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Frizzled proteins are colonic epithelial receptors for C. difficile toxin B

Abstract

Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR–Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

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Figure 1: Genome-wide CRISPR–Cas9-mediated screens to identify host factors for TcdB.
Figure 2: FZDs are functional receptors for TcdB.
Figure 3: FZDs versus CSPG4 in cell lines.
Figure 4: FZDs are receptors for TcdB in colonic organoids.
Figure 5: FZDs are TcdB receptors in the colonic epithelium.

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Acknowledgements

We thank members of the Dong laboratory, L. Peng, X. Zhong, Q. Ma and M. Waldor for discussions; B. Ding and Y. Jing for their assistance in data analysis; N. Renzette and T. Kowalik for their advice and access to the NGS sequencer; H. Tatge for assistance on constructing toxin-expression plasmids; J. Nathans for providing FZD7 and FZD8-CRD–Myc–GPI constructs. This study was supported by National Institutes of Health (NIH) grants R01NS080833 (M.D.), R01AI091786 (A.L.B.), R01AT006732 (J.H.), R01DK084056 (D.T.B.), R01CA095287 (W.B.S.), K99DK100539 (J.M.), R01GM057603, R01GM074241 and R01AR060359 (X.H.). We also acknowledge support from the Bill and Melinda Gates Foundation (P.M. and A.L.B.), the Timothy Murphy Fund (D.T.B.), the Harvard Digestive Diseases Center (NIH P30DK034854, X.H., D.T.B. and M.D.), and the Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center (NIH P30HD18655, X.H., D.T.B. and M.D.). X.H. is an American Cancer Society Research Professor. M.D. and A.L.B. both hold the Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.

Author information

Affiliations

Authors

Contributions

L.T., A.L.B. and M.D. conceived the project. L.T. designed and conducted the majority of the experiments. J.Z. and L.T. developed the colon loop ligation model and conducted in vivo experiments. P.M. and A.L.B. prepared the CRISPR library and cells for screening and contributed to the screen design and data analysis. A.T. and D.T.B. prepared colonic organoids, performed immunofluorescent experiments, and analysed data. J.M. prepared adenoviruses and assisted with knockdown experiments. X.Z. and X.H. assisted with the Wnt signalling inhibition experiments and data analysis. W.B.S. provided key reagents/advice on CSPG4. R.G., X.W. and J.G.H. provided key advice/reagents on TcdB and TcdA. L.T. and M.D. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Min Dong.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks J. Ballard, N. Fairweather and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Recombinant TcdB and TcdB1–1830.

a, Schematic drawings of TcdB and a truncated TcdB lacking the CROP region (TcdB1–1830). CPD, cysteine protease domain; GTD, glucosyltransferase domain; RBD, receptor-binding domain, including a putative receptor-binding region and the CROPs region; TD, translocation domain. b, Coomassie blue staining (left) and immunoblot (right; chicken polyclonal TcdB antibody) showing TcdB and TcdB1–1830 recombinantly expressed in Bacillus megaterium. We note that TcdB1–1830 contains a contaminating protein visible on Coomassie blue-stained gel. Mass spectrometry analysis confirmed that this band is not a fragment of TcdB. The top matching protein is the bacterial chaperone protein ClpB. c, Cytopathic toxicity of recombinant TcdB and TcdB1–1830 on HeLa cells was neutralized by anti-TcdB polyclonal antibody (pAb), confirming that the toxicity is from TcdB and TcdB1–1830 (error bars indicate mean ± s.d., two independent experiments). d, HeLa, CHO, HT-29, and Caco-2 cells were exposed to TcdB or TcdB1–1830 as indicated for 24 h. TcdB1–1830 induced cell rounding at picomolar concentrations. Scale bars: 25 μm (HT-29) or 50 μm (HeLa, CHO and Caco-2). Representative images are from one of three independent experiments.

