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The binary toxin CDT enhances Clostridium difficile virulence by suppressing protective colonic eosinophilia

Nature Microbiology volume 1, Article number: 16108 (2016) | Download Citation


Clostridium difficile is the most common hospital acquired pathogen in the USA, and infection is, in many cases, fatal. Toxins A and B are its major virulence factors, but expression of a third toxin, known as C. difficile transferase (CDT), is increasingly common. An adenosine diphosphate (ADP)-ribosyltransferase that causes actin cytoskeletal disruption, CDT is typically produced by the major, hypervirulent strains and has been associated with more severe disease. Here, we show that CDT enhances the virulence of two PCR-ribotype 027 strains in mice. The toxin induces pathogenic host inflammation via a Toll-like receptor 2 (TLR2)-dependent pathway, resulting in the suppression of a protective host eosinophilic response. Finally, we show that restoration of TLR2-deficient eosinophils is sufficient for protection from a strain producing CDT. These findings offer an explanation for the enhanced virulence of CDT-expressing C. difficile and demonstrate a mechanism by which this binary toxin subverts the host immune response.

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  1. 1.

    et al. Burden of Clostridium difficile infection in the United States. N. Engl. J. Med. 372, 825–834 (2015).

  2. 2.

    Clostridium difficile infection: epidemiology, risk factors and management. Nature Rev. Gastroenterol. Hepatol. 8, 17–26 (2010).

  3. 3.

    et al. The role of toxin A and toxin B in Clostridium difficile infection. Nature 467, 711–713 (2010).

  4. 4.

    , & The role of toxin A and toxin B in Clostridium difficile-associated disease. Gut Microbes 1, 58–64 (2010).

  5. 5.

    , , & Effects of Clostridium difficile toxins given intragastrically to animals. Infect. Immun. 47, 349–352 (1985).

  6. 6.

    et al. Clostridium difficile toxin A promotes dendritic cell maturation and chemokine CXCL2 expression through p38, IKK, and the NF-κB signaling pathway. J. Mol. Med. (Berl) 87, 169–180 (2009).

  7. 7.

    et al. MAPK-activated protein kinase 2 contributes to Clostridium difficile-associated inflammation. Infect. Immun. 81, 713–722 (2013).

  8. 8.

    , & Predicting recurrence of C. difficile colitis using bacterial virulence factors: binary toxin is the key. J. Gastrointest. Surg. 17, 118–125 (2013).

  9. 9.

    , , & Binary toxin and death after Clostridium difficile infection. Emerg. Infect. Dis. 17, 976–982 (2011).

  10. 10.

    et al. Clinical features of Clostridium difficile-associated infections and molecular characterization of strains: results of a retrospective study, 2000–2004. Infect. Control Hosp. Epidemiol. 28, 131–139 (2007).

  11. 11.

    , & Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nature Rev. Microbiol. 7, 526–536 (2009).

  12. 12.

    et al. Clostridium difficile infection in Europe: a hospital-based survey. Lancet 377, 63–73 (2011).

  13. 13.

    & Comparative analysis of Clostridium difficile clinical isolates belonging to different genetic lineages and time periods. J. Med. Microbiol. 53, 1129–1136 (2004).

  14. 14.

    , , & Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain. Infect. Immun. 56, 2299–2306 (1988).

  15. 15.

    et al. Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being. Toxins 6, 2626–2656 (2014).

  16. 16.

    et al. Interaction of the Clostridium difficile binary toxin CDT and its host cell receptor LSR. J. Biol. Chem. 290, 14031–14044 (2015).

  17. 17.

    et al. Lipolysis-stimulated lipoprotein receptor (LSR) is the host receptor for the binary toxin Clostridium difficile transferase (CDT). Proc. Natl Acad. Sci. USA 108, 16422–16427 (2011).

  18. 18.

    , , & Clostridium difficile binary toxin CDT. Gut Microbes 5, 15–27 (2014).

