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• W1971390030 abstract "F18-fimbriated Escherichia coli are associated with porcine postweaning diarrhea and edema disease. Adhesion of F18-fimbriated bacteria to the small intestine of susceptible pigs is mediated by the minor fimbrial subunit FedF. However, the target cell receptor for FedF has remained unidentified. Here we report that F18-fimbriated E. coli selectively interact with glycosphingolipids having blood group ABH determinants on type 1 core, and blood group A type 4 heptaglycosylceramide. The minimal binding epitope was identified as the blood group H type 1 determinant (Fucα2Galβ3GlcNAc), while an optimal binding epitope was created by addition of the terminal α3-linked galactose or N-acetylgalactosamine of the blood group B type 1 determinant (Galα3(Fucα2)Galβ3GlcNAc) and the blood group A type 1 determinant (GalNAcα3(Fucα2)-Galβ3GlcNAc). To assess the role of glycosphingolipid recognition by F18-fimbriated E. coli in target tissue adherence, F18-binding glycosphingolipids were isolated from the small intestinal epithelium of blood group O and A pigs and characterized by mass spectrometry and proton NMR. The only glycosphingolipid with F18-binding activity of the blood group O pig was an H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAc-β3Galβ4Glcβ1Cer). In contrast, the blood group A pig had a number of F18-binding glycosphingolipids, characterized as A type 1 hexaglycosylceramide (GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer), A type 4 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), A type 1 octaglycosylceramide (GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ3GlcNAcβ3Galβ4Glcβ1Cer), and repetitive A type 1 nonaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcα3-(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). No blood group antigen-carrying glycosphingolipids were recognized by a mutant E. coli strain with deletion of the FedF adhesin, demonstrating that FedF is the structural element mediating binding of F18-fimbriated bacteria to blood group ABH determinants. F18-fimbriated Escherichia coli are associated with porcine postweaning diarrhea and edema disease. Adhesion of F18-fimbriated bacteria to the small intestine of susceptible pigs is mediated by the minor fimbrial subunit FedF. However, the target cell receptor for FedF has remained unidentified. Here we report that F18-fimbriated E. coli selectively interact with glycosphingolipids having blood group ABH determinants on type 1 core, and blood group A type 4 heptaglycosylceramide. The minimal binding epitope was identified as the blood group H type 1 determinant (Fucα2Galβ3GlcNAc), while an optimal binding epitope was created by addition of the terminal α3-linked galactose or N-acetylgalactosamine of the blood group B type 1 determinant (Galα3(Fucα2)Galβ3GlcNAc) and the blood group A type 1 determinant (GalNAcα3(Fucα2)-Galβ3GlcNAc). To assess the role of glycosphingolipid recognition by F18-fimbriated E. coli in target tissue adherence, F18-binding glycosphingolipids were isolated from the small intestinal epithelium of blood group O and A pigs and characterized by mass spectrometry and proton NMR. The only glycosphingolipid with F18-binding activity of the blood group O pig was an H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAc-β3Galβ4Glcβ1Cer). In contrast, the blood group A pig had a number of F18-binding glycosphingolipids, characterized as A type 1 hexaglycosylceramide (GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer), A type 4 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), A type 1 octaglycosylceramide (GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ3GlcNAcβ3Galβ4Glcβ1Cer), and repetitive A type 1 nonaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcα3-(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). No blood group antigen-carrying glycosphingolipids were recognized by a mutant E. coli strain with deletion of the FedF adhesin, demonstrating that FedF is the structural element mediating binding of F18-fimbriated bacteria to blood group ABH determinants. Enterotoxigenic (ETEC) 4The abbreviations used are: ETEC, enterotoxigenic E. coli; F18R, receptor for F18-fimbriated E. coli; FAB, fast atom bombardment; LC/MS, liquid chromatography-mass spectrometry; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MS, mass spectrometry; MS2, tandem mass spectrometry. and verotoxigenic Escherichia coli are important causes of disease in man and animal (1Kaper J.B. Nataro J.P. Mobley H.L. Nat. Rev. Microbiol. 2004; 2: 123-140Crossref PubMed Scopus (3157) Google Scholar, 2Fairbrother J.M. Nadeau E. Gyles C.L. Anim. Health Res. Rev. 2005; 6: 17-39Crossref PubMed Scopus (569) Google Scholar). In newly weaned pigs, F18-fimbriated E. coli producing entero- and/or Shiga-like toxins induce diarrhea and/or edema disease, which accounts for substantial economical losses in the pig industry (3Bertschinger H. Gyles C.L. Gyles C.L. Escherichia coli in Domestic Animals and Humans. CAB, Wallingford, Oxon, UK1994: 193-219Google Scholar). Two virulence factors are of major importance, namely the F18 fimbriae, the adhesive polymeric protein surface appendages of F18-fimbriated E. coli, and the Shiga-like toxin (SLT-IIv), or enterotoxins (STa or STb), that are produced by the bacterium. F18 fimbriae are expressed by the fed (fimbriae associated with edema disease) gene cluster, with fedA encoding the major subunit, fedB the outer membrane usher, fedC the periplasmic chaperone, whereas fedE and fedF encode minor subunits (4Imberechts H. De Greve H. Schlicker C. Bouchet H. Pohl P. Charlier G. Bertschinger H. Wild P. Vandekerckhove J. Van Damme J. Van Montagu M. Lintermans P. Infect. Immun. 1992; 60: 1963-1971Crossref PubMed Google Scholar). FedF is the adhesive subunit and is presumably located at the tip of the fimbrial structure (5Imberechts H. Wild P. Charlier G. De Greve H. Lintermans P. Pohl P. Microb. Pathog. 1996; 21: 183-192Crossref PubMed Scopus (50) Google Scholar, 6Smeds A. Hemmann K. Jakava-Viljanen M. Pelkonen S. Imberechts H. Palva A. Infect. Immun. 2001; 69: 7941-7945Crossref PubMed Scopus (37) Google Scholar). Typically, tip adhesins consist of two domains: an N-terminal carbohydrate-specific lectin domain and a C-terminal pilin domain (7Hultgren S.J. Lindberg F. Magnusson G. Kihlberg J. Tennent J.M. Normark S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4357-4361Crossref PubMed Scopus (133) Google Scholar), which needs to be donor-strand-complemented to stabilize its incomplete Ig fold (8Choudhury D. Thompson A. Stojanoff V. Langermann S. Pinkner J. Hultgren S.J. Knight S.D. Science. 1999; 285: 1061-1066Crossref PubMed Scopus (512) Google Scholar, 9Sauer F.G. Fütterer K. Pinkner J.S. Dodson K.W. Hultgren S.J. Waksman G. Science. 1999; 285: 1058-1061Crossref PubMed Scopus (326) Google Scholar). Crystal structures of the lectin domains of type 1 pili, P pili, and F17 fimbrial adhesins reveal that they all have the immunoglobulin-like (Ig-like) fold in common, which is remarkable, because they show little to no sequence identity (10Buts L. Bouckaert J. De Genst E. Loris R. Oscarson S. Lahmann M. Messens J. Brosens E. Wyns L. De Greve H. Mol. Microbiol. 2003; 49: 705-715Crossref PubMed Scopus (86) Google Scholar). N-terminal truncation has enabled crystallization of the FedF adhesin, and elucidation of its crystal structure will shed light on the interaction with the natural receptor on the intestinal epithelium (11De Kerpel M. Van Molle I. Brys L. Wyns L. De Greve H. Bouckaert J. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006; 62: 1278-1282Crossref PubMed Scopus (11) Google Scholar). A crucial step in the pathogenesis of the F18-fimbriated E. coli-induced diarrhea/edema is the initial attachment of the bacteria to a specific receptor (F18R) on the porcine intestinal epithelium. However, some pigs are resistant to colonization by F18-fimbriated E. coli, due to lack of F18R expression. The F18R status of pigs is genetically determined (12Bertschinger H.U. Stamm M. Vögeli P. Vet. Microbiol. 1993; 35: 79-89Crossref PubMed Scopus (61) Google Scholar), with the gene controlling expression of F18R mapped to the halothane linkage group on pig chromosome 6 (13Vögeli P. Bertschinger H.U. Stamm M. Stricker C. Hagger C. Fries R. Rapacz J. Stranzinger G. Anim. Genet. 1996; 27: 321-328PubMed Google Scholar, 14Meijerink E. Fries R. Vögeli P. Masabanda J. Wigger G. Stricker C. Neuenschwander S. Bertschinger H.U. Stranzinger G. Mamm. Genome. 1997; 8: 736-741Crossref PubMed Scopus (139) Google Scholar). This locus contained two candidate genes, FUT1 and FUT2, both encoding α2-fucosyltransferases. Expression analysis of these two genes in the porcine small intestine revealed that the FUT2 gene is differentially expressed, whereas the FUT1 gene is expressed in all examined pigs (15Meijerink E. Neuenschwander S. Fries R. Dinter A. Bertschinger H.U. Stranzinger G. Vögeli P. Immunogenetics. 2000; 52: 129-136Crossref PubMed Scopus (107) Google Scholar). Sequencing of the FUT1 gene of pigs being either susceptible or resistant to infection by F18-fimbriated E. coli showed a polymorphism (G or A) at nucleotide 307. Presence of the A nucleotide on both alleles (FUT1A/A genotype) led to significantly reduced enzyme activity, corresponding to the F18-fimbriated E. coli-resistant genotype, whereas susceptible pigs had either the heterozygous FUT1G/A or the homozygous FUT1G/G genotype. These findings have led to the development of a PCR-restriction fragment length polymorphism test to differentiate between F18R-positive and F18R-negative pigs. Although substantial information exists regarding the genetics of F18R, the identity and nature of the F18R molecule have remained unclear. In this report, F18R was found to be of glycosphingolipid nature. Consequently, a collection of glycosphingolipids from various sources was tested for binding with F18-fimbriated E. coli, demonstrating a specific interaction of recombinant F18-expressing bacteria with blood group ABH determinants on type 1 core chains. The role of the FedF adhesin in this binding process was defined by using a FedF-negative mutant strain (5Imberechts H. Wild P. Charlier G. De Greve H. Lintermans P. Pohl P. Microb. Pathog. 1996; 21: 183-192Crossref PubMed Scopus (50) Google Scholar). Knowledge of the F18R structure could provide better insights into the interactions between F18-fimbriated E. coli and the porcine gut, and may, additionally, lead to the design of potent inhibitors of adherence for use in anti-adhesive therapy (16Wellens A. Garofalo C. Nguyen H. Van Gerven N. Slättegård R. Hernalsteens J.P. Wyns L. Oscarson S. De Greve H. Hultgren S. Bouckaert J. PLoS ONE. 2008; 3: e2040Crossref PubMed Scopus (204) Google Scholar). The wild-type verotoxigenic F18-positive E. coli reference strain 107/86 (serotype O139:K12:H1, F18ab+, SLT-IIv+) (17Bertschinger H.U. Bachmann M. Mettler C. Pospischil A. Schraner E.M. Stamm M. Sydler T. Wild P. Vet. Microbiol. 