Shiga toxin binding to glycolipids and glycans

Abstract

Background: Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable.

Methodology: We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells.

Results: By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells.

Conclusions: Stx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED(50)) of protein synthesis was about 10(-11) M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.

Figure 1. Binding of Stx1 and Stx2 to purified Pk trisaccharide and P tetrasaccharide by ITC.

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.

Figure 2. Glycan array results for Stx1.

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), N-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).

Figure 3. Stx binding to pure Gb3.

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.

Figure 4. Binding of Stx1 and Stx2 to purified glycolipids and mixtures.

Stx binding was assessed by ELISA at 10 nM for both Stx1 (white columns) and Stx2 (black columns) at 4°C. The RFU signal is the mean of three independent experiments and error bars indicate SD. Since different antibodies were used to detect Stx1 and Stx2, two axes are shown. (A) Binding of Stx1 and Stx2 to purified glycolipids and mixtures in absence of Ch and PC. Mixtures of glycolipids were prepared in methanol at a ratio of 1∶1 and added at 200 ng of total glycolipid per well. (B) Binding of Stx1 and Stx2 to purified glycolipids and mixtures in the presence of Ch and PC. Mixtures were prepared in methanol at a ratio of glycolipid 1, glycolipid 2, cholesterol, phosphatidylcholine 1∶1∶3∶3 and added at 200 ng of total glycolipid per well.

Figure 5. Stx binding to Gb3, Gb4 and Gb3/Gb4 mixture in the presence of cholesterol and phosphatidylcholine.

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.

Figure 6. Stx binding to Gb3 analogs.

Stx binding was assessed by ELISA at 10 nM for both Stx1 (A) and Stx2 (B) at 37°C. Gb3 −OH FA, with non-hydroxy Fatty Acid chain; +OH FA with hydroxy Fatty Acid chain. If not specified Gb3 is a standardized mixture that contains both variants with hydroxyl and nonhydroxyl fatty acid chains (Matreya Inc.). 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.

Figure 7. Stx binding to Lyso-Gb3.

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.

Figure 8. Comparison of Stx binding to Gb3 in absence of cholesterol or phosphatidylcholine.

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.

Figure 9. Stx binding to Gb3 in presence of a cholesterol analog.

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.

Figure 10. Vero protection studies.

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 Figure 5. 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 t-test using GraphPad Prism™ 5 (***, P = 0.0002).