SDS-PAGE of the supernatant, the samples were diluted to 2-4 mg of protein/ml (determined by BCA Protein Assay, Pierce), and 2-mercaptoethanol (5%) was added.

SDS-PAGE (gradient gel, 8-25%) and Coomassie R-350 (PhastGelBlue R, Pharmacia) staining were carried out with a Pharmacia Phast System(Pharmacia, Sweden) according to the protocols of the manufacturer. Briefly, samples were heated to 95 °C for 5 min and centrifuged at 10,000 g for 2 min before electrophoresis to remove unsolubilized material. After electrophoresis the gel was either stained for protein or sugar (Glycan detection kit, Boehringer, Germany), or electroblotted onto a nitrocellulose membrane (0.45 µm) in 20% methanol containing 192 m M glycine and 25 m M Tris at pH 8.3.

For incubation with bacteria, the nitrocellulose membrane with electroblotted proteins was preincubated in blocking solution (3% bovine serum albumin, 50 m M Tris-HCl, 200 m M NaCl, 0.1% NaN, pH 8.0) for 1.5 h. The membrane was then incubated with S-labeled E. coli in phosphate-buffered saline (0.14 M phosphate buffer, pH 7.2, 0.14 M NaCl, 5 m M KCl). After 1.5-2 h the membrane was washed in 50 m M Tris-HCl, 0.2 M NaCl, 0.05% Tween 20 (pH 8.0), dried at room temperature, and exposed to x-ray film overnight.

Chemical Analysis

Mass spectra were obtained with a ZAB-2F/HF (VG Analytical, Manchester, UK) and a Jeol SX 102A (Jeol, Tokyo, Japan), both sector instruments, either in the positive-EI or negative-FAB mode (Xe atom bombardment, 8 kV). Triethanolamine was used as matrix. Methylation was performed according to Ref. 28 (and references therein) and gas chromatography-mass spectroscopic analysis of partially methylated alditol acetates was done according to Refs. 29 and 30. For analysis of partially methylated alditol acetates, a quadropole MS (Trio-II, VG Masslab, Altricham, UK) was used. The capillary column was a DB-1, 15 m 0.25 mm, inner diameter, 0.2-µm film thickness (J& Scientific). FAB-MS analysis of glycolipids was performed directly from the TLC plate as described (31) with the first MS of a Jeol HX/HX110A instrument. The conditions for the aquisition of EI spectra were: 70 eV electron ionization potential, 10 kV accelerating voltage, 300 µA trap current, ion source temperature 375 °C, and scan time 26 s.

H NMR spectra on deuterium-exchanged glycolipid fractions were acquired at 11.74 telsa on a JEOL ALPHA-500 (Jeol, Tokyo, Japan). Samples were dissolved in 0.5 ml of MeSO/DO, 98:2, and spectra recorded at 30 °C with a digital resolution of 0.4 Hz. Chemical shifts are given relative to tetramethylsilane using the internal solvent peak. Nuclear Overhauser enhancements were measured using the standard software of the instrument. The recycle time was at least five times the longitudinal relaxation time ( T).

Growth and Labeling of Bacteria

Four different P-fimbriated E. coli strains were used, HB101/pPIL291-15 (a gift from Dr. I. van Die, Vrije Universiteit, Amsterdam, The Netherlands, and Dr. B. Westerlund, University of Helsinki, Finland (32) ), HB101/pPAP5 (33) , HB101/pDC1 (34) , HB101/pPAP601 (35) , and DS-17 (a gift from Dr. K. Tullus, S:t Görans barnsjukhus, Stockholm, Sweden (36) ). The bacteria were cultivated on colonization factor agar plates supplemented with [S]methionine (400 µCi/10 ml, Amersham International, UK) at 37 °C overnight. They were collected by centrifugation, washed twice with phosphate-buffered saline, and resuspended in phosphate-buffered saline to approximately 1 10colony-forming units/ml. The bacteria were diluted to an activity of approximately 1 10cpm/ml.