1990; 25: 267-281Crossref PubMed Scopus (122) Google Scholar), and the wild-type enterotoxigenic F4ac-positive E. coli reference strain GIS 26 (serotype O149:K91:F4ac, LT+, STa+, STb+), were cultured on BHI agar plates (Oxoid, Basingstoke, Hampshire, England) at 37 °C for 18 h. Subsequently, the bacteria were harvested by centrifugation and resuspended in phosphate-buffered saline (PBS, pH 7.3). The concentration of bacteria in the suspension was determined by measuring the optical density at 660 nm (A660). An optical density of 1 equals 109 bacteria per milliliter, as determined by counting colony forming units. Recombinant E. coli strains expressing whole F18 fimbriae (HB101(pIH120), or F18 fimbriae with deletion of the FedF adhesive subunit (HB101(pIH126)) (4Imberechts H. De Greve H. Schlicker C. Bouchet H. Pohl P. Charlier G. Bertschinger H. Wild P. Vandekerckhove J. Van Damme J. Van Montagu M. Lintermans P. Infect. Immun. 1992; 60: 1963-1971Crossref PubMed Google Scholar, 5Imberechts H. Wild P. Charlier G. De Greve H. Lintermans P. Pohl P. Microb. Pathog. 1996; 21: 183-192Crossref PubMed Scopus (50) Google Scholar) were grown on Iso Sensitest agar plates (Oxoid) supplemented with ampicillin (100 μg/ml) at 37 °C overnight. For metabolic labeling, the culture plates were supplemented with 10 μl of [35S]methionine (400 μCi, Amersham Biosciences). Bacteria were harvested, washed three times in PBS, and resuspended in PBS containing 2% (w/v) bovine serum albumin (BSA), 0.1% (w/v) NaN3, and 0.1% (w/v) Tween 20 (BSA/PBS/Tween) to a bacterial density of 1 × 108 colony forming units/ml. The specific activity of bacterial suspensions was ∼1 cpm per 100 bacteria. The same conditions (with omission of ampicillin) were used for culture and labeling of the background E. coli strain HB101. The physio-chemical properties of F18R on porcine small intestinal villous enterocytes was investigated using an in vitro villous adhesion inhibition assay (18Cox E. Houvenaghel A. Vet. Microbiol. 1993; 34: 7-18Crossref PubMed Scopus (41) Google Scholar, 19Verdonck F. Cox E. van Gog K. Van der Stede Y. Duchateau L. Deprez P. Goddeeris B.M. Vaccine. 2002; 20: 2995-3004Crossref PubMed Scopus (62) Google Scholar). Briefly, a 20-cm intestinal segment was collected from the mid jejunum of a euthanized pig, rinsed three times with ice-cold PBS, and fixed with Krebs-Henseleit buffer (160 mm, pH 7,4) containing 1% (v/v) formaldehyde for 30 min at 4 °C. Thereafter, the villi were gently scraped from the mucosae with a glass slide and stored in Krebs-Henseleit buffer at 4 °C. Treatment of villi with acetone, methanol, 1% Triton X-100, 10 mm NaIO4 in 0.2 m sodium acetate, pH 4.5, or 0.2 m sodium acetate, pH 4.5, without NaIO4, respectively, was performed at room temperature on a rotating wheel in a volume of 500 μl during 1 h. Next, the villi were washed six times with Krebs-Henseleit buffer followed by addition of 4 × 108 bacteria of the F18-positive reference E. coli strain (107/86), or the F4ac-expressing E. coli strain GIS 26, to an average of 50 villi in a total volume of 500 μl of PBS, supplemented with 1% (w/v) d-mannose to prevent adhesion mediated by type 1 pili. These mixtures were incubated at room temperature for 1 h while being gently shaken. Villi were examined by phase-contrast microscopy at a magnification of 600, and the number of bacteria adhering along a 50-μm brush border was quantitatively evaluated by counting the number of adhering bacteria at 20 randomly selected places, after which the mean bacterial adhesion was calculated. Total acid and non-acid glycosphingolipid fractions were prepared as described before (20Teneberg S. Willemsen P. de Graaf F.K. Stenhagen G. Pimlott W. Jovall P.-Å. Ångström J. Karlsson K.-A. J. Biochem. 1994; 116: 560-574Crossref PubMed Scopus (33) Google Scholar), and the individual glycosphingolipids were obtained by repeated chromatography on silicic acid columns and by high-performance liquid chromatography, and identified by mass spectrometry and proton NMR spectroscopy (20Teneberg S. Willemsen P. de Graaf F.K. Stenhagen G. Pimlott W. Jovall P.-Å. Ångström J. Karlsson K.