Extended Data Figure 2 Top-ranking sgRNAs.

a, Sequences of sgRNA were amplified by PCR after screening and subjected to NGS. The GeCKO v.2 sgRNA library is composed of two half libraries (library A and library B). Each half library contains three unique sgRNA per gene. These two half libraries were prepared and subjected to screens independently. be, Lists of top-ranking sgRNAs. See Source Data for lists of all identified sgRNAs.

Extended Data Figure 3 Assessing the sensitivity of HeLa knockout cells to TcdB and TcdB1–1830.

a, b, HeLa-Cas9 cells with the indicated genes mutated via CRISPR–Cas9, as well as wild-type (WT) Hela-Cas9 cells, were exposed to TcdB (a) or TcdB1–1830 (b) for 24 h. The percentages of rounded cells were quantified and plotted (error bars indicate mean ± s.d., three independent experiments). c, HeLa knockout cells were exposed to TcdB or TcdB1–1830 for 3 h. Cell lysates were subjected to immunoblot analysis for Rac1 and non-glucosylated (gluc.) Rac1. UGP2−/− cells retained high levels of non-glucosylated Rac1 after exposure to TcdB or TcdB1–1830. CSPG4−/− cells retained high levels of non-glucosylated Rac1 after exposure to TcdB. FZD2−/− and EMC4−/− cells showed slightly higher levels of non-glucosylated Rac1 compared to wild-type cells after exposure to TcdB1–1830. Representative blots are one from two independent experiments.

Extended Data Figure 4 CROPs are essential for TcdB binding to CSPG4, but not required for TcdB binding to FZDs.

a, Schematic drawings of rat CSPG4. Two pools 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 TcdB1–1830, binds directly to both EC P1 and EC P2 of CSPG4 in a microtitre 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), TcdB1–1830 (10 nM), or the receptor-binding domain of botulinum neurotoxin B (BoNT/BHC; 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 CSPG4 increased binding of TcdB, but not TcdB1–1830, whereas transfection of FZD2 increased binding of both TcdB and TcdB1–1830. One of three independent experiments is shown. d, The CROP 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 CROP domain reduced cytopathic toxicity of TcdB (5 pM) on wild-type (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, TcdB1–1830 and TcdB1501–2366, but not the CROPs (TcdB1831–2366). One of three independent experiments is shown.

Extended Data Figure 5 Characterizing TcdB binding to FZDs.

a, CSPG4−/− cells were transfected with 1D4-tagged FZD1, 2, 5, 7 and 9. Cells were exposed to TcdB (10 nM, 10 min), washed, fixed, permeabilized and subjected to immunostaining analysis. Scale bar, 20 μm. One of three independent experiments is shown. b, The CRD domains of human FZD1 (residues 102–235), FZD2 (residues 25–158) and FZD7 (residues 35–168) were aligned using the Vector NTI software. c, FZD7-CRD, but not FZD8-CRD, when expressed on the surface of CSPG4−/− cells via a GPI anchor, mediated binding of TcdB (10 nM, 10 min) to cells. One of three independent experiments is shown. d, Wild-type (WT) HeLa cells, FZD1/2/7−/− cells, and CSPG4−/− cells were exposed to TcdA and subjected to cytopathic cell-rounding assay. No reduction in sensitivity to TcdA was observed for FZD1/2/7−/− cells or CSPG4−/− cells, suggesting that TcdA does not use FZD1/2/7 or CSPG4 as its receptors (error bars indicate mean ± s.d., two independent experiments). e, f, Representative binding/dissociation curves for TcdB binding to Fc-tagged CRDs of FZD1, 2, 5 and 7 (e), and for TcdB1–1830 binding to FZD2-CRD-Fc (f). Binding parameters are listed in Supplementary Table 3. Representative curves are from one of three independent experiments. g, Wild-type and EMC4−/− cells were transfected with 1D4-tagged FZD1, 2 or 7. Cell lysates were subjected to immunoblot analysis. Expression of FZD1, 2 and 7 are reduced in EMC4−/− cells compared to wild-type cells (n = 6, *P < 0.005, one-way ANOVA). Representative blots are from one of three independent experiments. h, Expression levels of CSPG4 in EMC4−/− cells is similar to those in wild-type cells, suggesting that EMC is not required for single-pass transmembrane proteins. One of three independent experiments is shown.

