Carbohydrate recognition by enterohemorrhagic Escherichia coli: characterization of a novel glycosphingolipid from cat small intestine

Susann Teneberg1,2, Jonas Ångström2 and Åsa Ljungh3

2 Institute of Medical Biochemistry, Göteborg University, P.O. Box 440, SE 405 30 Göteborg, Sweden; and 3 Department of Medical Microbiology, Dermatology and Infection, Lund University, SE 223 62 Lund, Sweden

Received on September 2, 2003; revised on October 1, 2003; accepted on October 2, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
A key virulence trait of pathogenic bacteria is the ability to bind to receptors on mucosal cells. Here the potential glycosphingolipid receptors of enterohemorrhagic Escherichia coli were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two nonacid glycosphingolipids of cat small intestinal epithelium was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, identified as Gal{alpha}3Galß4Glcß1Cer (isoglobotriaosylceramide) and Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer. The latter glycosphingolipid has not been described before. The interaction was not based on terminal Gal{alpha}3 because the bacteria did not recognize the structurally related glycosphingolipids Gal{alpha}3Gal{alpha}4Galß4Glcß1Cer and Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer (B5 glycosphingolipid). However, further binding assays using reference glycosphingolipids showed that the enterohemorrhagic E. coli also bound to lactosylceramide with phytosphingosine and/or hydroxy fatty acids, suggesting that the minimal structural element recognized is a correctly presented lactosyl unit. Further binding of neolactotetraosylceramide, lactotetraosylceramide, the Lea-5 glycosphingolipid, as well as a weak binding to gangliotriaosylceramide and gangliotetraosylceramide, was found in analogy with binding patterns that previously have been described for other bacteria classified as lactosylceramide-binding.

Key words: carbohydrate binding / glycosphingolipids / enterohemorrhagic E. coli / mass spectrometry / microbial adhesion


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Infection with enterohemorrhagic Escherichia coli (EHEC) has been responsible for several large outbreaks of nonhemorrhagic colitis, hemorrhagic colitis, and hemolytic uremic syndrome in recent years (reviewed in Kaper, 1998Go; Nataro and Kaper, 1998Go). One major virulence factor of EHEC is the expression one or more toxins of the Shiga toxin (Stx) family. The Stxs are hetero-oligomeric proteins comprised of a single A subunit and five B subunits (Ling et al., 1998Go). The B subunit pentamer binds to cell surface receptors, and the receptors for most Stxs are glycosphingolipids with a terminal Gal{alpha}4Gal moiety, that is, galabiosylceramide (Gal{alpha}4Galß1Cer), globotriaosylceramide (Gal{alpha}4Galß4Glcß1Cer), and the P1 pentaglycosylceramide (Gal{alpha}4Galß4GlcNAcß3Galß4Glcß1Cer) (Lindberg et al., 1987Go; Ling et al., 1998Go; Lingwood et al., 1987Go) (see Table I for glycosphingolipid nomenclature).


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Table I. Binding of 35S-labeled enterohemorrhagic E. coli strain Bi 135 A to glycosphingolipids on thin-layer chromatograms

 
Although a substantial amount of information about the receptors for Stxs are available, less is known about the receptor(s) for EHEC bacterial cells. Binding to specific receptors on the target cells allows microorganisms to colonize and cause infection and leads to an efficient delivery of bacterial toxins (Beachey, 1981Go). In the present study the potential carbohydrate recognition by EHEC bacterial cells was investigated by binding of EHEC bacteria to glycosphingolipids from various sources on thin-layer chromatograms. A binding-active glycosphingolipid not previously described was found in the nonacid glycosphingolipid fraction of cat intestinal epithelium. This EHEC-binding compound was isolated and characterized by mass spectrometry (MS), degradation studies, and protonnuclear magnetic resonance (NMR) as Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer. In addition, as previously described for several other bacteria (Karlsson, 1989Go), EHEC interacted with lactosylceramide with phytosphingosine and/or hydroxy fatty acids, neolactotetraosylceramide, lactotetraosylceramide, the Lea-5 glycosphingolipid, and weakly with gangliotriaosylceramide and gangliotetraosylceramide.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Detection and isolation of EHEC-binding glycosphingolipids from cat intestinal epithelium
During the initial binding studies, mixtures of glycosphingolipids isolated from various species and organs were used to expose the bacteria to a large number of potentially binding-active carbohydrate structures. Thereby a selective binding of EHEC to compounds migrating in the tri- and tetraglycosylceramide region in the nonacid glycosphingolipid fraction from the epithelial cells of cat small intestine was detected (Figure 1, lane 7).