-A. J. Biochem. 1994; 116: 560-574Crossref PubMed Scopus (33) Google Scholar). Aluminum- or glass-backed silica gel 60 high-performance TLC plates (Merck, Darmstadt, Germany) were used for TLC and eluted with chloroform/methanol/water (60:35:8, by volume) as the solvent system. The different glycosphingolipids were applied to the plates in quantities of 0.002–4 μg of pure glycosphingolipids, and 40 μg of glycosphingolipid mixtures. Chemical detection was done with anisaldehyde (21Waldi D. Stahl E. Dünnschicht-Chromatographie. Springer-Verlag, Berlin1962: 496-515Google Scholar). Binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms was done as described before (20Teneberg S. Willemsen P. de Graaf F.K. Stenhagen G. Pimlott W. Jovall P.-Å. Ångström J. Karlsson K.-A. J. Biochem. 1994; 116: 560-574Crossref PubMed Scopus (33) Google Scholar), with minor modifications. Dried chromatograms were dipped in diethylether/n-hexane (1:5 v/v) containing 0.5% (w/v) polyisobutylmethacrylate for 1 min, dried, and then blocked with BSA/PBS/Tween for 2 h at room temperature. Thereafter, the plates were incubated with 35S-labeled bacteria (1–5 × 106 cpm/ml) diluted in BSA/PBS/Tween for another 2 h at room temperature. After washing six times with PBS, and drying, the thin-layer plates were autoradiographed for 12 h using XAR-5 x-ray films (Eastman Kodak, Rochester, NY). Chromatogram binding assays with monoclonal antibodies directed against the blood group A determinant (DakoCytomation Norden A/S, Glostrup, Denmark) were done as described (22Hansson G.C. Karlsson K-A. Larson G. McKibbin J.M. Blaszczyk M. Herlyn M. Steplewski Z. Koprowski H. J. Biol. Chem. 1983; 258: 4091-4097Abstract Full Text PDF PubMed Google Scholar), using 125I-labeled anti-mouse antibodies for detection. Non-acid glycosphingolipids were isolated from mucosal scrapings from porcine small intestines as described (20Teneberg S. Willemsen P. de Graaf F.K. Stenhagen G. Pimlott W. Jovall P.-Å. Ångström J. Karlsson K.-A. J. Biochem. 1994; 116: 560-574Crossref PubMed Scopus (33) Google Scholar). Briefly, the mucosal scrapings were lyophilized and then extracted in two steps in a Soxhlet apparatus with chloroform and methanol (2:1 and 1:9, by volume, respectively). The material obtained was subjected to mild alkaline hydrolysis and dialysis, followed by separation on a silicic acid column. Acid and non-acid glycosphingolipid fractions were obtained by chromatography on a DEAE-cellulose column. To separate the non-acid glycolipids from alkali-stable phospholipids, this fraction was acetylated and separated on a second silicic acid column, followed by deacetylation and dialysis. Final purifications were done by chromatographies on DEAE-cellulose and silicic acid columns. The total non-acid glycosphingolipid fractions obtained were thereafter separated, as described below. Throughout the separation procedures aliquots of the fractions obtained were analyzed by TLC, and fractions that were colored green by anisaldehyde were tested for binding of F18-fimbriated E. coli using the chromatogram binding assay. Blood Group O Pig Intestinal Mucosa-A total non-acid glycosphingolipid fraction (148 mg) from blood group O pig intestinal mucosa was first separated on a silicic acid column eluted with increasing volumes of methanol in chloroform. Thereby, an F18-fimbriated E. coli binding fraction containing tetraglycosylceramides and more slow-migrating compounds (22 mg) was obtained. This fraction was further separated on an Iatrobeads (Iatrobeads 6RS-8060; Iatron Laboratories, Tokyo) column (10 g), first eluted with chloroform/methanol/water (60:35:8, by volume), 10 × 5 ml, followed by chloroform/methanol/water (40:40:12, by volume), 2 × 10 ml. The F18-fimbriated E. coli binding compound eluted in fractions 3 and 4, and after