Antibodies

The following mouse monoclonal antibodies were used: anti-Le(BL-G15, Monosan, Bio-Zak, Järfälla, Sweden), three antibodies binding to terminal Gal4Gal sequences, pk002, P001, and MC2102 = 87:5 (all obtained from Accurate Chemical & Science Corp., New York); and anti-Forssman (MAS033b, Seralab, Göteborgs Termometerfabrik, Sweden). The secondary antibodies used (rabbit anti-mouse immunoglobulins, Z109, DAKO A/S, Denmark) were labeled with I (37) .

Molecular Modeling

Minimum energy conformers of the GL-III glycolipid, identified below as Gal4(Fuc3)GlcNAc6(Gal3)GalNAc3Gal4Gal4GlcCer, were calculated within the Biograf molecular modeling program (Molecular Simulations Inc.) using the Dreiding-II force field (38) on a Silicon Graphics 4D/35TG workstation. Partial atomic charges were generated using the charge equilibration method (39) , and a distance dependent dielectric constant = 3.5r was used for the Coulomb interactions. In addition a special hydrogen bonding term was used in which Dwas set to 4 kcal/mol.


RESULTS

Comparison of Glycolipid Pattern of Mouse Urinary Tract Organs

The acid and neutral glycosphingolipids of C3H/HeN mouse kidneys were isolated and purified separately. TLC separations of the neutral glycosphingolipids visualized chemically with anisaldehyde are shown in Fig. 1 A. For comparison, glycolipids from human kidneys, as well as purified globoside, were included in the chromatogram. The neutral glycolipids from mouse kidneys are dominated by a very slow-moving band with more than 6 sugars (GL-III, Fig. 1 ) although several more weakly-staining bands, particularly in the 3-4-sugar region, are also seen.


Figure 1: Binding of P-fimbriated E. coli to glycosphingolipids isolated from mouse urinary tract organs. Plate A, anisaldehyde staining of neutral glycolipids from human kidney, mouse kidney, mouse urether, mouse bladder plus urethra, and globoside. Plate B, binding of E. coli (HB101/pPIL291-15) to purified glycolipids, mixtures of neutral ( n), and mixtures of acid ( a) glycolipids as indicated. The amount of each glycolipid mixture is shown as µg/lane in parentheses. Numbers in italics indicate the approximate position of glycolipids with different numbers of sugars. The arrow shows the direction of elution.



The mixture of acid glycolipids from mouse kidneys and the crude preparations of lipids from urethers and bladders plus urethrae were used for bacterial binding studies. The latter preparations were not purified sufficiently for chemical staining (the bands seen in Fig. 1of urethers and bladders plus urethrae do not have the green color characteristic of glycoconjugates stained with anisaldehyde).

Mapping of Receptors for P-fimbriated E. coli

Initially P-fimbriated E. coli, HB101/pPIL291-15, which binds very strongly to Gal4Gal-containing glycolipids, was used to screen the glycolipid preparations as shown in Fig. 1 B. Globotriaosylceramide, globoside, and the Forssman glycolipid were applied in the first three lanes as references. The bacteria bound strongly to neutral glycolipids from mouse kidneys in the 4-5-sugar region. In addition, four slower moving bands were strongly bound by the bacteria (GL-I, GL-II, GL-III, and GL-IV). The urethers and bladders plus urethrae both contained binding glycolipids in the 4- and 5-sugar regions, while in the preparation of bladders plus urethrae weak binding is also seen to a band in the 3-sugar region and also to a band comigrating with GL-II. For the urether a weak binding band was obtained which had the same mobility as GL-III.

The acid glycolipids obtained from the mouse kidneys showed two distinct binding bands (Fig. 1 B) which had the same mobility as GL-II and GL-III. No chemical identification was made of these glycolipids.

Identification of Binding Glycosphingolipids

Antibodies were used to reveal the presence of terminally placed Gal4Gal structures in glycolipids from mouse kidneys. In the 2-sugar region, the binding of the antibody MC2001 showed that Gal4GalCer was present in mouse kidneys (Fig. 2 A). The three bands probably differed in ceramide composition. Dog intestine was included in the analysis as a positive reference.() In the 3-sugar region, the antibody pk002 bound weakly, Fig. 2 B, but strongly to the reference globotriaosylceramide. The lack of a clear binding by monoclonal antibody P001 indicated that the P1 antigen is absent from mouse kidneys (Fig. 2 C). Further investigation with an antibody specific for the Forssman glycolipid (Fig. 2 D), showed that this glycolipid was present in the mouse but not in human kidneys. A weak band was also detected in the preparation of mouse bladders plus urethrae but not in the urethers.