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Fig. 1. Detection of a enterohemorrhagic E. coli–binding compound in the nonacid glycosphingolipid fraction of cat small intestinal epithelium. The glycosphingolipids were chromatographed on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8 v/v/v) as solvent system, and visualized with anisaldehyde (A). Duplicate chromatograms were incubated with 35S-labeled enterohemorrhagic E. coli strain Bi 135A (B), followed by autoradiography for 12 h, as described under Materials and methods. Lane 1, total nonacid glycosphingolipids of human blood group A erythrocytes, 40 µg; lane 2, total nonacid glycosphingolipids of bovine intestine, 40 µg; lane 3, total acid glycosphingolipids of bovine intestine, 40 µg; lane 4, total nonacid glycosphingolipids of sheep intestine, 40 µg; lane 5, total acid glycosphingolipids of sheep intestine, 40 µg; lane 6, total acid glycosphingolipids of cat intestine, 40 µg; lane 7, total nonacid glycosphingolipids of cat intestine, 40 µg; lane 8, total acid glycosphingolipids of horse intestine, 40 µg; lane 9, total acid glycosphingolipids of human granulocytes, 40 µg.

 
To isolate the EHEC-binding glycosphingolipids, the nonacid fraction of cat small intestinal epithelium was first separated by silicic acid chromatography, followed by high-performance liquid chromatography. The glycosphingolipid-containing fractions were tested for binding of EHEC using the chromatogram binding assay, and binding of P-fimbriated E. coli was tested in parallel to detect Gal{alpha}4Gal-containing compounds. The fractions were pooled according to the mobility of the compounds on thin-layer chromatograms and their binding activities. Thereby, 1.8 mg EHEC-binding triglycosylceramides (Fraction C1; Figure 2, lane 2) and 10.2 mg tetraglycosylceramides were obtained. However, initial analysis with negative ion fast atom bombardement (FAB) MS showed that the tetraglycosylceramide fraction contained two compounds (data not shown). This fraction was therefore acetylated and further separated by Iatrobeads column chromatography. An early-eluting compound was recognized by EHEC but not by P-fimbriated E. coli, and after pooling 1.5 mg of this compound was obtained (fraction C2; Figure 2, lane 3), whereas subsequent tubes contained a mixture of the compounds recognized by EHEC and P-fimbriated E. coli. Pooling the contents of these tubes yielded 2.4 mg (fraction C3; Figure 2, lane 4).



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Fig. 2. Comparison of binding of enterohemorrhagic E. coli and P-fimbriated E. coli to glycosphingolipids of cat small intestinal epithelium on thin-layer chromatograms. The glycosphingolipids were chromatographed on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8 v/v/v) as solvent system, and visualized with anisaldehyde (A). Duplicate chromatograms were incubated with radiolabeled enterohemorrhagic E. coli strain Bi 135A (B) and P-fimbriated E. coli strain HB101/pPIL291-15 (C), followed by autoradiography for 12 h, as described under Materials and methods. Lane 1, total nonacid glycosphingolipids of cat small intestinal epithelium, 40 µg; lane 2, fraction C1 isolated from cat small intestinal epithelium, 4 µg; lane 3, fraction C2 isolated from cat small intestinal epithelium, 4 µg; lane 4, fraction C3 isolated from cat small intestinal epithelium, 4 µg; lane 5, globotetraosylceramide (GalNAcß3Gal{alpha}4Galß4Glcß1Cer) of human erythrocytes, 4 µg; lane 6, P1 glycosphingolipid (Gal{alpha}4Galß4GlcNAcß3 Galß4Glcß1Cer) of human erythrocytes, 4 µg; lane 7, B5 glycosphingolipid (Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer) of rabbit erythrocytes, 4 µg.

 
The EHEC-binding tri- and tetraglycosylceramides (fractions C1 and C2) were characterized by MS, proton NMR spectroscopy, gas chromatography–electron ionization (EI) MS after degradation, as follows.

EI MS of the EHEC-binding triglycosylceramide from cat intestinal epithelium (fraction C1)
The isolated triglycosylceramide (fraction C1) was permethylated, permethylated and LiAlH4-reduced, and analyzed with EI MS. Figure 3 shows the mass spectrum of the permethylated and reduced derivative, with a simplified formula for interpretation, representing the species with phytosphingosine and hydroxy 24:0 fatty acid.



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Fig. 3. EI mass spectrum of the permethylated and LiAlH4-reduced binding-active triglycosylceramide isolated from the epithelial cells of cat small intestine. Above the spectrum is a simplified interpretation formula representing the species with phytosphingosine and hydroxy 24:0 fatty acid. The analytical conditions were: electron energy 70 eV, trap current 300 µA, and acceleration voltage 10 kV. The temperature was raised from 150°C to 410°C, by increases of 10°C/min. The spectrum was recorded at 250°C.