pooling of these fractions 6.7 mg was obtained. This material was acetylated and further separated on an Iatrobeads column (2 g), eluted with increasing volumes of methanol in chloroform. After deacetylation and dialysis, 6.0 mg of pure F18-binding glycosphingolipid (designated fraction O-I) was obtained. Blood Group A Pig Intestinal Mucosa-A total non-acid glycosphingolipid fraction (183 mg) from blood group A pig intestinal mucosa was initially separated on a silicic acid column eluted with increasing volumes of methanol in chloroform. Pooling of fractions containing tetraglycosylceramides and more slowly migrating compounds yielded 51.2 mg. This material was further separated by high-performance liquid chromatography on a 1.0- × 25-cm silica column (Kromasil Silica, 10-μm particles, Skandinaviska Genetec, Kungsbacka, Sweden) eluted with a linear gradient of chloroform/methanol/water (70:25:4 to 40:40:12, by volume) during 180 min and with a flow of 2 ml/min. The fractions obtained were pooled according to mobility on thin-layer chromatograms and F18-fimbriated E. coli binding activity. Thereby, six F18-fimbriated E. coli binding fractions were obtained, designated fraction A-I (12.6 mg), A-II (3.6 mg), A-III (0.3 mg), A-IV (0.5 mg), A-V (0.2 mg), and A-VI (0.2 mg), respectively. Negative ion FAB mass spectra were recorded on a JEOL SX-102A mass spectrometer (JEOL, Tokyo, Japan). The ions were produced by 6 keV xenon atom bombardment using triethanolamine (Fluka, Buchs, Switzerland) as matrix and an accelerating voltage of –10 kV. Endoglycoceramidase II from Rhodococcus spp. (23Ito M. Yamagata T. J. Biol. Chem. 1989; 264: 9510-9519Abstract Full Text PDF PubMed Google Scholar) (Takara Bio Europe S.A., Gennevilliers, France) was used for hydrolysis of glycosphingolipids. Briefly, 50 μg of F18-fimbriated E. coli binding fraction O-I from blood group O porcine intestinal mucosa, H type 1 pentaglycosylceramide from human meconium, and H type 2 pentaglycosylceramide from human erythrocytes were resuspended in 100 μl of 0.05 m sodium acetate buffer, pH 5.0, containing 120 μg of sodium cholate, and sonicated briefly. Thereafter, 1 milliunit of endoglycoceramidase II was added, and the mixture was incubated at 37 °C for 48 h. The reaction was stopped by addition of chloroform/methanol/water to the final proportions 8:4:3 (by volume). The oligosaccharide-containing upper phase thus obtained was separated from detergent on a Sep-Pak QMA cartridge (Waters, Milford, MA). The eluant containing the oligosaccharides was dried under nitrogen and under vacuum. For LC/MS the glycosphingolipid-derived saccharides were separated on a column (200 × 0.180 mm) packed in-house with 5-μm porous graphite particles (Hypercarb, Thermo Scientific), and eluted with a linear gradient from 0%B to 45%B in 46 min (Solvent A: 8 mm NH4HCO3; Solvent B: 20% 8 mm NH4HCO3/80% acetonitrile (by volume)). Eluted saccharides were analyzed in the negative mode on an LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, CA), using Xcalibur software. 1H NMR spectra were acquired on a Varian 600-MHz spectrometer at 30 °C. Samples were dissolved in DMSO/D2O (98:2, by volume) after deuterium exchange. The ability of soluble oligosaccharides to interfere with the binding of F18-fimbriated E. coli to porcine small intestinal cells was evaluated in the in vitro villous adhesion assay. The F18-positive E. coli strain 107/86 (8 × 107 bacteria) was incubated with different concentrations (10 mg/ml, 1 mg/ml, 100 μg/ml) of blood group H type 1 pentasaccharide (Fucα2Galβ3GlcNAcβ3-Galβ4Glc) or lacto-N-tetraose saccharide (Galβ3GlcNAcβ3Galβ4Glc) (Glycoseparations, Moscow, Russia) in a final volume of 100 μl of PBS, for 1 h at room temperature, while being gently shaken. The mixtures were then added to the