Figure 2: TLC separation of glycolipids from various sources and analysis with monoclonal antibodies. The following antibodies were used: A, MC2102, which detects Gal4GlcCer; B, pk002, which binds globotriaosylceramide, and more weakly the P1 antigen; C, P001, which binds the P1 antigen; and D, MAS033b, which binds the Forssman glycolipid. The amount of each glycolipid mixture is shown as µg/lane in parentheses. Numbers in italics indicate the approximate position of glycolipids with different numbers of sugars.



To elucidate the structure of GL-II, GL-III, and GL-IV in the mouse kidneys, the neutral glycolipid mixture was subjected to HPLC fractionation on a silica column. The separation conditions were chosen to optimize separation of slow-moving compounds. Analysis by TLC of the fractions obtained is shown in Fig. 3. Fractions 4 and 5 were analyzed by FAB-MS in two ways after separation by TLC. First, the 3- and 4-sugar regions were scraped off the plates separately, extracted in chloroform/methanol, 2:1, and their mass spectra were collected. Second, they were analyzed by direct TLC FAB-MS. The 2-sugar region gave spectra consistent with (Hex)Cer, the 3-sugar region with (Hex)Cer, and the 4-sugar region gave spectra showing that the glycolipid was HexNAcHexHexHexCer. All three compounds had similar ratios of hydroxylated to unhydroxylated ceramide (1:1).

HPLC fractions 8 (GL-II), 11 (GL-III), and 15 (GL-IV) were subjected to negative-ion FAB-MS analysis (Fig. 4, A-C, respectively). The glycolipid in fraction 8 was composed of five hexoses (of which at least one was terminal) and two internal N-acetylhexosamines. One of the N-acetylhexosamines was located next to a terminal hexose. The FAB-MS obtained from fraction 11 (Fig. 4 B) showed the presence of five hexoses, two N-acetylhexosamines, and one Fuc. The Fuc and at least one hexose were terminally placed. This glycolipid had two dominating ceramide species which differed by two carbon atoms. The mass spectrum of fraction 15 closely resembled that of fraction 11. However, fraction 15 contained one dominating ceramide species with six carbons less than the light ceramide species of fraction 11. This conclusion was also consistent with the difference in mobility on the TLC plate. Because of the high intensity of m/z 1736.8 and 1387.8 relative to m/z 1590.8 and 1225.7, it is probable that the molecule contained two branching points. This irregular intensity of ions is also seen for fraction 11 (Fig. 4 B).


Figure 4: MS analysis of mouse kidney glycolipids. Negative-ion FAB-MS of native glycolipids are shown in A-C, and the positive-ion EI-MS of a methylated glycolipid in D. The HPLC fractions analyzed were: A, 8; B, 11; C, 15; and D, 11-12. Peaks labeled with an asterisk originated from the matrix, triethanolamine. Tentative theoretical fragmentation patterns and molecular weight are shown above each spectrum.



Fractions 11 and 12 were pooled and analyzed by EI-MS as the methylated derivative (Fig. 4 D). Molecular ions of m/z 2347.1 and 2375.2 were obtained which correspond to ceramides with sphingosine and 22- and 24-carbon fatty acids, respectively. These ceramides are also seen at m/z 632.6 and 660.6. Large peaks appear at 638.3, 1087.5, and 1291.5, which correspond to the following oxonium ions: HexHexNAcFuc, (Hex)(HexNAc)Fuc, and (Hex)(HexNAc)Fuc, respectively. These three oxonium ions, [Ox], lose either Fuc [Ox-Fuc+H]or Hex [Ox-Hex+H]giving rise to the following sets of ions: m/z 450.2 and 420.2 from 638.3, m/z 899.4 and 869.4 from 1087.5, and m/z 1073.5 and 1103.5 from 1291.5. Ions from terminal Hex (219.1 and 219.1-32 = 187.1) and Fuc (189.1 and 189.1-32 = 157.1) were also obtained. The presence of a set of m/z at 692.6 (Cer), 2173.4 (M-Hex), and 2203.1 (M-Fuc), indicates that minor amounts of the glycolipid might carry phytosphingosine.

