IgA and IgG serum antibody responses to CS5 and CS6 in individual patients.

<p>The responses to CS5 (broken lines) and CS6 (solid lines) were measured by ELISA in sera from patients infected with strains E1777, E1785 and E1779, on different days after hospitalization. Serum from the patient infected with strain E2265 was not available.</p

Strains used in the study and results from culturing^a of clinical stool specimens.

a<p>Culture was performed on MacConkey agar plates to detect <i>E. coli</i> and on Taurocholate-tellurite-gelatin agar (TTGA) for detection of vibrios; all cultures were performed overnight at 37°C.</p>b<p>Department of Microbiology and Immunology, University of Gothenburg, enterotoxigenic <i>Escherichia coli</i> (ETEC) strain collection number.</p

Primers used for PCR and real-time Reverse Transcriptase PCR (rt RT-PCR).

a<p>CS5 minor subunit<b>;</b> GenBank accession number <u>AJ224079</u>.</p>b<p>CS6 structural subunit CssB; GenBank accession number <u>UO4846</u>.</p>c<p>Primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035827#pone.0035827-Nicklasson1" target="_blank">[44]</a>.</p>d<p><i>E. coli</i> D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH); GenBank accession number <u>U014639.1.</u></p

Phenotypic CS5 levels in LB alone and LB supplemented with bile or individual bile salts.

<p>CS5 expression was quantified by inhibition ELISA after overnight culture to stationary phase of strains E1777, E1779, E1785, E2265, and E3003. CS5 surface expression tifwas induced by NaGCH, but not by the corresponding unconjugated bile salt NaCH or its tauro-conjugated counterpart TCA (representative data).</p

Dose-dependent induction of phenotypic CS5 expression by NaGCH.

<p>Expression of CS5 was determined by inhibition ELISA in strain E1777 after overnight culture to stationary phase in LB medium supplemented with NaGCH or crude bile. Bars indicate means and standard errors of the means of two measurements in one experiment.</p

Levels of <i>csfD</i> and <i>cssB</i> transcription compared to in LB alone after one hour of culture.

<p>Level of <i>csfD</i> (A) and <i>cssB</i> (B) transcription in LB supplemented with crude bile or individual bile salts standardized to the level of transcription in LB medium alone, after one hour of culture. Transcription was measured by reverse transcriptase real time PCR. Bars show means and standard errors of the means of three separate experiments (strains E1785, E2265, and E3003, respectively). *, P = <0.05</p

Transcriptional <i>csfD</i>:<i>ccsB</i> ratio <i>in vivo</i> and <i>in vitro</i>.

<p>Transcription of <i>csfD</i> and <i>cssB</i> was quantified by real time reverse transcriptase PCR (rt RT-PCR) in <i>in vivo</i> bacterial samples (strains E1777, E1785, E2265; closed bar) and after one (strains E1785, E2265, E3003; open bars) and two (E1777, E1779; hatched bars) hours of <i>in vitro</i> culture in LB medium alone and in LB supplemented with 0.15% crude bile or 0.2% individual bile salts. Bars show the means and standard errors of the means of two (after two hours of culture) or three (<i>in vivo</i> and after one hour of culture) separate experiments.</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 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