 
A series of intense immonium ions were found at m/z 954–1066 in the spectrum of the permethylated and reduced glycosphingolipid. These ions contain the complete carbohydrate chain together with the fatty acid, and in the present case they gave evidence of a saccharide part composed of three hexoses, in combination with hydroxy 16:0–24:0 fatty acids. In the spectrum of the permethylated sample (not shown), immonium ions corresponding to a trihexosylceramide with hydroxy 22:0 and hydroxy 24:0 fatty acids were found at m/z 1052 and 1080, respectively.

The spectra of both derivatives had carbohydrate sequence ions at m/z 219 and 187 (terminal hexose) and m/z 423 (terminal hexose-hexose). The rearrangement fragment at m/z 700, seen in both spectra, containing the saccharide chain plus part of the fatty acid, confirmed a hexose-hexose-hexose sequence.

The spectrum of the permethylated and reduced glycosphingolipid (Figure 3) had molecular ions of trihexosylceramide with phytosphingosine and hydroxy 22:0 and 24:0 fatty acids at m/z 1323 and 1351, respectively.

The rearrangement ions at m/z 616 and 644 and at m/z 820 and 848 present in the spectrum of the permethylated and reduced derivative (explained below the formula in Figure 3) gave further evidence for the deduced carbohydrate sequence and fatty acid composition.

The conclusion from MS thus was a triglycosylceramide with a hexose-hexose-hexose sequence having phytosphingosine as long-chain base and mainly hydroxy 22:0 and 24:0 fatty acids.

EI MS of the EHEC-binding tetraglycosylceramide from cat intestinal epithelium (fraction C2)
EI mass spectra of the permethylated and the permethylated and LiAlH4-reduced derivatives of the isolated tetraglycosylceramide (fraction C2) are reproduced in Figure 4. Above the spectra are simplified formulae for interpretation, representing the species with phytosphingosine and hydroxy 24:0 fatty acid.




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Fig. 4. EI mass spectra of the permethylated (A) and permethylated and LiAlH4-reduced (B) binding-active tetraglycosylceramide isolated from the epithelial cells of cat small intestine. Above the spectra are simplified interpretation formulae representing the species with phytosphingosine and hydroxy 24:0 fatty acid. The analytical conditions were: electron energy 70 eV, trap current 300 µA, and acceleration voltage 10 kV. The temperature was raised from 150°C to 410°C, by increases of 10°C/min. The spectrum in (A) was recorded at 290°C, and the spectrum in (B) at 310°C.

 
Carbohydrate sequence ions were found in the mass spectrum of the permethylated derivative (Figure 4A) at m/z 219 and 187 (terminal hexose), m/z 423 (terminal hexose-hexose), m/z 627 (terminal hexose-hexose-hexose), and m/z 831 (terminal hexose-hexose-hexose-hexose). The ions at m/z 219 and 187 and at m/z 423 were also present in the spectrum of the permethylated and reduced deivative (Figure 4B). The suggested hexose-hexose-hexose-hexose sequence was confirmed by the rearrangement fragment at m/z 904, present in both spectra. In Figure 4B a series of intense immonium ions at m/z 1158 to 1270, corresponding to the complete saccharide chain and the fatty acid, were found, demonstrating four hexoses and a series of hydroxy 16:0–24:0 fatty acids. The corresponding immonium ions were found at m/z 1172–1284 in the spectrum of the permethylated glycosphingolipid, along with ions at m/z 1300 and 1328, indicating phytosphingosine in combination with hydroxy 22:0 (1541 minus 241) and hydroxy 24:0 (1569 minus 241) fatty acids. The ions in the molecular region at m/z 1541 and 1569 (Figure 4A), and m/z 1527 and 1555 (Figure 4B), indicated the same carbohydrate composition and hydroxy 22:0 and 24:0 fatty acids, together with phytosphingosine long chain base.

The rearrangement ions at m/z 616 and 644, m/z 820 and 848, and m/z 1024 and 1052, produced by the permethylated and reduced derivative (explained below the formula in Figure 4B) further corroborated the proposed carbohydrate sequence and fatty acid composition.

The conclusion from MS of the EHEC-binding tetraglycosylceramide from cat intestine was thus a tetrahexosylceramide with phytosphingosine as long chain base and mainly hydroxy 22:0 and 24:0 fatty acids.

Degradation studies
The binding positions between the carbohydrate residues were obtained by degradation of the permethylated tri- and tetraglycosylceramide, that is, the samples were subjected to acid hydrolysis, followed by reduction and acetylation. The resulting partially methylated alditol acetates were analyzed by gas chromatography–EI MS, and the components were identified by comparison of retention times and mass spectra of partially methylated alditol acetates obtained from reference glycosphingolipids. The reconstructed ion chromatograms obtained from the triglycosylceramide (fraction C1; Figure 5A) and the tetraglycosylceramide (fraction C2; Figure 5B) were very similar, and both had three carbohydrate peaks. In both cases the acetate of 2,3,4,6-tetramethyl-galactitol identified a terminal galactose, and the presence of the acetates of 2,4,6-trimethyl-galactitol and 2,3,6-trimethyl-glucitol identified 3-substituted galactose and 4-substituted glucose, respectively.



