<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

Purified recombinant CS6, CssA and CssB proteins.

<p>The protein preparations were separated on 10% (A) and 12% (B) NuPAGE BisTris gels, and stained by Coomassie Brilliant Blue R-250. The lanes on A were 1, molecular mass standards; 2, CS6 protein, and the lanes on B were 1, molecular mass standards; 2, CssA protein (with polyhistidine tag); 3, CssB protein (fused to glutathione-S-transferase (26 kDa) and with polyhistidine tag).</p

Binding of ¹²⁵I-labeled native CS6 protein, ³⁵S-labeled recombinant CS6-expressing <i>Escherichia coli</i> (TOP10-CS6) and ³⁵S-labeled background <i>E. coli</i> strain TOP10 to glycosphingolipids on thin-layer chromatograms.

a<p>Binding is defined as follows: + denotes a binding when 4 µg of the glycosphingolipid was applied on the thin-layer chromatogram, while − denotes no binding even at 4 µg.</p

Effects of incubation of CS6 protein and recombinant CS6-expressing <i>E. coli</i> with dextran and dextran sulfate.

<p>Radiolabeled CS6 protein and recombinant CS6-expressing bacteria were incubated with dextran or dextran sulfate (0.01–1 mg/ml) for 1 h at room temperature. Thereafter the suspensions were utilized in the chromatogram binding assay or the microtiter well assay as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#s4" target="_blank">Materials and Methods</a>”. Autoradiograms obtained by binding of ³⁵S-labeled <i>E. coli</i> TOP10-CS6 strain incubated with dextran (A), and with dextran sulfate (B). Autoradiography was for 12 h. The lanes were: Lane 1, sulfatide (SO₃-3Galβ1Cer), 4 µg; Lane 2, sulfatide, 2 µg; Lane 3, sulfatide, 1 µg. Binding of ¹²⁵I-labeled CS6 protein, and ¹²⁵I-labeled CS6 protein incubated with dextran sulfate, to pure glycosphingolipids in microtiter wells (C).</p

Binding of ¹²⁵I-labeled CS6 adhesin to mixtures of glycosphingolipids on thin-layer chromatograms.

<p>Chemical detection by anisaldehyde (A), and autoradiogram obtained by binding of ¹²⁵I-labeled CS6 protein (B). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60∶35∶8, by volume) as solvent system, and the binding assay was performed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#s4" target="_blank">Materials and methods</a>”. Autoradiography was for 12 h. The lanes were: Lane 1, acid glycosphingolipids of human hepatoma, 40 µg; Lane 2, acid glycosphingolipids of human small intestine, 40 µg; Lane 3, acid glycosphingolipids of guinea pig erythrocytes, 40 µg; Lane 4, acid glycosphingolipids of guinea pig stomach, 40 µg; Lane 5, acid glycosphingolipids of human meconium, 40 µg; Lane 6, acid glycosphingolipids of human colon cancer, 40 µg; Lane 7, glucosylceramide (Glcβ1Cer), 4 µg. The band in marked with an X in lane 3 was stained blue by anisaldehyde, and thus a non-glycosphingolipid contaminant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#pone.0004487-Waldi1" target="_blank">[36]</a>.</p

Binding of ¹²⁵I-labeled CS6 protein to serial dilutions of pure glycosphingolipids in microtiter wells.

<p>The assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#s4" target="_blank">Materials and methods</a> section. The results from one representative experiment out of three is shown.</p

Binding of CS6 protein, vector <i>E. coli</i> TOP10 strain, and recombinant <i>E. coli</i> TOP10-CS6 strain to pure glycosphingolipids on thin-layer chromatograms.

