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: Recombinant human proteins were purchased from ACRO Biosystems (IgG1 Fc and FZD2-CRD-Fc), R&D Systems (FZD1-CRD-Fc, FZD5-CRD-Fc, and FZD7-CRD-Fc), and Sino Biologics (PVRL3-EC).

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

Journal: PLoS Biology

Article Title: Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B

doi: 10.1371/journal.pbio.3001589

Figure Lengend Snippet: (A) Binding kinetics between TcdB and receptors are measured by BLI. The calculated binding affinities of TcdB to the 2 receptors, under pH 7.5 or pH 5, are labeled above each diagram. This measurement was calculated based on 2 biological replicates with standard deviations listed for each sample (raw data in ). (B) The structure comparisons of the TcdB-CRD2 complex at pH 7.5 and the 2 states at pH 5. (C) The helices (α2 and α4) of CRD2 that interact with TcdB are colored for each state. Dashed curves label the CRD-binding groove. Stars label the CRD-binding loop. Dark gray color labels regions in α-helices 2 and 4 that lack the cryo-EM density. The buried surface areas, calculated based on the model for the complex of TcdB and CRD2 under pH 7.5, state 1 and state 2 are around 700, 150, and 80 Å 2 , respectively. (D) The movement of the β-sheet (residues 1,812–1,827) in the D97 region are shown and color coded for each state. Tyr1819 of TcdB and its interaction partner Pro485 of CSPG4 are shown. (E) The same region rotated 90° from Panel D. BLI, bio-layer interferometry; CRD, cysteine-rich domain; CRD2, cysteine-rich domain of frizzled-2; TcdB, Toxin B.

Article Snippet: The purified TcdB (0.3 mg/mL) was preincubated with FZD2-CRD-Fc (R&D Systems, Minneapolis, MN) in a 50-mM PBS buffer at 1:1 ratio under room temperature and pH 7.5 prior to the pH change.

Techniques: Binding Assay, Labeling, Cryo-EM Sample Prep

Journal: PLoS Biology

Article Title: Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B

doi: 10.1371/journal.pbio.3001589

Figure Lengend Snippet: (A) TcdB (shown as a ribbon model) binds to the receptors (gray cartoon), CSPG4 and FZD2, before endosomal acidification. The proposed pore-forming helices of TcdB are shown as orange ribbon models. (B) TcdB dissociates from the receptors and the pore-forming helices rearrange. Gray arrows indicate the dissociation of TcdB. Orange arrows suggest the insertion of pore-forming helices into the endosomal membrane. CSPG4, chondroitin sulfate proteoglycan 4; FZD2, frizzled-2; TcdB, Toxin B.

Article Snippet: The purified TcdB (0.3 mg/mL) was preincubated with FZD2-CRD-Fc (R&D Systems, Minneapolis, MN) in a 50-mM PBS buffer at 1:1 ratio under room temperature and pH 7.5 prior to the pH change.

Techniques: Membrane