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Fig. 5. Reconstructed ion chromatograms obtained after degradation of the permethylated triglycosylceramide (A) and tetraglycosylceramide (B) of cat small intestinal epithelium. For analytical conditions, see Materials and methods.

 
In combination with the data from MS, this suggested that the triglycosylceramide had a carbohydrate chain with the sequence Gal1-3Gal1-4Glc1 and the tetraglycosylceramide had a Gal1-3Gal1-3Gal1-4Glc1 sequence.

Proton NMR spectroscopy
Both the tri- and tetraglycosylceramide structures were analyzed by 1H NMR spectroscopy at 500 MHz and 30°C. Starting with the smaller compound (data not shown), the spectrum revealed three H1 anomeric signals at 4.84 ppm ({alpha}), 4.29 ppm (ß), and 4.21 ppm (ß), thus identifying this structure as Gal{alpha}3Galß4Glcß1Cer (isoglobotriaosylceramide) through comparison with earlier published spectra of compounds having a terminal Gal{alpha}3 (Dabrowski et al., 1988aGo,bGo). The identity of the Gal{alpha}3 H1 signal was further corroborated in the double quantum-filtered correlated spectroscopy (DQF-COSY) spectrum by its connectivity to the H2 resonance at 3.60 ppm and the connectivities between the H5 resonance (3.99 ppm) and the H6,6' resonances (3.43 ppm and 3.52 ppm, respectively) (Dabrowski et al., 1988aGo). These values differ from those of a terminal Gal{alpha}4, which reveals H1, H2, and H5 signals at 4.81 ppm, 3.65 ppm, and 4.05 ppm, respectively (Dabrowski et al., 1988bGo).

The four anomeric signals of the tetraglycosylceramide structure were located at 4.89 ppm ({alpha}), 4.83 ppm ({alpha}), 4.31 ppm (ß), and 4.21 ppm (ß), of which the latter two correspond to Galß4 and Glcß1 as previously (Figure 6). Regarding the two former signals, the one at 4.83 ppm is indicative of a terminal Gal{alpha}3 as also found above (the H2 signal is located at 3.61 ppm), whereas the signal at 4.89 ppm can be indentified as an internal Gal{alpha}3 (the H2 signal is now found at 3.71 ppm) from the COSY spectrum (not shown) and through comparison with earlier data on related compounds such as isoglobotetraosylceramide (GalNAcß3Gal{alpha}3Galß4Glcß1Cer) (Hansson et al., 1987Go). An internal Gal{alpha}4 as in, for example, globoside can be excluded because the H1 and H2 resonances in this case are found at 4.82 ppm and 3.82 ppm, respectively (Scarsdale et al., 1986Go). Considered together, these data (including the degradation studies) strongly suggest that the tetraglycosylceramide structure is Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer.



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Fig. 6. Anomeric region of the 500 MHz 1H NMR spectrum (30°C) of fraction C2 containing the EHEC-binding-active tetraglycosylceramide (Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer) from cat small intestinal epithelium. For analytical conditions, see Materials and methods.

 
From all the data combined, the structures of the EHEC-binding glycosphingolipids from cat small intestinal epithelium were established as Gal{alpha}3Galß4Glcß1Cer (isoglobotriaosylceramide) and Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer. The latter glycosphingolipid has not been described previously.

Comparison of binding preferences
Binding to Gal{alpha}3Galß4Glcß1Cer and Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer was obtained with all 10 EHEC strains (representing 5 different serotypes) listed in Table II.


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Table II. Characteristics and serotypes of the enterohemorrhagic E. coli strains used in this study

 
The EHEC binding activity of glycosphingolipids structurally related to Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer was thereafter examined (exemplified in Figures 7 and 8, and summarized in Table I). No binding of EHEC to structurally related compounds as the tetrahexosylceramide of rat intestine (Gal{alpha}3Gal{alpha}4Galß4Glcß1Cer (Figure 7, lane 4), the B5 glycosphingolipid (Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer; Figure 2, lane 7, and Figure 7, lane 5), or isoglobotetraosylceramide (GalNAcß3Gal{alpha}3Galß4Glcß 1Cer, not shown), was obtained.