  19. 19.

    et al. Analysis of the ‘angulin’ proteins LSR, ILDR1 and ILDR2—tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis. J. Cell Sci. 126, 966–977 (2013).

  20. 20.

    , , & Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins. Microbiol. Mol. Biol. Rev. 68, 373–402 (2004).

  21. 21.

    et al. Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence. Proc. Natl Acad. Sci. USA 111, 2313–2318 (2014).

  22. 22.

    et al. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathogens 5, e1000626 (2009).

  23. 23.

    , , & Intestinal inflammatory biomarkers and outcome in pediatric Clostridium difficile infections. J. Pediatr. 163, 1697–1704 (2013).

  24. 24.

    et al. Innate immune defenses mediated by two ILC subsets are critical for protection against acute clostridium difficile infection. Cell Host Microbe 18, 27–37 (2015).

  25. 25.

    et al. Interleukin-22 regulates the complement system to promote resistance against pathobionts after pathogen-induced intestinal damage. Immunity 41, 620–632 (2014).

  26. 26.

    et al. Role of IL-23 signaling in Clostridium difficile colitis. J. Infect. Dis. 208, 917–920 (2013).

  27. 27.

    et al. Inflammasome activation contributes to interleukin-23 production in response to Clostridium difficile. mBio 6, e02386–14 (2015).

  28. 28.

    et al. The importance of toxin A, toxin B and CDT in virulence of an epidemic Clostridium difficile strain. J. Infect. Dis. 209, 83–86 (2013).

  29. 29.

    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, e00551–15 (2015).

  30. 30.

    et al. A mouse model of Clostridium difficile-associated disease. Gastroenterology 135, 1984–1992 (2008).

  31. 31.

    et al. Selection of nanobodies that block the enzymatic and cytotoxic activities of the binary Clostridium difficile toxin CDT. Sci. Rep. 5, 7850 (2015).

  32. 32.

    et al. Microbiota-regulated IL-25 increases eosinophil number to provide protection during Clostridium difficile infection. Cell Rep. (2016).

  33. 33.

    et al. Granulocyte macrophage colony-stimulating factor-activated eosinophils promote interleukin-23 driven chronic colitis. Immunity 43, 187–199 (2015).

  34. 34.

    , & Eosinophils: changing perspectives in health and disease. Nature Rev. Immunol. 13, 9–22 (2013).

  35. 35.

    & Roles and regulation of gastrointestinal eosinophils in immunity and disease. J. Immunol. 193, 999–1005 (2014).

  36. 36.

    & Eosinophil progenitors in allergy and asthma—do they matter?. Pharmacol. Ther. 121, 174–184 (2009).

  37. 37.

    et al. Allergen-induced fluctuation in CC chemokine receptor 3 expression on bone marrow CD34+ cells from asthmatic subjects: significance for mobilization of haemopoietic progenitor cells in allergic inflammation. Immunology 109, 536–546 (2003).

  38. 38.

    et al. Mechanisms of acute eosinophil mobilization from the bone marrow stimulated by interleukin 5: the role of specific adhesion molecules and phosphatidylinositol 3-kinase. J. Exp. Med. 188, 1621–1632 (1998).

  39. 39.

    & Eosinophil progenitors in airway diseases: clinical implications. Chest 134, 1037–1043 (2008).

  40. 40.

    , & Haemopoietic processes in allergic disease: eosinophil/basophil development. Clin. Exp. Allergy 39, 1297–1306 (2009).

  41. 41.

    et al. Thymic stromal lymphopoietin and IL-33 modulate migration of hematopoietic progenitor cells in patients with allergic asthma. J. Allergy Clin. Immunol. 135, 1594–1602 (2015).

  42. 42.

    Behavior of eosinophil leukocytes in acute inflammation. II. Eosinophil dynamics during acute inflammation. J. Clin. Invest. 56, 870–879 (1975).

  43. 43.

    & Experimental observations on the eosinopenia induced by acute infection. Br. J. Exp. Pathol. 52, 214–220 (1971).

  44. 44.