Gas chromatography-mass spectroscopic analysis of partially methylated alditol acetates was performed on the pooled fractions 13-15 in parallel to analysis of the reference compounds gangliotetraosylceramide and Le. The following monosaccharides were identified; terminal Gal and Fuc, 4-substituted Gal and Glc, 3-substituted Gal, and 3,4-substituted GlcNAc. A second di-substituted HexNAc, with a longer retention time, but similar MS, was obtained.

The anomeric region of the H NMR spectrum (Fig. 5 C, Table I) of GL-III (HPLC fraction 10) shows two -signals ( J 4 Hz) at 4.82 and 4.80 ppm ( a and b in Fig. 5 C) corresponding to Fuc3 and Gal4, respectively. These signals can be conclusively assigned from the nuclear Overhauser enhancement (NOE, magnetic dipole coupling through space) experiments. The NOE between the 4.80 ppm signal and a typical Gal H4 signal at 3.82 ppm (40, 41) , as well as a quartet at 3.76 ppm (H2 of Gal), are shown in Fig. 5 B. The Fuc H5 gave rise to a quartet of 4.67 ppm ( c in Fig. 5 C). Of the two HexNAc signals at 4.61 and 4.49 ppm ( d and e in Fig. 5C) the former showed NOEs (Fig. 5 A) to Gal H5 (4.19 ppm) and Gal H4 (3.98 ppm) and also a large NOE to a signal at 3.59 ppm. This corresponds well with published values for the Gal H3 (40, 41) , and thus confirms the 4.61 ppm signal as arising from the GalNAc3 of a globo-core structure. The signal at 4.49 ppm is within the region for GlcNAc shifts, see for example, Ref. 42. Two well separated Gal signals were seen at 4.29 and 4.26 ppm ( f and g in Fig. 5C), respectively, the latter arises from the internal Gal4 linked to Glc, which in turn was seen at 4.19 ppm, overlapping two other signals. The former Gal is consistent with a terminal Gal4 of a Leterminal (43) . The other signals at 4.19 ppm (overlapping the Glc anomer) were triplets from the H5 of Gal and a terminal Gal3 (41) . NOEs were measured from all anomeric protons (not shown), and they confirmed the saccharide sequence as determined above. Since most non-anomeric signals are unassigned, the NOE analysis was based on the multiplicity of signals and their chemical shifts compared to model structures. For most glycosidic linkages there was no uncertainty. Only the linkage between the terminal Lefragment and the globo-core was unclear. There were NOEs (not shown) into the region of H6 protons but also an NOE to a typical Gal H4 signal, all of moderate strength. Thus, the results from the NMR indicate that the structure is Gal4(Fuc3)GlcNAc6/4(Gal3)GalNAc3Gal4Gal4GlcCer. This spectrum corresponds well with that published by Sekine et al. (44) . However, molecular modeling indicates that a 4-linked Ledeterminant most likely can be excluded since the strong NOE expected between the GlcNAc H1 and H4 of GalNAc is in this case not observed, whereas in the 6-linked form several conformers are compatible with the NOE effects.


Figure 5: Proton-NMR spectra of glycolipid fraction 10. C, the normal spectrum; B, NOE difference spectrum after irradiation at the 4.80 ppm signal; and A, NOE difference spectrum after irradiation at the 4.61 ppm signal. In C the following letters are used to assign signals: a, Fuc3 H1; b, Gal4 H1; c, Fuc3 H5; d, GalNAc3 H1; e, GlcNAc4/6 H1; f, Gal4 H1; g, Gal4 (internal) H1; h-j, Gal3 H1, Glc1 H1, and Gal4 H5, and k, Gal4 H4. All spectra were recorded at 30 °C, and in the NOE difference spectra the dispersion-like remains of the residual water peak have been baseline corrected.