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Fig. 7. Binding of 35S-labeled enterohemorrhagic E. coli to reference glycosphingolipids on thin-layer chromatograms. The glycosphingolipids were chromatographed on aluminum-backed silica gel plates and visualized with anisaldehyde (A). Duplicate chromatograms were incubated with radiolabeled enterohemorrhagic E. coli strain Bi 135A (B), followed by autoradiography for 12 h, as described under Materials and methods. The solvent system used was chloroform/methanol/water (60:35:8 v/v/v). Lane 1, fraction C1 isolated from cat small intestinal epithelium, 4 µg; lane 2, fraction C2 isolated from cat small intestinal epithelium, 4 µg; lane 3, fraction C3 isolated from cat small intestinal epithelium, 4 µg; lane 4, tetrahexosylceramide (Gal{alpha}3Gal{alpha}4Galß4Glcß1Cer) of rat small intestine, 4 µg; lane 5, B5 glycosphingolipid (Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer) of rabbit erythrocytes, 4 µg.

 


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Fig. 8. Binding of 35S-labeled enterohemorrhagic E. coli to reference glycosphingolipids on thin-layer chromatograms. The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60:35:8 v/v/v) as solvent system. The chromatogram in (A) was visualized with anisaldehyde. Duplicate chromatograms were incubated with radiolabeled enterohemorrhagic E. coli strain Bi 135A (B), followed by autoradiography for 12 h, as described under Materials and methods. Lane 1, lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy 16:0–24:0 fatty acids of rabbit intestine, 4 µg; lane 2, neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer) of human granulocytes, 4 µg; lane 3, lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer) of human meconium, 4 µg; lane 4, Lea-5 pentaglycosylceramide (Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer) of human intestine, 4 µg; lane 5, Leb-6 hexaglycosylceramide (Fuc{alpha}2Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer) of human meconium, 4 µg; lane 6, B7 type 1 heptaglycosylceramide (Gal{alpha}3(Fuc{alpha}2)Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer) of monkey intestine, 4 µg; lane 7, A6 type 2 hexaglycosylceramide (GalNAc{alpha}3(Fuc{alpha}2)Galß4GlcNAcß3Galß4Glcß1Cer) of human erythrocytes, 4 µg.

 
However, further testing of EHEC binding with pure reference glycosphingolipids revealed binding patterns very similar to the patterns obtained with bacteria classified as lactosylceramide-binding (Ångström et al., 1998Go; Hugosson et al., 1998Go; Karlsson, 1989Go; Strömberg and Karlsson, 1990aGo,bGo). Thus the bacteria selectively interacted with lactosylceramide with phytosphingosine and/or hydroxy fatty acid (no. 6 in Table I; Figure 8, lane 1), neolactotetraosylceramide (no. 12; Figure 8, lane 2), lactotetraosylceramide (no. 21; Figure 8, lane 3), the Lea-5 glycosphingolipid (no. 22; Figure 8, lane 4), gangliotriaosylceramide (no. 10), and gangliotetraosylceramide (no. 11). Similar binding patterns have thus been reported for a number of other bacteria, for example, Propionibacterium granulosum (Strömberg and Karlsson, 1990aGo) and Helicobacter pylori (Ångström et al., 1998Go). It should be noted, however, that although the binding of EHEC to lactosylceramide, neolactotetraosylceramide, lactotetraosylceramide, and the Lea-5 glycosphingolipid was highly reproducible, binding to gangliotriaosylceramide and gangliotetraosylceramide was only occasionally obtained.

All other glycosphingolipids tested, including those having a Gal{alpha}4Gal sequence (nos. 8, 14, 24, 26, 27 in Table I), were nonbinding in the chromatogram binding assay.

Binding to reference glycosphingolipids was tested with the EHEC strains Bi 135A, Bi 110, RL 66, H10, H13, and Bi 44/91. All five strains displayed the same glycosphingolipid binding pattern.


    Discussion
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 Abstract
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 Discussion
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 References
 
Intimate attachment to the host cells, followed by the attaching and effacing (A/E) lesions on the surfaces of intestinal epithelial cells, is a characteristic feature of both EHEC infection and infection with enteropathogenic E. coli (EPEC). The A/E lesion formation involves degeneration of intestinal epithelial microvilli and formation of actin-rich pedestals in the cell beneath the adherent bacteria. A number of recent studies have shown that these effects are mediated primarily by the bacterial outer membrane protein intimin and a second bacterial protein Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell membrane. Subsequent binding of intimin to Tir triggers a signal transduction cascade followed by cytoskeletal rearrangements (reviewed in Campellone and Leong, 2003Go).

Based on homologies in the C-terminal parts of the proteins, five antigenically distinct subtypes of intimin (designated {alpha}, ß, {gamma}, {sigma}, and {varepsilon}) have been identified (Abu-Bobie et al., 1998Go; Oswald et al., 2000Go). Intimin-{alpha} and intimin-{sigma} are found in human EPEC strains, intimin-ß and intimin-{gamma} are produced both by EPEC and EHEC strains, and intimin-{varepsilon} was found in EHEC strains.