villi, and again incubated for 1 h at room temperature with gentle shaking. Thereafter, the villi were examined by phase-contrast microscopy, and adhering bacteria were quantitated as described above. Villi of two different F18R-positive pigs were used, and the counts were performed in triplicate. The pigs used were found to be blood group H type 1 and 2 positive, and blood group A type 2 negative, by indirect immunofluorescence using blood group-specific monoclonal antibodies (clone 17-206, GeneTex, Inc., San Antonio, TX; clone 92FR-A2, Abcam, Cambridge, UK; clone 29.1 Sigma-Aldrich), and fluorescein isothiocyanate-labeled secondary anti-mouse antibody (Sigma-Aldrich). To examine the physiochemical characteristics of F18R, intestinal villi isolated from four pigs were treated with different agents that affect lipids or carbohydrates on the cell membrane. Treatment was followed by assessment of adhesion of F18-fimbriated E. coli to the villus epithelium. The adhesion of F18-fimbriated E. coli to porcine intestinal villi was completely abolished after treatment with acetone, methanol, 1% Triton X-100, and NaIO4 (Fig. 1), suggesting that F18R is a glycolipid. In contrast, the adhesion of the control F4ac-fimbriated E. coli strain, having a glycoprotein receptor (24Erickson A.K. Baker D.R. Bosworth B.T. Casey T.A. Benfield D.A. Francis D.H. Infect. Immun. 1994; 62: 5404-5410Crossref PubMed Google Scholar), was only abolished by incubation with NaIO4, whereas treatment with acetone, methanol, and 1% Triton X-100 had no or only little effect. The suggested glycolipid binding of F18-fimbriated E. coli was next investigated by binding to glycosphingolipids separated on thin-layer plates. The initial screening for F18-fimbriated E. coli carbohydrate-binding activity was done by using mixtures of glycosphingolipids from various sources, to expose the bacteria to a large number of potentially binding-active carbohydrate structures. Thereby, a distinct binding of the F18-expressing E. coli strain HB101(pIH120) to slow migrating minor non-acid glycosphingolipids from black and white rat intestine was obtained (Fig. 2B, lane 4). Notably, these compounds were not recognized by the E. coli strain HB101(pIH126), having a deletion of the FedF adhesin (Fig. 2C). A weak binding of both the F18-expressing strain HB101(pIH120) and the FedF deletion strain HB101(pIH126) to the major compound (gangliotriaosylceramide) of the non-acid glycosphingolipid fraction of guinea pig erythrocytes (lane 3) was also observed. Binding to this gangliotriaosylceramide was also obtained with the HB101 background strain (not shown) and was most likely due to the relatively large amounts of this compound on the thin-layer chromatograms. A characteristic feature of black and white rat intestine is the presence of glycosphingolipids with blood group A determinants on type 1 (Galβ3GlcNAc) core chains, whereas in white rat intestine blood group A-terminated glycosphingolipids are absent (25Breimer M.E. Hansson G.C. Karlsson K.-A. Leffler H. J. Biol. Chem. 1981; 257: 557-568Abstract Full Text PDF Google Scholar). When binding of the F18-positive strain HB101-(pIH120) to non-acid glycosphingolipids from white rat intestine was examined (Fig. 3, lane 5), no binding of the bacteria to this fraction (lane 5) occurred, in contrast to the distinct binding of the bacteria to the non-acid glycosphingolipid fraction from black and white rat intestine (Fig. 3B, lane 4). A further observation was the absence of binding of the F18-fimbriated bacteria to the non-acid glycosphingolipids from human blood group A, B, or O erythrocytes (lanes 1–3), where the predominant glycosphingolipids with blood group A, B, and H determinants have type 2 (Galβ4GlcNAc) core chains (26Koscielak J. Plasek A. Górniak H. Gardas A. Gregor A. Eur. J. Biochem. 