GL-II (HPLC fraction 8) was available in very small amounts and therefore only a single H NMR spectrum could be acquired. The spectrum obtained (not shown) contained the same globo-core signals () as GL-III, but the Fuc signals (H1, H5, and methyl) were missing. In accordance with this, the GlcNAc (to which the Fuc was linked in fraction 10) anomer signal had shifted upfield to 4.39 ppm, whereas the terminal Gal4 had shifted upfield to 4.21 ppm (45) . An additional component was present in fraction 8, which had a HexNAc signal at 4.57 ppm. Also the EI-MS of methylated fraction 8 (not shown) contained an ion ( m/z = 260, typical for terminal HexNAc) that supports the presence of a second component.

Testing with an anti-Leantibody showed strong staining of one band in fractions 10 and 15 (Fig. 3 C). A second weak band was also seen in fraction 15 and two weak bands in fraction 8. Several binding bands were present in the Le-containing reference, human liver tumor. The antibody did not cross-react with Lc, nLc, Le, Le, or Le(not shown). When testing the mouse kidney gangliosides, neither the mixture of acid glycolipids as such, nor the desialylated mixture, were bound by the anti-Leantibody (data not shown).


Figure 3: HPLC separation of mouse kidney glycolipids. A, TLC separation of glycolipids before HPLC; B and C after HPLC fractionation. In A and B chemical detection was used (anisaldehyde), and in C binding by anti-Leantibody.



Binding of E. coli with Class I, II, and III Adhesins to Mouse Glycolipids

E. coli strains expressing the three different classes of P-adhesins were tested for binding to mouse urinary tract glycolipids together with human kidney glycolipids and purified globotriaosylceramide, globoside, and the Forssman glycolipid (Fig. 6, A-C, and Tables II and III). E. coli carrying the class I adhesin bound strongly to globotriaosylceramide of human kidney and to globoside of human and mouse kidney (Fig. 6 A). It also bound to a TLC band in the 4-sugar region of the mouse bladders plus urethrae preparation. Weaker binding to GL-I and GL-III in mouse kidney was also found. The class II bacteria, which bound equally strongly to globotriaosylceramide, globoside, and the Forssman glycolipid on TLC, bound to the same structures as the class I carrying bacteria. In addition, it bound to bands in the mouse kidney and in the bladders plus urethrae that had slightly lower mobility than globoside. The binding to the GL-I, GL-II, GL-III, and GL-IV glycolipids seen for HB101/pPIL291-15 was also observed for this class II E. coli.


Figure 6: Binding of P-fimbriated E. coli to glycolipid mixtures from urinary tract organs of C3H/HeN mice. The glycolipids were separated by TLC before incubation with P-fimbriated bacteria. The following E. coli isolates were used: A, class I, HB101/pPAP5; B, class II, HB101/pDC1; C, class III, HB101/pPAP601; and D, the clinical isolate DS-17. The amount of each glycolipid mixture is shown as µg/lane in parentheses. Numbers in italics indicate the approximate position for glycolipids with different numbers of sugars.



The class III adhesin carrying E. coli, however, showed a very different binding pattern (Fig. 6 C). It clearly preferred the Forssman glycolipid to the shorter Gal4Gal-containing glycolipids. In addition to the Forssman glycolipid of the mouse kidneys, it also bound to bands with the same mobility in both the urether and the bladder plus urethrae preparations. However, GL-II, GL-III, and GL-IV of mouse kidneys were only very weakly bound.

The glycolipid mixtures from the mouse were tested for binding with a human clinical isolate of uropathogenic E. coli (DS17) as shown in Fig. 6 D. Binding was seen with DS17 to all three mouse organs, giving the same pattern as the class II bacteria (Figs. 1 B and 6 B). DS17-8, an isogenic strain with a frameshift mutation in the adhesin gene, did not bind to any glycolipid (not shown).

Binding of E. coli to Proteins of Mouse Kidneys

Proteins extracted from mouse and monkey kidneys were resolved by SDS-PAGE and stained with Coomassie Blue (Fig. 7 A). Specific staining of sugars indicated that all protein bands were glycosylated (not shown). Incubation of the blotting membrane with E. coli pDC1, which carries a class II adhesin (Fig. 7 B), showed that bacterial binding to components in both the monkey and the mouse preparations occurred. However, when the SDS extracts were further treated with butanol to extract remaining lipids, only binding to the mouse kidney proteins was seen.