<p>Chemical detection by anisaldehyde (A), and autoradiograms obtained by binding of ¹²⁵I-labeled CS6 protein (B), ³⁵S-labeled <i>E. coli</i> TOP10 strain (C) and ³⁵S-labeled <i>E. coli</i> TOP10-CS6 strain (D). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60∶35∶8, by volume) as solvent system, and the binding assays were performed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#s4" target="_blank">Materials and methods</a>”. Autoradiography was for 12 h. The lanes were: Lane 1, sulfatide (SO₃-3Galβ1Cer), 4 µg, and fucosyl-gangliotetraosylceramide (Fucα2Galβ3GalNAcβ4Galβ4Glcβ1Cer), 4 µg; Lane 2, sulfatide (SO₃-3Galβ1Cer), 4 µg, and Forssman glycosphingolipid (GalNAcα3GalNAcβ3Galα4Galβ4Glcβ1Cer), 4 µg; Lane 3, galactosylceramide (Galβ1Cer), 4 µg, and blood group H type 2 pentaglycosylceramide (Fucα2Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 4, blood group B type 2 hexaglycosylceramide (Galα3(Fucα2)Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 5, blood group A type 2 hexaglycosylceramide (GalNAcα3(Fucα2)Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 6, blood group A type 2 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer), 4 µg; Lane 7, globotriaosylceramide, (Galα4Galβ4Glcβ1Cer), 4 µg.</p

Binding of recombinant CS6-expressing <i>E. coli</i> to intestinal acid glycosphingolipids on thin-layer chromatograms.

<p>Chemical detection by anisaldehyde (A and C), and autoradiograms obtained by binding of ³⁵S-labeled CS6-expressing <i>E. coli</i> (TOP10-CS6) (B and D). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60∶35∶8, by volume) as solvent system, and the binding assays were performed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#s4" target="_blank">Materials and methods</a>”. Autoradiography was for 12 h. The lanes on A and B were: Lane 1, acid glycosphingolipids of human small intestine (Individual No. 1), 40 µg; Lane 2, acid glycosphingolipids of human small intestine (Individual No. 2), 40 µg; Lane 3, acid glycosphingolipids of human small intestine (Individual No. 3), 40 µg; Lane 4, acid glycosphingolipids of mouse small intestine, 40 µg; Lane 5, acid glycosphingolipids of rabbit small intestine, 40 µg. The arrow denotes a band stained blue by anisaldehyde, and thus a non-glycosphingolipid contaminant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#pone.0004487-Waldi1" target="_blank">[36]</a>. The lanes on C and D were: Lane 1, galactosylceramide (Galβ1Cer), 4 µg; Lane 2, acid glycosphingolipids of human small intestinal epithelium (Individual No. 4), 40 µg; Lane 3, sulfatide (SO₃-3Galβ1Cer) with phytosphingosine and hydroxy 16∶0 fatty acid from human small intestinal epithelium, 4 µg; Lane 4, sulfatide (SO₃-3Galβ1Cer) with phytosphingosine and hydroxy 24∶1 fatty acid from human small intestinal epithelium, 4 µg; Lane 5, sulf-gangliotetraosylceramide (SO₃-3Galβ3GalNAcβ4Galβ4Glcβ1Cer), 4 µg.</p

Binding of ¹²⁵I-labeled CS6 protein, and CssA and CssB subunits, to mixtures of glycosphingolipids on thin-layer chromatograms.

<p>Chemical detection by anisaldehyde (A), and autoradiograms obtained by binding of ¹²⁵I-labeled CS6 (B), CssA (C) and CssB (D). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60∶35∶8, by volume) as solvent system, and the binding assay was performed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004487#s4" target="_blank">Materials and methods</a>”. The lanes were: Lane 1, non-acid glycosphingolipids of human erythrocytes, 40 µg; Lane 2, non-acid glycosphingolipids of human small intestine (Individual No. 1), 40 µg; Lane 3, non-acid glycosphingolipids of human small intestine (Individual No. 2), 40 µg; Lane 4, non-acid glycosphingolipids of rat intestine, 40 µg; Lane 5, non-acid glycosphingolipids of human meconium, 40 µg; Lane 6, calf brain gangliosides, 40 µg; Lane 7, acid glycosphingolipids of human erythrocytes, 40 µg; Lane 8, acid glycosphingolipids of piglet intestine, 40 µg. Autoradiography was for 12 h.</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