1973; 37: 214-225Crossref PubMed Scopus (124) Google Scholar, 27Stellner K. Watanabe K. Hakomori S.-i. Biochemistry. 1973; 12: 656-661Crossref PubMed Scopus (138) Google Scholar, 28Clausen H. Levery S.B. Nudelman E. Baldwin M. Hakomori S.-i. Biochemistry. 1986; 25: 7075-7085Crossref PubMed Scopus (86) Google Scholar). Once again, no glycosphingolipid was recognized by the FedF deletion mutant HB101(pIH126) (Fig. 3C). In summary, the binding of the F18-fimbriated bacteria to slow migrating minor non-acid glycosphingolipids from black and white rat intestine suggested a specific recognition of blood group determinants on type 1 core chains. Furthermore, the absence of binding of the FedF deletion mutant to these compounds indicated an involvement of the FedF protein in the interaction. Binding assays using pure reference glycosphingolipids in defined amounts confirmed the suggested binding of F18-fimbriated E. coli to blood group determinants on type 1 core chains. The results are exemplified in Fig. 4 and summarized in Table 1. Thus, the F18-expressing E. coli bound to all glycosphingolipids with blood group A, B, or H determinants on type 1 core chains, as the H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer, 5The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for Lipids (65Chester M.A. Eur. J. Biochem. 1998; 257: 293-298Crossref PubMed Scopus (175) Google Scholar)). It is assumed that Gal, Glc, GlcNAc, GalNAc, NeuAc, and NeuGc are of the D-configuration, Fuc of the L-configuration, and all sugars are present in the pyranose form. Fig. 4, lane 1), the B type 1 hexaglycosylceramide (Galα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fig. 4, lane 3), the A type 1 hexaglycosylceramide (GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fig. 4, lane 5), the A type 1 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer, Fig. 4, lane 7), and the B type 1 heptaglycosylceramide (Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer, Fig. 4, lane 9). In contrast, no type 2 core counterparts of these compounds were recognized by the F18-fimbriated bacteria, as the H type 2 pentaglycosylceramide (Fucα2Galβ4GlcNAcβ3Galβ4Glcβ1Cer, Fig. 4, lane 2), the B type 2 hexaglycosylceramide (Galα3(Fucα2)Galβ4GlcNAcβ3Galβ4Glcβ1Cer, Fig. 4, lane 4), the A type 2 hexaglycosylceramide (GalNAcα3(Fucα2)Galβ4GlcNAcβ3 Galβ4Glcβ1Cer, Fig. 4, lane 6), or the A type 2 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ4(Fucα4)GlcNAcβ3 Galβ4Glcβ1Cer, Fig. 4, lane 8).TABLE 1Binding of 35S-labeled recombinant F18 fimbriae-expressing E. coli (pIH120) and recombinant E. coli expressing F18 fimbriae with deletion of the tip subunit (pIH126), to glycosphingolipids on thin-layer chromatogramsNo. and trivial nameStructurepIH120pIH126Simple compounds1) GalactosylceramideGalβ1Cer-aBinding is defined as follows: +++ denotes a binding when <0.1 μg of the glycosphingolipid was applied on the thin-layer chromatogram, whereas ++ denotes a binding when <1 μg of the glycosphingolipid was applied, + denotes a binding at 1-2 μg, and - denotes no binding even at 4 μg.-2) GlucosylceramideGlcβ1Cer--3) SulfatideSO3-3Galβ1Cer--4) LacCer (d18:1-16:0-24:0)bIn the shorthand nomenclature for fatty acids and bases, the number before the colon refers to the carbon chain length, and the number after" @default.
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• W1971390030 date "2009-04-01" @default.
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• W1971390030 title "Recognition of Blood Group ABH Type 1 Determinants by the FedF Adhesin of F18-fimbriated Escherichia coli" @default.
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• W1971390030 doi "https://doi.org/10.1074/jbc.m807866200" @default.
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