Extended Data Figure 6 TcdB can bind to both FZD and CSPG4 simultaneously.

a, Rat CSPG4-EC was immobilized on microtitre 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 CSPG4-EC on the microtitre plate. FZD2-CRD did not bind to CSPG4-EC without TcdB, and FZD5-CRD showed no detectable binding to CSPG4–TcdB in this assay (error bars indicate mean ± s.d., two independent experiments). b, Experiments are described in Fig. 3d 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.

Extended Data Figure 7 PVRL3 failed to mediate binding and entry of TcdB in HeLa and Caco-2 cells.

a, CSPG4−/− HeLa cells transfected with the indicated constructs were exposed to TcdB in medium for 10 min. Cell lysates were collected and subjected to immunoblot analysis. Expression of PVRL3 was confirmed using an anti-PVRL3 antibody. Transfection of FZD2, but not PVRL-3, increased binding of TcdB to CSPG4−/− cells. One of three independent experiments is shown. b, Cells were challenged with TcdB (300 pM). Ectopic expression of PVRL3 failed to restore the sensitivity of CSPG4−/− HeLa cells towards TcdB, while expression of FZD2 restored entry of TcdB and resulted in rounding of transfected cells. Co-transfected GFP marked transfected cells. Scale bar, 50 μm. One of three independent experiments is shown. c, Recombinant extracellular domain of PVRL3 (PVRL3-EC) did not reduce TcdB entry into Caco-2 cells, analysed by the cytopathic cell-rounding assay. In contrast, FZD2-CRD prevented entry of TcdB into Caco-2 cells. Scale bar, 50 μm. One of three independent experiments is shown.

Extended Data Figure 8 Colonic organoids showed similar levels of sensitivity to TcdB and TcdB1–1830, and validation of FZD1 and FZD2 knockdown efficiency.

a, Colonic organoids were cultured from wild-type mice. They were exposed to a gradient of TcdB or TcdB1–1830. Viability of organoids was quantified using the MTT assay. TcdB and TcdB1–1830 showed similar IC50 values, suggesting that wild-type organoids are equally susceptible to TcdB and TcdB1–1830 (n = 8, error bars indicate mean ± s.d., two independent experiments). NS, not significant. b, Immunoblot analysis of CSPG4 expression in mouse brain, colonic organoids, mouse whole colon tissue, and isolated mouse colonic epithelium (200 μg cell/tissue lysates). The colonic epithelium was isolated from colon tissues by EDTA treatment (10 mM, 2 h at 4 °C). One of three independent experiments is shown. c, d, shRNA sequences targeting FZD1 and FZD2 were validated by measuring knockdown efficiency of transfected 1D4-tagged FZD1 and FZD2 in 293T cells. shRNAs marked with asterisks (shRNA2 for FZD1 and shRNA5 for FZD2) were used to generate adenoviruses. Actin served as the loading control. One of two independent experiments is shown.

Extended Data Figure 9 TcdB1114–1835 inhibits Wnt signalling and induces death of colonic organoids.

