Skip to main content
SpringerLink
Log in

A human gut ecosystem protects against C. difficile disease by targeting TcdA

  • Original Article—Alimentary Tract
  • Published:
Journal of Gastroenterology Aims and scope Submit manuscript

Abstract

Background

A defined Microbial Ecosystem Therapeutic (MET-1, or “RePOOPulate”) derived from the feces of a healthy volunteer can cure recurrent C. difficile infection (rCDI) in humans. The mechanisms of action whereby healthy microbiota protect against rCDI remain unclear. Since C. difficile toxins are largely responsible for the disease pathology of CDI, we hypothesized that MET-1 exerts its protective effects by inhibiting the effects of these toxins on the host.

Methods

A combination of in vivo (antibiotic-associated mouse model of C. difficile colitis, mouse ileal loop model) and in vitro models (FITC-phalloidin staining, F actin Western blots and apoptosis assay in Caco2 cells, transepithelial electrical resistance measurements in T84 cells) were employed.

Results

MET-1 decreased both local and systemic inflammation in infection and decreased both the cytotoxicity and the amount of TcdA detected in stool, without an effect on C. difficile viability. MET-1 protected against TcdA-mediated damage in a murine ileal loop model. MET-1 protected the integrity of the cytoskeleton in cells treated with purified TcdA, as indicated by FITC-phalloidin staining, F:G actin assays and preservation of transepithelial electrical resistance. Finally, co-incubation of MET-1 with purified TcdA resulted in decreased detectable TcdA by Western blot analysis.

Conclusions

MET-1 intestinal microbiota confers protection against C. difficile and decreases C. difficile-mediated inflammation through its protective effects against C. difficile toxins, including enhancement of host barrier function and degradation of TcdA. The effect of MET-1 on C. difficile viability seems to offer little, if any, contribution to its protective effects on the host.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Rohlke F, Stollman N. Fecal microbiota transplantation in relapsing Clostridium difficile infection. Ther Adv Gastroenterol. 2012;5(6):403–20.

    Article  Google Scholar 

  2. Lozupone CA, Stombaugh JI, Gordon JI, et al. Diversity, stability and resilience of the human gut microbiota. Nature. 2012;489(7415):220–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gough E, Shaikh H, Manges AR. Systematic review of intestinal microbiota transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection. Clin Infect Dis. 2011;53:994–1002.

    Article  PubMed  Google Scholar 

  4. van Nood E, Vrieze A, Nieuwdorp M, et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med. 2013;368(5):407–15.

    Article  PubMed  Google Scholar 

  5. Britton RA, Young VB. Interaction between the intestinal microbiota and host in Clostridium difficilecolonization resistance. Trends Microbiol. 2012;20(7):313–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Seekatz AM, Theriot CM, Molloy CT, et al. Fecal microbiota transplantation eliminates Clostridium difficile in a murine model of relapsing disease. Infect Immun. 2015;83(10):3838–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wilson KH. The microecology of Clostridium difficile. Clin Infect Dis. 1993;16(Suppl 4):S214–8.

    Article  PubMed  Google Scholar 

  8. Li M, Liang P, Li Z, et al. Fecal microbiota transplantation and bacterial consortium transplantation have comparable effects on the re-establishment of mucosal barrier function in mice with intestinal dysbiosis. Front Microbiol. 2015;6:692.

    PubMed  PubMed Central  Google Scholar 

  9. Buffie CG, Bucci V, Stein RR, et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature. 2015;517(7533):205–8.

    Article  CAS  PubMed  Google Scholar 

  10. Sorg JA, Sonenshein AL. Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol. 2008;190(7):2505–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ng KM, Ferreyra JA, Higginbottom SK, et al. Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. Nature. 2013;502(7469):96–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kuehne SA, Cartman ST, Heap JT, et al. The role of toxin A and toxin B in Clostridium difficile infection. Nature. 2010;467(7316):711–3.

    Article  CAS  PubMed  Google Scholar 

  13. Kuehne SA, Collery MM, Kelly ML, et al. Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J Infect Dis. 2014;209(1):83–6.

