Clostridium difficile toxin A (TcdA) is a major exotoxin contributing to disruption of the colonic epithelium during C. difficile infection. TcdA contains a carbohydrate-binding combined repetitive oligopeptides (CROPs) domain that mediates its attachment to cell surfaces, but recent data suggest the existence of CROPs-independent receptors. Here, we carried out genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)-mediated screens using a truncated TcdA lacking the CROPs, and identified sulfated glycosaminoglycans (sGAGs) and low-density lipoprotein receptor (LDLR) as host factors contributing to binding and entry of TcdA. TcdA recognizes the sulfation group in sGAGs. Blocking sulfation and glycosaminoglycan synthesis reduces TcdA binding and entry into cells. Binding of TcdA to the colonic epithelium can be reduced by surfen, a small molecule that masks sGAGs, by GM-1111, a sulfated heparan sulfate analogue, and by sulfated cyclodextrin, a sulfated small molecule. Cells lacking LDLR also show reduced sensitivity to TcdA, although binding between LDLR and TcdA are not detected, suggesting that LDLR may facilitate endocytosis of TcdA. Finally, GM-1111 reduces TcdA-induced fluid accumulation and tissue damage in the colon in a mouse model in which TcdA is injected into the caecum. These data demonstrate in vivo and pathological relevance of TcdA–sGAGs interactions, and reveal a potential therapeutic approach of protecting colonic tissues by blocking these interactions.
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The data that support the findings of this study are available from the corresponding authors upon request.
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We thank Y. Matsuura (Osaka University) and A. Jonathan (Harvard Medical School) for providing cDNA and cell lines, H. Tatge (Hannover Medical School) for toxin purification, J. Savage (Glycomira) for providing GM-1111 and C. Araneo (Harvard Medical School) for assisting flow cytometry analysis. This study was partially supported by National Institute of Health (NIH) grants (R01NS080833, R01AI132387, R01AI139087, and R21NS106159 to M.D.). R.G. acknowledges support by the Federal State of Lower Saxony, Niedersächsisches Vorab (VWZN2889, VWZN3215 and VWZN3266). M.D. and D.T.B. acknowledge support by the NIH-funded Harvard Digestive Disease Center (P30DK034854) and Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center (P30HD18655). L.T. acknowledges support by the National Natural Science Foundation of China (Grant no. 31800128). M.D. and S.P.J.W hold the Investigator in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund.
The authors declare no competing interests.
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Tao, L., Tian, S., Zhang, J. et al. Sulfated glycosaminoglycans and low-density lipoprotein receptor contribute to Clostridium difficile toxin A entry into cells. Nat Microbiol 4, 1760–1769 (2019). https://doi.org/10.1038/s41564-019-0464-z