a, TcdB1114–1835 blocked WNT3A-mediated signalling in 293T cells in a dose-dependent manner. Increasing concentrations of WNT3A restored Wnt reporter activity blocked by TcdB1114–1835. Wnt signalling activity was analysed using the TOPFLASH/TK-Renilla dual luciferase reporter assay (error bars indicate mean ± s.d., two independent experiments). We note that 1.25 nM WNT3A equals 50 ng ml−1 concentration used in Fig. 4c. b, 293T cells in 24-well plates were exposed to WNT3A (50 ng ml−1) and TcdB1114-1835 in culture medium for 6 h. Cell lysates were harvested and subjected to immunoblot analysis for detecting phosphorylated DVL2 and LRP6. Wnt signalling activation results in phosphorylation of DVL2 and LRP6. Phosphorylated DVL2 is marked with an asterisk. One of three independent experiments is shown. c, Mouse colonic organoids were exposed to TcdB or TcdB1114–1835 for 12 h. Cell lysates were subjected to immunoblot analysis. No glucosylation (gluc.) of Rac1 was observed in organoids treated with TcdB1114–1835. One of two independent experiments is shown. d, Colonic organoids were exposed to TcdB1114–1835 for 72 h, with or without CHIR99021 (5 μM). Normal organoids (green arrow), growth inhibited organoids (red arrow), and disrupted/dead organoids (asterisk) are indicated. Scale bar, 200 μm. One of three independent experiments is shown. e, Time-course images of colonic organoids exposed to CHIR99021 (5 μM), TcdB1114–1835 (25 nM) or a combination of TcdB1114–1835 plus CHIR99021, at 0, 2, 4 and 6 days. Normal organoids (green arrow), growth inhibited organoids (red arrow), and disrupted/dead organoids (asterisk) are indicated. Scale bar, 500 μm. One of four independent experiments is shown.

Extended Data Figure 10 FZDs are receptors for TcdB in the colonic epithelium.

ac, Human colon cryosections were obtained from a commercial vendor and subjected to IHC analysis for detecting FZD7 (a), FZD2 (b) and CSPG4 (c). Ep, epithelial cells; Mf, sub-epithelial myofibroblasts. Scale bar, 50 μm. Representative images are from one of three independent experiments. d, Expression of FZD1 is not detectable in mouse or human colonic tissues. One of three independent experiments is shown. e, FZD7 antibody labelled wild-type colonic sections, but showed no signals on colonic tissues from FZD7−/− mice in IHC analysis, confirming the specificity of this antibody. One of three independent experiments is shown. f, Immunostaining of FZD2 (green) is reduced in FZD2-knockdown colonic organoids compared to control organoids, confirming the specificity of FZD2 antibody. Cell nuclei were labelled by DAPI (blue). Scale bar, 30 μm. One of three independent experiments is shown. g, Experiments are described in Fig. 5g. Representative images from one of three independent experiments are shown. Scale bar, 100 μm. h, Experiments were carried out as described in Fig. 5h. Low-magnification images of immunofluorescent staining of the cell–cell junction markers claudin-3 (green) and ZO-1 (red) were stitched together to show an overview of the colon tissue. The middle panel (WT/TcdB1–1830) showed disruption of the normal staining pattern for claudin-3 and ZO-1, indicating a loss of epithelial integrity, compared with both control and FZD7−/−/TcdB1–1830. Scale bar, 200 μm. Representative images are from one of three independent experiments. i, A schematic overview of cellular factors identified in the CRISPR–Cas9 screen. Validated and plausible cellular factors identified in our unbiased genome-wide screens were grouped based on their presence in the same protein complexes and/or signalling pathways. The colour of the gene names reflects the number of unique sgRNAs identified. The arrows link these genes to either confirmed or plausible roles in four major steps of TcdB action: (1) receptor-mediated endocytosis; (2) low pH in the endosomes triggers conformational changes of the TD, which translocates the GTD across endosomal membranes; (3) GTD is later released via auto-proteolysis by the CPD, which is activated by the cytosolic co-factor inositol hexakisphosphate (InsP6); (4) released GTD glucosylates small GTPases such as Rho, Rac, and CDC42.

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Tao, L., Zhang, J., Meraner, P. et al. Frizzled proteins are colonic epithelial receptors for C. difficile toxin B. Nature 538, 350–355 (2016). https://doi.org/10.1038/nature19799

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