    Article  CAS  PubMed  Google Scholar 

  14. Lyras D, O’Connor JR, Howarth PM, et al. Toxin B is essential for virulence of Clostridium difficile. Nature. 2009;458(7242):1176–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Planche TD, Davies KA, Coen PG, et al. Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C. difficile infection. Lancet Infect Dis. 2013;13(11):936–45.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Polage CR, Gyorke CE, Kennedy MA, et al. Overdiagnosis of Clostridium difficile infection in the molecular test era. JAMA Intern Med 2015;175(11):1792–801.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Gerding DN, Meyer T, Lee C, et al. Administration of spores of nontoxigenic Clostridium difficile strain M3 for prevention of recurrent C. difficile infection: a randomized clinical trial. JAMA. 2015;313(17):1719–27.

    Article  PubMed  Google Scholar 

  18. Lawley TD, Clare S, Walker AW, et al. Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. PLoS Pathog. 2012;8(10):e1002995.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Petrof EO, Gloor GB, Vanner SJ, et al. Stool substitute transplant therapy for the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut. Microbiome. 2013;1(1):3.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Bidet P, Barbut F, Lalande V, et al. Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett. 1999;175(2):261–6.

    Article  CAS  PubMed  Google Scholar 

  21. Sullivan NM, Pellett S, Wilkins TD. Purification and characterization of toxins A and B of Clostridium difficile. Infect Immun. 1982;35(3):1032–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Chen X, Katchar K, Goldsmith JD, Nanthakumar N, et al. A mouse model of Clostridium difficile-associated disease. Gastroenterology. 2008;135(6):1984–92.

    Article  PubMed  Google Scholar 

  23. Martz SL, McDonald JA, Sun J, et al. Administration of defined microbiota is protective in a murine Salmonella infection model. Sci Rep 2015;5:16094.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Castagliuolo I, Riegler M, Pasha A, et al. Neurokinin-1 (NK-1) receptor is required in Clostridium difficile-induced enteritis. J Clin Invest. 1998;101(8):1547–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen X, Kokkotou EG, Mustafa N, et al. Saccharomyces boulardii inhibits ERK1/2 mitogen-activated protein kinase activation both in vitro and in vivo and protects against Clostridium difficile toxin A-induced enteritis. J Biol Chem. 2006;281(34):24449–54.

    Article  CAS  PubMed  Google Scholar 

  26. Ishida Y, Maegawa T, Kondo T, et al. Essential involvement of IFN-gamma in Clostridium difficile toxin A-induced enteritis. J Immunol. 2004;172(5):3018–25.

    Article  CAS  PubMed  Google Scholar 

  27. McDonald JA, Fuentes S, Schroeter K, et al. Simulating distal gut mucosal and luminal communities using packed-column biofilm reactors and an in vitro chemostat model. J Microbiol Methods. 2015;108:36–44.

    Article  CAS  PubMed  Google Scholar 

  28. Gloor GB, Hummelen R, Macklaim JM, et al. Microbiome profiling by illumina sequencing of combinatorial sequence-tagged PCR products. PLoS One. 2010;5:e15406.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Caporaso JG, Lauber CL, Walters WA, et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012;6(8):1621–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Masella AP, Bartram AK, Truszkowski JM, et al. PANDAseq: paired-end assembler for illumina sequences. BMC Bioinf. 2012;13:31.

    Article  CAS  Google Scholar 

  31. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26(19):2460–1.

    Article  CAS  PubMed  Google Scholar 

  32. Edgar RC, Haas BJ, Clemente JC, et al. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27(16):2194–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schloss PD, Westcott SL, Ryabin T, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009;75(23):7537–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yilmaz P, Parfrey LW, Yarza P, et al. The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks. Nucl Acid Res. 2014;42:D643–8.

    Article  CAS  Google Scholar 

  35. Aitchison J. The statistical analysis of compositional data. New Jersey: Chapman and Hall; 1986.

    Book  Google Scholar 

  36. Lovell D, Pawlowsky-Glahn V, Egozcue JJ, et al. Proportionality: a valid alternative to correlation for relative data. PLoS Comput Biol 2015;11(e):1004075.

