Bacterial adherence to mucosal surfaces is an important virulence trait of pathogenic bacteria. Adhesion of enterotoxigenic Escherichia coli (ETEC) to the intestine is mediated by a number of antigenically distinct colonization factors (CFs). One of the most common CFs is CFA/I. This has a fimbrial structure composed of a major repeating subunit, CfaB, and a single tip subunit, CfaE. The potential carbohydrate recognition by CFA/I was investigated by binding CFA/I-fimbriated bacteria and purified CFA/I fimbriae to a large number of variant glycosphingolipids separated on thin-layer chromatograms. For both fimbriated bacteria and purified fimbriae, specific interactions could be identified with a number of nonacid glycosphingolipids. These included glucosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, neolactotetraosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, the H5 type 2 pentaglycosylceramide, the Le ^a -5 glycosphingolipid, the Le ^x -5 glycosphingolipid, and the Le ^y -6 glycosphingolipid. These glycosphingolipids were also recognized by recombinant E. coli expressing CFA/I in the absence of tip protein CfaE, as well as by purified fimbriae from the same strain. This demonstrates that the glycosphingolipid-binding capacity of CFA/I resides in the major CfaB subunit.

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.

The first step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections is adhesion of the bacterium to the small intestinal epithelium. Adhesion of ETEC is mediated by a number of antigenically distinct colonization factors, and among these, one of the most commonly detected is the non-fimbrial adhesin coli surface antigen 6 (CS6). The potential carbohydrate recognition by CS6 was investigated by binding of recombinant CS6-expressing E. coli and purified CS6 protein to a large number of variant glycosphingolipids separated on thin-layer chromatograms. Thereby, a highly specific binding of the CS6-expressing E. coli, and the purified CS6 protein, to sulfatide (SO₃-3Galβ1Cer) was obtained. The binding of the CS6 protein and CS6-expressing bacteria to sulfatide was inhibited by dextran sulfate, but not by dextran, heparin, galactose 4-sulfate or galactose 6-sulfate. When using recombinantly expressed and purified CssA and CssB subunits of the CS6 complex, sulfatide binding was obtained with the CssB subunit, demonstrating that the glycosphingolipid binding capacity of CS6 resides within this subunit. CS6-binding sulfatide was present in the small intestine of species susceptible to CS6-mediated infection, e.g. humans and rabbits, but lacking in species not affected by CS6 ETEC, e.g. mice. The ability of CS6-expressing ETEC to adhere to sulfatide in target small intestinal epithelium may thus contribute to virulence.

Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated. To investigate the structural requirements for H. pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing H. pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to N -acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the N -acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N -acetyllactosamine core chain. While the J99/NAP ⁻ mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA ⁻ mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H. pylori bacterial cells to gangliosides.

Isolation and characterization of glycosphingolipids is of importance in many aspects of glycobiology, but is difficult to achieve due to the high degree of heterogeneity and isomerism present in these compounds. In this study, oligosaccharides obtained from non-acid glycosphingolipids by enzymatic digestion with endoglycoceramidase II of Rhodococcus sp. were analyzed by liquid chromatography/electrospray ionization mass spectrometry using graphitized carbon columns. Resolution of isomeric oligosaccharides was achieved, and the MS² analyses gave complete sequence information and allowed differentiation of linkage positions. Diagnostic cross-ring ^0,2A-type fragments have previously been described for GlcNAc substituted on C-4 and for 4-substituted Glc. Diagnostic cross-ring ^0,2A-type fragments were present in the MS² spectrum of the H type 2 (Fucα2Galβ4GlcNAcβ4Galβ4Glc) pentasaccharide, but not in the MS² spectrum of H type 1 pentasaccharide (Fucα2Galβ3GlcNAcβ4Galβ4Glc). Cross-ring ^0,2A-type fragments were also obtained from the 4-substituted Glc at the reducing end of the glycosphingolipid-derived oligosaccharides. Oligosaccharides of the globo-series (globotriaose (Galα4Galβ4Glc) and globotetraose (GalNAcβ3Galα4Galβ4Glc)) and the isoglobo-series (isoglobotriaose (Galα3Galβ4Glc) and isoglobotetraose (GalNAcβ3Galα3Galβ4Glc)) were also chromatographically resolved on the graphitized carbon column. Furthermore, diagnostic fragment ions from cross-ring ^0,2A-type cleavages were present in the MS² spectra of the globo-series oligosaccharides, having a Gal substituted on C-4. The applicability of this method on tissue-derived samples was demonstrated using a non-acid glycosphingolipid fraction from human gastric epithelium and a partially purified non-acid glycosphingolipid fraction from 8 × 10⁷ bone marrow-derived mouse dendritic cells. Here, liquid chromatography/mass spectrometry of the oligosaccharides released by endoglycoceramidase allowed tentative identification of a number of glycosphingolipids ranging from tri- to nonaglycosylceramides.

Adhesion of Helicobacter pylori to the gastric mucosa is a prerequisite for the pathogenesis of H. pylori related diseases. In this study, we investigated the ganglioside composition of human stomach as the target for attachment mediated by H. pylori SabA (sialic acid binding adhesin). Acid glycosphingolipids were isolated from human stomach and separated into subfractions, which were characterized by mass spectrometry and by binding of antibodies, bacteria, and Solanum tuberosum lectin. H. pylori SabA binding gangliosides were characterized as Neu5Acα3-neolactohexaosylceramide and Neu5Acα3-neolactooctaosylceramide, while the other acid human stomach glycosphingolipids characterized (sulfatide and the gangliosides GM3, GD3, GM1, Neu5Acα3-neolactotetraosylceramide, GD1a and GD1b) were not recognized by the bacteria. Defining H. pylori binding glycosphingolipids of the human gastric mucosa will be useful to specifically target this microbe-host interaction for therapeutic intervention.