Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a, The sensitivities of the indicated HeLa KO cells to TcdB and TcdB 1-1830 were quantified using the cytopathic cell-rounding assay (see ) and normalized to WT HeLa cells as fold-of-resistance. The experiments have been repeated three times. b–c, Immunostaining analysis showed that TcdB binding (10 nM, 10 min) to CSPG4 −/− cells was reduced ( b ). Ectopic expression of NG2 (rat CSPG4) increased binding of TcdB. Transfection of FZD2 also increased TcdB binding to CSPG4 −/− cells ( c ). Scale bar = 20 μm. DIC: differential interference contrast. d, Ectopic expression of CSPG4 or FZD2 restored TcdB entry into CSPG4 −/− cells, resulting in cell-rounding (5 pM, 3 h). GFP marked transfected cells. Scale bar = 50 μm. e, A schematic illustration of FZD (upper panel). Fc-tagged FZD2-CRD binds to GST-tagged TcdB 1501-2366 , but not to GST-tagged CROPs. f–g, FZD2-CRD prevented TcdB (300 pM, 3 h) entry into CSPG4 −/− cells, measured by the cell-rounding assay ( f ) and glucosylation of Rac1 ( g ). Human IgG1-Fc (hIgG1-Fc) is a control. h, Transfection of FZD1, 2, and 7 each increased TcdB binding (10 nM, 10 min) to CSPG4 −/− cells, assayed by immunoblot analysis of cell lysates. Actin is a loading control. i, The sensitivities of FZD1 −/− , FZD2 −/− , FZD7 −/− , and FZD1/2/7 −/− cells to TcdB and TcdB 1-1830 were analyzed as described in panel a. j, Ectopic expression of FZD1, 2, or 7 restored TcdB 1-1830 entry into FZD1/2/7 −/− cells (300 pM, 3 h). Scale bar = 50 μm. k, Characterization of TcdB binding to Fc-tagged CRDs of FZD1, 2, 5, and 7 using the bio-layer interferometry (BLI) assay (see for K D analysis). Representative images are from one of three independent experiments. Error bars indicate mean ± s.d., n = 6, *P < 0.005, t -test.

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Immunostaining, Binding Assay, Expressing, Transfection, Western Blot

Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a, Schematic drawings of NG2 (rat CSPG4). Two fractions of recombinant extracellular domain (EC) fragments were used: one that does not contain chondroitin sulfate (CS) chains (EC P1), and the other that contains CS (EC P2). TMD-cyto: transmembrane and cytoplasmic domain. b, TcdB, but not TcdB 1-1830 , binds directly to both EC P1 and EC P2 of NG2 in a microtiter plate-based binding assay (error bars indicate mean ± s.d., two independent experiments). c, CSPG4 −/− cells transfected with the indicated constructs were exposed to TcdB (10 nM), TcdB 1-1830 (10 nM), or the receptor-binding domain of botulinum neurotoxin B (BoNT/B, 100 nM) for 10 min. Cell lysates were collected and subjected to immunoblot analysis. IL1RAPL2 and synaptotagmin II (Syt II, a receptor for BoNT/B) served as controls. Transfection of NG2 increased binding of TcdB, but not TcdB 1-1830 , whereas transfection of FZD2 increased binding of both TcdB and TcdB 1-1830 . One of three independent experiments is shown. d, The CROPs domain binds to CSPG4 on cell surfaces in a dose-dependent manner. High concentrations of recombinant CROPs reduced CSPG4-dependent binding of TcdB to cell surfaces, indicating that the CROPs can compete with TcdB for binding to CSPG4 on cell surfaces. One of three independent experiments is shown. e, The CROPs domain reduced cytopathic toxicity of TcdB (5 pM) on WT HeLa cells (error bars indicate mean ± s.d., two independent experiments). f, CSPG4 −/− cells were transfected with FZD2 and then exposed to TcdB or indicated TcdB fragments. FZD2 mediated binding of TcdB, TcdB 1-1830 , and TcdB 1501-2366 , but not the CROPs (TcdB 1831-2366 ). One of three independent experiments is shown.

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Recombinant, Binding Assay, Transfection, Construct, Western Blot

Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a, NG2-EC was immobilized on microtiter plates, followed by binding of TcdB, washing away unbound TcdB, and addition of FZD-CRD. FZD2-CRD binds robustly to TcdB that is pre-bound by NG2-EC on the microtiter plate. FZD2-CRD did not bind to NG2-EC without TcdB, and FZD5-CRD showed no detectable binding to NG2-TcdB in this assay (error bars indicate mean ± s.d., two independent experiments). b, Experiments are described in on HeLa (5 pM TcdB), HT-29 (50 pM TcdB), and Caco-2 cells (150 pM TcdB). Scale bars = 50 μm (HeLa and Caco-2) or 25 μm (HT-29). Representative images are from one of four independent experiments.

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Binding Assay

Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a–b, FZD2-CRD protected HT-29 ( a ) and Caco-2 cells ( b ) from TcdB 1-1830 (300 pM, 3 h). Representative images are from one of three independent experiments. Scale bars = 25 μm ( a ) or 50 μm ( b ). c, Expression of CSPG4 in HeLa, HT-29, and Caco-2 cells was examined via immunoblot analysis of cell lysates. One experiment from four is shown. d, Protection from TcdB using FZD2-CRD and NG2-EC on HeLa (5 pM TcdB), HT-29 (50 pM TcdB), and Caco-2 (150 pM TcdB) cells was quantified by the cytopathic cell-rounding assay. Representative images are shown in . Error bars indicate mean ± s.d..

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Expressing, Western Blot