Abstract
Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.
Highlights
E. coli Shiga like toxin type 2 (Stx2a) is responsible for serious clinical outcomes
The Crystal Structure of Stx2a with Bound Ligand 2—Stx2a was co-crystallized with a PkNAc disaccharide methyl glycoside 2 (Fig. 2C), and the structure of the complex was determined by x-ray crystallography (Table 1)
In previous studies we showed that PolyBAIT-Pk protects human serum amyloid P-component (HuSAP)-tg mice against Stx1 at concentrations ϳ10-fold lower (3.15 g/g mouse), but treatment with PolyBAIT-Pk at a concentration as high as 100 g/g mouse afforded only modest protection against Stx2a infection [32]
Summary
E. coli Shiga like toxin type 2 (Stx2a) is responsible for serious clinical outcomes. In this Gb3 analog, ␣-GalNAc replaces the terminal ␣-Gal residue This co-crystal structure confirms previous inferences that two of the primary binding sites identified in the B5 pentamer of Stx are functional in Stx2a. The intrinsic affinity of Stx and Stx2a binding to Gb3 is millimolar at best and among the lowest measured protein-carbohydrate interactions [18], toxin binding to cells expressing Gb3 is translated into nanomolar avidity due to the fluidity of glycolipids in the lipid bilayer This permits lateral translation of receptors into an array that allows for optimally spaced multivalent binding via the numerous saccharide binding sites of the B5 pentamer [19]. We report here a solved crystal structure of Stx2a with bound disaccharide 2 (Fig. 2C) and the activity of inhibitor 3 (Fig. 2D) of Shigatoxemia in transgenic mice designed based on inferences drawn from this crystal structure
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