<i>Helicobacter pylori</i> SabA binding gangliosides of human stomach

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.</p

<i>Aeromonas salmonicida</i> binds α2-6 linked sialic acid, which is absent among the glycosphingolipid repertoires from skin, gill, stomach, pyloric caecum, and intestine

Carbohydrates can both protect against infection and act as targets promoting infection. Mucins are major components of the slimy mucus layer covering the fish epithelia. Mucins can act as decoys for intimate pathogen interaction with the host afforded by binding to glycosphingolipids in the host cell membrane. We isolated and characterized glycosphingolipids from Atlantic salmon skin, gill, stomach, pyloric caeca, and intestine. We characterized the glycosphingolipids using liquid chromatography – mass spectrometry and tandem mass spectrometry and the glycan repertoire was compared with the glycan repertoire of mucins from the same epithelia. We also investigated Aeromonas salmonicida binding using chromatogram and microtiter well based binding assays. We identified 29 glycosphingolipids. All detected acid glycans were of the ganglio-series (unless shorter) and showed a high degree of polysialylation. The non-acid glycans were mostly composed of the neolacto, globo, and ganglio core structures. The glycosphingolipid repertoire differed between epithelia and the proportion of the terminal moieties of the glycosphingolipids did not reflect the terminal moieties on the mucins from the same epithelia. A. salmonicida did not bind the Atlantic salmon glycosphingolipids. Instead, we identified that A. salmonicida binding to sialic acid occurred to α2–6 Neu5Ac but not to α2–3 Neu5Ac. α2–6 Neu5Ac was present on mucins whereas mainly α2–3 Neu5Ac was found on the glycosphingolipids, explaining the difference in A. salmonicida binding ability between these host glycoconjugates. A. salmonicida´s ability to bind to Atlantic salmon mucins, but not the glycosphingolipids, is likely part of the host defence against this pathogen.</p

Characterization of El Tor binding fraction TH-I from rabbit thymus.

<p>(A) Base peak chromatogram from LC-ESI/MS of the oligosaccharide obtained by digestion of fraction TH-I with endoglycoceramidase II. (B) MS² spectrum of the [M-H⁺]⁻ ion at <i>m/z</i> 909 (retention time 32.2 min). (C) Anomeric region of the 600 MHz ¹H NMR spectrum of fraction TH-I (30°C). The designation B refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053999#pone-0053999-t001" target="_blank">Table 1</a>.</p

Presence or absence of mucin specific binding of pathogens.

Presence or absence of mucin specific binding of pathogens.</p

Binding of <i>V. cholerae</i> El Tor and <i>E. cristagalli</i> lectin to glycosphingolipids of rabbit thymus.

<p>(A and D) Chemical detection by anisaldehyde. (B and E) Autoradiograms obtained by binding of <i>V. cholerae</i> JBK 70. (C and F) Autoradiograms obtained by binding of <i>E. cristagalli</i> lectin. The lanes on A–C were: Lane 1, neolactotetraosylceramide (Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 2, fraction TH-I isolated from rabbit thymus, 1 µg; Lane 3, fraction TH-II from rabbit thymus, 1 µg; Lane 4, Lane 4, sialylneolactohexaosylceramide (NeuGcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 1 µg. The lanes on D–F were: Lane 1, total non-acid glycosphingolipids of rabbit thymus, 40 µg; B5 pentaosylceramide (Galα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lanes 3 and 4, subfractions isolated from rabbit thymus, 1 µg/lane; Lane 5, fraction TH-II from rabbit thymus, 1 µg; Lane 6, fraction TH-III, 1 µg; Lane 7, fraction TH-IV, 1 µg.</p

Chemical shift data (ppm) for anomeric resonances from proton NMR spectra at 600 MHz of <i>V. cholerae</i> binding glycosphingolipids, and co-migrating glycosphingolipids, obtained in DMSO-d₆/D₂O (98∶2, by volume) at 30°C.

<p>Chemical shift data (ppm) for anomeric resonances from proton NMR spectra at 600 MHz of <i>V. cholerae</i> binding glycosphingolipids, and co-migrating glycosphingolipids, obtained in DMSO-d₆/D₂O (98∶2, by volume) at 30°C.</p

Comparison of the glycosphingolipid binding of classical and El Tor <i>V. cholerae,</i> and an El Tor GbpA deletion mutant strain.

