Article | Published:

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

Nature volume 538, pages 350355 (20 October 2016) | Download Citation

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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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

Author notes

    • Jie Zhang
    •  & Paul Meraner

    These authors contributed equally to this work.

Affiliations

  1. Department of Urology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Liang Tao
    • , Jie Zhang
    •  & Min Dong
  2. Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Liang Tao
    • , Jie Zhang
    •  & Min Dong
  3. Department of Microbiology and Physiological Systems (MaPS), University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

    • Paul Meraner
    •  & Abraham L. Brass
  4. Division of Endocrinology, Boston Children’s Hospital, Boston, Massachusetts 02115, USA.

    • Alessio Tovaglieri
    • , Ji Miao
    •  & David T. Breault
  5. Center for Infectious and Inflammatory Diseases, Texas A & M Health Science Center, Houston, Texas 77030, USA.

    • Xiaoqian Wu
    •  & Julian G. Hurdle
  6. Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.

    • Ralf Gerhard
  7. The F. M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Xinjun Zhang
    •  & Xi He
  8. Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Xinjun Zhang
    •  & Xi He
  9. Tumor Microenvironment and Cancer Immunology Program, Sanford-Burnham Prebys Medical Discovery Institute, Cancer Center, La Jolla, California 92037, USA.

    • William B. Stallcup
  10. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Ji Miao
    •  & David T. Breault
  11. Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA.

    • David T. Breault
  12. Gastroenterology Division, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

    • Abraham L. Brass

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Min Dong.

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

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DOI

https://doi.org/10.1038/nature19799

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