Although both EPEC and EHEC are capable of forming A/E lesions, they have different target tissues. EPEC infects the small intestine, but the target tissue of EHEC is the large intestine (Levine, 1987Go). Thus the presence of a host-encoded intimin receptor has been suggested (reviewed in Frankel et al., 2001Go). A role for carbohydrate recognition in intimin-mediated adhesion of bacterial cells has also been suggested by the identification of a C-type lectin-like moiety at the carboxy-terminus of the intimin polypeptide by an NMR study of the C-terminal 280 amino acids of EPEC intimin-{alpha} (Kelly et al., 1999Go).

In the present study, the potential carbohydrate recognition of EHEC bacteria was investigated by binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a specific recognition of lactosylceramide phytosphingosine and/or hydroxy fatty acids, neolactotetraosylceramide, lactotetraosylceramide, the Lea-5 glycosphingolipid, gangliotriaosylceramide, and gangliotetraosylceramide was demonstrated. Similar binding specificities have been previously been described for other bacteria with different target tissues (Ångström et al., 1998Go; Hugosson et al., 1998Go; Karlsson, 1989Go; Strömberg and Karlsson, 1990aGo,bGo).

In addition, a specific interaction of EHEC to two nonacid glycosphingolipids of cat small intestinal epithelium was detected during the course of the study. The binding-active glycosphingolipids were isolated, and on the basis of MS, gas chromatography–MS after degradation, and 1H NMR characterized as Gal{alpha}3Galß4Glcß1Cer (isoglobotriaosylceramide) and Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer. The latter glycosphingolipid is a novel structure.

The absence of interaction of EHEC with the rat intestinal tetrahexosylceramide (Gal{alpha}3Gal{alpha}4Galß4Glcß1Cer) and the B5 glycosphingolipid (Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer) shows that a terminal 3-linked Gal{alpha} is not sufficient for the binding process to occur. More likely the basic element recognized in the novel glycosphingolipid is the Gal{alpha}3Galß4Glcß sequence of isoglobotriaosylceramide or even the Galß4Glcß sequence of lactosylceramide. The terminal 3-linked Gal{alpha} of Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer is thus a tolerated substitution, whereas the terminal GalNAcß3 of isoglobotetraosylceramide (GalNAcß3Gal{alpha}3Galß4Glcß1Cer) blocks the binding. However, the latter glycosphingolipid may also be nonbinding due to the absence of the correct ceramide species.

The binding of EHEC and many other bacteria to lactosylceramide is ceramide-dependent, that is, the presence of phytosphingosine and/or hydroxy fatty acids is necessary for binding to occur. Selective binding to liposomes with lactosylceramide having phytosphingosine and hydroxy fatty acids has also been demonstrated (Ångström et al., 1998Go), suggesting that this selectivity may be present also in vivo. Previous molecular modeling studies have indicated that the selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the fatty acid hydroxyl group forms a hydrogen bond with the hydroxy methyl group of the glucose (Ångström et al., 1998Go).

Inspection of the binding-active and nonbinding glycosphingolipids summarized in Table I shows that not all glycosphingolipids with phytosphingosine and hydroxy fatty acids are recognized by EHEC bacteria. In these cases (nos. 16, 22, 23, and 27 in Table I) the extensions of the carbohydrate chains most likely limit the access to the lactosyl element.

Furthermore the requirement of phytosphingosine and/or hydroxy fatty acids is restricted to lactosylceramide/ isoglobotriaosylceramide, and vanishes upon is elongation into ganglio and neolacto series compounds (nos. 10–12). For other lactosylceramide-binding bacteria, such as H. pylori, Neisseria meningitidis, and Haemophilus influenzae, the binding to gangliotria- and gangliotetraosylceramide is abolished after conversion of the acetamido group of GalNAc to an amine (Ångström et al., 1998Go; Hugosson et al., 1998Go), suggesting that the binding to these two glycosphingolipids is a specificity separate from lactosylceramide recognition. Our current hypothesis is that the binding to lactosylceramide/isoglobotriaosylceramide, gangliotria-/gangliotetraosylceramide, and N-acetyllactosamine-terminated glycosphingolipids correspond to three different adhesins that are frequently coexpressed by the lactosylceramide-binding bacteria.

The relevance of the Gal{alpha}3Galß4Glcß1Cer/Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer binding capacity for adhesion of EHEC to host cells and colonization has yet to be determined. Isoglobotriasylceramide is present in, for instance, dog intestine (Hansson et al., 1983Go), but has hitherto not been found in human tissues. EHEC are strict large intestinal pathogens (Levine, 1987Go). In the epithelial cells of human large intestine the Lea-5 glycosphingolipid is a major compound, and there are also trace amounts of lactosylceramide with phytosphingosine and/or hydroxy fatty acids (Holgersson et al., 1988Go). These may thus function as targets for EHEC adherence.

