Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin

Abstract

AB(5) toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB(5) toxin secreted by Shiga toxigenic Escherichia coli (STEC), which causes serious gastrointestinal disease in humans. SubAB causes haemolytic uraemic syndrome-like pathology in mice through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin's receptor is generated by metabolic incorporation of an exogenous factor derived from food.

Figure 1. Structural analysis of SubB-sialic acid interactions and comparison with other AB₅ toxins

a, Structures of Neu5Gc and Neu5Ac, showing additional O at C11 of the former. b, SubB protomer (orange) superimposed upon Ptx S2 (blue). N and C termini of SubB and Ptx S2 are designated with subscript S and P, respectively. c, Cartoon representation of the pentameric SubB-Neu5Gc structure, with each protomer colour-coded. Cyan sticks represent the sugar, with blue sticks representing nitrogen atoms and red sticks representing oxygen atoms. d. Neu5Gc in sialic acid receptor binding site of SubB. The extra hydroxyl of Neu5Gc points interacts with Tyr78^OH and also hydrogen bonds with the main chain of Met10. e, Trisaccharide Neu5Gcα2–3Galβ1–3GlcNAcβProN₃ binding to SubB. ProN3 refers to the linker used in the synthesis. f, Neu5Acα2–3-Gal binding site of Ptx S2/3, which shares similarity to the subB binding site, namely: Ser12 in SubB, Ser104 in Ptx S2/3; Gln36 in SubB, Arg125 in Ptx S2/3; Phe1 1 in SubB, Tyr103 in Ptx S2/3. Ptx does not have the equivalent of Tyr78 and Asp8 g, Side-on view of SubB: Neu5Gcα2–3Galβ1-GlcNAcβProN₃. h, Side-on view of CtxB:GM₁. In panels d-h, cyan sticks represent ligands, with dark blue sticks representing nitrogen atoms and red sticks oxygen atoms. Yellow sticks represent key residues in the protein backbone. Black dotted lines represent hydrogen bonds.

Figure 2. Fluorescence microscopy and Neu5Gc-dependent cytotoxicity

a, Binding of SubAB mutants to Vero cells. Vero cells growing on coverslips were incubated with 1 µg/ml OG-SubAB, OG-SubAB_A12 or Texas Red-SubAB_F78 for 60 min at 37°C (scale bar, 25 µm). b, Binding of SubAB to kidney sections from wild type and Cmah null mice. Frozen kidney sections were incubated with 1 µg/ml OG-SubAB for 60 min at 37°C, washed, and examined by epifluorescence microscopy (scale bar, 100 µm). c, Neu5Gc-dependent cytotoxicity. Cytotoxicity of SubAB for MDA-MB-231 cells or 293 cells after growth in medium supplemented with 3 mM Neu5Gc (Gc) or Neu5Ac (Ac) was determined as described in the Methods Summary. Data shown are from a single experiment. Analysis of data pooled from triplicate experiments yielded CD₅₀ values of 3.92 ± 1.58 pg (mean ± standard error) and 13.17 ± 2.46 pg for MDA cells fed Neu5Gc or Neu5Ac, (P = 0.034; 2-tailed t-test). For 293 cells fed Neu5Gc or Neu5Ac, the CD₅₀ values are 1.33 ± 0.30 pg and 11.58 ± 3.30 pg, respectively (P = 0.036). The mean fold increase in SubAB susceptibility (± SE) for cells fed Neu5Gc vs Neu5Ac is 5.56 ± 2.71 for MDA cells and 8.35 ± 0.88 for 293 cells.

Figure 3. Neu5Gc-dependent binding of SubAB to human tissues and toxicity of SubAB in wild type and Cmah null mice

a, Frozen sections of human colon were stained with chicken anti-Neu5Gc or control IgY at 5ug/ml, followed by anti-chicken IgY-HRP conjugate, and examined by immunohistochemistry (scale bar, 100 µm). b, Similar human colon sections were overlaid with or without 1 µg/ml SubAB or SubAB_A12 and bound toxin was detected using rabbit anti-SubA and Cy3-labeled goat anti-rabbit IgG and examined by epifluorescence microscopy (see Methods) (scale bar, 50 µm). c, Human colon sections were overlaid first with anti-Neu5Gc or control IgY at 5ug/ml, followed by 1 µg/ml SubAB, and bound toxin was detected as for panel b. Background control sections received only rabbit anti-SubAB, followed by Cy3-labeled anti-rabbit IgG (scale bar, 100 µm). d & e, Human kidney sections were overlaid 1 µg/ml SubAB, SubAB_A12, or SubAB_F78, and in the presence or absence of 10% human or chimpanzee serum, as indicated. Bound toxin was detected as for panel b (scale bar, 50 µm). f, Wild type and Cmah null mice (n = 8 each) were injected intraperitoneally with 200 ng/g purified SubAB in a total of 100 µl PBS, and survival time was recorded. Kaplan-Meier survival curves were plotted and statistical analysis (Wilcoxon-Gehan test) was performed in GraphPad Prism. g & h, Inhibition of SubAB binding to immobilized Neu5Gcα2–3Lac-HSA by wild type versus Cmah null mouse serum (f), or human versus chimpanzee serum (g) was assayed by ELISA as described in the Methods. Data are expressed as % of a control with no serum and are the mean ± SE of triplicate wells and representative of three independent experiments.