Molecular Modeling

The glycosidic dihedral angles for the globoside part of GL-III were found to be very similar to earlier published values (46) as were those for the terminal Ledeterminant (47) . In the case of the GlcNAc6(Gal3)GalNAc structural element, energy minima were located by varying the dihedral angles of GlcNAc6GalNAc while in turn keeping constant one of the three staggered conformations of the C5-C6 bond of GalNAc as well as dihedral angles of Gal3GalNAc. The global minimum energy structure was found to be backfolded with the Ledeterminant pointing toward the ceramide. However, this conformer may be excluded on the grounds that the Ledeterminant would be unavailable for antibody binding as well as being too close to the membrane surface in a membrane-bound environment. The next energetically favored conformer (0.8 kcal/mol above the global minimum and 2.6 kcal/mol below subsequent conformers) shows an extended conformation (see Fig. 8 A) consistent with both binding and H NMR data given above.


DISCUSSION

Identification of galabiosylceramide, globotriaosylceramide, and the Forssman glycolipid in mouse kidneys was achieved using monoclonal antibodies in combination with TLC mobility (). The specificity of the antibody against Gal4GalCer (MC2102) and globotriaosylceramide (pk002) has been studied() and the anti-Forssman antibody has a strict specificity (48) . Gal4GalCer has earlier been identified (the anomeric configuration, however, was not determined) in kidneys of some mouse strains (49) . The presence of globotriaosylceramide and globoside was confirmed by FAB-MS analysis of the 3- and 4-sugar region. In the 2-sugar region, lactosylceramide was probably present in addition to the galabiosylceramide. Due to the low abundance of GL-I, no chemical identification was performed. The anti-Leantibody bound to GL-II, GL-III, and GL-IV. Since both MS and NMR data showed that GL-II lacked Fuc, this antibody seems to be able to cross-react with Gal4GlcNAc- when present in Gal4GlcNAc6(Gal3)GalNAc3Gal4Gal4GlcCer but not in nLc. The minor, unidentified, component that was present in this sample could also be responsible for this antibody binding, although being devoid of Fuc.

The MS and NMR data confirm the suggested structure for GL-III, as being Gal4(Fuc3)GlcNAc6(Gal3)GalNAc3Gal4Gal4GlcCer. FAB analysis of native GL-III showed uneven appearance of fragment ions that indicate the presence of two branches. Further evidence for such branching was obtained from EI-MS, where fragment ions from terminal Hex, HexHexNAcFuc, and (Hex)(HexNAc)Fuc were obtained while ions from HexHexNAc and Hex(HexNAc)Fuc or (Hex)HexNAcFuc were absent. The binding position x in -GlcNAcx(Gal3)GalNAc3- was, however, more difficult to identify. But the combined H NMR and molecular modeling results point rather strongly to a 6-linked branch in agreement with the suggestion of Sekine et al. (44) , who based their conclusion on data from partially methylated alditol acetates.

The glycolipid GL-II was suggested by Sekine et al. (44) to be defucosylated GL-III, a suggestion that was confirmed both with FAB-MS analysis and by H NMR in the present study.

Sekine et al. (45, 50) have shown that DBA mice lack a dominant gene that BALB mice carry. This means that DBA mice cannot synthesize GL-III but accumulate Gal3GbCer. Apparently the mice used here, C3H/HeN, resemble BALB mice in their expression of GL-III.

The mixture of acid glycolipids from C3H/HeN mouse kidneys contained two TLC bands that were bound by P-fimbriated E. coli. The observation that anti-Leantibodies did not bind to these compounds excludes the possibility that the separation of the kidney lipids into neutral and acid components was incomplete. Mono- and disialylated Gal3GbCer have been identified both in DBA mouse kidneys (51) and in human vagina (52) . However, Sekine et al. (51) found a correlation between the presence of sialylated Gal3GbCer and absence of Le-elongated Gal3GbCer. Therefore, sialylated Gal3GbCer would not be expected to exist in C3H/HeN mice. In addition, it has been shown that gangliosides that are bound by P-fimbriated E. coli are present in monkey urinary tract tissues.()

In mouse bladder plus urethra and urether the dominating E. coli-binding activity was found in the 4-5-sugar region, suggesting the presence of globoside and the Forssman glycolipid. The presence of the latter was confirmed by antibody binding. These organs contain one late eluting band, each of which bound E. coli carrying the class II adhesin (shown in Fig. 1 ). The mobilities of these two bands coincided with those of GL-II and GL-III in the mouse kidney and also with two acid glycolipids from mouse kidney.