Nucleolin was recently identified as the receptor for intimin-{gamma} on Hep-2 cells (Sinclair and O'Brien, 2002Go). Obviously it would be of interest to examine the carbohydrate binding of purified intimin, both from EHEC and EPEC, to determine if there is a correlation with the carbohyrate binding specificities found in this study or if the carbohydrate binding is caused by other bacterial adhesins.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Bacterial strains, culture conditions, and labeling
The serotypes and characteristics of the EHEC isolates used are summarized in Table II. The strains were isolated from fecal specimens of patients with hemorrhagic colitis. RL 66 was from Robert Koch Institut, Berlin; B1409C and C984 from Center for Vaccine Development, Baltimore, MD; and the other strains from Clinical Microbiology and Immunology, Lund University Hospital, Lund, Sweden. Most of the binding experiments were done with the strain Bi 135A S; two or three of the other strains were used in parallel.

The Gal{alpha}4Gal-binding recombinant E. coli strain HB101/pPIL291–15, carrying a plasmid-born pap gene cluster with a class II papG allele, was a kind gift from Dr. Benita Westerlund (University of Helsinki, Finland).

All E. coli strains were cultured on Luria agar with the addition of 10 µl 35S-methionine (400 µCi; Amersham Pharmacia Biotech, Little Chalfont, UK) at 37°C for 12 h. The bacteria were harvested by scraping, washed three times with phosphate-buffered saline (PBS), pH 7.3, and thereafter resuspended in PBS containing 1% mannose (w/v) to 1 x 108 CFU/ml. The specific activities of the suspensions were approximately 1 cpm per 100 bacteria.

Reference glycosphingolipids
Total acid and nonacid glycosphingolipid fractions were obtained by standard procedures (Karlsson, 1987Go). The individual glycosphingolipids were isolated by repeated chromatography on silicic acid columns of the native glycosphingolipid fractions or acetylated derivatives thereof. The identity of the purified glycosphingolipids was confirmed by MS (Samuelsson et al., 1990Go), proton NMR spectroscopy (Koerner et al., 1983Go), and degradation studies (Stellner et al., 1973Go; Yang and Hakomori, 1971Go).

Thin-layer chromatography
Thin-layer chromatography of glycosphingolipids was performed on glass- or aluminum-backed silica gel 60 high-performance thin-layer chromatography plates (Merck, Darmstadt, Germany), using chloroform/methanol/water (60:35:8 v/v/v) as solvent system. Chemical detection was done with anisaldehyde (Waldi, 1962Go).

Chromatogram binding assays
Binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms was done as reported (Hansson et al., 1985Go). Dried chromatograms were dipped for 1 min in diethylether/n-hexane (1:5 v/v) containing 0.5% (w/v) polyisobutylmethacrylate (Aldrich, Milwaukee, WI). After drying, the chromatograms were soaked in PBS containing 2% bovine serum albumin (w/v), 0.1% NaN3 (w/v), and 0.1% Tween 20 (v/v) for 2 h at room temperature. The chromatograms were subsequentely covered with radiolabeled bacteria diluted in PBS (2–5 x 106 cpm/ml). Incubation was done for 2 h at room temperature, followed by repeated washings with PBS. The chromatograms were thereafter exposed to XAR-5 X-ray films (Eastman Kodak, Rochester, NY) for 12 h.

Isolation of EHEC-binding nonacid glycosphingolipids from epithelial cells of cat small intestine
A total nonacid glycosphingolipid fraction (64 mg) was obtained from pooled epithelial cell scrapings from the small intestines of 12 cats by standard methods (Karlsson, 1987Go). Briefly, the tissue was lyophilized, followed by extraction in two steps with chloroform-methanol (2:1 and 9:1, v/v) in a Soxhlet apparatus. The material obtained was pooled and subjected to mild alkaline hydrolysis and dialysis, followed by separation on a silicic acid column. Acid and nonacid glycosphingolipids were separated on a DEAE column.

The isolated nonacid glycosphingolipid fraction (50 mg) was first separated on a silicic acid column stepwise eluted with increasing amounts of methanol in chloroform. The fractions containing triglycosylceramides and larger glycosphingolipids were pooled, giving 45 mg, which were further separated by high-performance liquid chromatography on a Kromasil 5 Silica column (2.12 x 25 cm inner diameter, particle size 5 µm; Skandinaviska Genetec, Kungsbacka, Sweden) eluted with a linear gradient of chloroform/methanol/water (80:20:1 to 40:40:12 v/v/v) over 180 min, with a flow rate of 4 ml/min. Each 4-ml fraction was analyzed by thin-layer chromatography using anisaldehyde for detection.

