Stx binding to Gb3 in presence of a cholesterol analog.

<p>Stx binding was assessed by ELISA at 10 nM for both Stx1 (A) and Stx2 (B) at 37°C. As negative controls, toxin was incubated in methanol-coated wells. The RFU signal is the mean of three independent experiments and error bars indicate SD. Statistical differences were calculated by the two-tailed Student's t-test using GraphPad Prism™ 5.</p

Primers used in this study.

<p>Primers used in this study.</p

Glycan array results for Stx1.

<p>Binding of Stx1 (2.84 µM) toxoid to the Consortium for functional Glycomics Mammalian Array Version 4.1 with 465 different natural and synthetic mammalian glycans was assessed by ELISA. Displayed are the top three hits for Stx1 (glycans 331, 402, and 120). For comparison, also displayed is native Pk (glycan 121), glycan 119 which is attached using the same linker as native Pk, and glycan 120, which is attached with a different linker from 119. The symbolic representation of the compounds follows the CFG standards: galactose (Gal, white circle), glucose, (Glc, black circle), <i>N</i>-acetyl-glucosamine (GlcNAc, black square), mannose (Man, gray circle). X corresponds to β1-4GlcNAcβ1-4GlcNAcβ-LVANKT. Spacers used to couple the glycans to the array surface matrix: Sp0, -CH₂CH₂NH₂; Sp8, -CH₂CH₂CH₂NH₂; LVANKT, peptide (Leucine, L; valine, V; alanine, A; asparagine, N; lysine, K; threonine, T). Relative fluorescence units (RFU) signal is the mean of four independent experiments and error bars indicate Standard Deviation (SD).</p

Stx binding to pure Gb3.

<p>Stx1 (black squares, ▪) and Stx2 (black circles, •) toxoid binding affinity to Gb3 alone was assessed by ELISA at 4°C. Stx1 binding as fitted to a one-site specific binding model with Hill coefficients. Symbols represent experimental data, while lines represent the fitted model for that data analyzed with Prism5 (GraphPad software, La Jolla, CA). Values for Stx2 were not determined due to poor binding. The RFU signal is the mean of three independent experiments and error bars indicate SD.</p

Binding of Stx1 and Stx2 to purified Pk trisaccharide and P tetrasaccharide by ITC.

<p>Glycans (50 mM) were titrated into a microcalorimeter cell containing 238–300 µM of Stx B-subunits. Stx binding interaction with Pk (A, B) and P tetrasaccharide (C, D). Both Stx1 B-subunits (A–C) and Stx2 B-subunits (B–D) raw heat signals (top) and integrated data from titrations (bottom) are shown.</p

Stx binding to Gb3, Gb4 and Gb3/Gb4 mixture in the presence of cholesterol and phosphatidylcholine.

<p>Stx1 (A) and Stx2 (B) toxoid binding was assessed by ELISA at 37°C. As negative controls, toxin was incubated in methanol, PC, Ch, or PC+Ch coated wells. In all experiments, background RFU values obtained in methanol were subtracted from each value. Binding curves were fitted to a one-site specific binding model with Hill coefficients. Symbols represent experimental data, while lines represent the fitted model for that data analyzed with Prism5 (GraphPad software, La Jolla, CA). The RFU signal is the mean of three independent experiments and error bars indicate SD.</p

Vero protection studies.

<p>Stx cellular toxicity was assessed using luciferase activity of Luc2p Vero cells treated with dilutions of Stx1 (A) or Stx2 (B) pre-incubated with glycolipid mixtures as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030368#pone-0030368-g005" target="_blank">Figure 5</a>. As negative controls, toxin was untreated or incubated in methanol-coated wells or PC+Ch. The results are the average of three independent experiments. Statistical difference was calculated between untreated control and Gb3+PC+Ch treatment by the two-tailed Student's <i>t</i>-test using GraphPad Prism™ 5 (***, <i>P</i> = 0.0002).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Glycolipids used in this study.

<p>Glycolipids used in this study.</p

Comparison of Stx binding to Gb3 in absence of cholesterol or phosphatidylcholine.

<p>Stx binding was assessed by ELISA at 10 nM for both Stx1 and Stx2 at 37°C as described in Experimental Procedures. As negative controls, toxin was incubated in methanol, PC, Ch or PC+Ch coated wells. In all experiments, RFU values obtained in methanol were subtracted from each value in order to define a base level. The RFU signal is the mean of three independent experiments and error bars indicate SD. Statistical differences were calculated by the two-tailed Student's t-test using GraphPad Prism™ 5.</p