The binding of P-fimbriated E. coli to glycoproteins extracted from mouse kidneys was demonstrated, although it is not clear if this binding is to carbohydrate or peptide (53) . The presence of receptor-active glycoproteins could be of relevance for the use of mice as model animals for urinary tract infection (16, 17, 18) . Such proteins, if secreted into the urine (54) or added in experimental situations (18) , may act as competitive inhibitors for E. coli adherence to epithelial cells of the urinary tract. In this respect, it was shown that Gal4Gal structures were apparently absent from secreted material in human urine (54) , indicating that E. coli may have selected for this specificity to assure adhesion to cell membranes. Based on the results of Fig. 7, it is not unlikely that receptor-active glycoproteins are secreted into mouse urine.


Figure 7: Binding of P-fimbriated E. coli to protein extracts from mouse and monkey kidneys. In A, the SDS-PAGE gel was stained with Coomassie Brilliant Blue, and in B, the autoradiogram after binding of S-labeled bacteria (HB101/pDC1) to an electrophoretic blot on nitrocellulose is shown. 2-4 µg of protein was applied per lane. The numbers to the left denote apparent molecular weights (kDa).



When using animal models for experiments with human pathogens it is important to know if binding-active glycoconjugates are present in the target tissue. Differences in glycoconjugate composition of the same organ from different species (20, 55, 56) and also between strains of the same species are well known (49, 57) . It may be more difficult to demonstrate the presence of receptors on individual target cells with chemical techniques. However, alternative methods exist, using tissue sections and binding with antibodies, bacteria, or lectins (58, 59, 60) .

Differences in glycolipid composition between epithelial and subepithelial cells have been demonstrated in several cases, e.g. for human urethers (61) , human intestine (Ref. 21 and references therein), mouse intestine (62) , and rat intestine (56) . The difference in degree of ceramide hydroxylation as found for mouse kidney glycolipids (higher for lipids with 4 or less sugars and lower for lipids with more than 4 sugars) are analogous to data previously obtained for human kidney (63, 64) . This may mean that the short-chain glycolipids predominate in kidney epithelial membranes rather than in the subepithelial tissue.

E. coli carrying class II adhesin showed a good binding to GL-II, GL-III, and GL-IV (I). The class III adhesin, however, behaved differently. The bacteria with this adhesin bound strongly to the Forssman glycolipid (12) but did not bind to GL-II, GL-III, and GL-IV even though the latter are also elongated globoside structures. A comparison between the minimum energy conformers of GL-III, the Forssman glycolipid, and Globo-A reveals the reasons behind these observations (Fig. 8). Optimal binding of the class III adhesin is to the Forssman glycolipid (Fig. 8 B) involving the terminal trisaccharide GalNAc3GalNAc3Gal4 (12, 65) . The strong binding of the class III adhesin (12) to Globo-A (I) is due to the fact that the terminal blood group A determinant does not occlude the binding epitope. Furthermore, the Fuc2 residue of Globo-A occupies approximately the same position as the GalNAc3, including the acetamido moiety, in the Forssman glycolipid, thus giving rise to only minor steric interferences. For the GL-III glycolipid it is seen that the Gal3 residue is not expected to be the major (66) cause for the loss of adhesin binding since it occupies a position close to that of the GalNAc3 residue in the Forssman glycolipid, a conclusion which is supported by the relatively small reduction in binding affinity seen for Gal3-substituted globoside (12) . The Ledeterminant of GL-III (Fig. 8 A), however, interferes directly with adhesin accessibility due to its attachment to the central residue of the trisaccharide epitope and thus explains the complete loss of binding in this case. The most probable conformation of GL-III, as found here, is also compatible with retained binding of the class II adhesin whose optimal binding epitope includes the Gal4Gal4 segment of globoside with some involvement of the terminal GalNAc3 (12) .