The glycosphingolipid-containing fractions were tested for binding of EHEC and P-fimbriated E. coli using the chromatogram binding assay. The EHEC-binding compound migrating in triglycosylceramide region eluted in tubes 78–88, whereas tetraglycosylceramides were collected in tubes 89–106. After pooling, 1.8 mg of triglycosylceramides (designated fraction C1) and 10.2 mg of tetraglycosylceramides were obtained. The tetraglycosylceramide fraction was acetylated (Handa, 1963Go), and further separated on a 10-g Iatrobeads column (Iatron Laboratories, Tokyo) eluted with a linear gradient of methanol in chloroform (0–5%, v/v). Fractions of 1 ml were collected and, after deacetylation, analyzed by thin-layer chromatography and tested for binding activities. The compound collected in tubes 120–140 was recognized by EHEC but not by P-fimbriated E. coli, and after pooling, 1.5 mg of this compound was obtained (designated fraction C2). Tubes 141–195 contained a mixture of compounds recognized by EHEC and P-fimbriated E. coli. Pooling of these tubes yielded 2.4 mg (designated fraction C3).

MS
Before EI MS, aliquots of the isolated glycosphingolipids were permethylated (Larson et al., 1987Go) or permethylated and reduced with LiAlH4 (Karlsson, 1974Go). The derivatized samples were analyzed on a JEOL SX-102A mass spectrometer (JEOL, Tokyo), using the in-beam technique (Breimer et al., 1980Go). The analyses of both derivatives were performed with an electron energy of 70 eV, trap current of 300 µA, and acceleration voltage of 10 kV. The temperature was raised from 150°C to 410°C, by increases of 10°C/min.

Degradation studies
The permethylated glycosphingolipids from cat small intestinal epithelium were hydrolyzed, reduced, and acetylated (Stellner et al., 1973Go; Yang and Hakomori, 1971Go), and the partially methylated alditol acetates obtained were analyzed by capillary gas chromatography on a Hewlett-Packard 5890A gas chromatograph using a BPX-35 0.25 µm column (30 m x 0.22 mm ID) (SGE, Perth, Australia), with split injection and with helium as carrier gas. The samples were dissolved in ethylacetate. The temperature was raised from 150°C to 280°C by increases of 10°C/min.

Gas chromatography–EI MS was performed on a Hewlett-Packard 5890-II gas chromatograph coupled to a JEOL SX-102A mass spectrometer. The chromatographic conditions, as well as the capillary column, were the same as for the analyses by gas chromatography, and the conditions for MS were: interface temperature 260°C, ion source temperature 200°C, electron energy 70 eV, trap current 300 µA, acceleration voltage 10 kV, mass range scanned 50–500, total cycle time 1.5 s, resolution 1200 (10% valley definition), and pressure in the ion source region 10–5 Pa.

Proton NMR spectroscopy
1H NMR spectra were acquired on a Varian 500 MHz spectrometer at 30°C. Samples were dissolved in dimethyl sulfoxide/D2O (98/2) after deuterium exchange. Two-dimensional DQF-COSY spectra were recorded by the standard pulse sequence (Marion and Wüthrich, 1983Go) using 32 scans per t1 increment in 2K * 256 point matrices.

The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for lipids: Eur. J. Biochem. [1977] 79, 11–21, J. Biol. Chem. [1982] 257, 3347–3351, and J. Biol. Chem. [1987] 262, 13–18). It is assumed that Gal, Glc, GlcNAc, GalNAc, and NeuAc are of the D-configuration, Fuc of the L-configuration, and all sugars present in the pyranose form.


    Acknowledgements
 
This study was supported by the Swedish Medical Research Council (Grant No. 12628), the Swedish Cancer Foundation, the Wallenberg Foundation and Lund University Hospital, Lund, Sweden. Gal{alpha}3Gal{alpha}4Galß4Glcß1Cer from rat small intestine was a kind gift from Dr. G. C. Hansson at the Institute of Medical Biochemistry, Göteborg University. The use of the Varian 500 MHz machine at the Swedish NMR Centre, Hasselblad Laboratory, Göteborg University, is gratefully acknowledged.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: susann.teneberg{at}medkem.gu.se Back


    Abbreviations
 
A/E, attaching and effacing; DQF-COSY, double quantum filtered correlation spectroscopy; EHEC, enterohemorrhagic Escherichia coli; EI, electron ionization; EPEC, enteropathogenic Escherichia coli; FAB fast atom bombardment; MS, mass spectrometry; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; Stx, Shiga toxin


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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