Abstract
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.
References
Theriot, C. M. & Young, V. B. Interactions between the gastrointestinal microbiome and Clostridium difficile. Annu. Rev. Microbiol. 69, 445–461 (2015).
Collins, J. et al. Dietary trehalose enhances virulence of epidemic Clostridium difficile. Nature 553, 291–294 (2018).
McDonald, L. C. et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N. Engl. J. Med. 353, 2433–2441 (2005).
Hunt, J. J. & Ballard, J. D. Variations in virulence and molecular biology among emerging strains of Clostridium difficile. Microbiol. Mol. Biol. Rev. 77, 567–581 (2013).
Lessa, F. C. et al. Burden of Clostridium difficile infection in the United States. N. Engl. J. Med. 372, 825–834 (2015).
Lyras, D. et al. Toxin B is essential for virulence of Clostridium difficile. Nature 458, 1176–1179 (2009).
Carter, G. P. et al. Defining the roles of TcdA and TcdB in localized gastrointestinal disease, systemic organ damage, and the host response during Clostridium difficile infections. mBio 6, e00551 (2015).
Kuehne, S. A. et al. The role of toxin A and toxin B in Clostridium difficile infection. Nature 467, 711–713 (2010).
Kuehne, S. A. et al. Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J. Infect. Dis. 209, 83–86 (2014).
Aktories, K., Schwan, C. & Jank, T. Clostridium difficile toxin biology. Annu. Rev. Microbiol. 71, 281–307 (2017).
Cowardin, C. A. et al. The binary toxin CDT enhances Clostridium difficile virulence by suppressing protective colonic eosinophilia. Nat. Microbiol. 1, 16108 (2016).
Chumbler, N. M. et al. Crystal structure of Clostridium difficile toxin A. Nat. Microbiol. 1, 15002 (2016).
Krivan, H. C., Clark, G. F., Smith, D. F. & Wilkins, T. D. Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Galα1-3Galβ1-4GlcNAc. Infect. Immun. 53, 573–581 (1986).
Tucker, K. D. & Wilkins, T. D. Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y. Infect. Immun. 59, 73–78 (1991).
Teneberg, S. et al. Molecular mimicry in the recognition of glycosphingolipids by Galα3 Galβ4 GlcNAcβ-binding Clostridium difficile toxin A, human natural anti α-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor. Glycobiology 6, 599–609 (1996).
Genisyuerek, S. et al. Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B. Mol. Microbiol. 79, 1643–1654 (2011).
Olling, A. et al. The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A. PLoS ONE 6, e17623 (2011).
Yuan, P. et al. Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B. Cell Res. 25, 157–168 (2015).
LaFrance, M. E. et al. Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity. Proc. Natl Acad. Sci. USA 112, 7073–7078 (2015).
Tao, L. et al. Frizzled proteins are colonic epithelial receptors for C. difficile toxin B. Nature 538, 350–355 (2016).
Chen, P. et al. Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B. Science 360, 664–669 (2018).
Pothoulakis, C. et al. Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A. J. Clin. Invest. 98, 641–649 (1996).
Na, X., Kim, H., Moyer, M. P., Pothoulakis, C. & LaMont, J. T. gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A. Infect. Immun. 76, 2862–2871 (2008).
Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Kreuger, J. & Kjellen, L. Heparan sulfate biosynthesis: regulation and variability. J. Histochem. Cytochem. 60, 898–907 (2012).
Chaves-Olarte, E. et al. UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii. J. Biol. Chem. 271, 6925–6932 (1996).
Barth, H. et al. Low pH-induced formation of ion channels by Clostridium difficile toxin B in target cells. J. Biol. Chem. 276, 10670–10676 (2001).
Qa’Dan, M., Spyres, L. M. & Ballard, J. D. pH-induced conformational changes in Clostridium difficile toxin B. Infect. Immun. 68, 2470–2474 (2000).
Smith, R. D. & Lupashin, V. V. Role of the conserved oligomeric Golgi (COG) complex in protein glycosylation. Carbohydr. Res. 343, 2024–2031 (2008).
Foulquier, F. et al. TMEM165 deficiency causes a congenital disorder of glycosylation. Am. J. Hum. Genet. 91, 15–26 (2012).
Jae, L. T. et al. Deciphering the glycosylome of dystroglycanopathies using haploid screens for Lassa virus entry. Science 340, 479–483 (2013).