    , , , & TLR2 agonist ameliorates murine experimental allergic conjunctivitis by inducing CD4 positive T-cell apoptosis rather than by affecting the Th1/Th2 balance. Biochem. Biophys. Res. Commun. 339, 1048–1055 (2006).

  45. 45.

    et al. TLR-2 activation induces regulatory T cells and long-term suppression of asthma manifestations in mice. PLoS ONE 8, e55307 (2013).

  46. 46.

    et al. TLR2 agonist ameliorates established allergic airway inflammation by promoting Th1 response and not via regulatory T cells. J. Immunol. 174, 7558–7563 (2005).

  47. 47.

    et al. Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator. J. Bacteriol. 189, 7290–7301 (2007).

  48. 48.

    , , , & Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile. PLoS One 8, e79666 (2013).

  49. 49.

    , , , & Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. Infect. Immun. 65, 1402–1407 (1997).

  50. 50.

    Measuring the inflammasome. Methods Mol. Biol. 844, 199–222 (2012).

  51. 51.

    et al. Murine model of Clostridium difficile infection with aged gnotobiotic C57BL/6 mice and a BI/NAP1 strain. J. Infect. Dis. 202, 1708–1712 (2010).

  52. 52.

    et al. Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J. Immunol. 181, 4004–4009 (2008).

  53. 53.

    , , , & Eosinophil adoptive transfer system to directly evaluate pulmonary eosinophil trafficking in vivo. Proc. Natl Acad. Sci. USA 110, 6067–6072 (2013).

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The authors thank the UVA Research Histology and Flow Cytometry Cores for their assistance with sample preparation and analysis. The authors acknowledge TechLab for providing purified toxins A and B and TOX A/B ELISA kits. The authors thank A. Criss, J. Casanova, U. Lorenz and M. Kendall for discussions. C.A.C. was supported by the Robert R. Wagner Fellowship from the University of Virginia School of Medicine and by NIH training grant 5T32AI07046-38. E.L.B. was supported by NIH grants T32AI07496 and F31AI114203. M.M.S. was supported by NIH grant 2T32GM008715-16. D.L. was supported by Future Fellowship FT120100779 (from the Australian Research Council). This work was supported by NIH grants R01AI124214, R01AI026649 and R21AI114734 to W.A.P.

Author information


  1. Departments of Microbiology, Immunology and Cancer Biology, University of Virginia Health System, Charlottesville, Virginia 22908 USA

    • Carrie A. Cowardin
    • , Erica L. Buonomo
    • , Mahmoud M. Saleh
    • , Madeline G. Wilson
    • , Stacey L. Burgess
    •  & William A. Petri Jr
  2. Clostridia Research Group, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK

    • Sarah A. Kuehne
    •  & Nigel P. Minton
  3. Institute of Experimental and Clinical Pharmacology and Toxicology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany

    • Carsten Schwan
    •  & Klaus Aktories
  4. Institute of Immunology, University Medical Center Hamburg-Eppendorf, D20246 Hamburg, Germany

    • Anna M. Eichhoff
    •  & Friedrich Koch-Nolte
  5. Department of Microbiology, Infection and Immunity Program, Monash Biomedicine Discovery Institute, and Monash University, Victoria 3800, Australia

    • Dena Lyras
  6. Departments of Medicine, University of Virginia Health System, Charlottesville, Virginia 22908 USA

    • William A. Petri Jr
  7. Departments of Pathology, University of Virginia Health System, Charlottesville, Virginia 22908, USA

    • William A. Petri Jr


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C.A.C. conceived and designed the experiments, performed the experiments, analysed the data and wrote the paper. E.L.B. performed the experiments, provided valuable advice and contributed materials. M.M.S., M.G.W. and S.L.B performed the experiments. S.A.K., C.S., A.M.E., F.K.-N., D.L., K.A. and N.P.M. contributed materials and valuable advice on experimental design. W.A.P. assisted with the experimental design and edited the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to William A. Petri Jr.

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