<p>(A, C, E) Chemical detection by anisaldehyde. (B, D, G) Autoradiograms obtained by binding of El Tor <i>V. cholerae</i> strain JBK 70. (F) Autoradiogram obtained by binding of classical <i>V. cholerae</i> strain CVD103. (H) Autoradiogram obtained by binding of El Tor GbpA deletion mutant strain 1382. The lanes on (A and B) were: Lane 1, galactosylceramide (Galβ1Cer), 4 µg, and gangliotriaosylceramide (GalNAcβ4Galβ4Glcβ1Cer), 4 µg; Lane 2, glucosylceramide (Glcβ1Cer), 4 µg, and globotriaosylceramide (Galα4Galββ4Glcβ1Cer), 4 µg; Lane 3, lactosylceramide (Galβ4Glcβ1Cer) with sphingosine and non-hydroxy fatty acids, 4 µg, and Forssman pentaosylceramide (GalNAcα3GalNAcβ3Galα4Galβ4Glcβ1Cer), 4 µg; Lane 4, lactosylceramide (Galβ4Glcβ1Cer) with phytosphingosine and hydroxy fatty acids, 4 µg, and globotetraosylceramide (GalNAcβ3Galα4Galβ4Glcβ1Cer), 4 µg; Lane 5, isoglobotriaosylceramide (Galα3Galβ4Glcβ1Cer), 4 µg, and Le^a pentaosylceramide (Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 6, Le^x pentaosylceramide (Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; lane 7, H type 1 pentaosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), 4 µg. The lanes on (C and D) were: Lane 1, B5 pentaosylceramide (Galα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 2, non-acid glycosphingolipids of rabbit erythrocytes, 40 µg; Lane 3, Galα3Galα3Galβ4Glcβ1Cer, 1 µg; Lane 4, B type 2 hexaosylceramide (Galα3(Fucα2)Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg. The lanes on (E-H) were: Lane 1, non-acid glycosphingolipids of rabbit thymus, 20 µg; Lane 2, lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer), 2 µg; Lane 3, Galα3Galα3Galβ4Glcβ1Cer, 4 µg; Lane 4, B5 pentaosylceramide, 4 µg; Lane 5, gangliotriaosylceramide, 4 µg; Lane 6, H type 1 pentaosylceramide, 4 µg.</p

Binding of <i>V. cholerae</i> El Tor to mixtures of glycosphingolipids.

<p>(A) Chemical detection by anisaldehyde. (B) Autoradiograms obtained by binding of <i>V. cholerae</i> JBK 70. The lanes were: Lane 1, non-acid glycosphingolipids of human small intestine, 20 µg; Lane 2, non-acid glycosphingolipids of dog erythrocytes, 20 µg; Lane 3, non-acid glycosphingolipids of cod intestine, 20 µg; Lane 4, A type 2 heptaosylceramide (GalNAcα3(Fucα2)Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 5, globotriaosylceramide (Galα4Galβ4Glcβ1Cer), 4 µg; Lane 6, acid glycosphingolipids of human small intestine, 40 µg; Lane 7, non-acid glycosphingolipids of cat erythrocytes, 40 µg; Lane 8, non-acid glycosphingolipids of rabbit thymus, 40 µg; Lane 9, non-acid glycosphingolipids of rabbit erythrocytes, 40 µg; Lane 10, non-acid glycosphingolipids of chicken erythrocytes, 40 µg.</p

Quantitative analysis of pathogen binding to Atlantic salmon mucins.

Mucin containing fractions from individual fish and tissue sites were pooled according to the method shown in Fig 1A (n = 5 for each tissue). Pathogen binding to each of these 25 samples was analyzed using the Bac Titer-Glo method, and the luminescence signals were transformed to CFU/cm2 according to standard curves for each pathogen (Fig 2) to allow comparison of binding levels between pathogens. A. A. hydrophila binding to gill mucins was higher compared to the proximal intestinal mucins: p≤0.05; n = 5). B. V. harveyi bound with no distinguishable organ preference (p = n.s.). C. The level of M. viscosa binding differed between mucin groups (distal intestine vs. skin and gill: p≤0.05). D. Y. ruckeri bound to proximal and distal intestinal mucins more than to gill mucins (p≤0.01 and p≤0.05). Bars denote median ± interquartile range of biological replicates, after subtracting the background signal. The results were reproduced twice. Statistics: Kruskal-Wallis test by ranks with Dunn´s Post Hoc test to compare binding to mucins from different epithelial sites. The numerical p values on the graphs show the result of the test, without the post hoc test. Abbreviations: Pyloric = pyloric cecal mucins; Proximal = proximal intestinal mucins; Distal = distal intestinal mucins.</p

Characterization of the El Tor binding glycosphingolipid of rabbit erythrocytes.

<p>(A) Chemical detection by anisaldehyde. (B) Autoradiogram obtained by binding of <i>V. cholerae</i> strain JBK 70. The lanes were: Lane 1, total non-acid glycosphingolipids of rabbit thymus, 40 µg; Lane 1, total non-acid glycosphingolipids of rabbit erythrocytes, 40 µg; Lane 3, B5 pentaosylceramide (Galα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 4, fraction RE-I of rabbit erythrocytes, 2 µg. (C) Base peak chromatogram from LC-ESI/MS of the oligosaccharides derived from fraction RE-I by hydrolysis with endoglycoceramidase II. (D) MS² spectrum of the [M-H⁺]⁻ ion at <i>m/z</i> 868 (retention time 14.0 min). (E) MS² spectrum of the [M-H⁺]⁻ ion at <i>m/z</i> 1030 (retention time 14.7 min). (F) Anomeric region of the 600 MHz ¹H NMR spectrum of fraction RE-I (30^oC). The designations A and D refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053999#pone-0053999-t001" target="_blank">Table 1</a>.</p