Figure 8: Minimum energy conformers of Gal4(Fuc3)GlcNAc6(Gal3)GalNAc3Gal4Gal4GlcCer ( A), GalNAc3GalNAc3Gal4Gal4GlcCer ( B), and GalNAc3(Fuc2)Gal3GalNAc3Gal4Gal4GlcCer ( C). Both side and top views are shown in which the methyl carbons of Fuc, GalNAc, and GlcNAc are black for orientation. A comparison of the terminal trisaccharide (GalNAc3GalNAc3Gal4) of the optimal receptor for the class III adhesin (the Forssman glycolipid, B), which also constitutes the binding epitope, with the corresponding part of Globo-A ( C) shows that the fucose residue assumes a position very close to that of the terminal GalNAc3 in the Forssman glycolipid, resulting in only a minor steric hindrance, thus explaining the strong binding found for this isoreceptor. In GL-III ( A), however, the 6-linked Ledeterminant interferes with the central part of the binding epitope causing complete loss of class III binding. The class II binding epitope (GalNAc3Gal4Gal4) is not affected, on the other hand, since an extended conformation of the 6-linked branch leaves the epitope essentially unobstructed.



  
Table: Proton-NMR data of GL-II, GL-III, and references

Chemical shifts, in ppm relative to TMS, of anomeric protons in the eight-sugar fraction (GL-III) and the non-fucosylated 7-sugar fraction (GL-II). Spectra were run at 30 °C.


  
Table: Glycosphingolipids in C3H/HeN mouse kidneys, urethers, and bladders plus urethra that are bound by P-fimbriated E. coli of class II

The method of identification of each compound and tissue is indicated with subscripts.


  
Table: Comparison of class I, II, and III adhesins in their relative affinities for Gal4Gal-containing glycosphingolipids on TLC plates

Data are based on the present report and published results (12, 65, 67). -, denotes weak or no binding; +, intermediate binding; and ++, strong binding.



FOOTNOTES

*
This work was supported by grants from Symbicom Ltd. and The Swedish Medical Research Council (No. 3967). Grants for equipment were obtained from The Research Councils, The Knut and Alice Wallenberg Foundation, The Inga-Britt and Arne Lundberg Foundation, and Svenska Handelsbanken. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 46-31-413190; Tel.: 46-31-7733487.

Present address: Pharmacia AB, Hospital Care, Franzéngatan 9, S-112 87 Stockholm, Sweden.

**
Present address: Dept. of Microbiology, University of Umeå, S-901 87 Umeå, Sweden.

The abbreviations and trivial names used are: globoside, Gb, GalNAc3Gal4Gal4GlcCer; Cer, ceramide; d18:1-24:0, ceramide with sphingosine and a saturated fatty acid chain; galabiosylceramide, Gal4GalCer; globotriaosylceramide, Gb, Gal4Gal4GlcCer; Forssman glycolipid, GalNAc3GalNAc3Gal4Gal4GlcCer; P1 antigen, Gal4Gal4GlcNAc3Gal4GlcCer; Globo-A, GalNAc3(Fuc2)Gal3GalNAc3Gal4Gal4GlcCer; gangliotetraosylceramide, GgO, Gal3GalNAc4Gal4GlcCer; lactotetraosylceramide, Lc, Gal3GlcNAc3Gal4GlcCer; lactoneotetraosylceramide, nLc, Gal4GlcNAc3Gal4GlcCer; Le, Gal4(Fuc3)GlcNAc3Gal4GlcCer; Le, Fuc2Gal4(Fuc3)GlcNAc3Gal4GlcCer; Le, Gal3(Fuc4)GlcNAc3Gal4GlcCer; Le, Fuc2Gal3(Fuc4)GlcNAc3Gal4GlcCer; HPLC, high-pressure liquid chromatography; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; MS, mass spectrometry; FAB, fast-atom bombardment; EI, electron impact; GC, gas chromatography; PAGE, polyacrylamide gel electrophoresis.

H. Linder, unpublished data.

K.-A. Karlsson, unpublished data.

B. Lanne, unpublished data.

J. Andziak, R. Möllby, C.-E. Nord, B.-I. Marklund, P. Falk, D. Ilner, S. Teneberg, I. Leonardsson, M. Abul Milh, J. Bergström, K.-A. Karlsson, S. Normark, J. Winberg, unpublished data.


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