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

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 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 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 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 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

Development of antibody responses over the course of the infection experiment.

<p>For each treatment group and sampling occasion, the mean (with 95% confidence interval) of the birds' antibody responses (mOD/min) to membrane proteins of the (A) human <i>C. jejuni</i> isolate 00F4382 and (B) the Song Thrush isolate 00-4:268 are given. The control birds are shown in blue, TR1 birds in green, and TR2 birds in yellow. Samples were taken at days 0, 6, 13, and 25 post-infection.</p

Body mass changes in European Robins <i>Erithacus rubecula</i> over the course of the infection experiment.

<p>Body mass is presented as the mean (±2 SE) of all individuals in each treatment group for each day over the course of the experiment. Challenge time points are marked with stars (*) for the human <i>C. jejuni</i> isolate 00F4382 and a bracket (#) for the Song Thrush isolate 00-4:268. Blood sampling time points are noted with hatched reference lines at days 0, 6, 13, and 25 post-infection.</p