  37. Fernandes AD, Reid JN, Macklaim JM, et al. Unifying the analysis of high-throughput sequencing datasets: characterizing RNA-seq, 16S rRNA gene sequencing and selective growth experiments by compositional data analysis. Microbiome. 2014;2:15.

    Article  PubMed  PubMed Central  Google Scholar 

  38. van den Boogaart K, Tolosana-Delgado R. Analyzing compositional data with R. New York: Springer; 2013.

    Book  Google Scholar 

  39. R Core Team. R: a language and environment for statistical computing. Vienna, Austria. 2015; Available from: http://www.R-project.org/.

  40. Palarea-Albaladejo J, Martín-Fernández JA. zCompositions: R package for multivariate imputation of left-censored data under a compositional approach. Chemometr Intell Lab. 2015;143:85–96.

    Article  CAS  Google Scholar 

  41. Rao K, Erb-Downward JR, Walk ST, et al. The systemic inflammatory response to Clostridium difficile infection. PLoS One. 2014;9(3):e92578.

    Article  PubMed  PubMed Central  Google Scholar 

  42. George RH, Symonds JM, Dimock F, et al. Identification of Clostridium difficile as a cause of pseudomembranous colitis. Br Med J. 1978;1(6114):695.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Bartlett JG, Chang TW, Gurwith M, et al. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med. 1978;298(10):531–4.

    Article  CAS  PubMed  Google Scholar 

  44. Chang TW, Lauermann M, Bartlett JG. Cytotoxicity assay in antibiotic-associated colitis. J Infect Dis. 1979;140(5):765–70.

    Article  CAS  PubMed  Google Scholar 

  45. Hecht G, Pothoulakis C, LaMont JT, et al. Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers. J Clin Invest. 1988;82(5):1516–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Liu TS, Musch MW, Sugi K, et al. Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction. Am J Physiol Cell Physiol. 2003;284(4):C1073–82.

    Article  CAS  PubMed  Google Scholar 

  47. Moore R, Pothoulakis C, LaMont JT, et al. C. difficile toxin A increases intestinal permeability and induces Cl- secretion. Am J Physiol. 1990;259(2 Pt 1):G165–72.

    CAS  PubMed  Google Scholar 

  48. Musch MW, Petrof EO, Kojima K, et al. Bacterial superantigen-treated intestinal epithelial cells upregulate heat shock proteins 25 and 72 and are resistant to oxidant cytotoxicity. Infect Immun. 2004;72(6):3187–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hirota SA, Iablokov V, Tulk SE, et al. Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure. Infect Immun. 2012;80(12):4474–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sanders DS. Mucosal integrity and barrier function in the pathogenesis of early lesions in Crohn’s disease. J Clin Pathol. 2005;58(6):568–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Sun Z, Wang X, Wallen R, et al. The influence of apoptosis on intestinal barrier integrity in rats. Scand J Gastroenterol. 1998;33(4):415–22.

    Article  CAS  PubMed  Google Scholar 

  52. Zeissig S, Bojarski C, Buergel N, et al. Downregulation of epithelial apoptosis and barrier repair in active Crohn’s disease by tumour necrosis factor alpha antibody treatment. Gut. 2004;53(9):1295–302.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Castagliuolo I, LaMont JT, Nikulasson ST, et al. Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum. Infect Immun. 1996;64(12):5225–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Castagliuolo I, Riegler MF, Valenick L, et al. Saccharomyces boulardii protease inhibits the effects of Clostridium difficile toxins A and B in human colonic mucosa. Infect Immun. 1999;67(1):302–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Surawicz CM, McFarland LV, Greenberg RN, et al. The search for a better treatment for recurrent Clostridium difficile disease: use of high-dose vancomycin combined with Saccharomyces boulardii. Clin Infect Dis. 2000;31(4):1012–7.

    Article  CAS  PubMed  Google Scholar 

  56. Islam J, Taylor AL, Rao K, et al. The role of the humoral immune response to Clostridium difficile toxins A and B in susceptibility to C. difficile infection: a case-control study. Anaerobe. 2014;27:82–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lyerly DM, Saum KE, MacDonald DK, et al. Effects of clostridium difficile toxins given intragastrically to animals. Infect Immun. 1985;47(2):349–52.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Savidge TC, Pan WH, Newman P, et al. Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine. Gastroenterology. 2003;125(2):413–20.