Tanaka, A. et al. Genome-wide screening uncovers the significance of N-sulfation of heparan sulfate as a host cell factor for chikungunya virus infection. J. Virol. 91, e00432-17 (2017).
Tian, S. et al. Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation. PLoS Biol. 16, e2006951 (2018).
Schuksz, M. et al. Surfen, a small molecule antagonist of heparan sulfate. Proc. Natl Acad. Sci. USA 105, 13075–13080 (2008).
Zhang, J. et al. Novel sulfated polysaccharides disrupt cathelicidins, inhibit RAGE and reduce cutaneous inflammation in a mouse model of rosacea. PLoS ONE 6, e16658 (2011).
Yamamoto, S. et al. Lipoprotein receptors redundantly participate in entry of hepatitis C virus. PLoS Pathog. 12, e1005610 (2016).
Fisher, C., Beglova, N. & Blacklow, S. C. Structure of an LDLRRAP complex reveals a general mode for ligand recognition by lipoprotein receptors. Mol. Cell 22, 277–283 (2006).
Finkelshtein, D., Werman, A., Novick, D., Barak, S. & Rubinstein, M. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc. Natl Acad. Sci. USA 110, 7306–7311 (2013).
Schorch, B. et al. LRP1 is a receptor for Clostridium perfringens TpeL toxin indicating a two-receptor model of clostridial glycosylating toxins. Proc. Natl Acad. Sci. USA 111, 6431–6436 (2014).
Griffin, C. C. et al. Isolation and characterization of heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin. Carbohydr. Res. 276, 183–197 (1995).
Bode, L. et al. Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function. J. Clin. Investig. 118, 229–238 (2008).
Yamamoto, S. et al. Heparan sulfate on intestinal epithelial cells plays a critical role in intestinal crypt homeostasis via Wnt/β-catenin signaling. Am. J. Physiol. Gastrointest. Liver Physiol. 305, G241–G249 (2013).
Sauerborn, M., Leukel, P. & von Eichel-Streiber, C. The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality. FEMS Microbiol. Lett. 155, 45–54 (1997).
Zhang, Y. et al. The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model. Anaerobe 48, 249–256 (2017).
Lindahl, U., Couchman, J., Kimata, K. & Esko, J. D. Essentials of Glycobiology 3rd edn (eds Varki, A. et al.) Ch. 17 (Cold Spring Harbor Laboratory Press, 2015).
Kamhi, E., Joo, E. J., Dordick, J. S. & Linhardt, R. J. Glycosaminoglycans in infectious disease. Biol. Rev. Camb. Phil. Soc. 88, 928–943 (2013).
Jeon, H. & Blacklow, S. C. Structure and physiologic function of the low-density lipoprotein receptor. Annu. Rev. Biochem. 74, 535–562 (2005).
Agnello, V., Abel, G., Elfahal, M., Knight, G. B. & Zhang, Q. X. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl Acad. Sci. USA 96, 12766–12771 (1999).
Gustafsen, C. et al. Heparan sulfate proteoglycans present PCSK9 to the LDL receptor. Nat. Commun. 8, 503 (2017).
Bomsel, M. & Alfsen, A. Entry of viruses through the epithelial barrier: pathogenic trickery. Nat. Rev. Mol. Cell Biol. 4, 57–68 (2003).
Acknowledgements
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.
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L.T. and M.D. initiated and designed the project. L.T. and S.T. carried out the CRISPR–Cas9 screen. L.T., S.T. and J.Z. carried out colon loop ligation assays. S.T. and J.Z. carried out caecum-injection assays. Z.L., L.R.-M. and S.P.J.W. generated heparan sulfate-deficient cells, analysed cell surface heparan sulfate levels and provided related reagents. S.M. purified LDLR–Fc. R.G. provided TcdA and performed the experiment on CHO cells. D.T.B. and S.O. provided key reagents and advice. L.T. and M.D. wrote the manuscript with input from all co-authors.
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Supplementary Figures 1–9, raw images used in figures and legend for Supplementary Dataset.
Supplementary Data 1
Lists of all sgRNA sequences and target genes identified from CRISPR–Cas9 screen.
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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
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DOI: https://doi.org/10.1038/s41564-019-0464-z
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