    Article  CAS  PubMed  Google Scholar 

  59. Kim H, Riley TV, Kim M, et al. Increasing prevalence of toxin A-negative, toxin B-positive isolates of Clostridium difficile in Korea: impact on laboratory diagnosis. J Clin Microbiol. 2008;46(3):1116–7.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Rupnik M, Kato N, Grabnar M, et al. New types of toxin A-negative, toxin B-positive strains among Clostridium difficile isolates from Asia. J Clin Microbiol. 2003;41(3):1118–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Shin BM, Kuak EY, Yoo HM, et al. Multicentre study of the prevalence of toxigenic Clostridium difficile in Korea: results of a retrospective study 2000–2005. J Med Microbiol. 2008;57(Pt 6):697–701.

    Article  CAS  PubMed  Google Scholar 

  62. Cairns MD, Preston MD, Lawley TD, et al. Genomic epidemiology of a protracted hospital outbreak caused by a toxin A-negative clostridium difficile sublineage PCR Ribotype 017 strain in London, England. J Clin Microbiol. 2015;53(10):3141–7.

    Article  CAS  PubMed  Google Scholar 

  63. Munoz S, Guzman-Rodriguez M, Sun J, et al. Rebooting the Microbiome. Gut Microb. 2016;13:1–11.

    Google Scholar 

  64. Wirtz S, Neufert C, Weigmann B, et al. Chemically induced mouse models of intestinal inflammation. Nat Protoc. 2007;2(3):541–6.

    Article  CAS  PubMed  Google Scholar 

  65. Aitchison J, Greenacre M. Biplots of compositional data. J Ry Stat Soc: Ser C (Appl Stat). 2002;51:375–92.

    Article  Google Scholar 

Download references

Acknowledgments

This study was supported by the Canada Foundation for Innovation and the Southeastern Ontario Academic Medical Organization (E.O.P) by NIH Grant No R21AI121575 (E.O.P, E.A.V., G.B.G) and by a Queen’s University CTAQ award (E.O.P and D.J.H). M.G.R. was supported by postdoctoral fellowship CONACYT-263618.

Author information

Authors and Affiliations

  1. Division of Infectious Diseases/GI Diseases Research Unit Wing, Department of Medicine, Kingston General Hospital, Queen’s University, 76 Stuart Street, Kingston, ON, K7L 2V7, Canada

    Sarah Lynn Martz, Mabel Guzman-Rodriguez, Shu-Mei He, Curtis Noordhof & Elaine Olga Petrof

  2. Department of Pathology and Molecular Medicine, Queen’s University, Kingston, ON, K7L 2V7, Canada

    David John Hurlbut

  3. Department of Biochemistry, University of Western Ontario, London, ON, N6A 5C1, Canada

    Gregory Brian Gloor

  4. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada

    Christian Carlucci, Scott Weese & Emma Allen-Vercoe

  5. Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA

    Jun Sun

  6. Departments of Pediatrics and Medicine, University of Chicago, Chicago, IL, 60637, USA

    Erika Chiong Claud

Authors
  1. Sarah Lynn Martz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mabel Guzman-Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shu-Mei He
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Curtis Noordhof
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David John Hurlbut
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gregory Brian Gloor
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christian Carlucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Scott Weese
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Emma Allen-Vercoe
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jun Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Erika Chiong Claud
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Elaine Olga Petrof
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elaine Olga Petrof.

Ethics declarations

Conflict of interest

E.O.P and E.A-V. are co-founders of Nubiyota and have filed a patent for MET-1 through Parteq Innovations (Queen’s University). The other authors have no conflict of interests to declare.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Martz, S.L., Guzman-Rodriguez, M., He, SM. et al. A human gut ecosystem protects against C. difficile disease by targeting TcdA. J Gastroenterol 52, 452–465 (2017). https://doi.org/10.1007/s00535-016-1232-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00535-016-1232-y

Keywords

Search

Navigation